WO2023030180A1 - Biological angstrom pore system based on mechanosensitive channel of small conductance - Google Patents

Biological angstrom pore system based on mechanosensitive channel of small conductance Download PDF

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WO2023030180A1
WO2023030180A1 PCT/CN2022/115022 CN2022115022W WO2023030180A1 WO 2023030180 A1 WO2023030180 A1 WO 2023030180A1 CN 2022115022 W CN2022115022 W CN 2022115022W WO 2023030180 A1 WO2023030180 A1 WO 2023030180A1
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medium
angstrom
pore
insulating film
mscs
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French (fr)
Chinese (zh)
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耿佳
包锐
柯博文
陈路
赵长健
李开菊
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四川大学
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
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    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
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    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Definitions

  • the invention belongs to the field of nanopore detection, and in particular relates to a biological angstrom pore system based on a small conductance mechanical force sensitive channel.
  • Nanopore single-molecule detection technology is a sensing and detection technology that has the advantages of simple operation, high sensitivity, fast detection speed, and no need for labeling. It is widely used in protein detection, gene sequencing, and marker detection. At present, the cost, sensitivity and precision of genetic testing are the main problems to be solved in the development of this testing technology, so the development of new nanoporous materials is the key means to solve these problems.
  • a biological nanopore is a naturally occurring nanoscale pore with a pore size similar to that of many important biomolecules. As molecules pass through the channels inside the nanopore, specific blockage currents and translocation events arise. According to the blocking current and translocation frequency of molecules, qualitative and quantitative analysis of target molecules can be achieved. Therefore, the channel pore size is the dominant factor affecting the detection ability and application range of nanopores.
  • Some protein nanopores with suitable channel pore sizes have been used for nanobiotechnology applications, such as ⁇ -hemolysin ( ⁇ -HL), MspA, CsgG, Aerolysin, phi29-linked device etc.
  • biological nanopores are mainly derived from bacterial porins or viral phyla, and have pore diameters (1.0 nm-3.6 nm) about the size of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Therefore, they are suitable for detecting nucleic acids and have been used in DNA/RNA sequencing, nucleic acid biomarker detection, and biomolecular interaction studies.
  • biological nanopores need to be locally modified according to specific application requirements, such as site-directed mutagenesis or modification of specific aptamers, etc., in order to adapt to a wider range of sequencing.
  • ⁇ -HL its limited pore size is about 1.4nm, so the scope of application is limited to the analysis of ssDNA, RNA or other molecules.
  • cyclodextrin (cyclodextrin) modification it can be used to directly detect monophosphate deoxyribose nucleus Glycoside dNMPs without fluorescent labeling.
  • changing the pore size of biological nanopores through modification requires a lot of bioengineering technology assistance.
  • protein pores are much less flexible in adjusting the size. In this sense, it is urgent to find a nanopore with a flexible structure to efficiently detect molecules of various sizes.
  • the present invention provides a novel biological angstrom pore system based on a small conductance mechanosensitive channel.
  • the angstrom pore is a pore size smaller than a nanopore Protein, this novel protein angstrompore system does not require aptamers or modifications, providing a low-cost, highly versatile new approach for real-time molecular sensing, genetic detection, and DNA computing.
  • the present invention provides an application of an angstrom hole system in detecting charged molecules, characterized in that, the angstrom hole system comprises an angstrom hole, an insulating film, a first medium, and a second medium; An angstrom hole is embedded in the insulating film, the insulating film separates the first medium from the second medium, the angstrom hole provides communication between the first medium and the second medium channel, after applying a driving force between the first medium and the second medium, the charged molecules located in the first medium interact with the angstrom pore; the angstrom pore is MscS Mipore, the Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening.
  • the charge properties and/or pore size of the openings are adjustable.
  • the way of adjusting the opening includes subjecting the insulating film to mechanical stimulation and/or changing the physical state of the insulating film.
  • the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure.
  • the change in the physical state of the insulating film includes a change in the thickness of the insulating film, a change in the composition of the insulating film, a change in the curvature of the surface of the insulating film, and the like.
  • the aperture of the opening can be adjusted in the following manner:
  • the emipore is derived from bacillus.
  • the pore is derived from one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
  • the emipore is a variant of MscS.
  • the MscS variants include side hole volume variants and/or side hole charge variants.
  • the insulating film includes a phospholipid film and/or a polymer film.
  • the charged molecules include one or more of nucleotides, amino acids, peptides, and drug molecules.
  • the emipore is a variant of PaMscS.
  • the angstrompore includes one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
  • the molar mass of the drug molecule is less than 1000 g/mol.
  • the drug molecule may be pyrophosphate, gentamicin sulfate, neomycin sulfate, sisomicin, glutamic acid and the like.
  • first medium and/or the second medium include one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
  • the present invention also provides a biological angstrompore system, characterized in that the biological angstrompore system includes an angstrompore, an insulating film, a first medium, and a second medium, and the In the insulating film, the insulating film separates the first medium from the second medium, and the Angstrom hole provides a channel connecting the first medium and the second medium; the Angstrom hole It is a MscS variant angstrom pore, and the angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening.
  • Angstrom pore is a side pore volume variant and/or a side pore charge variant of MscS.
  • the insulating film includes a phospholipid film and/or a polymer film.
  • the phospholipid membrane includes DPHPC, DOPC, E.coli lipid; the polymer membrane includes a triblock copolymer polymer membrane.
  • the emipore is derived from bacillus.
  • the pore is derived from one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
  • the emipore is a variant of PaMscS.
  • the angstrompore includes one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
  • the mutation sites of the above-mentioned variants are located at the side opening of the cytoplasmic end, specifically involving changes in the volume and charge properties of amino acids.
  • the pore diameter of the mutated side hole (also can be understood as "pore size") can be changed, thereby improving the detection ability of molecules with a specific molecular volume; the local charge characteristics of the mutated side hole channel can also be changed , and then improve the detection ability of specific charged molecules; it can also enhance the stability of the protein channel current of the mutant PaMscS angstrom pore.
  • the charge properties and/or pore size of the openings are adjustable.
  • the manner of adjusting the opening includes subjecting the insulating film to mechanical force stimulation and/or changing the physical state of the insulating film.
  • the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
  • the aperture of the opening can be adjusted in the following manner:
  • the present invention also provides the application of the above-mentioned biological angstrompore system in the detection of charged molecules, characterized in that the charged molecules include one or more of nucleotides, amino acids, peptides, and drug molecules .
  • the invention provides an application of an angstrom hole system in detecting charged molecules, wherein the angstrom hole system comprises a MscS angstrom hole.
  • the present invention creatively forms a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS) into an angstrom pore system, and uses the characteristics of the mechanosensitive channel protein to detect charged molecules, specifically embodied as follows:
  • the pore size of the MscS pore is narrow.
  • the pore size of the MscS angstrompore is estimated to be in the range of ⁇ 6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the ⁇ -hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy).
  • the pore size of the MscS angstrompore is adjustable (it can also be understood as a flexible structure).
  • MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration.
  • the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e.
  • 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to achieve optimal pairing.
  • Protein nanopores in the prior art usually have a fixed channel structure, which requires additional protein engineering modification or chemical modification to achieve channel structure adjustment.
  • the pore diameter of the MscS angstrompore provided by the present invention can be reversibly adjusted in situ only by changing the external conditions, and is suitable for direct detection of molecules of various types and sizes.
  • the angstrom pore system provided by the present invention can be applied to the sensing and detection of single molecules.
  • the angstrom pore system provided by the present invention is applicable to a variety of charged molecules (in theory, as long as the molecules with a size smaller than the pore diameter of the MscS pore can be sensed and detected), for example, nucleotides, amino acids, peptides, drug molecules etc.; while larger-sized nucleic acids (such as ssDNA) and proteins (such as proteins in whole blood samples) cannot enter the channel of the MscS angstrompore and will not interfere with the detection molecules.
  • the angstrom system provided by the present invention has wide application scenarios.
  • mutations can be introduced into the side pore of the MscS angstrompore, adjusting the volume (e.g., W to A, S, P) and charge (e.g., W to H, K to R) of the amino acids at the side pore. ), to achieve better detection of specific charged molecules and molecules of specific sizes.
  • the angstrom pore system provided by the present invention can directly detect single nucleotides, and can also be used with consumption strategies (for example, detecting the remaining nucleotides of the nucleic acid amplification system) to identify the presence or absence of the target nucleic acid in the sample, For example, in the diagnosis of SARS-CoV-2 samples, it exhibited good specificity and sensitivity.
  • the angstrom system provided by the present invention can detect the presence or absence of drug molecules in complex samples (such as whole blood), and can also directly measure the drug concentration in whole blood with molar sensitivity and the effect on blood drug concentration in living animals. Continuous, real-time monitoring exhibits robustness and sensitivity.
  • the angstrom pore system provided by the present invention can also detect amino acids and short peptides (eg, dipeptides).
  • the present invention also provides a method for detecting nucleotides in a sample, which is characterized by comprising the following steps:
  • the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the angstrom hole is embedded in the insulating film, the The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, the angstrom hole is a MscS angstrom hole,
  • the Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening; the sample is added to the first medium;
  • S2 applies a driving force to the first medium and the second medium, and the nucleotides in the sample interact with the angstrom pores and generate electrical signals;
  • S3 analyzes the electrical signal, and then identifies nucleotides in the sample.
  • the charge properties and/or pore size of the openings are adjustable.
  • the way of adjusting the opening includes subjecting the insulating film to mechanical stimulation and/or changing the physical state of the insulating film.
  • the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
  • the aperture of the opening can be adjusted in the following manner:
  • the osmotic pressure difference between the first medium and the second medium is adjusted by the concentration difference between the first medium and the second medium.
  • the concentration difference between the first medium and the second medium is about 0-270 mM.
  • Amypore is a MscS variant Amypore.
  • the MscS variants include side hole volume variants and/or side hole charge variants.
  • the emipore is derived from bacillus.
  • the pore includes one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
  • the Amipore is a PaMscS variant Amipore.
  • the mutation site of the angmipore of the PaMscS variant is located at the side opening of the cytoplasmic region of the PaMscS.
  • the PaMscS variant angstrompore includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
  • nucleotides include one or more of dGTP, dATP, dTTP, dCTP, dUTP, GTP, ATP, TTP, CTP, UTP.
  • the insulating film includes a phospholipid film and/or a polymer film.
  • first medium and/or the second medium include one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
  • the present invention also provides a rapid detection kit for nucleotides, characterized in that the kit includes:
  • the MscS Angstrom pore includes a side pore volume variant and/or a side pore charge variant of MscS.
  • the insulating film includes a phospholipid film and/or a polymer film.
  • the conductive solution includes one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
  • the MscS pore includes a PaMscS variant pore.
  • the PaMscS variant angstrompore includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
  • the invention provides a method for detecting nucleotides in a sample by using an angstrom hole system, wherein the angstrom hole system includes a MscS angstrom hole.
  • the present invention creatively utilizes the characteristics of a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS) to detect nucleotides in a sample, specifically embodied as follows:
  • the pore size of the MscS pore is narrow.
  • the pore size of the MscS angstrompore is estimated to be in the range of ⁇ 6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the ⁇ -hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy).
  • the pore size of the MscS angstrompore is adjustable (it can also be understood as a flexible structure).
  • MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration.
  • the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e.
  • 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to achieve optimal pairing.
  • Protein nanopores in the prior art usually have a fixed channel structure, and the direct detection of nucleotides (for example, dNTP and other similar molecules) usually requires the introduction of additional protein engineering modifications or chemical modifications.
  • the pore diameter of the MscS angstrom hole involved in the present invention can realize reversible in-situ adjustment only by changing the external conditions, and is suitable for direct single-molecule sensing and recognition of nucleotides (also can be understood as direct detection of nucleotides ).
  • the method provided by the present invention can directly detect and distinguish one or more nucleotides, and can also be used in conjunction with other strategies to further detect the presence of the target nucleic acid in the sample.
  • mutations can be introduced into the side pore of the MscS angstrompore, adjusting the volume (e.g., W to A, S, P) and charge (e.g., W to H, K to R) of the amino acids at the side pore. ), to achieve better detection of specific charged molecules and molecules of specific sizes.
  • the present invention also provides a method for detecting drug molecules in a sample, which is characterized in that it includes the following steps:
  • the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the angstrom hole is embedded in the insulating film, the The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, the angstrom hole is a MscS angstrom hole,
  • the Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening; the sample is added to the first medium;
  • S3 analyzes the electrical signal, and then identifies drug molecules in the sample.
  • the charge properties and/or pore size of the openings are adjustable.
  • the manner of adjusting the opening includes subjecting the insulating film to mechanical force stimulation and/or changing the physical state of the insulating film.
  • the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
  • the aperture of the opening can be adjusted in the following manner:
  • Amypore is a MscS variant Amypore.
  • the MscS variants include side hole volume variants and/or side hole charge variants.
  • the emipore is derived from bacillus.
  • the pore includes one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
  • the Amipore is a PaMscS variant Amipore.
  • the angstrompore includes one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
  • the molecular weight of the drug molecule is less than 1000 g/mol.
  • the molecular weight of the drug molecule is 177.98-712.72 g/mol.
  • the concentration of the drug molecule is greater than 10 nM.
  • sample is a body fluid sample.
  • the body fluid samples include urine, blood, serum, plasma, lymph fluid, cyst fluid, pleural fluid, ascitic fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tear fluid, saliva , One or more of sputum.
  • sample volume of the body fluid sample is greater than 10 ⁇ L.
  • the concentration of drug molecules in the body fluid sample is greater than 10 nM.
  • the method further includes S4: connecting a dialysis device with the first medium through a catheter, so that the blood sample enters the angstrom pore system through the dialysis device, wherein S4 is prior to S1.
  • the insulating film includes a phospholipid film and/or a polymer film.
  • the invention provides a method for detecting drug molecules in a sample by using an angstrom hole system, wherein the angstrom hole system includes a MscS angstrom hole.
  • the present invention creatively utilizes the characteristics of a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS) to detect drug molecules in a sample, specifically embodied as follows:
  • the pore size of the MscS pore is narrow.
  • the pore size of the MscS angstrompore is estimated to be in the range of ⁇ 6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the ⁇ -hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy).
  • the pore size of the MscS angstrompore is adjustable (it can also be understood as a flexible structure).
  • MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration.
  • the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e.
  • 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to realize the analysis Optimize the selectivity of analytes and improve the discrimination of analytes.
  • Protein nanopores in the prior art usually have a fixed channel structure, which requires additional protein engineering modification or chemical modification to achieve channel structure adjustment.
  • the pore diameter of the MscS angstrompore involved in the present invention can be reversibly adjusted in situ only by changing the external conditions, and is suitable for the direct detection of various types and sizes of drug molecules.
  • drug molecules such as aminoglycoside antibiotics and glutamic acid can cause corresponding blocking current signals in the MscS pore, and the MscS pore can detect drug molecules at the single-molecule level.
  • the MscS pore can realize the quantitative analysis of drug molecules.
  • the gradient concentration measurement of drug molecules shows a good linear relationship between the signal frequency and drug concentration, so the MscS pore can not only detect drug molecules but also detect the concentration of drug molecules (quantitative analysis).
  • MscS pore has strong anti-interference ability.
  • the cytoplasmic end of MscS is a sieve-like structure, with one bottom opening at the bottom and seven side openings at the side, and the channels of each opening (pore) are narrow. Molecular substances, such as proteins, are blocked out of the channel, so these biomacromolecules cannot enter and block the channel. Therefore, MscS exhibits strong anti-interference ability and can be directly detected in body fluid samples (such as whole blood samples). More specifically, the method of the present invention can also be used in conjunction with devices such as dialysis devices to realize real-time and continuous monitoring of blood drug concentration.
  • the present invention also provides a method for detecting the presence of target nucleic acid in a sample, characterized in that it comprises the following steps:
  • the single-channel electrophysiological detection system includes: a transmembrane pore, an insulating membrane, a first medium, and a second medium, wherein the transmembrane pore is covered Embedded in the insulating film, the insulating film separates the first medium from the second medium, the transmembrane pores provide channels for communicating the first medium and the second medium, the The nucleic acid amplification product of the sample is added to the first medium;
  • S3 applies a driving force between the first medium and the second medium, and the remaining nucleotides in the nucleic acid amplification product of the sample interact with the transmembrane pore and generate an electrical signal;
  • S4 quantifies the electrical signal to obtain the quantity of the remaining nucleotides
  • S5 compares the quantity of the remaining nucleotides with the quantity of the substrate nucleotides to determine whether the target nucleic acid exists in the sample.
  • transmembrane pore is an MscS variant pore.
  • the MscS variants include side hole volume variants and/or side hole charge variants.
  • the charge properties and/or pore size of the opening of the angstrom pore of the MscS variant are adjustable.
  • the way of adjusting the opening includes subjecting the insulating film to mechanical stimulation and/or changing the physical state of the insulating film.
  • the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
  • the aperture of the opening can be adjusted in the following manner:
  • the osmotic pressure difference between the first medium and the second medium is adjusted by the concentration difference between the first medium and the second medium.
  • the concentration difference between the first medium and the second medium is about 0-270 mM.
  • first medium and/or the second medium include one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
  • MscS variant emicon is derived from Bacillus.
  • the MscS variant Emipore includes one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
  • MscS variant emipore is a PaMscS variant emipore.
  • the PaMscS variant angstrompore includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
  • the nucleic acid amplification is performed by one or more of polymerase chain reaction, ligase chain reaction, strand displacement amplification technique, transcription-mediated amplification technique, and loop-mediated isothermal amplification technique.
  • nucleotides include ribonucleotides and/or deoxyribonucleotides.
  • nucleotides include one or more of dGTP, dATP, dTTP, dCTP, dUTP, GTP, ATP, TTP, CTP, UTP.
  • the nucleic acid amplification system further includes:
  • a probe the probe includes a complementary region and a repeating region, the complementary region includes a sequence that is complementary to the target nucleic acid, and the repeating region includes an oligonucleotide sequence that repeats the same base, so
  • the bases include A, T, C, G, U; or
  • target nucleic acid is coronavirus nucleic acid.
  • the coronavirus includes one or more of SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
  • the insulating film includes a phospholipid film and/or a polymer film.
  • the present invention also provides a virus rapid detection kit, comprising:
  • the MscS Angstrom pore includes a side pore volume variant and/or a side pore charge variant of MscS.
  • the insulating film includes a phospholipid film and/or a polymer film.
  • the conductive solution includes one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
  • the Amipore is a PaMscS variant Amipore.
  • the Angstrom hole includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
  • the present invention provides a method for detecting the presence of a target nucleic acid in a sample using a single-channel electrophysiological detection system.
  • the method provided by the present invention can rapidly detect the presence of target nucleic acid in a sample.
  • Traditional nucleic acid detection methods usually require fluorescent labeling or staining of target nucleic acids, therefore, they rely on expensive fluorescence monitoring equipment or staining systems.
  • Current nanopore-based nucleic acid detection methods often require complex systems such as auxiliary polynucleotide binding proteins (such as helicases and polymerases).
  • the method provided by the present invention utilizes transmembrane pores, and only needs to detect the consumption of substrate dNTPs in an in vitro nucleic acid amplification system (such as substrate dNTPs, polymerase, reverse transcriptase), and then judge whether there is a target nucleic acid in the nucleic acid amplification system , which has the advantages of rapidity, low cost, easy high-throughput detection, and good specificity and sensitivity.
  • the transmembrane pore involved in the present invention can detect (or distinguish) different nucleotides, and is not interfered by other substances (eg, amplified nucleic acid, enzyme) in the nucleic acid amplification system.
  • the transmembrane pore of the present invention is a MscS (small conductance mechanosensitive channel) Angstrom pore, which has a narrow pore size and an adjustable pore size (it can also be understood as a flexible structure).
  • the pore size of the MscS angstrompore is estimated to be in the range of ⁇ 6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the ⁇ -hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy).
  • MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration.
  • the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification.
  • the concentration of the first medium and the second medium i.e. 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl
  • the concentration of the first medium and the second medium can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to achieve optimal pairing.
  • Selectivity of dNTPs and improved discrimination of dNTPs can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane.
  • Protein nanopores in the prior art usually have a fixed channel structure, and the direct detection of nucleotides (for example, dNTP and other similar molecules) usually requires the introduction of additional protein engineering modifications or chemical modifications.
  • the pore diameter of the MscS angstrom hole involved in the present invention can realize reversible in-situ adjustment only by changing the external conditions, and is suitable for direct single-molecule sensing and recognition of nucleotides (also can be understood as direct detection of nucleotides ).
  • the term "derived from” refers not only to proteins produced by the strain of organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences.
  • charged molecule refers to a substance with a net charge and a size smaller than or equal to the Angstrom pore size to which the present invention relates.
  • exemplary charged molecules include nucleotides, amino acids, peptides, drug molecules, and/or other charged small molecules (eg, short peptides).
  • Figure 1 shows the electrophysiological test and dNTP detection based on PaMscS angstrompore
  • Figure 2 shows the translocation frequency of dNTPs through PaMscS1 pores under different osmotic pressure conditions
  • Figure 3 shows the detection of SARS-CoV-2 nucleic acid based on real-time monitoring of dNTPs consumption by PaMscS2;
  • Figure 4 shows the detection of AFP aptamers and miR21 by PaMscS1 through dNTPs depletion
  • FIG. 5 shows the SDS-PAGE results of PaMscS protein (1: wild-type PaMscS; 2: W130A mutant; 3: K180R mutant; 4: marker);
  • Figure 6 shows the current signal or current distribution of wild-type or mutant PaMscS
  • Figure 7 shows the current trajectory through a single PaMscS1 angstrom hole under a ramp voltage from 0 mV to +100 mV;
  • Figure 8 shows the transport capacity of different ions through the pores of PaMscS1;
  • Figure 10 shows the current trajectory and residence time distribution of PaMscS1 detecting single nucleotides
  • Figure 11 shows that single-stranded DNA cannot translocate through the PaMscS1 pore
  • Figure 12 shows the results of the Angstrom hole detection of the SARS-CoV-2 orf1ab gene by loop-mediated isothermal amplification (LAMP);
  • Figure 13 shows the result of the non-denaturing (native) PAGE electrophoresis of the PCR reagent mixed with miR21 and AFP aptamer
  • Figure 14 shows the drug single-molecule biosensing experiment based on PaMscS3 (V271I) pore;
  • Figure 15 shows drug concentration measurements of whole blood samples
  • Figure 16 shows a proof-of-concept experiment for continuous monitoring of drug concentration in living rats via an emipore
  • Figure 17 shows the continuous current trace of the PaMscS3(V271I) angstrom pore of gentamicin sulfate
  • Figure 18 shows the continuous current trace of the PaMscS3 (V271I) angstrom pore of neomycin sulfate
  • Figure 19 shows that high concentrations of gentamicin sulfate and neomycin sulfate can block PaMscS3 (V271I) angstrom pores for a long time;
  • Figure 20 shows that the MspA-2NNN angstrom pore can be frequently blocked by whole blood samples (10 ⁇ L whole blood sample to the 1 mL end);
  • Figure 21 shows the continuous current trace of the PaMscS3(V271I) angstrom pore in a blood sample
  • Figure 22 shows the current trace of direct measurement of rat whole blood samples by PaMscS3 (V271I) angstrom hole;
  • Figure 23 shows the current signal of gentamicin sulfate from -50mV to -80mV through PaMscS3 (V271I) angstrompore;
  • Fig. 24 shows the current signal of the sisomi star through the PaMscS3 (V271I) emipore from -50mV to -80mV;
  • Figure 25 shows a single channel embedded current track of wild-type EcMscS (voltage+100mV, conductive solution 30mM:300mM NaCl);
  • Figure 26 shows the channel sweep voltage (-100mV to 100mV) of wild-type EcMscS
  • FIG. 27 shows the conductance distribution of wild-type EcMscS
  • Figure 28 shows a sequence alignment of PaMscS with MscS of other bacteria
  • Figure 29 shows dNTP detection based on wild-type PaMscS angiopores
  • Figure 30 shows the current trace of the detection of glutamate by PaMscS1
  • Figure 31 shows the PaMscS Angstrompore-based amino acid detection scheme and different amino acid blocking current distributions.
  • the term "about” typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value /-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.
  • Figure 1 A. Schematic diagram of the electrophysiological measurement chamber.
  • D. Conductance distribution of PaMscS1 and PaMscS2 angstrom pores (N 18 respectively) (buffer conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl).
  • Figure 2 The translocation frequency of dCTP (indicated in orange) and dGTP (indicated in blue) was tested under different osmotic pressure differences, symmetrical (A, 300mM NaCl: 300mM NaCl for cis end: trans end) , low osmotic pressure difference (LOD) (B, 300 mM NaCl: 100 mM NaCl for cis end: trans end), and high osmotic pressure difference (HOD) (C, 300 mM NaCl: 30 mM NaCl for cis end: trans end).
  • A 300mM NaCl: 300mM NaCl for cis end: trans end
  • LOD low osmotic pressure difference
  • HOD high osmotic pressure difference
  • dNTPs concentrations 0.5 mM, 1.0 mM, 1.5 mM and 2.0 mM, were used to test the translocation of dCTP and dGTP.
  • E The increase rate of f dCTP and f dGTP under 3 different osmotic pressure difference conditions.
  • Figure 3 A. Schematic diagram of the detection of SARS-CoV-2 by angstrom.
  • Test results of 22 clinical samples including 15 positive samples (patient numbers: 1-15) and 7 negative samples (patient numbers: 16-22), Amypore test results of 21 samples and hospital qPCR test results Consistent (Patient No.: 1-15, 17-22), 1 negative sample (Patient No.: 16) was diagnosed as positive by Amypore (buffer conditions were -cis end: 300mM NaCl, -trans end: 100mM NaCl, voltage is +50mV).
  • Monitoring dNTPs depletion via MscS angstropores can be combined with nucleic acid amplification techniques (NAAT) such as polymerase chain reaction (PCR) and strand displacement amplification (SDA).
  • NAAT nucleic acid amplification techniques
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • Figure 4 A. Schematic of the detection strategy.
  • B. Current traces of no target control group, miR21 group, AFP aptamer group, and both miR21 and AFP aptamer groups.
  • C. Current distribution of the 4 test groups. Relative increase in dATP and dGTP signals in group D.4.
  • Figure 6 A. The background signal frequency of wild-type PaMscS and mutant PaMscS1, PaMscS2, the background noise frequency of PaMscS1 and PaMscS2 is lower than that of wild-type PaMscS (voltage +50mv, n ⁇ 3).
  • Figure 7 Current traces through a single PaMscS1 angstrom pore under a ramped voltage from 0 mV to +100 mV: Voltage gating was observed when the voltage was raised above +90 mV (buffer conditions at the -cis end: 300 mM NaCl, - Trans end: 30mM NaCl, sampling frequency: 4999hz).
  • Figure 8 The buffer conditions are: -cis side 300mM NaCl, -trans side 30mM NaCl, each data point n ⁇ 3, mean ⁇ SD.
  • Figure 10 Residence time distribution: dGTP(A), dATP(B), dTTP(C) and dCTP(D); the concentration of each nucleotide is 2mM and the buffer condition is -cis end: 300mM NaCl, -trans end : 30mM NaCl, the voltage is +50mV.
  • Figure 11 Voltage: +50mV; buffer conditions: 300mM NaCl on the cis side, 30mM NaCl on the trans side.
  • the final concentration of ssDNA was 5 ⁇ M and the sequence was 5'TAGCTTATCAGACTGATGTTGA 3' (SEQ ID NO:5).
  • Figure 12 Samples containing 10 ⁇ 3 copies/mL to 10 ⁇ 11 copies/mL of the Orf1ab gene can be detected.
  • Figure 13 1. DNA template 1 (containing poly T); 2. DNA template 2 (containing poly C); 3. PCR reagents with miR21 and AFP aptamers; 4. Control group (without miR21 and AFP aptamers ).
  • the electrolyte conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl, 10mM HEPES, pH 7.0, and the voltage for drug detection is -50mV.
  • E Comparison of detection results for 1.5 ⁇ M gentamicin sulfate between LC-MS and PaMscS3 angstrompores.
  • the electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, and the drug detection voltage is -50mV.
  • Figure 15 A. Direct detection of whole blood samples through the PaMscS3(V271I) angstrompore, the PaMscS3 angstrompore remained open after addition of the whole blood sample. Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV. B. After adding 20 ⁇ L of rat blood, the conductivity buffer at the -cis end turns red. C. Percentage of channel opening for whole blood samples. D. The quantitative standard curve of gentamicin sulfate ranges from 0 to 3 ⁇ M. E.
  • Electrolyte conditions for whole blood Amypore detection are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV, N ⁇ 3.
  • Figure 16 A. Setup of the Rat Drug Concentration Monitoring System.
  • D. Results of continuous drug blood concentration monitoring of rats with different doses of gentamicin sulfate through the PaMscS3 angstrom pore (N 1). Gray data points indicate drug blocking signal frequencies higher than the highest signal frequency within the range of the standard curve, double-step signals occur frequently and make quantitation inaccurate.
  • Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV.
  • Figure 19 At high drug concentrations, the blocking signal of the drug is difficult to count and the pore of PaMscS3(V271I) can be blocked for a long time, making quantitative calculation impossible.
  • the electrolyte conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl, 10mM HEPES, pH 7.0, and the voltage for drug detection is -50mV.
  • Figure 21 Current traces in the background of the angstrom hole and traces after the addition of whole blood samples.
  • Figure 22 Adding 20 ⁇ L of rat whole blood to the -cis end (1 mL), the PaMscS3(V271I) angstrom pore can work well in the presence of whole blood samples.
  • Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0. Notably, two peaks appeared in the gradient voltage.
  • Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0.
  • the structure of sisomicin is close to the C1a component of gentamicin sulfate. Under the gradient voltage, only one blocking current peak was observed.
  • Figure 28 Sequence alignment of the MscS family. Residues highlighted in red in Figure 28D are identical across the 4 sequences; columns above the sequences designate ⁇ -helices and ⁇ -strands.
  • the angstrom pore used in the present invention is a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS), preferably PaMscS (Pseudomonas aeruginosa small conductance mechanosensitive channel) or a variant thereof.
  • the variant also understood as “mutant” may be a naturally occurring variant expressed by an organism such as Pseudomonas aeruginosa. Variants also include non-naturally occurring variants produced by recombinant techniques.
  • “PaMscS variant”, “mutant PaMscS”, “mutant PaMscS”, and “PaMscS mutant” have the same meaning unless otherwise specified.
  • the angstrompore may be a MscS variant.
  • Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, for example single or multiple amino acid substitutions. Substitutions may be conservative or non-conservative. Preferably, non-conservative substitutions can be made to one or more positions of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, wherein the substituted amino acid residue is replaced by Amino acids that differ significantly in chemical properties and/or physical size are substituted.
  • the MscS variants can be divided into side hole volume variants and side hole charge variants.
  • the side pore volume variant refers to a variant in which the mutation site is located at the side opening (also known as "side pore") at the cytoplasmic end and the volume of the side pore is changed by changing the amino acid at this site.
  • the side hole charge variant refers to a variant in which the mutation site is located at the side opening of the cytoplasmic end and the side hole charge is changed by changing the amino acid at this site.
  • the side pore volume variant may be the substitution of a larger amino acid (e.g. tryptophan (W)) for a smaller amino acid (e.g.
  • Side hole charge variants can be the substitution of a certain charged amino acid for an oppositely charged or neutral amino acid, or the substitution of a neutral amino acid for a charged amino acid.
  • non-limiting examples of positively charged amino acids include histidine, arginine, and lysine; non-limiting examples of negatively charged amino acids include aspartic acid and glutamic acid; neutral, non-limiting Examples include glycine, alanine, phenylalanine, valine, leucine, isoleucine, cysteine, asparagine, glutamine, serine, threonine, tyrosine, Methionine, Proline and Tryptophan.
  • Amino acid conservative substitutions or non-conservative substitutions as well as many different types of amino acid modifications (deletion, substitution, addition) and other modifications are well known in the art, and those skilled in the art can modify MscS according to the actual situation to obtain the corresponding MscS Variants.
  • the means of modification include modifying the corresponding DNA sequence (for example, directly synthesizing the corresponding protein after modifying the DNA sequence information or using PCR to perform site-directed mutation on the DNA sequence), and then obtain the corresponding variant (and its corresponding DNA sequence).
  • said MscS variant may be a PaMscS variant.
  • the PaMscS variants include, for example, one or more of 130A, 130H, 180R, 271I, 130S and 130P.
  • the side pore volume mutants of PaMscS include, for example, 130A, 130S, and 130P, and the side pore charge variants of PaMscS, for example, include 130H, 180R, and 271I.
  • Such modification can change the pore diameter of the modified side hole (also can be understood as "pore size"), thereby improving the detection ability of the analyte with a specific molecular volume; it can also change the local charge characteristics of the modified side hole channel, Further, the detection ability of specific charged analytes is improved; the stability of the protein channel current of the PaMscS variant can also be enhanced.
  • the angstrompore may be wild-type PaMscS, which has the ability to detect analytes despite its high background noise.
  • the angstrom pore may be wild-type EcMscS (Escherichia coli small conductance mechanosensitive channel) or a variant thereof.
  • EcMscS and PaMscS are highly similar, and they can also form a stable channel current and have the ability to detect analytes.
  • the sequence similarity between PaMscS and EcMscS is 60%.
  • Conservative substitutions or non-conservative substitutions of amino acids, as well as many different types of modifications (deletion, substitution, addition) to amino acids are well known in the art, and those skilled in the art can modify EcMscS according to actual conditions to obtain Corresponding EcMscS variant.
  • the emipore in addition to Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa (Pseudomonas aeruginosa), can also be derived from other bacilli, such as Tengchong thermophilic anaerobic bacteria (Thermoanaerobacter tengcongensis ) and Helicobacter pylori.
  • the structures of PaMscS and TtMscS and HpMscS are also highly similar, and the sequence similarities are 55% and 44%, respectively.
  • MscS can be used as an analyte in the angmipore.
  • Conservative substitutions or non-conservative substitutions of amino acids, as well as many different types of modifications (deletion, substitution, addition) to amino acids are well known in the art, and those skilled in the art can modify MscS according to actual conditions to obtain Corresponding MscS variant.
  • the analyte is a charged species. An analyte is charged if it has a net charge.
  • the analyte can be negatively or positively charged.
  • An analyte is negatively charged if it has a net negative charge.
  • An analyte is positively charged if it has a net positive charge.
  • Suitable analytes should be substances with a size smaller than or equal to the angstrom pore diameter, preferably nucleotides, amino acids, peptides, drug molecules.
  • the analyte may be a nucleotide.
  • Nucleotide refers to a monomeric unit consisting of a heterocyclic base, a sugar and a phosphate group. It is to be understood that heterocyclic bases include naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T) and uracil (U)) as well as non-naturally occurring bases base analogs. Sugars include naturally occurring sugars (deoxyribose and ribose) and non-naturally occurring sugar analogs.
  • the nucleotides include deoxyribonucleotides and ribonucleotides such as ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, GMP, UMP, TMP, CMP, dGMP, dAMP, dTMP, dCMP, dUMP, ADP, GDP, TDP, UDP, CDP, dADP, dGDP, dTDP, dUDP, dCDP.
  • the nucleotides include naturally occurring nucleotides and non-naturally occurring nucleotide analogs that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • the nucleotides are free (or, it may be understood as “single”).
  • the nucleotides are ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP.
  • the analyte may be an amino acid.
  • amino acid refers to any of the 20 naturally occurring amino acids found in proteins, the D-stereoisomers of naturally occurring amino acids (eg, D-threonine), unnatural amino acids, and chemically modified amino acids. Each of these amino acid types are not mutually exclusive.
  • alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine amino acid (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I ), Leucine (Leu; L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F), Proline (Pro; P), Serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
  • the analyte may be a short peptide, such as a dipeptide.
  • the analyte may be a drug molecule.
  • a drug molecule can be a compound. More specifically, a "drug molecule" may be a drug having a molecular weight of 1000 g/mol or lower (eg, lower than 800, 700, 600, 500, 400, 300 or 200 g/mol).
  • the drug molecule can be an aminoglycoside antibiotic.
  • the drug molecules include amino acids and their salts (including non-druggable amino acids) and peptides.
  • the "angstrom hole system” includes a hole having a size in the angstrom order (abbreviated as "angstrom hole”), an insulating film, a first medium, and a second medium.
  • the pore with a size in the Angstrom order is a small conductance mechanosensitive channel (MscS) Angstrom pore.
  • the pores with angstrom size are preferably heptamer structures with radial symmetry and cylindrical shape, and the heptamer structures include 7 side openings and 1 bottom opening.
  • the pore with Angstrom size has a typical heptamer structure with radial symmetry and cylindrical shape, and the heptamer structure contains 8 openings, 7 of which are equal The openings are distributed on the side, and the eighth opening is distributed on the bottom and formed by 7 subunits; the pore size of the above 8 openings can be adjusted.
  • the pores having Angstrom dimensions allow translocation of the analyte from one side of the insulating membrane to the other.
  • the hole with the angstrom-scale size is embedded in the insulating film, and the insulating film (also can be understood as, the hole with the angstrom-scale size and the insulating film Composite) separates the first medium from the second medium, and the channels with pores in the Angstrom order provide passages connecting the first medium and the second medium; After a driving force is applied between the first medium and the second medium, the analyte located in the first medium interacts with the MscS pore to form a current (ie, an electrical signal).
  • a current ie, an electrical signal
  • first medium refers to the medium in which the analyte is added to the angstrom pore system
  • second medium refers to the two parts of the medium separated by the insulating film. , the other side of the "first medium”.
  • the driving force refers to the force driving the interaction between the analyte and the angstrom pore by means of potential, electroosmotic flow, concentration gradient and the like.
  • the first medium and the second medium may be the same or different, and the first medium and the second medium may comprise electrically conductive fluids.
  • the conductive liquid is an aqueous alkali metal halide solution, specifically sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl), and sodium bromide (NaBr).
  • the concentrations of the conductive liquid contained in the first medium and the second medium are different, in other words, the concentrations of the conductive liquid in the first medium and the second medium exist The difference, and then there is a difference in the osmotic pressure on both sides of the insulating film.
  • the first medium and/or the second medium may also comprise a buffer, such as HEPES.
  • the concentration range of the first medium and/or the second medium may be 30mM-3M.
  • An insulating film refers to a film that has the ability to host angstrompores (or nanopores) and block ionic currents passing through non-angstrompores (or nanopores).
  • the insulating film may include a phospholipid film and/or a polymer film.
  • Exemplary phospholipid membranes include DPHPC, DOPC, E.coli lipid, and exemplary polymer membranes include triblock copolymer polymer membranes.
  • the present pore system can comprise any of the small conductance mechanosensitive channels described herein, such as wild-type PaMscS (SEQ ID NO: 1), wild-type EcMscS (SEQ ID NO: 2), wild-type TtMscS (SEQ ID NO :3) and wild-type HpMscS (SEQ ID NO: 4) and its corresponding variants, the specific sequence information of the above four MscSs is shown in Table 4.
  • the small conductance mechanosensitive channel can be mutant PaMscS1 (W130A), mutant PaMscS2 (K180R), mutant PaMscS3 (V271I).
  • the Angstrom pore system includes two electrolyte chambers, which are separated by an insulating membrane to form a trans (-trans) compartment and a cis (-cis) compartment, so The holes of the above-mentioned angstrom holes are embedded in the insulating film, and there are only small conductance mechanical force-sensitive channel angmometer holes on the insulating film to communicate with the above-mentioned two electrolyte chambers.
  • electrolyte ions in solution in the electrolyte chambers move by electrophoresis and pass through the Angstrom pores.
  • the small conductance mechanosensitive channel (MscS) angstrom hole can be embedded in the insulating film, but it retains the response to the mechanical stimulation of the insulating film and the change of the physical state of the insulating film The ability to change the structure of a protein.
  • mechanical force stimulation includes osmotic pressure changes on both sides of the insulating membrane, direct physical stimulation of micro-targeting on the insulating membrane, stimulation of the insulating membrane by negative air pressure, and the like.
  • the physical change of the insulating film includes the change of the thickness of the insulating film, the change of the composition of the insulating film, and the change of the surface curvature of the insulating film.
  • Said altering the protein structure comprises altering the charge properties and/or pore size of the openings of MscS. Further, the charge properties and/or pore size of the altered opening of the MscS angstrompore can be utilized to detect different analytes.
  • the pore diameter of the angstrom hole involved in the present invention can be adjusted in the range of 5-15 angstrom.
  • the analyte may be in contact with the Angstrom pore on either side of the insulating film.
  • the analyte may be in contact with either side of the insulating film such that the analyte passes through the passage of the Angstrom pore to the other side of the insulating film.
  • the analyte interacts with the Angstrom pore as it passes through the insulating membrane via the passage of the pore.
  • the analyte may be in contact with the side of the insulating film that allows the analyte to interact with the Angstrom pore, separate it from the Angstrom pore, and reside in the Angstrom pore. on the same side as the insulating film.
  • the analyte can interact with the pore in any manner and at any site.
  • the analyte may also impinge on the Angstrompore, interact with the Angstrompore, separate it from the Angstrompore and reside on the same side of the insulating membrane.
  • the analyte affects the current flowing through the pore in a manner specific to the analyte, i.e. the current flowing through the pore
  • the current is characteristic of a particular analyte.
  • Control experiments can be performed to determine the effect of a particular analyte on the current flowing through the angstrom pore, and then to identify the particular analyte in the sample or to determine the presence or absence of the particular analyte in the sample. More specifically, the presence or absence or concentration of the analyte can be identified based on the comparison of the current pattern obtained by detecting the analyte with the known current pattern obtained using the known analyte under the same conditions.
  • the angstrompore system of the present invention may also include one or more measuring devices that measure the current flowing through the angstrompore, such as patch clamp amplifiers or data acquisition devices.
  • the analyte can be present in any suitable sample.
  • the invention is generally performed on samples known to contain or suspected to contain the analyte.
  • the invention can be performed on samples containing one or more analytes of unknown type.
  • the present invention may identify the species of said one or more analytes known to be present or predicted to be present in said sample.
  • the sample can be a biological sample.
  • the invention may be performed in vitro on a sample obtained or extracted from any organism or microorganism.
  • the invention can also be performed in vitro on samples obtained or extracted from any virus.
  • the sample is a fluid sample.
  • the sample typically includes bodily fluids.
  • the sample may be a body fluid sample, such as urine, blood, serum, plasma, lymph fluid, cyst fluid, pleural fluid, ascitic fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tear fluid, Saliva, sputum, or a combination thereof.
  • the sample can be derived from humans or from other mammals.
  • the sample can be a non-biological sample.
  • the non-biological samples are preferably fluid samples such as drinking water, sea water, river water and reagents for laboratory tests.
  • the sample may not be processed prior to analysis, eg, the analyte is detected directly in whole blood.
  • the sample may also be treated prior to analysis, eg, by centrifugation, filtration, dilution, sedimentation, or other physical or chemical means known in the art.
  • the sample is a whole blood sample.
  • the sample is a nucleic acid amplification product.
  • the invention also provides a method of detecting the presence of nucleic acid in a sample.
  • the method includes: S1 placing the sample in a nucleic acid amplification system and performing nucleic acid amplification, determining the number of substrate nucleotides in the nucleic acid amplification system, and obtaining a nucleic acid amplification product of the sample; S2 placing the The nucleic acid amplification product of the sample is added to the angstrom hole system, and the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the protein angstrom hole is embedded in the insulating film, The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, and the angstrom hole is MscS angstrom A hole, the Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, the hepta
  • S1 and S2 can be performed simultaneously or in the same system.
  • a threshold can also be set, for example, only when the quantity of at least one of the remaining nucleotides is lower than the threshold, it is considered that the target nucleic acid exists in the sample; or, only the quantity of all kinds of remaining nucleotides is high When this threshold is reached, it is considered that the target nucleic acid does not exist in the sample.
  • transmembrane pore is a structure that passes through a membrane to some extent. It allows the analyte to flow through or within the membrane driven by an applied driving force. Transmembrane pores typically span the entire length of the membrane, allowing analytes to flow from one side of the membrane to the other. However, the transmembrane pore does not necessarily have to pass through the membrane. It can be closed at one end.
  • a pore may be a well, gap, channel, groove or slit in a membrane along which or into which analyte may flow.
  • transmembrane pores can be biological or artificial. Suitable pores may be protein pores, polynucleotide pores and solid state pores.
  • the transmembrane pore in the present invention should at least have the ability to detect and distinguish multiple nucleotides, preferably a transmembrane protein pore.
  • the present invention relates to transmembrane protein pores that allow the flow of analytes from one side of the membrane to the other, driven by a driving force.
  • Nucleic acid refers to a polymer of deoxyribonucleotides or ribonucleotides in single- or double-stranded form.
  • Nucleic acid amplification of a target nucleic acid refers to the process of constructing a nucleic acid chain in vitro that is at least partially identical or complementary to a target nucleic acid sequence, and the nucleic acid amplification process can only occur when the target nucleic acid is present in a sample.
  • nucleic acid amplification enzymes (such as nucleic acid polymerase, transcriptase) are usually used to generate multiple copies of a target nucleic acid or a fragment thereof, or multiple copies of a sequence complementary to the target nucleic acid or a fragment thereof.
  • the substrate nucleotides of the nucleic acid amplification system will decrease correspondingly with the increase of the copy number.
  • Figure 3 and Figure 4 are examples of nucleic acid amplification.
  • the principle of the method of the present invention is based on in vitro nucleic acid amplification technology, to consume the substrate nucleotide in the nucleic acid amplification system, so common in vitro nucleic acid amplification technology, such as polymerase chain reaction (PCR), ligase chain reaction (LCR) ), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Loop-Mediated Isothermal Amplification (LAMP) can all be used in conjunction with the method provided by the invention.
  • PCR polymerase chain reaction
  • LCR Ligase chain reaction
  • SDA Strand Displacement Amplification
  • TMA Transcription-Mediated Amplification
  • LAMP Loop-Mediated Isothermal Amplification
  • the method provided by the present invention can detect whether there is a new coronavirus nucleic acid in a sample.
  • the method provided by the present invention can detect whether there is a single-stranded nucleic acid in a sample.
  • the present invention constructs suitable primers (for example, specific primers for SARS-CoV-2 nucleic acid) and introduces the primers into the nucleic acid amplification system (including substrate dNTPs, polymerase, reverse transcriptase). If the new coronavirus nucleic acid exists in the sample, the new coronavirus nucleic acid is amplified under suitable conditions to consume substrate dNTPs and generate multiple copies of the nucleic acid.
  • suitable primers for example, specific primers for SARS-CoV-2 nucleic acid
  • the nucleic acid amplification product of the sample is added to the angstrom pore system provided by the present invention, macromolecular substances (such as enzymes, polynucleotides, etc.) in the nucleic acid amplification system cannot pass through the angstrom pore, nor That is to say, only free single nucleotides in the nucleic acid amplification system can pass through the angstrompore and generate a specific current, and then determine the number of remaining nucleotides to determine whether there is a target new coronavirus nucleic acid in the sample (That is, if there is no target new coronavirus nucleic acid in the sample, the number of remaining nucleotides is closer to the number of substrate nucleotides before nucleic acid amplification; if there is new coronavirus nucleic acid in the sample, the number of remaining nucleotides is significantly lower than The number of substrate nucleotides before nucleic acid amplification, more specifically, at least one of the substrate nucleo
  • the present invention constructs a suitable probe (for example, the probe includes a sequence complementary to the target nucleic acid sequence and a polynucleotide sequence) and introduces the probe into the nucleic acid Amplification system (including substrate dNTPs, polymerase).
  • a suitable probe for example, the probe includes a sequence complementary to the target nucleic acid sequence and a polynucleotide sequence
  • the probe introduces the probe into the nucleic acid Amplification system (including substrate dNTPs, polymerase).
  • the target nucleic acid sequence is amplified under suitable conditions, consumes substrate dNTPs and generates multiple copies of the target nucleic acid sequence; more specifically, since the probe also has multiple Polynucleotide sequence (for example, Poly T, Poly A, Poly C, Poly G), therefore, after described target nucleic acid sequence is combined with described probe, also can consume a large amount of corresponding to polynucleotide sequence Substrate nucleotides, therefore, the presence or absence of target nucleic acid sequences in samples can be judged from the specific consumption of certain substrate nucleotides.
  • Polynucleotide sequence for example, Poly T, Poly A, Poly C, Poly G
  • the nucleic acid amplification product of the sample is added to the angstrom pore system provided by the present invention, macromolecular substances (such as enzymes, polynucleotides, etc.) in the nucleic acid amplification system cannot pass through the angstrom pore, that is, Said, in the nucleic acid amplification system, only free single nucleotides can pass through the angstrom hole and generate a specific current, and then determine the number of remaining nucleotides to judge whether there is a target nucleic acid sequence in the sample (i.e.
  • the number of remaining nucleotides is closer to the number of substrate nucleotides before nucleic acid amplification; if there is a target nucleic acid sequence in the sample, the number of remaining nucleotides is significantly lower than that of nucleic acid amplification
  • the number of substrate nucleotides in the front and the corresponding polynucleotides in the probe are consumed in large quantities. Based on this, different probes can be designed to simultaneously detect different target nucleic acid sequences in the same nucleic acid amplification system.
  • the present invention also provides a method for detecting drug molecules in a sample, the method comprising: S1 adding the sample to an angstrom hole system, the angstrom hole system comprising: an angstrom hole, an insulating film, a first medium, a second medium, wherein the Angstrom hole is embedded in the insulating film separating the first medium from the second medium, the Angstrom hole providing communication with the first medium With the channel of the second medium, the angstrom hole is a MscS angstrom hole, the angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 sides Opening and 1 bottom opening; the sample is added to the first medium; S2 applies a driving force to the first medium and the second medium, and the drug molecule in the sample interacts with the angstrom hole function and generate an electrical signal; S3 analyzes the electrical signal, and then identifies the drug molecule in the sample.
  • S1 adding the sample to an ang
  • the sample is a body fluid sample.
  • the body fluid sample can be urine, blood, serum, plasma, lymph fluid, cystic fluid, pleural fluid, ascitic fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tear fluid, saliva, sputum or a combination thereof.
  • the sample may not be processed prior to analysis, eg, the analyte is detected directly in whole blood.
  • the sample can also be processed before analysis, such as by centrifugation, filtration, dilution, precipitation or other physical or chemical means known in the art.
  • the samples referred to in the present invention include untreated samples and processed samples.
  • the detectable range of the drug molecule may be greater than 10 nM (it can also be understood that the detection limit is 10 nM).
  • the detectable range of the drug molecule may be 10nM-1mM. If the concentration of the drug molecule is much greater than 10 nM (eg 10 mM), its concentration can be diluted to 10 nM-1 mM.
  • said drug molecule is a compound. More specifically, a "drug molecule” may be a drug having a molecular weight of 1000 g/mol or lower (eg, lower than 800, 700, 600, 500, 400, 300 or 200 g/mol).
  • the drug molecule can be an aminoglycoside antibiotic, such as gentamicin sulfate, neomycin sulfate, sisomicin and the like.
  • the drug molecules include amino acids and their salts (including non-druggable amino acids) and peptides.
  • the present invention detects drug molecules in body fluid samples, wherein the detection limit of drug molecules is 10 nM.
  • the detectable range of the drug molecule in the bodily fluid sample may be greater than 10 nM.
  • the present invention detects drug molecules in whole blood samples (also known as "blood samples").
  • whole blood samples also known as "blood samples”.
  • the cells for example, red blood cells, white blood cells, platelets
  • macromolecular substances such as proteins
  • the drug molecules present in the whole blood sample can pass through the angstrom pore and generate a specific current.
  • the angstrom hole provided by the present invention can sensitively recognize the drug molecule at a lower concentration, and then judge the whole blood The presence and concentration of the drug molecule in the sample.
  • the method provided by the present invention can be used for continuous monitoring of the blood drug concentration of the drug molecule in the subject.
  • PaMscS2 K180R
  • PaMscS3 V271I angmipores are used to detect drug molecules in whole blood samples, but other MscS and their corresponding variants are also included in the protection scope of the present invention , based on the fact that the above variants all have the ability to sense and detect drug molecules.
  • Sodium Chloride NaCl, >99.0%, CAS#7647-14-5
  • dNTP Mixture >99.0%
  • dATP >97%, CAS#1927-31-7
  • dCTP >98%, CAS# 102783-51-7
  • dGTP >98%, CAS#93919-41-6
  • dTTP >98%, CAS#18423-43-3
  • Yeast Extract (CAS#8013-01-2), Trypsin (CAS#73049-73-7), Ampicillin Sodium Salt ( ⁇ 98.5%, CAS#69-52-3), Tris ( ⁇ 99.9%, CAS #77-86-1), Imidazole ( ⁇ 99%, CAS#288-32-4), Dodecyl- ⁇ -D-maltoside (n-Dodecyl- ⁇ -D-Maltopyranoside, DDM) ( ⁇ 99%, CAS#69227-93-6), isopropyl- ⁇ -D-thiogalactoside (IPTG) ( ⁇ 99%, CAS#367-93-1 ), phenylmethylsulfonyl fluoride (PMSF) ( ⁇ 99.%, CAS#329-98-6), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (4-(2 -Hydroxyethyl)piperazine-1-ethanesulfonic acid, HEPES) (>99.5%,
  • Phospholipids extracted from Escherichia coli were purchased from Avanti.
  • PrimeSTAR HS DNA polymerase was purchased from TaKaRa.
  • the pUC57 vector plasmid, DNA template, miRNA-21, and AFP aptamer were synthesized by Sangon Biotech, and their sequence information is listed in Table 1.
  • the gene from PaMscS in Pseudomonas aeruginosa genomic DNA was amplified by polymerase chain reaction (PCR) using gene-specific primers. Genes were inserted into plasmids using the ClonExpress II One Step Cloning Kit (Vazyme).
  • the Escherichia coli BL21 (DE3) cells containing the plasmid of the PaMscS gene were cultured at 37° C. in Luria-Bertani (LB) medium in the presence of 50 ⁇ g/mL ampicillin, and expressed and purified. Peaks were identified by SDS-PAGE analysis.
  • the expression and purification steps of the wild-type and mutant proteins in the present invention are the same, but there are differences in the plasmid synthesis stage due to sequence differences between the wild-type protein and the mutant protein.
  • the experimental team of the present invention revealed the structure of the protein angstrom pore through modeling, which is a typical heptamer that is radially symmetrical and resembles a cylinder. It contains 8 openings, 7 on the sides and 1 on the bottom. N-terminal residues 1-13 are too flexible to be resolved in the model. Topologically, PaMscS can be divided into 2 parts, the transmembrane region and the large cytoplasmic part. Each monomer generates three N-terminal transmembrane helices, including TM1 (residues 17-52), TM2 (residues 58-83) and TM3 (residues 90-122).
  • the C-terminal cytoplasmic region can be divided into an intermediate ⁇ domain and a COOH terminal domain.
  • TM1 and TM2 in each subunit are aligned together in an antiparallel orientation, with TM1 passing through the bilayer membrane on the outside of the channel and TM2 forming the central layer, thus forming a permeation pathway around the channel axis.
  • the TM3 helix can be described as two helical segments, TM3a and TM3b, separated by a distinct kink ⁇ 53° at Gly108, residues that are conserved in homology.
  • TM3a like TM1, crosses the membrane with a different deflection, while TM3b returns to the cytoplasm, where it interacts with the cytoplasmic domain.
  • 7 subunits form a radius of The central pore of , which senses tension and is involved in conformational changes.
  • the slant angles of TM1 and TM2 of the former are smaller than those of the latter, which leads to a large deflection of the TM region, especially the loop between TM1 and TM2.
  • the middle ⁇ -domain contains 5 ⁇ -strands that are tightly connected to ⁇ -strands of other different subunits.
  • the C-terminal domain (177-273 residues) is composed of 5 ⁇ -strands and 2 ⁇ -helices, which is a mixed structure. Between these two domains of adjacent monomers, there are seven equal openings on the sides, clearly visible, with a radius of approx. It has been proposed to be responsible for ion permeation in EcMscS.
  • PaMscS2 In addition to these entrances, an 8th opening exists at the bottom of the protein, which is expressed by 7 ⁇ -strands with a narrowest radius of In all sizes, extends to PaMscS2 is parallel to the heptad axis and has a vertical width of
  • the structure of PaMscS is similar to that of EcMscS in the closed state (PDB:2OAU), and the TM domain has more than 101 rmsd as C ⁇ atoms, but in the open state (PDB:2VV5), there is a large difference in the TM region, the rmsd is
  • Mutant PaMscS protein Amypore muteins may include 130A, 130H, 180R, 271I, 130S or 130P.
  • Different sets of amplification products were detected by MscS angstrompores. Different samples were added to the -cis end, recorded at +50 mV and observed for 20 min. When a stable PaMscS1 angstrom hole is formed on the planar phospholipid membrane, the single nucleotide to be detected is added to the -cis end of the sample hole, and then a voltage is applied to record the current signal.
  • the SARS-CoV-2 RNA reverse transcription amplification system of the present invention is as follows: random hexamer (60 ⁇ M) and anchor poly T (23): 1 ⁇ L, dNTP mixture (each 10 mM): 1 ⁇ L, RNA sample: 11 ⁇ L. Reactions were incubated in a thermal cycler at 65°C for 5 minutes. Immediately place samples on ice to rapidly cool >1 min. Then, in a clean pre-PCR hood, mix the following reagents with the sample: 5X SuperScript IV Buffer: 4 ⁇ L, DTT (100 mM): 1 ⁇ L, RNaseOUT RNase Inhibitor: 1 ⁇ L, Superscript IV Reverse Transcriptase: 1 ⁇ L.
  • cDNA was obtained after incubating the samples in a thermal cycler using a program of 42 °C for 50 min and 70 °C for 10 min.
  • the cDNA PCR amplification system in the experiment of the present invention is as follows: ddH2O: 26.5 ⁇ L, 5 ⁇ PrimeSTAR buffer (Mg2+Plus): 10 ⁇ L, dNTP mixture (2.5mM): 4 ⁇ L, primer 1 (forward primer ORF P1, 10 ⁇ M): 2 ⁇ L, primer 2 (reverse primer ORF P2, 10 ⁇ M): 2 ⁇ L, PrimeSTARHS DNA polymerase (2.5U/ ⁇ L): 0.5 ⁇ L, cDNA sample: 5 ⁇ L.
  • the amplification program was as follows: preheating at 95°C for 5 minutes, heat denaturation at 98°C for 10 seconds. Refractive annealing at 55°C for 15 seconds, followed by extension at 72°C for 12 seconds. The cycle was repeated for a total of 35 times.
  • the above primers were independently synthesized by our research team, and the sequences are as follows:
  • ORF P1 TTGTTTGAATAGTAGTTGTCTGA (SEQ ID NO:7)
  • ORF P2 TCAACTCAATATGAGTATGGTACTG (SEQ ID NO:8)
  • RTP-LAMP Reverse Transcriptase Loop-mediated Isothermal Amplification
  • the nucleic acid amplification system of the present invention is as follows: ddH2O: 64.6 ⁇ L, 5 ⁇ PrimeSTAR buffer (Mg2+Plus): 20 ⁇ L, dNTP mixture (each 10 mM): 6 ⁇ L, primer 1 (miRNA21, 100 ⁇ M): 0.4 ⁇ L, primer 2 ( AFP aptamer, 10 ⁇ M): 4 ⁇ L, template 1 (extracted, poly T): 2 ⁇ L, template 2 (poly C, 1 ⁇ M): 2 ⁇ L, PrimeSTAR HS DNA polymerase (2.5U/ ⁇ L): 1 ⁇ L.
  • the amplification procedure was as follows: preheating at 95°C for 5 minutes and thermal denaturation at 98°C for 10s. Refractive annealing at 60°C for 15 seconds, followed by extension at 68°C for 23 seconds. A total of 30 cycles were repeated, and the nucleic acid amplification results are shown in FIG. 13 .
  • electrobiological data were processed by Clampfit software and plotted by Origin software.
  • MscS The basic function of MscS is a rapid on/off switch in response to mechanical stimuli such as changes in membrane tension during osmolarity.
  • the cytoplasmic domain of MscS functions as a molecular sieve that balances the loss of osmolytes during osmoadaptation.
  • the seven side pores from the cytoplasmic region play a key role in the translocation of ions and solutes. Therefore, side hole mutants PaMscS1(W130A) and PaMscS2(K180R) were selected for subsequent studies due to low background noise (Fig. 5, Fig. 6A-C).
  • the purified protein was added to the -cis end of the electrophysiological device ( Figure 1A).
  • Figure 1A When the PaMscS mutant channel was embedded in a bilayer lipid membrane (BLM, a type of insulating membrane), a steady channel current jump could be observed at a voltage of +50 mV (Fig. 1B).
  • the channel conductance of PaMscS1 remained stable at voltages ranging from -50 mV to +50 mV (Fig. 1C), and the gating probability of PaMscS1 increased when the voltage was higher than +90 mV (Fig. 7).
  • the ion transport results of PaMscS1 showed that PaMscS1 had better selectivity for Br- (Fig. 8).
  • PaMscS1 and PaMscS2 angstrom Pores present a different profile for the distribution of dNTPs blocking currents. Specifically, PaMscS1 angstrompores showed three peaks for the blocking rate of the four dNTPs mixtures, while PaMscS2 angstrompores showed two peaks for the blocking rate of the four dNTPs mixtures. Because the difference between PaMscS1 and PaMscS2 mutations lies in the amino acid difference of the side hole, it is speculated that the detection signal of dNTPs is related to the side hole.
  • PaMscS1 Since PaMscS1 has a better discrimination effect on dNTP mixtures, it is more suitable for the discrimination of dNTPs mixtures. As for PaMscS2, it exhibited more stable channel conductance and relatively higher membrane fusion efficiency, so it was more suitable for subsequent rapid diagnosis (Fig. 6A-C). The blocking rate of the wild-type PaMscS pore for the four dNTPs mixtures showed two peaks (Fig. 29). The current traces and residence time distributions of PaMscS1 detecting single nucleotides are shown in Fig. 10A-D.
  • ssDNA single-stranded DNA
  • 50 ⁇ M ssDNA was detected under buffer conditions of 30 mM NaCl/300 mM NaCl and a bias voltage of +50 mV, and no translocation events were observed due to its narrow channel size ( Figure 11). Therefore, the PaMscS mutant emipore has the potential to be a useful small molecule sensor.
  • the experimenters tuned the selectivity of PaMscS1 angstrompores by applying different osmotic pressure differences.
  • the experimenters kept the conductivity buffer concentration at the -cis end at 300 mM and changed the conductivity buffer concentration at the -trans end to change the osmotic pressure difference.
  • the PaMscS1 angstrompore was tested for its ability to detect macromolecular dGTP and small molecular dCTP under three osmotic pressure differential conditions, including symmetric conditions (Fig.
  • Figure 2E summarizes the detection of dGTP and dCTP, and it concludes that low osmolality conditions showed the highest increase in dCTP translocation events, while high osmolality conditions showed the highest increase in dGTP translocation events (Figure 2E).
  • Low osmolarity differential conditions showed a balanced capture capacity for both dCTP and dGTP compared to the reduced capture efficiency of dCTP for high osmolarity conditions.
  • the channel size of the MscS family (such as EcMscS, HpMscS, AtMsL1 proteins, etc.) can vary under different pressure, osmotic pressure conditions or membrane potentials.
  • the experimenters can conclude that the difference in the selectivity of the PaMscS1 angstrompore to dNTPs is caused by the variation of the channel size under different osmotic pressure difference conditions.
  • the synthetic SARS-CoV-2 Orf1ab gene could be detected in the concentration range from 10 ⁇ 3 copies/mL to 10 ⁇ 11 copies/mL (Fig. 3B). Then, 22 clinical samples were tested, including 15 samples from confirmed patients and 7 samples from healthy controls. All 15 positive samples and 6 negative samples (patient numbers: 1-15, 17-22) tested by Amipore showed results consistent with clinical testing (Table 3), and 1 negative sample was diagnosed as a false positive Results (patient number: 16). The method had a specificity of 86% and a sensitivity of 100% (Fig. 3C).
  • the PaMscS mutant angstrom system can be combined with various NAAT (Nucleic Acid Amplification Tests, nucleic acid amplification detection) such as polymerase chain reaction and chain displacement amplification ( Figure 12),
  • NAAT Nucleic Acid Amplification Tests, nucleic acid amplification detection
  • Figure 12 polymerase chain reaction and chain displacement amplification
  • the well also has the potential to monitor the reverse transcription process, which enables rapid and amplification-free detection of the target RNA.
  • probe A includes a barcode sequence complementary to miR21 and a polyT
  • AFP aptamer probe includes a sequence complementary to the AFP aptamer and a poly C barcode sequence.
  • LC-MS and LC-MS/MS analyzes were performed on a Shimadzu ultrafast liquid chromatography system (UFLC, Shimadzu) and an AB SCIEX Qtrap 5500 mass spectrometer equipped with a Turbo Spray ion source.
  • UFLC Shimadzu ultrafast liquid chromatography system
  • AB SCIEX Qtrap 5500 mass spectrometer equipped with a Turbo Spray ion source.
  • the collection and analysis of chromatographic and mass spectrometric data were completed by Analyst 1.6.2 software (AB SCIEX, USA).
  • Chromatographic separation was achieved on a WatersACQUITYUPLC BEH C18 column (2.1mm ⁇ 100mm I.D., 1.7 ⁇ m).
  • the mobile phase consisted of water (A) and acetonitrile (B), and the gradient elution was as follows: 0-1.0 minutes, 10-90% B; 1-2.0 minutes, 90% B.
  • the flow rate was 0.5 mL/min.
  • the temperature of the column and the autosampler were maintained at 35°C and 15°C, respectively.
  • the injection volume is 1 ⁇ L.
  • MS/MS analysis the positive ionization mode was used for sample detection, and the mass spectrometry parameters were optimized as follows: ion spray voltage, 5500V; declustering voltage, 100V; temperature, 500°C. Select the MRM (Multiple Reaction Monitoring) mode to quantify gentamicin sulfate and IS (internal standard), and the ion pairs are 450.2-160.1, 464.2-160.1, 478.2-157.1 and 265.2-232.2, respectively.
  • MRM Multiple Reaction Monitoring
  • an initial 0.4 mL of heparin solution 250 U/mL was infused through the catheter, followed by 0.1 mL every 40 minute cycle to prevent clot formation during monitoring.
  • the left femoral artery was then isolated, catheterized, and immediately connected to the device through a pre-designed tubing with a dialysis membrane. After the air in the device is expelled by the blood flow, the tube is connected to an IV catheter, creating a steady cycle.
  • the baseline signal in the absence of the drug of interest was first recorded, and then a specific concentration of gentamicin sulfate was infused at a slow rate through the venous catheter.
  • a similar cycle is set up, without the device and without the dialysis membrane.
  • Blood samples at 0, 15, 30, 45, and 60 min were collected from the catheter arteriosus and drug concentrations were measured using a PaMscS3(V271I) angstrom. After each experiment, the rats were sacrificed by cervical dislocation.
  • small molecule drugs (molar mass less than 1000 g/mol) were selected for detection.
  • the detection experiments of gentamicin sulfate and neomycin sulfate were carried out under the electrolyte conditions of 300mM NaCl (-cis end) and 30mM NaCl (-trans end), 10mM HEPES, pH 7.0, and the voltage of drug detection was -50mV.
  • PaMscS emipores can also single-molecule sense other drugs, such as sisomicin (MW: 447.53), pyrophosphate (MW: 177.975).
  • LC-MS was used to measure the concentration of gentamicin sulfate.
  • concentration of gentamicin sulfate For the 1.5 ⁇ M gentamicin sulfate sample, PaMscS3(V271I) angstrompore and LC-MS showed similar detection results, indicating that the detection of PaMscS3(V271I) angstrompore had good precision ( FIG. 14E ).
  • the drug concentration trend measured by the PaMscS3(V271I) angiopore conforms to the law of pharmacokinetics, indicating that the PaMscS angiopore can accurately measure the change of the drug concentration in the living rat.
  • the blocking current profile of gentamicin sulfate showed two peaks at higher negative voltages in a buffer of 130 mM NaCl (Fig. 23), while the monocomponent sisomicin ) showed a blocking current peak under the same conditions (Fig. 24), which indicated that the two blocking current peaks of gentamicin sulfate may be related to its multi-component.
  • gentamicin sulfate In a feasibility verification experiment in live rats, based on a simple dialysis device, a clear signal of gentamicin sulfate could be continuously observed until 3 hours after injection ( FIG. 16C ). Different doses of gentamicin sulfate to rats, including 4 mg/kg and 20 mg/kg, could be differentiated by the emipore monitoring device (Fig. 16D). These results demonstrate that the system can continuously monitor drug concentrations in living animals with minimal loss.
  • Figures 28a-c show the structures of EcMscS, TtMscS and HpMscS, respectively, which are highly similar to the structure of PaMscS, that is, they are all heptamers with radial symmetry and cylindrical shape.
  • Figure 28a-c and Figure 28d further compared the sequences of PaMscS and EcMscS, TtMscS, HpMscS, and the results showed that EcMscS, TtMscS, HpMscS and PaMscS have certain homology, but this homology is not highly homologous.
  • EcMscS and PaMscS are only 60% similar, but both have the ability to detect analytes.
  • PaMscS1 is taken as an example to detect amino acids.
  • the detection experiment of glutamic acid (10mM) was carried out under the electrolyte conditions of 300mM NaCl (-cis end) and 30mM NaCl (-trans end), 10mM HEPES, pH 7.0, and the voltage of drug detection was -50mV.
  • the current trajectory of glutamate is shown in Figure 30.
  • the Angstrompores contemplated by the present invention can also detect short peptides (eg, dipeptides).
  • short peptides eg, dipeptides
  • the amino acid to be tested is dehydrated and condensed with the aspartic acid carrier to form a dipeptide, which is detected under the electrolyte conditions of 300mM NaCl (-cis end) and 30mM NaCl (-trans end), 10mM HEPES, pH 7.0
  • the dipeptide is formed, and the current signal generated by it is compared with the specific current signal of the detected amino acid to determine the type of amino acid to be tested.
  • Maksaev, G. & Haswell, E.S. MscS-Like10 is a stretch-activated ion channel from Arabidopsis thaliana with a preference for anions. Proceedings of the National Academy of Sciences 109, 19015–19020 (2012).

Abstract

A biological angstrom pore system based on a mechanosensitive channel of small conductance (MscS), and the application of the angstrom pore system to the detection of charged molecules, which belong to the field of nanopore detection. The angstrom pore system includes MscS angstrom pores, an insulating film, a first medium and a second medium, wherein the pore size of the MscS angstrom pore can be reversibly adjusted in situ simply by means of changing external conditions, and is applicable to direct detection of molecules of various types and sizes, e.g. nucleotides, amino acids, peptides and drug molecules.

Description

基于小电导机械力敏感性通道的生物埃米孔***Biological angstrom pore system based on small conductance mechanosensitive channels
本申请要求2021年08月30日提交的中国发明专利申请【CN2021110062606】、名称为“基于PaMscS的用于dNTPs和新冠病毒检测的生物纳米孔***”的优先权，以及，2021年08月30日提交的中国发明专利申请【CN2021110042496】、名称为“基于PaMscS的用于小分子药物检测和全血检测的生物纳米孔***”的优先权，两个优先权发明专利申请以引用方式全文并入。This application claims the priority of the Chinese invention patent application [CN2021110062606] filed on August 30, 2021, entitled "PaMscS-based biological nanopore system for dNTPs and novel coronavirus detection", and, August 30, 2021 The priority of the submitted Chinese invention patent application [CN2021110042496] titled "PaMscS-based biological nanopore system for small molecule drug detection and whole blood detection", the two priority invention patent applications are incorporated by reference in their entirety.
技术领域technical field
本发明属于纳米孔检测领域，具体涉及一种基于小电导机械力敏感性通道的生物埃米孔***。The invention belongs to the field of nanopore detection, and in particular relates to a biological angstrom pore system based on a small conductance mechanical force sensitive channel.
背景技术Background technique
纳米孔单分子检测技术是一种集操作简单、灵敏度高、检测速度快、无需标记等优点的传感检测技术，广泛应用于蛋白质检测、基因测序和标志物检测等领域。目前，基因检测的费用、灵敏度和精度是该检测技术发展中亟待解决的主要问题，因此开发新型纳米孔材料是解决这些问题的关键手段。Nanopore single-molecule detection technology is a sensing and detection technology that has the advantages of simple operation, high sensitivity, fast detection speed, and no need for labeling. It is widely used in protein detection, gene sequencing, and marker detection. At present, the cost, sensitivity and precision of genetic testing are the main problems to be solved in the development of this testing technology, so the development of new nanoporous materials is the key means to solve these problems.
生物纳米孔是一种自然形成纳米尺度的孔，其孔尺寸与许多重要的生物分子的大小相似。当分子通过纳米孔内部的通道时，特定的阻塞电流和易位事件产生。根据分子的阻塞电流和易位频率，可以实现对目标分子的定性和定量分析。因此，通道孔径大小是影响纳米孔的检测能力和应用范围的主导因素。一些具有合适通道孔径的蛋白质纳米孔已被用于纳米生物技术的应用，如α-溶血素(α-hemolysin，α-HL)、MspA、CsgG、气单胞菌溶素(Aerolysin)、phi29连接器等。这些生物纳米孔主要来自细菌孔蛋白或病毒门，并且有大约为单链DNA(ssDNA)或双链DNA(dsDNA)大小的孔径(1.0nm-3.6nm)。因此，它们适用于检测核酸，并已被用于DNA/RNA测序、核酸生物标记物检测和生物分子相互作用研究。然而，目前需要根据特定的应用需求对生物纳米孔进行局部修饰，如定点诱变或修饰特定的适配体等，才能适应更广泛的测序范围。以α-HL为例，其有限的孔径约1.4nm，因此应用范围仅限制在ssDNA、RNA或其他分子的分析中，通过利用环糊精(cyclodextrin)修饰，可用于直接检测单磷酸脱氧核糖核苷dNMPs，无需荧光标记。但通过修饰手段改变生物纳米孔的孔径需要大量的生物工程技术辅助，此外，与固态纳米孔相比，蛋白质孔在调节尺寸方面的灵活性要差很多。在此意义上，迫切需要找到一种具有灵活结构的纳米孔来高效地检测各种尺寸的分子。A biological nanopore is a naturally occurring nanoscale pore with a pore size similar to that of many important biomolecules. As molecules pass through the channels inside the nanopore, specific blockage currents and translocation events arise. According to the blocking current and translocation frequency of molecules, qualitative and quantitative analysis of target molecules can be achieved. Therefore, the channel pore size is the dominant factor affecting the detection ability and application range of nanopores. Some protein nanopores with suitable channel pore sizes have been used for nanobiotechnology applications, such as α-hemolysin (α-HL), MspA, CsgG, Aerolysin, phi29-linked device etc. These biological nanopores are mainly derived from bacterial porins or viral phyla, and have pore diameters (1.0 nm-3.6 nm) about the size of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Therefore, they are suitable for detecting nucleic acids and have been used in DNA/RNA sequencing, nucleic acid biomarker detection, and biomolecular interaction studies. However, currently, biological nanopores need to be locally modified according to specific application requirements, such as site-directed mutagenesis or modification of specific aptamers, etc., in order to adapt to a wider range of sequencing. Taking α-HL as an example, its limited pore size is about 1.4nm, so the scope of application is limited to the analysis of ssDNA, RNA or other molecules. By using cyclodextrin (cyclodextrin) modification, it can be used to directly detect monophosphate deoxyribose nucleus Glycoside dNMPs without fluorescent labeling. However, changing the pore size of biological nanopores through modification requires a lot of bioengineering technology assistance. In addition, compared with solid-state nanopores, protein pores are much less flexible in adjusting the size. In this sense, it is urgent to find a nanopore with a flexible structure to efficiently detect molecules of various sizes.
综上所述，为改善现有技术的不足，本发明提供了一种新型的基于小电导机械力敏感性通道的生物埃米孔***，埃米孔是一种尺寸比纳米孔更小的孔径蛋白质，这种新型蛋白质埃米孔***无需适配体或修饰，为实时分子传感、基因检测和DNA计算提供了一条低成本、高通用的新途径。In summary, in order to improve the deficiencies of the prior art, the present invention provides a novel biological angstrom pore system based on a small conductance mechanosensitive channel. The angstrom pore is a pore size smaller than a nanopore Protein, this novel protein angstrompore system does not require aptamers or modifications, providing a low-cost, highly versatile new approach for real-time molecular sensing, genetic detection, and DNA computing.
发明内容Contents of the invention
一方面，本发明提供了一种埃米孔***在检测带电荷分子中的应用，其特征在于，所述埃米孔***包含埃米孔、绝缘膜、第一介质和第二介质；所述埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，向所述第一介质和所述第二介质之间施加驱动力后，位于所述第一介质的所述带电荷分子与所述埃米孔相互作用；所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口。In one aspect, the present invention provides an application of an angstrom hole system in detecting charged molecules, characterized in that, the angstrom hole system comprises an angstrom hole, an insulating film, a first medium, and a second medium; An angstrom hole is embedded in the insulating film, the insulating film separates the first medium from the second medium, the angstrom hole provides communication between the first medium and the second medium channel, after applying a driving force between the first medium and the second medium, the charged molecules located in the first medium interact with the angstrom pore; the angstrom pore is MscS Mipore, the Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening.
进一步地，所述开口的电荷性质和/或孔径大小是可调节的。Further, the charge properties and/or pore size of the openings are adjustable.
进一步地，所述开口的调节方式包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的 物理状态变化。Further, the way of adjusting the opening includes subjecting the insulating film to mechanical stimulation and/or changing the physical state of the insulating film.
进一步地，所述机械力刺激包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。所述的绝缘膜的物理状态变化包括所述绝缘膜的厚度变化、所述绝缘膜的组成成分变化、所述绝缘膜的表面曲率变化等。Further, the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind. The change in the physical state of the insulating film includes a change in the thickness of the insulating film, a change in the composition of the insulating film, a change in the curvature of the surface of the insulating film, and the like.
进一步地，所述开口的孔径可以根据以下方式来调节：Further, the aperture of the opening can be adjusted in the following manner:
(1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
(2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
进一步地，所述埃米孔源自杆菌。Further, the emipore is derived from bacillus.
进一步地，所述埃米孔源自铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。Further, the pore is derived from one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
进一步地，所述埃米孔为MscS变体。Further, the emipore is a variant of MscS.
进一步地，所述MscS变体包括侧孔体积变体和/或侧孔电荷变体。Further, the MscS variants include side hole volume variants and/or side hole charge variants.
进一步地，所述绝缘膜包括磷脂膜和/或高分子膜。Further, the insulating film includes a phospholipid film and/or a polymer film.
进一步地，所述带电荷分子包括核苷酸、氨基酸、肽、药物分子中的一种或多种。Further, the charged molecules include one or more of nucleotides, amino acids, peptides, and drug molecules.
进一步地，所述埃米孔为PaMscS变体。Further, the emipore is a variant of PaMscS.
进一步地，所述埃米孔包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。Further, the angstrompore includes one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
进一步地，所述药物分子的摩尔质量小于1000g/mol。具体地，所述药物分子可以为焦磷酸、硫酸庆大霉素、硫酸新霉素、西索米星、谷氨酸等。Further, the molar mass of the drug molecule is less than 1000 g/mol. Specifically, the drug molecule may be pyrophosphate, gentamicin sulfate, neomycin sulfate, sisomicin, glutamic acid and the like.
进一步地，所述第一介质和/或所述第二介质包括氯化钠溶液、氯化锂溶液、氯化铯溶液、氯化钾溶液和溴化钠溶液中的一种或多种。Further, the first medium and/or the second medium include one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
另一方面，本发明还提供一种生物埃米孔***，其特征在于，所述生物埃米孔***包括埃米孔、绝缘膜、第一介质和第二介质，所述埃米孔被嵌入绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道；所述埃米孔为MscS变体埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口。On the other hand, the present invention also provides a biological angstrompore system, characterized in that the biological angstrompore system includes an angstrompore, an insulating film, a first medium, and a second medium, and the In the insulating film, the insulating film separates the first medium from the second medium, and the Angstrom hole provides a channel connecting the first medium and the second medium; the Angstrom hole It is a MscS variant angstrom pore, and the angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening.
进一步地，所述埃米孔为MscS的侧孔体积变体和/或侧孔电荷变体。Further, the Angstrom pore is a side pore volume variant and/or a side pore charge variant of MscS.
进一步地，所述绝缘膜包括磷脂膜和/或高分子膜。所述磷脂膜包括DPHPC、DOPC、E.coli lipid；所述高分子膜包括三嵌段共聚物高分子膜。Further, the insulating film includes a phospholipid film and/or a polymer film. The phospholipid membrane includes DPHPC, DOPC, E.coli lipid; the polymer membrane includes a triblock copolymer polymer membrane.
进一步地，所述埃米孔源自杆菌。Further, the emipore is derived from bacillus.
进一步地，所述埃米孔源自铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。Further, the pore is derived from one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
进一步地，所述埃米孔为PaMscS变体。Further, the emipore is a variant of PaMscS.
进一步地，所述埃米孔包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。上述几个变体的突变位点位于胞质端的侧面开口处，具体涉及氨基酸的体积以及电荷性质的改变。通过突变，可以改变被突变的侧孔的孔径(也可以理解为“孔道尺寸”)，进而提高对特定分子体积的分子的检测能力；还可以改变被突变的所述侧孔通道的局部电荷特性，进而提高对特定带电荷分子的检测能力；还可以增强所述突变型PaMscS埃米孔的蛋白通道电流的稳定性。Further, the angstrompore includes one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P. The mutation sites of the above-mentioned variants are located at the side opening of the cytoplasmic end, specifically involving changes in the volume and charge properties of amino acids. Through mutation, the pore diameter of the mutated side hole (also can be understood as "pore size") can be changed, thereby improving the detection ability of molecules with a specific molecular volume; the local charge characteristics of the mutated side hole channel can also be changed , and then improve the detection ability of specific charged molecules; it can also enhance the stability of the protein channel current of the mutant PaMscS angstrom pore.
进一步地，所述开口的电荷性质和/或孔径大小是可调节的。Further, the charge properties and/or pore size of the openings are adjustable.
进一步地，所述开口的调节方式包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的物理状态变化。Further, the manner of adjusting the opening includes subjecting the insulating film to mechanical force stimulation and/or changing the physical state of the insulating film.
进一步地，所述机械力刺激包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。Further, the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
进一步地，所述开口的孔径可以根据以下方式来调节：Further, the aperture of the opening can be adjusted in the following manner:
(1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
(2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
另一方面，本发明还提供上述生物埃米孔***在检测带电荷分子中的应用，其特征在于，所述带电荷分子包括核苷酸、氨基酸、肽、药物分子中的一种或多种。On the other hand, the present invention also provides the application of the above-mentioned biological angstrompore system in the detection of charged molecules, characterized in that the charged molecules include one or more of nucleotides, amino acids, peptides, and drug molecules .
与现有技术相比，本发明的有益效果：Compared with prior art, the beneficial effect of the present invention:
本发明提供一种埃米孔***在检测带电荷分子中的应用，其中埃米孔***包含MscS埃米孔。本发明创造性地将小电导机械力敏感性通道(Mechanosensitive channel of small conductance,MscS)形成埃米孔***，并利用机械力敏感性通道蛋白的特性来检测带电荷分子，具体体现为：The invention provides an application of an angstrom hole system in detecting charged molecules, wherein the angstrom hole system comprises a MscS angstrom hole. The present invention creatively forms a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS) into an angstrom pore system, and uses the characteristics of the mechanosensitive channel protein to detect charged molecules, specifically embodied as follows:
1)MscS埃米孔的孔径狭窄。据估计，MscS埃米孔的孔径范围为～6-16埃米，远小于现有技术中常用的纳米孔(例如，α-溶血素纳米孔的孔径约为1.4-2.4nm，即14–24埃米)。1) The pore size of the MscS pore is narrow. The pore size of the MscS angstrompore is estimated to be in the range of ~6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the α-hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy).
2)MscS埃米孔的孔径可调(也可以理解为结构灵活)。MscS埃米孔可在毫秒内将机械刺激转化为电信号或生化信号，从而引发通道构型的调节。利用MscS埃米孔对绝缘膜所受到的机械力刺激和/或绝缘膜的物理状态变化的敏感性，可以通过影响绝缘膜来实现对MscS埃米孔孔径的调整，无需繁复的化学修饰。例如，可以调整第一介质和第二介质的浓度(即30mM NaCl/300mM NaCl、100mM NaCl/300mM NaCl和300mM NaCl/300mM NaCl)来调整绝缘膜两侧的渗透压差进而调节孔径，实现优化对dNTPs的选择性并提高对dNTPs的区分。现有技术中的蛋白质纳米孔通常具有固定的通道结构，需要额外的蛋白质工程修饰或者化学修饰等才能实现通道结构调整。而本发明提供的MscS埃米孔的孔径只需改变外界条件即可实现可逆性原位调整，适用于多种类型、尺寸的分子的直接检测。2) The pore size of the MscS angstrompore is adjustable (it can also be understood as a flexible structure). MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration. Utilizing the sensitivity of the MscS angstrompore to the mechanical stimulation of the insulating film and/or the change of the physical state of the insulating film, the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e. 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to achieve optimal pairing. Selectivity of dNTPs and improved discrimination of dNTPs. Protein nanopores in the prior art usually have a fixed channel structure, which requires additional protein engineering modification or chemical modification to achieve channel structure adjustment. However, the pore diameter of the MscS angstrompore provided by the present invention can be reversibly adjusted in situ only by changing the external conditions, and is suitable for direct detection of molecules of various types and sizes.
3)本发明提供的埃米孔***可应用于单分子的传感检测。本发明提供的埃米孔***适用于多种带电荷分子(理论上只要尺寸小于MscS埃米孔的孔径的分子都可以实现传感与检测)，例如，核苷酸、氨基酸、肽、药物分子等；而较大尺寸的核酸(例如ssDNA)和蛋白(例如，全血样本中的蛋白质)无法进入MscS埃米孔的通道中并且不会对检测分子造成干扰。3) The angstrom pore system provided by the present invention can be applied to the sensing and detection of single molecules. The angstrom pore system provided by the present invention is applicable to a variety of charged molecules (in theory, as long as the molecules with a size smaller than the pore diameter of the MscS pore can be sensed and detected), for example, nucleotides, amino acids, peptides, drug molecules etc.; while larger-sized nucleic acids (such as ssDNA) and proteins (such as proteins in whole blood samples) cannot enter the channel of the MscS angstrompore and will not interfere with the detection molecules.
4)基于上述特点，本发明提供的埃米孔***的应用场景广阔。例如，可以向MscS埃米孔的侧孔引入突变，调整侧孔处氨基酸的体积(例如，将W替换为A、S、P)和电荷(例如，将W替换为H、将K替换为R)，以实现对特定带电荷分子和特定尺寸的分子的更优检测。例如，本发明提供的埃米孔***可直接对单核苷酸进行检测，也可以搭配消耗策略(例如，检测核酸扩增体系的剩余核苷酸)来鉴定样本中目标核酸的存在与否，例如在SARS-CoV-2样本的诊断中，展现出良好的特异性和灵敏度。例如，本发明提供的埃米孔***可以检测复杂样本(例如全血)中的药物分子的存在与否，还能够以摩尔的灵敏度直接测量全血中的药物浓度以及对活体动物血药浓度的连续、实时监测，展现出稳健性和敏感性。例如，本发明提供的埃米孔***还可对氨基酸和短肽(例如，二肽)进行检测。4) Based on the above characteristics, the angstrom system provided by the present invention has wide application scenarios. For example, mutations can be introduced into the side pore of the MscS angstrompore, adjusting the volume (e.g., W to A, S, P) and charge (e.g., W to H, K to R) of the amino acids at the side pore. ), to achieve better detection of specific charged molecules and molecules of specific sizes. For example, the angstrom pore system provided by the present invention can directly detect single nucleotides, and can also be used with consumption strategies (for example, detecting the remaining nucleotides of the nucleic acid amplification system) to identify the presence or absence of the target nucleic acid in the sample, For example, in the diagnosis of SARS-CoV-2 samples, it exhibited good specificity and sensitivity. For example, the angstrom system provided by the present invention can detect the presence or absence of drug molecules in complex samples (such as whole blood), and can also directly measure the drug concentration in whole blood with molar sensitivity and the effect on blood drug concentration in living animals. Continuous, real-time monitoring exhibits robustness and sensitivity. For example, the angstrom pore system provided by the present invention can also detect amino acids and short peptides (eg, dipeptides).
另一方面，本发明还提供一种检测样本中核苷酸的方法，其特征在于，包括如下步骤：On the other hand, the present invention also provides a method for detecting nucleotides in a sample, which is characterized by comprising the following steps:
S1将所述样本加入埃米孔***，所述埃米孔***包括：埃米孔、绝缘膜、第一介质、第二介质，其中所述埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口；所述样本被加入到所述第一介质；S1 adding the sample to the angstrom hole system, the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the angstrom hole is embedded in the insulating film, the The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, the angstrom hole is a MscS angstrom hole, The Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening; the sample is added to the first medium;
S2向所述第一介质和所述第二介质施加驱动力，所述样本中的核苷酸与所述埃米孔相互作用并产生电信号；S2 applies a driving force to the first medium and the second medium, and the nucleotides in the sample interact with the angstrom pores and generate electrical signals;
S3分析所述电信号，进而识别所述样本中的核苷酸。S3 analyzes the electrical signal, and then identifies nucleotides in the sample.
进一步地，所述开口的电荷性质和/或孔径大小是可调节的。Further, the charge properties and/or pore size of the openings are adjustable.
进一步地，所述开口的调节方式包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的 物理状态变化。Further, the way of adjusting the opening includes subjecting the insulating film to mechanical stimulation and/or changing the physical state of the insulating film.
进一步地，所述机械力刺激包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。Further, the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
进一步地，所述开口的孔径可以根据以下方式来调节：Further, the aperture of the opening can be adjusted in the following manner:
(1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
(2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
进一步地，所述第一介质与所述第二介质之间渗透压差是通过所述第一介质与所述第二介质之间的浓度差来调节的。Further, the osmotic pressure difference between the first medium and the second medium is adjusted by the concentration difference between the first medium and the second medium.
进一步地，所述第一介质与所述第二介质之间的浓度差为大约0-270mM。Further, the concentration difference between the first medium and the second medium is about 0-270 mM.
进一步地，所述埃米孔为MscS变体埃米孔。Further, the Amypore is a MscS variant Amypore.
进一步地，所述MscS变体包括侧孔体积变体和/或侧孔电荷变体。Further, the MscS variants include side hole volume variants and/or side hole charge variants.
进一步地，所述埃米孔源自杆菌。Further, the emipore is derived from bacillus.
进一步地，所述埃米孔包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。Further, the pore includes one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
进一步地，所述埃米孔为PaMscS变体埃米孔。所述PaMscS变体埃米孔的突变位点位于所述PaMscS的胞质区的侧面开口。Further, the Amipore is a PaMscS variant Amipore. The mutation site of the angmipore of the PaMscS variant is located at the side opening of the cytoplasmic region of the PaMscS.
进一步地，所述PaMscS变体埃米孔包括130A、130H、180R、271I、130S和130P中的一种或多种。Further, the PaMscS variant angstrompore includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
进一步地，所述核苷酸包括dGTP、dATP、dTTP、dCTP、dUTP、GTP、ATP、TTP、CTP、UTP中的一种或多种。Further, the nucleotides include one or more of dGTP, dATP, dTTP, dCTP, dUTP, GTP, ATP, TTP, CTP, UTP.
进一步地，所述绝缘膜包括磷脂膜和/或高分子膜。Further, the insulating film includes a phospholipid film and/or a polymer film.
进一步地，所述第一介质和/或所述第二介质包括氯化钠溶液、氯化锂溶液、氯化铯溶液、氯化钾溶液和溴化钠溶液中的一种或多种。Further, the first medium and/or the second medium include one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
另一方面，本发明还提供了一种核苷酸快速检测试剂盒，其特征在于，所述试剂盒包括：On the other hand, the present invention also provides a rapid detection kit for nucleotides, characterized in that the kit includes:
(1)MscS埃米孔；(1) MscS Amipore;
(2)绝缘膜；(2) insulating film;
(3)电导液。(3) Conductive fluid.
进一步地，所述MscS埃米孔包括MscS的侧孔体积变体和/或侧孔电荷变体。Further, the MscS Angstrom pore includes a side pore volume variant and/or a side pore charge variant of MscS.
进一步地，所述绝缘膜包括磷脂膜和/或高分子膜。Further, the insulating film includes a phospholipid film and/or a polymer film.
进一步地，所述电导液包括氯化钠溶液、氯化锂溶液、氯化铯溶液、氯化钾溶液和溴化钠溶液中的一种或多种。Further, the conductive solution includes one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
进一步地，所述MscS埃米孔包括PaMscS变体埃米孔。Further, the MscS pore includes a PaMscS variant pore.
进一步地，所述PaMscS变体埃米孔包括130A、130H、180R、271I、130S和130P中的一种或多种。Further, the PaMscS variant angstrompore includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
与现有技术相比，本发明的有益效果：Compared with prior art, the beneficial effect of the present invention:
本发明提供了一种利用埃米孔***检测样本中核苷酸的方法，其中埃米孔***包含MscS埃米孔。本发明创造性地利用小电导机械力敏感性通道(Mechanosensitive channel of small conductance,MscS)的特性来检测样本中核苷酸，具体体现为：The invention provides a method for detecting nucleotides in a sample by using an angstrom hole system, wherein the angstrom hole system includes a MscS angstrom hole. The present invention creatively utilizes the characteristics of a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS) to detect nucleotides in a sample, specifically embodied as follows:
1)MscS埃米孔的孔径狭窄。据估计，MscS埃米孔的孔径范围为～6-16埃米，远小于现有技术中常用的纳米孔(例如，α-溶血素纳米孔的孔径约为1.4-2.4nm，即14–24埃米)。1) The pore size of the MscS pore is narrow. The pore size of the MscS angstrompore is estimated to be in the range of ~6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the α-hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy).
2)MscS埃米孔的孔径可调(也可以理解为结构灵活)。MscS埃米孔可在毫秒内将机械刺激转化为电信号或生化信号，从而引发通道构型的调节。利用MscS埃米孔对绝缘膜所受到的机械力刺激和/或绝缘膜的物理状态变化的敏感性，可以通过影响绝缘膜来实现对MscS埃米孔孔径的调整，无需繁复的化学修饰。例如，可以调整第一介质和第二介质的浓度(即30mM NaCl/300mM NaCl、100mM NaCl/300mM NaCl和300mM NaCl/300mM NaCl)来 调整绝缘膜两侧的渗透压差进而调节孔径，实现优化对dNTPs的选择性并提高对dNTPs的区分。现有技术中的蛋白质纳米孔通常具有固定的通道结构，对核苷酸(例如，dNTP等类似分子)的直接检测通常需要额外的蛋白质工程修饰或者化学修饰引入。而本发明涉及的MscS埃米孔的孔径只需改变外界条件即可实现可逆性原位调整，适用于对核苷酸直接地单分子传感识别(也可以理解为对核苷酸的直接检测)。2) The pore size of the MscS angstrompore is adjustable (it can also be understood as a flexible structure). MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration. Utilizing the sensitivity of the MscS angstrompore to the mechanical stimulation of the insulating film and/or the change of the physical state of the insulating film, the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e. 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to achieve optimal pairing. Selectivity of dNTPs and improved discrimination of dNTPs. Protein nanopores in the prior art usually have a fixed channel structure, and the direct detection of nucleotides (for example, dNTP and other similar molecules) usually requires the introduction of additional protein engineering modifications or chemical modifications. The pore diameter of the MscS angstrom hole involved in the present invention can realize reversible in-situ adjustment only by changing the external conditions, and is suitable for direct single-molecule sensing and recognition of nucleotides (also can be understood as direct detection of nucleotides ).
3)本发明提供的方法可以直接检测并区分一种或多种核苷酸，还可以与其他策略联用，以进一步检测样本中是否有目标核酸的存在。此外，可以向MscS埃米孔的侧孔引入突变，调整侧孔处氨基酸的体积(例如，将W替换为A、S、P)和电荷(例如，将W替换为H、将K替换为R)，以实现对特定带电荷分子和特定尺寸的分子的更优检测。3) The method provided by the present invention can directly detect and distinguish one or more nucleotides, and can also be used in conjunction with other strategies to further detect the presence of the target nucleic acid in the sample. In addition, mutations can be introduced into the side pore of the MscS angstrompore, adjusting the volume (e.g., W to A, S, P) and charge (e.g., W to H, K to R) of the amino acids at the side pore. ), to achieve better detection of specific charged molecules and molecules of specific sizes.
另一方面，本发明还提供一种检测样本中的药物分子的方法，其特征在于，包括如下步骤：On the other hand, the present invention also provides a method for detecting drug molecules in a sample, which is characterized in that it includes the following steps:
S1将所述样本加入埃米孔***，所述埃米孔***包括：埃米孔、绝缘膜、第一介质、第二介质，其中所述埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口；所述样本被加入到所述第一介质；S1 adding the sample to the angstrom hole system, the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the angstrom hole is embedded in the insulating film, the The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, the angstrom hole is a MscS angstrom hole, The Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening; the sample is added to the first medium;
S2向所述第一介质和所述第二介质施加驱动力，所述样本中的药物分子与所述埃米孔相互作用并产生电信号；S2 applying a driving force to the first medium and the second medium, and drug molecules in the sample interact with the angstrom pores and generate electrical signals;
S3分析所述电信号，进而识别所述样本中的药物分子。S3 analyzes the electrical signal, and then identifies drug molecules in the sample.
进一步地，所述开口的电荷性质和/或孔径大小是可调节的。Further, the charge properties and/or pore size of the openings are adjustable.
进一步地，所述开口的调节方式包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的物理状态变化。Further, the manner of adjusting the opening includes subjecting the insulating film to mechanical force stimulation and/or changing the physical state of the insulating film.
进一步地，所述机械力刺激包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。Further, the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
进一步地，所述开口的孔径可以根据以下方式来调节：Further, the aperture of the opening can be adjusted in the following manner:
(1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
(2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
进一步地，所述埃米孔为MscS变体埃米孔。Further, the Amypore is a MscS variant Amypore.
进一步地，所述MscS变体包括侧孔体积变体和/或侧孔电荷变体。Further, the MscS variants include side hole volume variants and/or side hole charge variants.
进一步地，所述埃米孔源自杆菌。Further, the emipore is derived from bacillus.
进一步地，所述埃米孔包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。Further, the pore includes one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
进一步地，所述埃米孔为PaMscS变体埃米孔。Further, the Amipore is a PaMscS variant Amipore.
进一步地，所述埃米孔包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。Further, the angstrompore includes one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
进一步地，所述药物分子的分子量为小于1000g/mol。Further, the molecular weight of the drug molecule is less than 1000 g/mol.
进一步地，所述药物分子的分子量为177.98～712.72g/mol。Further, the molecular weight of the drug molecule is 177.98-712.72 g/mol.
进一步地，所述药物分子的浓度为大于10nM。Further, the concentration of the drug molecule is greater than 10 nM.
进一步地，所述样本为体液样本。Further, the sample is a body fluid sample.
进一步地，所述体液样本包括尿液、血液、血清、血浆、淋巴液、囊肿液、胸膜液、腹水液、腹膜液、羊水、***、脑脊液、支气管肺泡灌洗液、母乳、泪液、唾液、痰中的一种或多种。Further, the body fluid samples include urine, blood, serum, plasma, lymph fluid, cyst fluid, pleural fluid, ascitic fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tear fluid, saliva , One or more of sputum.
进一步地，所述体液样本的样本量为大于10μL。Further, the sample volume of the body fluid sample is greater than 10 μL.
进一步地，所述体液样本中的药物分子的浓度为大于10nM。Further, the concentration of drug molecules in the body fluid sample is greater than 10 nM.
进一步地，所述方法进一步包括S4：将透析装置通过导管与所述第一介质连通，使得所 述血液样本通过所述透析装置进入所述埃米孔***，其中S4先于S1。Further, the method further includes S4: connecting a dialysis device with the first medium through a catheter, so that the blood sample enters the angstrom pore system through the dialysis device, wherein S4 is prior to S1.
进一步地，所述绝缘膜包括磷脂膜和/或高分子膜。Further, the insulating film includes a phospholipid film and/or a polymer film.
与现有技术相比，本发明的有益效果：Compared with prior art, the beneficial effect of the present invention:
本发明提供了一种利用埃米孔***检测样本中药物分子的方法，其中埃米孔***包含MscS埃米孔。本发明创造性地利用小电导机械力敏感性通道(Mechanosensitive channel of small conductance,MscS)的特性来检测样本中的药物分子，具体体现为：The invention provides a method for detecting drug molecules in a sample by using an angstrom hole system, wherein the angstrom hole system includes a MscS angstrom hole. The present invention creatively utilizes the characteristics of a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS) to detect drug molecules in a sample, specifically embodied as follows:
1)MscS埃米孔的孔径狭窄。据估计，MscS埃米孔的孔径范围为～6-16埃米，远小于现有技术中常用的纳米孔(例如，α-溶血素纳米孔的孔径约为1.4-2.4nm，即14–24埃米)。1) The pore size of the MscS pore is narrow. The pore size of the MscS angstrompore is estimated to be in the range of ~6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the α-hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy).
2)MscS埃米孔的孔径可调(也可以理解为结构灵活)。MscS埃米孔可在毫秒内将机械刺激转化为电信号或生化信号，从而引发通道构型的调节。利用MscS埃米孔对绝缘膜所受到的机械力刺激和/或绝缘膜的物理状态变化的敏感性，可以通过影响绝缘膜来实现对MscS埃米孔孔径的调整，无需繁复的化学修饰。例如，可以调整第一介质和第二介质的浓度(即30mM NaCl/300mM NaCl、100mM NaCl/300mM NaCl和300mM NaCl/300mM NaCl)来调整绝缘膜两侧的渗透压差进而调节孔径，实现对分析物的选择性的优化并提高对分析物的区分。现有技术中的蛋白质纳米孔通常具有固定的通道结构，需要额外的蛋白质工程修饰或者化学修饰等才能实现通道结构调整。而本发明涉及的MscS埃米孔的孔径只需改变外界条件即可实现可逆性原位调整，适用于多种类型、尺寸的药物分子的直接检测。具体地，例如氨基糖苷类抗生素和谷氨酸等药物分子都可以引起MscS埃米孔相应的阻塞电流信号，MscS埃米孔能在单分子水平上检测药物分子。2) The pore size of the MscS angstrompore is adjustable (it can also be understood as a flexible structure). MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration. Utilizing the sensitivity of the MscS angstrompore to the mechanical stimulation of the insulating film and/or the change of the physical state of the insulating film, the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e. 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to realize the analysis Optimize the selectivity of analytes and improve the discrimination of analytes. Protein nanopores in the prior art usually have a fixed channel structure, which requires additional protein engineering modification or chemical modification to achieve channel structure adjustment. However, the pore diameter of the MscS angstrompore involved in the present invention can be reversibly adjusted in situ only by changing the external conditions, and is suitable for the direct detection of various types and sizes of drug molecules. Specifically, drug molecules such as aminoglycoside antibiotics and glutamic acid can cause corresponding blocking current signals in the MscS pore, and the MscS pore can detect drug molecules at the single-molecule level.
3)MscS埃米孔可以实现对药物分子的定量分析。药物分子的梯度浓度测量在信号频率和药物浓度之间呈现出良好的线性关系，因此MscS埃米孔不仅能够检测药物分子还能够实现对药物分子的浓度的检测(定量分析)。3) The MscS pore can realize the quantitative analysis of drug molecules. The gradient concentration measurement of drug molecules shows a good linear relationship between the signal frequency and drug concentration, so the MscS pore can not only detect drug molecules but also detect the concentration of drug molecules (quantitative analysis).
4)MscS埃米孔具有较强的抗干扰能力。MscS胞质端为类似筛状的结构，底部1个底部开口、侧部7个侧面开口，且各个开口(孔)的通道狭窄，这种结构有利于离子和小分子的通过，却能够将大分子物质(例如蛋白质)阻挡在通道外，因此这些生物大分子无法进入通道并无法对通道造成阻塞。因此，MscS展现出较强的抗干扰能力，并能够对体液样本(例如全血样本)进行直接检测。更具体地，本发明方法还可以与透析装置等装置联用，实现对血药浓度的实时、连续监测。4) MscS pore has strong anti-interference ability. The cytoplasmic end of MscS is a sieve-like structure, with one bottom opening at the bottom and seven side openings at the side, and the channels of each opening (pore) are narrow. Molecular substances, such as proteins, are blocked out of the channel, so these biomacromolecules cannot enter and block the channel. Therefore, MscS exhibits strong anti-interference ability and can be directly detected in body fluid samples (such as whole blood samples). More specifically, the method of the present invention can also be used in conjunction with devices such as dialysis devices to realize real-time and continuous monitoring of blood drug concentration.
另一方面，本发明还提供了一种检测样本中目标核酸的存在的方法，其特征在于，包括如下步骤：On the other hand, the present invention also provides a method for detecting the presence of target nucleic acid in a sample, characterized in that it comprises the following steps:
S1将样本置于核酸扩增体系并进行核酸扩增，确定所述核酸扩增体系中底物核苷酸的数量，获得所述样本的核酸扩增产物；S1 placing the sample in a nucleic acid amplification system and performing nucleic acid amplification, determining the number of substrate nucleotides in the nucleic acid amplification system, and obtaining a nucleic acid amplification product of the sample;
S2将所述样本的核酸扩增产物加入单通道电生理检测***，所述单通道电生理检测***包括：跨膜孔、绝缘膜、第一介质、第二介质，其中所述跨膜孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述跨膜孔提供连通所述第一介质与所述第二介质的通道，所述样本的核酸扩增产物被加入到所述第一介质；S2 Add the nucleic acid amplification product of the sample into a single-channel electrophysiological detection system, the single-channel electrophysiological detection system includes: a transmembrane pore, an insulating membrane, a first medium, and a second medium, wherein the transmembrane pore is covered Embedded in the insulating film, the insulating film separates the first medium from the second medium, the transmembrane pores provide channels for communicating the first medium and the second medium, the The nucleic acid amplification product of the sample is added to the first medium;
S3向所述第一介质和所述第二介质之间施加驱动力，所述样本的核酸扩增产物中剩余核苷酸与所述跨膜孔相互作用并产生电信号；S3 applies a driving force between the first medium and the second medium, and the remaining nucleotides in the nucleic acid amplification product of the sample interact with the transmembrane pore and generate an electrical signal;
S4对所述电信号进行量化，获得所述剩余核苷酸的数量；S4 quantifies the electrical signal to obtain the quantity of the remaining nucleotides;
S5将所述剩余核苷酸的数量与所述底物核苷酸的数量进行对比，确定所述样本中所述目标核酸是否存在。S5 compares the quantity of the remaining nucleotides with the quantity of the substrate nucleotides to determine whether the target nucleic acid exists in the sample.
进一步地，所述跨膜孔为MscS变体埃米孔。Further, the transmembrane pore is an MscS variant pore.
进一步地，所述MscS变体包括侧孔体积变体和/或侧孔电荷变体。Further, the MscS variants include side hole volume variants and/or side hole charge variants.
进一步地，所述MscS变体埃米孔的开口的电荷性质和/或孔径大小是可调节的。Further, the charge properties and/or pore size of the opening of the angstrom pore of the MscS variant are adjustable.
进一步地，所述开口的调节方式包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的 物理状态变化。Further, the way of adjusting the opening includes subjecting the insulating film to mechanical stimulation and/or changing the physical state of the insulating film.
进一步地，所述机械力刺激包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。Further, the mechanical stimulation includes one or more of changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film, and stimulation of the insulating film by negative pressure pressure. kind.
进一步地，所述开口的孔径可以根据以下方式来调节：Further, the aperture of the opening can be adjusted in the following manner:
(1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
(2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
进一步地，所述第一介质与所述第二介质之间的渗透压差是通过所述第一介质与所述第二介质之间的浓度差来调节的。Further, the osmotic pressure difference between the first medium and the second medium is adjusted by the concentration difference between the first medium and the second medium.
进一步地，所述第一介质与所述第二介质之间的浓度差为大约0-270mM。Further, the concentration difference between the first medium and the second medium is about 0-270 mM.
进一步地，所述第一介质和/或所述第二介质包括氯化钠溶液、氯化锂溶液、氯化铯溶液、氯化钾溶液和溴化钠溶液中的一种或多种。Further, the first medium and/or the second medium include one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
进一步地，所述MscS变体埃米孔源自杆菌。Further, the MscS variant emicon is derived from Bacillus.
进一步地，所述MscS变体埃米孔包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。Further, the MscS variant Emipore includes one or more of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori.
进一步地，所述MscS变体埃米孔为PaMscS变体埃米孔。Further, the MscS variant emipore is a PaMscS variant emipore.
进一步地，所述PaMscS变体埃米孔包括130A、130H、180R、271I、130S和130P中的一种或多种。Further, the PaMscS variant angstrompore includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
进一步地，通过聚合酶链反应、连接酶链反应、链置换扩增技术、转录介导的扩增技术、环介导等温扩增技术中的一种或多种进行所述核酸扩增。Further, the nucleic acid amplification is performed by one or more of polymerase chain reaction, ligase chain reaction, strand displacement amplification technique, transcription-mediated amplification technique, and loop-mediated isothermal amplification technique.
进一步地，所述核苷酸包括核糖核苷酸和/或脱氧核糖核苷酸。Further, the nucleotides include ribonucleotides and/or deoxyribonucleotides.
进一步地，所述核苷酸包括dGTP、dATP、dTTP、dCTP、dUTP、GTP、ATP、TTP、CTP、UTP中的一种或多种。Further, the nucleotides include one or more of dGTP, dATP, dTTP, dCTP, dUTP, GTP, ATP, TTP, CTP, UTP.
进一步地，所述核酸扩增体系进一步包括：Further, the nucleic acid amplification system further includes:
(1)探针，所述探针包括互补区和重复区，所述互补区包括与所述目标核酸互补配对的序列，所述重复区包括重复同一个碱基的寡核苷酸序列，所述碱基包括A、T、C、G、U；或者(1) a probe, the probe includes a complementary region and a repeating region, the complementary region includes a sequence that is complementary to the target nucleic acid, and the repeating region includes an oligonucleotide sequence that repeats the same base, so The bases include A, T, C, G, U; or
(2)所述目标核酸的特异性引物。(2) Specific primers for the target nucleic acid.
进一步地，所述目标核酸为冠状病毒核酸。Further, the target nucleic acid is coronavirus nucleic acid.
进一步地，所述冠状病毒包括SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV中的一种或多种。Further, the coronavirus includes one or more of SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
进一步地，所述绝缘膜包括磷脂膜和/或高分子膜。Further, the insulating film includes a phospholipid film and/or a polymer film.
另一方面，本发明还提供一种病毒快速检测试剂盒，包括：On the other hand, the present invention also provides a virus rapid detection kit, comprising:
(1)MscS埃米孔；(1) MscS Amipore;
(2)绝缘膜；(2) insulating film;
(3)电导液；(3) conductive liquid;
(4)所述病毒的核酸的特异性引物或探针。(4) A specific primer or probe for nucleic acid of the virus.
进一步地，所述MscS埃米孔包括MscS的侧孔体积变体和/或侧孔电荷变体。Further, the MscS Angstrom pore includes a side pore volume variant and/or a side pore charge variant of MscS.
进一步地，所述绝缘膜包括磷脂膜和/或高分子膜。Further, the insulating film includes a phospholipid film and/or a polymer film.
进一步地，所述电导液包括氯化钠溶液、氯化锂溶液、氯化铯溶液、氯化钾溶液和溴化钠溶液中的一种或多种。Further, the conductive solution includes one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
进一步地，所述埃米孔为PaMscS变体埃米孔。Further, the Amipore is a PaMscS variant Amipore.
进一步地，所述埃米孔包括130A、130H、180R、271I、130S和130P中的一种或多种。Further, the Angstrom hole includes one or more of 130A, 130H, 180R, 271I, 130S and 130P.
与现有技术相比，本发明的有益效果：Compared with prior art, the beneficial effect of the present invention:
本发明提供了一种利用单通道电生理检测***检测样本中目标核酸的存在的方法。The present invention provides a method for detecting the presence of a target nucleic acid in a sample using a single-channel electrophysiological detection system.
1)本发明提供的方法可以快速检测样本中目标核酸的存在。传统的核酸检测方法通常需要对目标核酸进行荧光标记或染色，因此，其依赖昂贵的荧光监测设备或染色体系。目前 基于纳米孔的核酸检测方法，往往需要辅助性的多核苷酸结合蛋白(例如解旋酶、聚合酶)等复杂的体系。本发明提供的方法利用跨膜孔，仅需检测体外核酸扩增体系(例如底物dNTPs、聚合酶、反转录酶)中底物dNTPs的消耗，进而判断核酸扩增体系里是否存在目标核酸，具有快速、成本低、易于高通量检测、特异性和敏感性较佳的优势。本发明涉及的跨膜孔能够检测(或区分)不同核苷酸，且不受核酸扩增体系中的其他物质(例如，扩增出的核酸、酶)的干扰。1) The method provided by the present invention can rapidly detect the presence of target nucleic acid in a sample. Traditional nucleic acid detection methods usually require fluorescent labeling or staining of target nucleic acids, therefore, they rely on expensive fluorescence monitoring equipment or staining systems. Current nanopore-based nucleic acid detection methods often require complex systems such as auxiliary polynucleotide binding proteins (such as helicases and polymerases). The method provided by the present invention utilizes transmembrane pores, and only needs to detect the consumption of substrate dNTPs in an in vitro nucleic acid amplification system (such as substrate dNTPs, polymerase, reverse transcriptase), and then judge whether there is a target nucleic acid in the nucleic acid amplification system , which has the advantages of rapidity, low cost, easy high-throughput detection, and good specificity and sensitivity. The transmembrane pore involved in the present invention can detect (or distinguish) different nucleotides, and is not interfered by other substances (eg, amplified nucleic acid, enzyme) in the nucleic acid amplification system.
2)作为优选，本发明的跨膜孔为MscS(小电导机械力敏感性通道)埃米孔，其孔径狭窄、孔径可调(也可以理解为结构灵活)。据估计，MscS埃米孔的孔径范围为～6-16埃米，远小于现有技术中常用的纳米孔(例如，α-溶血素纳米孔的孔径约为1.4-2.4nm，即14–24埃米)。MscS埃米孔可在毫秒内将机械刺激转化为电信号或生化信号，从而引发通道构型的调节。利用MscS埃米孔对绝缘膜所受到的机械力刺激和/或绝缘膜的物理状态变化的敏感性，可以通过影响绝缘膜来实现对MscS埃米孔孔径的调整，无需繁复的化学修饰。例如，可以调整第一介质和第二介质的浓度(即30mM NaCl/300mM NaCl、100mM NaCl/300mM NaCl和300mM NaCl/300mM NaCl)来调整绝缘膜两侧的渗透压差进而调节孔径，实现优化对dNTPs的选择性并提高对dNTPs的区分。现有技术中的蛋白质纳米孔通常具有固定的通道结构，对核苷酸(例如，dNTP等类似分子)的直接检测通常需要额外的蛋白质工程修饰或者化学修饰引入。而本发明涉及的MscS埃米孔的孔径只需改变外界条件即可实现可逆性原位调整，适用于对核苷酸直接地单分子传感识别(也可以理解为对核苷酸的直接检测)。2) As a preference, the transmembrane pore of the present invention is a MscS (small conductance mechanosensitive channel) Angstrom pore, which has a narrow pore size and an adjustable pore size (it can also be understood as a flexible structure). The pore size of the MscS angstrompore is estimated to be in the range of ~6-16 angstrom, much smaller than the nanopores commonly used in the prior art (for example, the α-hemolysin nanopore has a pore size of about 1.4-2.4 nm, i.e. 14–24 Amy). MscS angiopores can convert mechanical stimuli into electrical or biochemical signals within milliseconds, eliciting modulation of channel configuration. Utilizing the sensitivity of the MscS angstrompore to the mechanical stimulation of the insulating film and/or the change of the physical state of the insulating film, the pore size of the MscS angstrompore can be adjusted by affecting the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e. 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference on both sides of the insulating membrane and then adjust the pore size to achieve optimal pairing. Selectivity of dNTPs and improved discrimination of dNTPs. Protein nanopores in the prior art usually have a fixed channel structure, and the direct detection of nucleotides (for example, dNTP and other similar molecules) usually requires the introduction of additional protein engineering modifications or chemical modifications. The pore diameter of the MscS angstrom hole involved in the present invention can realize reversible in-situ adjustment only by changing the external conditions, and is suitable for direct single-molecule sensing and recognition of nucleotides (also can be understood as direct detection of nucleotides ).
如本文所使用，术语“源自”不仅是指所讨论的生物菌株产生的蛋白质，还指由分离自此类菌株的DNA序列编码且在含有此类DNA序列的宿主生物中产生的蛋白质。As used herein, the term "derived from" refers not only to proteins produced by the strain of organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences.
如本文所使用，术语“带电荷分子”是指带有净电荷的、尺寸小于或等于本发明涉及的埃米孔孔径的物质。示例性的带电荷分子包括核苷酸、氨基酸、肽、药物分子和/或其他带电荷小分子(例如短肽)。As used herein, the term "charged molecule" refers to a substance with a net charge and a size smaller than or equal to the Angstrom pore size to which the present invention relates. Exemplary charged molecules include nucleotides, amino acids, peptides, drug molecules, and/or other charged small molecules (eg, short peptides).
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the description of the embodiments or the prior art. Apparently, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.
图1示出了基于PaMscS埃米孔的电生理测试和dNTP检测；Figure 1 shows the electrophysiological test and dNTP detection based on PaMscS angstrompore;
图2示出了不同渗透压差条件下dNTP通过PaMscS1孔的转运频率；Figure 2 shows the translocation frequency of dNTPs through PaMscS1 pores under different osmotic pressure conditions;
图3示出了基于通过PaMscS2实时监测dNTPs消耗的SARS-CoV-2核酸检测；Figure 3 shows the detection of SARS-CoV-2 nucleic acid based on real-time monitoring of dNTPs consumption by PaMscS2;
图4示出了由PaMscS1通过dNTPs消耗检测AFP适配体和miR21；Figure 4 shows the detection of AFP aptamers and miR21 by PaMscS1 through dNTPs depletion;
图5示出了PaMscS蛋白(1：野生型PaMscS；2：W130A突变体；3：K180R突变体；4：标记物)的SDS-PAGE结果；Figure 5 shows the SDS-PAGE results of PaMscS protein (1: wild-type PaMscS; 2: W130A mutant; 3: K180R mutant; 4: marker);
图6示出了野生型或突变型的PaMscS的电流信号或电流分布；Figure 6 shows the current signal or current distribution of wild-type or mutant PaMscS;
图7示出了在0mV到+100mV的斜坡电压下，通过单个PaMscS1埃米孔的电流轨迹；Figure 7 shows the current trajectory through a single PaMscS1 angstrom hole under a ramp voltage from 0 mV to +100 mV;
图8示出了不同离子通过PaMscS1孔中的转运能力；Figure 8 shows the transport capacity of different ions through the pores of PaMscS1;
图9示出了不同电压下dNTPs通过PaMscS1埃米孔的易位频率统计(n＝3)；Figure 9 shows the translocation frequency statistics of dNTPs passing through the PaMscS1 angstrom hole at different voltages (n=3);
图10示出了PaMscS1检测单核苷酸的电流轨迹和停留时间分布；Figure 10 shows the current trajectory and residence time distribution of PaMscS1 detecting single nucleotides;
图11示出了单链DNA不能通过PaMscS1孔易位；Figure 11 shows that single-stranded DNA cannot translocate through the PaMscS1 pore;
图12示出了通过环介导等温扩增(LAMP)的SARS-CoV-2orf1ab基因的埃米孔检测结果；Figure 12 shows the results of the Angstrom hole detection of the SARS-CoV-2 orf1ab gene by loop-mediated isothermal amplification (LAMP);
图13示出了与miR21和AFP适配体混合的PCR试剂的非变性(native)PAGE电泳的结果；Figure 13 shows the result of the non-denaturing (native) PAGE electrophoresis of the PCR reagent mixed with miR21 and AFP aptamer;
图14示出了基于PaMscS3(V271I)埃米孔的药物单分子生物传感实验；Figure 14 shows the drug single-molecule biosensing experiment based on PaMscS3 (V271I) pore;
图15示出了全血样品的药物浓度测量；Figure 15 shows drug concentration measurements of whole blood samples;
图16示出了通过埃米孔连续监测活体大鼠体内药物浓度的概念验证实验；Figure 16 shows a proof-of-concept experiment for continuous monitoring of drug concentration in living rats via an emipore;
图17示出了硫酸庆大霉素的PaMscS3(V271I)埃米孔的连续电流轨迹；Figure 17 shows the continuous current trace of the PaMscS3(V271I) angstrom pore of gentamicin sulfate;
图18示出了硫酸新霉素的PaMscS3(V271I)埃米孔的连续电流轨迹；Figure 18 shows the continuous current trace of the PaMscS3 (V271I) angstrom pore of neomycin sulfate;
图19示出了高浓度硫酸庆大霉素和硫酸新霉素可以长时间阻塞PaMscS3(V271I)埃米孔；Figure 19 shows that high concentrations of gentamicin sulfate and neomycin sulfate can block PaMscS3 (V271I) angstrom pores for a long time;
图20示出了MspA-2NNN埃米孔可被全血样本(10μL全血样本到加入1mL端)频繁阻塞；Figure 20 shows that the MspA-2NNN angstrom pore can be frequently blocked by whole blood samples (10 μL whole blood sample to the 1 mL end);
图21示出了血液样本中PaMscS3(V271I)埃米孔的连续电流轨迹；Figure 21 shows the continuous current trace of the PaMscS3(V271I) angstrom pore in a blood sample;
图22示出了通过PaMscS3(V271I)埃米孔直接测量大鼠全血样本的电流轨迹；Figure 22 shows the current trace of direct measurement of rat whole blood samples by PaMscS3 (V271I) angstrom hole;
图23示出了从-50mV至-80mV通过PaMscS3(V271I)埃米孔的硫酸庆大霉素的电流信号；Figure 23 shows the current signal of gentamicin sulfate from -50mV to -80mV through PaMscS3 (V271I) angstrompore;
图24示出了从-50mV到-80mV通过PaMscS3(V271I)埃米孔的西索米星的电流信号；Fig. 24 shows the current signal of the sisomi star through the PaMscS3 (V271I) emipore from -50mV to -80mV;
图25示出了野生型EcMscS的单个通道嵌入电流轨迹(电压+100mV，电导液30mM:300mM NaCl)；Figure 25 shows a single channel embedded current track of wild-type EcMscS (voltage+100mV, conductive solution 30mM:300mM NaCl);
图26示出了野生型EcMscS的通道扫描电压(-100mV到100mV)；Figure 26 shows the channel sweep voltage (-100mV to 100mV) of wild-type EcMscS;
图27示出了野生型EcMscS的电导分布；Figure 27 shows the conductance distribution of wild-type EcMscS;
图28示出了PaMscS与其他细菌的MscS的序列比对；Figure 28 shows a sequence alignment of PaMscS with MscS of other bacteria;
图29示出了基于野生型PaMscS埃米孔的dNTP检测；Figure 29 shows dNTP detection based on wild-type PaMscS angiopores;
图30示出了PaMscS1检测谷氨酸的电流轨迹；Figure 30 shows the current trace of the detection of glutamate by PaMscS1;
图31示出了基于PaMscS埃米孔的氨基酸检测方案和不同氨基酸阻塞电流分布。Figure 31 shows the PaMscS Angstrompore-based amino acid detection scheme and different amino acid blocking current distributions.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。It should be noted that, in this document, the term "comprising", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, It also includes other elements not expressly listed, or elements inherent in the process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising that element.
如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value /-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.
在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围1～6的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description "within a certain range" is merely for convenience and brevity, and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range 1 to 6 should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc. , and individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the scope.
详细附图说明Detailed Description of Drawings
图1：A.电生理测量室示意图。B.在+50mv电压下，PaMscS1和PaMscS2埃米孔的单孔***。C.PaMscS1和PaMscS2埃米孔在电压范围-50mv到+50mv下的I-V关系。D.PaMscS1 和PaMscS2埃米孔的电导分布(分别为N＝18)(缓冲条件为-cis端：300mM NaCl，-trans端：30mM NaCl)。E.通过PaMscS突变体埃米孔检测dNTPs(缓冲条件为-cis端：300mM NaCl，-trans端：30mM NaCl，并且电压为+50mv)。Figure 1: A. Schematic diagram of the electrophysiological measurement chamber. B. Single pore insertion of PaMscS1 and PaMscS2 angstrompores at +50mv voltage. C. I-V relationship of PaMscS1 and PaMscS2 Angstrompores in the voltage range -50mV to +50mV. D. Conductance distribution of PaMscS1 and PaMscS2 angstrom pores (N=18 respectively) (buffer conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl). E. Detection of dNTPs through the angstrom hole of the PaMscS mutant (buffer conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl, and the voltage is +50mv).
图2：在不同的渗透压差的条件下测试了dCTP(以橙色表示)和dGTP(以蓝色表示)的易位频率，对称(A，300mM NaCl：300mM NaCl用于cis端：trans端)，低渗透压差(LOD)(B，300mM NaCl：100mM NaCl用于cis端：trans端)，以及高渗透压差(HOD)(C，300mM NaCl：30mM NaCl用于cis端：trans端)。四组dNTPs浓度，0.5mM、1.0mM、1.5mM和2.0mM，以测试了dCTP和dGTP的易位。D.在对称、低渗透压差和高渗透压差条件下，易位频率与dCTP/dGTP浓度之间的关系(每个数据点n＝3)。E.在3种不同的渗透压差条件下，f dCTP和f dGTP的增加率。 Figure 2: The translocation frequency of dCTP (indicated in orange) and dGTP (indicated in blue) was tested under different osmotic pressure differences, symmetrical (A, 300mM NaCl: 300mM NaCl for cis end: trans end) , low osmotic pressure difference (LOD) (B, 300 mM NaCl: 100 mM NaCl for cis end: trans end), and high osmotic pressure difference (HOD) (C, 300 mM NaCl: 30 mM NaCl for cis end: trans end). Four sets of dNTPs concentrations, 0.5 mM, 1.0 mM, 1.5 mM and 2.0 mM, were used to test the translocation of dCTP and dGTP. D. The relationship between translocation frequency and dCTP/dGTP concentration under symmetric, low osmolarity and high osmolarity conditions (n=3 for each data point). E. The increase rate of f dCTP and f dGTP under 3 different osmotic pressure difference conditions.
图3：A.埃米孔检测SARS-CoV-2的示意图。B.梯度拷贝数下SARS-CoV-2 Orf1ab基因检测结果(缓冲液条件为-cis端：300mM NaCl，-trans端：100mM NaCl，电压+50mV，阴性对照(NC)组：n＝4，10^11拷贝/mL组：n＝4，10^8拷贝/mL组：n＝4，10^5拷贝/mL组：n＝4，10^3拷贝/mL组：n＝3)。C.22份临床样本检测结果，包括15个阳性样本(患者编号：1～15)和7个阴性样本(患者编号：16～22)，21份样本的埃米孔检测结果与医院qPCR检测结果一致(患者编号：1～15，17～22)，1个阴性样本(患者编号：16)被埃米孔诊断为阳性(缓冲条件为-cis端：300mM NaCl，-trans端：100mM NaCl，电压为+50mV)。D.通过MscS埃米孔监测dNTPs的消耗可以结合核酸扩增技术(NAAT)，如聚合酶链反应(PCR)和链式置换扩增(SDA)。Figure 3: A. Schematic diagram of the detection of SARS-CoV-2 by angstrom. B. Detection results of SARS-CoV-2 Orf1ab gene under gradient copy number (buffer conditions are -cis end: 300mM NaCl, -trans end: 100mM NaCl, voltage +50mV, negative control (NC) group: n=4, 10 ^11 copy/mL group: n=4, 10^8 copy/mL group: n=4, 10^5 copy/mL group: n=4, 10^3 copy/mL group: n=3). C. Test results of 22 clinical samples, including 15 positive samples (patient numbers: 1-15) and 7 negative samples (patient numbers: 16-22), Amypore test results of 21 samples and hospital qPCR test results Consistent (Patient No.: 1-15, 17-22), 1 negative sample (Patient No.: 16) was diagnosed as positive by Amypore (buffer conditions were -cis end: 300mM NaCl, -trans end: 100mM NaCl, voltage is +50mV). D. Monitoring dNTPs depletion via MscS angstropores can be combined with nucleic acid amplification techniques (NAAT) such as polymerase chain reaction (PCR) and strand displacement amplification (SDA).
图4：A.检测策略示意图。B.无靶标对照组、miR21组、AFP适配体组和既有miR21又有AFP适配体组的电流轨迹。C.4个检测组的电流分布。D.4组中dATP和dGTP信号的相对增加。没有miR21和AFP适配体的样本不会导致dNTPs消耗；有miR21的样本会导致更多的dATP消耗和相对更低的dATP易位频率；有AFP适配体的样本会导致更多的dGTP消耗和相对更低的dGTP易位频率；既有miR21又有AFP适配体的样本将导致更多的dATP和dGTP消耗以及相对更低的dATP和dGTP易位频率(缓冲条件为-cis端：300mM NaCl，-trans端：100mM NaCl，电压为+50mV，每个实验n＝3)。Figure 4: A. Schematic of the detection strategy. B. Current traces of no target control group, miR21 group, AFP aptamer group, and both miR21 and AFP aptamer groups. C. Current distribution of the 4 test groups. Relative increase in dATP and dGTP signals in group D.4. Samples without miR21 and AFP aptamers did not result in dNTPs depletion; samples with miR21 resulted in more dATP consumption and relatively lower dATP translocation frequency; samples with AFP aptamers resulted in more dGTP depletion and relatively lower dGTP translocation frequency; samples with both miR21 and AFP aptamer will result in more dATP and dGTP consumption and relatively lower dATP and dGTP translocation frequency (buffer condition is -cis end: 300mM NaCl, -trans end: 100mM NaCl, the voltage is +50mV, each experiment n=3).
图6：A.野生型PaMscS和突变型PaMscS1、PaMscS2的背景信号频率，PaMscS1和PaMscS2的背景噪音频率低于野生型PaMscS(电压为+50mv，n≥3)。B.PaMscS1和PaMscS2埃米孔***的时间，PaMscS2埃米孔具有比PaMscS1更高的膜融合效率(n≥3)。C.PaMscS1和PaMscS2埃米孔的dNTPs阻塞电流分布。Figure 6: A. The background signal frequency of wild-type PaMscS and mutant PaMscS1, PaMscS2, the background noise frequency of PaMscS1 and PaMscS2 is lower than that of wild-type PaMscS (voltage +50mv, n≥3). B. Insertion time of PaMscS1 and PaMscS2 angstrompores, PaMscS2 angstrompores have higher membrane fusion efficiency than PaMscS1 (n≥3). C. Blockage current distribution of dNTPs in PaMscS1 and PaMscS2 angstrompores.
图7：在0mV到+100mV的斜坡电压下，通过单个PaMscS1埃米孔的电流轨迹：当电压升高到+90mV以上时，观察到电压门控(缓冲条件为-cis端:300mM NaCl，-trans端:30mM NaCl，采样频率：4999hz)。Figure 7: Current traces through a single PaMscS1 angstrom pore under a ramped voltage from 0 mV to +100 mV: Voltage gating was observed when the voltage was raised above +90 mV (buffer conditions at the -cis end: 300 mM NaCl, - Trans end: 30mM NaCl, sampling frequency: 4999hz).
图8：缓冲条件为：-cis侧300mM NaCl，-trans侧30mM NaCl，每个数据点n≥3，平均值±SD。Figure 8: The buffer conditions are: -cis side 300mM NaCl, -trans side 30mM NaCl, each data point n≥3, mean ± SD.
图10：停留时间分布：dGTP(A)、dATP(B)、dTTP(C)和dCTP(D)；每个核苷酸的浓度为2mM且缓冲条件为-cis端：300mM NaCl，-trans端：30mM NaCl，电压为+50mV。Figure 10: Residence time distribution: dGTP(A), dATP(B), dTTP(C) and dCTP(D); the concentration of each nucleotide is 2mM and the buffer condition is -cis end: 300mM NaCl, -trans end : 30mM NaCl, the voltage is +50mV.
图11：电压：+50mV；缓冲条件：cis侧300mM NaCl，trans侧30mM NaCl。ssDNA的最终浓度为5μM，序列为5′TAGCTTATCAGACTGATGTTGA 3′(SEQ ID NO:5)。Figure 11: Voltage: +50mV; buffer conditions: 300mM NaCl on the cis side, 30mM NaCl on the trans side. The final concentration of ssDNA was 5 μM and the sequence was 5'TAGCTTATCAGACTGATGTTGA 3' (SEQ ID NO:5).
图12：包含10^3拷贝/mL至10^11拷贝/mL的Orf1ab基因的样本可被检测。Figure 12: Samples containing 10^3 copies/mL to 10^11 copies/mL of the Orf1ab gene can be detected.
图13：1.DNA模板1(含poly T)；2.DNA模板2(含poly C)；3.有miR21和AFP适配体的PCR试剂；4.对照组(没有miR21和AFP适配体)。Figure 13: 1. DNA template 1 (containing poly T); 2. DNA template 2 (containing poly C); 3. PCR reagents with miR21 and AFP aptamers; 4. Control group (without miR21 and AFP aptamers ).
图14：A.通过PaMscS3(V271I)埃米孔检测硫酸庆大霉素的结果，包括代表性电流轨迹和阻塞信号。B.通过PaMscS3埃米孔检测硫酸新霉素的结果。C.硫酸庆大霉素的定量标准曲线(N＝3)和阻塞信号的热图(右，878个阻塞事件)。D.硫酸新霉素的定量标准曲线(中间，N＝4) 和阻塞信号的热图(右，883个阻塞事件)。电解质条件为-cis端：300mM NaCl，-trans端：30mM NaCl，10mM HEPES，pH 7.0，药物检测的电压为-50mV。E.比较LC-MS和PaMscS3埃米孔之间对1.5μM硫酸庆大霉素的检测结果。电解质条件为-cis端：130mMNaCl，-trans端：130mM NaCl，10mM HEPES，pH 7.0，药物的检测电压为-50mV。Figure 14: A. Detection of gentamicin sulfate through PaMscS3(V271I) angmipore, including representative current traces and blocking signals. B. Results of detection of neomycin sulfate by PaMscS3 angstrompore. C. Quantitative standard curve (N=3) and heat map of the blocking signal for gentamicin sulfate (right, 878 blocking events). D. Quantitative standard curve for neomycin sulfate (middle, N=4) and heat map of the blocking signal (right, 883 blocking events). The electrolyte conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl, 10mM HEPES, pH 7.0, and the voltage for drug detection is -50mV. E. Comparison of detection results for 1.5 μM gentamicin sulfate between LC-MS and PaMscS3 angstrompores. The electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, and the drug detection voltage is -50mV.
图15：A.通过PaMscS3(V271I)埃米孔直接检测全血样品，加入全血样品后PaMscS3埃米孔保持开放。电解质条件为-cis端：130mM NaCl，-trans端：130mM NaCl，10mM HEPES，pH 7.0，电压为-50mV。B.加入20μL大鼠血液后，-cis端的电导缓冲液变成红色。C.全血样本的通道开放的百分比。D.硫酸庆大霉素的定量标准曲线范围为0至3μM。E.通过PaMscS3埃米孔测量注射庆大霉素后不同时间间隔的大鼠的药物浓度。全血埃米孔检测的电解质条件是-cis端：130mM NaCl，-trans端：130mM NaCl，10mM HEPES，pH 7.0，电压为-50mV，N≥3。Figure 15: A. Direct detection of whole blood samples through the PaMscS3(V271I) angstrompore, the PaMscS3 angstrompore remained open after addition of the whole blood sample. Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV. B. After adding 20 μL of rat blood, the conductivity buffer at the -cis end turns red. C. Percentage of channel opening for whole blood samples. D. The quantitative standard curve of gentamicin sulfate ranges from 0 to 3 μM. E. Drug concentrations in rats at different time intervals after injection of gentamicin were measured by PaMscS3 angstrompore. Electrolyte conditions for whole blood Amypore detection are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV, N≥3.
图16：A.大鼠药物浓度监测***的装置。B.硫酸庆大霉素的定量标准曲线范围从0到30μM。C.在注射4mg/kg和20mg/kg硫酸庆大霉素的药物浓度监测期间，PaMscS3(V271I)埃米孔的典型电流轨迹。D.以不同剂量的硫酸庆大霉素、通过PaMscS3埃米孔对大鼠的连续药物血液浓度监测结果(N＝1)。灰色数据点表明药物阻塞信号频率高于标准曲线范围内的最高信号频率，双步信号频繁出现并使定量不准确。电解质条件为-cis端：130mM NaCl，-trans端：130mM NaCl，10mM HEPES，pH 7.0，电压为-50mV。Figure 16: A. Setup of the Rat Drug Concentration Monitoring System. B. Quantitative standard curve for gentamicin sulfate ranging from 0 to 30 μM. C. Typical current traces of PaMscS3(V271I) angstropores during drug concentration monitoring of 4 mg/kg and 20 mg/kg gentamicin sulfate injections. D. Results of continuous drug blood concentration monitoring of rats with different doses of gentamicin sulfate through the PaMscS3 angstrom pore (N=1). Gray data points indicate drug blocking signal frequencies higher than the highest signal frequency within the range of the standard curve, double-step signals occur frequently and make quantitation inaccurate. Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV.
图17：埃米孔背景的电流轨迹和加入硫酸庆大霉素后的轨迹。电解质条件为-cis端：300mM NaCl，-trans端：30mM NaCl，10mM HEPES，pH 7.0，电压为-50mV，N＝3。Figure 17: Current trajectories in the background of the pore and the trajectories after adding gentamicin sulfate. Electrolyte conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV, N=3.
图18：埃米孔的背景电流轨迹和添加硫酸新霉素后的轨迹。电解质条件为-cis端：300mM NaCl，-trans端：30mM NaCl，10mM HEPES，pH 7.0，电压为-50mV，N＝3。Figure 18: Background current traces of the angmipore and traces after addition of neomycin sulfate. Electrolyte conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl, 10mM HEPES, pH 7.0, voltage -50mV, N=3.
图19：在高浓度药物下，药物的阻塞信号难以统计且PaMscS3(V271I)埃米孔可被长时间阻塞，导致无法定量计算。电解质条件为-cis端：300mM NaCl，-trans端：30mM NaCl，10mM HEPES，pH 7.0，药物检测的电压为-50mV。Figure 19: At high drug concentrations, the blocking signal of the drug is difficult to count and the pore of PaMscS3(V271I) can be blocked for a long time, making quantitative calculation impossible. The electrolyte conditions are -cis end: 300mM NaCl, -trans end: 30mM NaCl, 10mM HEPES, pH 7.0, and the voltage for drug detection is -50mV.
图20：电解质条件为-cis端：300mM NaCl，-trans端：300mM NaCl，10mM HEPES，pH 7.0，电压为+100mV，N＝3。Figure 20: Electrolyte conditions are -cis end: 300mM NaCl, -trans end: 300mM NaCl, 10mM HEPES, pH 7.0, voltage +100mV, N=3.
图21：埃米孔背景的电流轨迹和全血样品加入后的轨迹。Figure 21: Current traces in the background of the angstrom hole and traces after the addition of whole blood samples.
图22：将20μL大鼠全血加入-cis端(1mL)，PaMscS3(V271I)埃米孔可以在全血样品存在下工作良好。Figure 22: Adding 20 μL of rat whole blood to the -cis end (1 mL), the PaMscS3(V271I) angstrom pore can work well in the presence of whole blood samples.
图23：电解质条件为-cis端：130mM NaCl，-trans端：130mM NaCl，10mM HEPES，pH 7.0。值得注意的是，梯度电压中出现了两个峰值。Figure 23: Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0. Notably, two peaks appeared in the gradient voltage.
图24：电解质条件为-cis端：130mM NaCl，-trans端：130mM NaCl，10mM HEPES，pH 7.0。西索米星的结构接近硫酸庆大霉素的C1a组分。在梯度电压下，只观察到一个阻塞电流峰值。Figure 24: Electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0. The structure of sisomicin is close to the C1a component of gentamicin sulfate. Under the gradient voltage, only one blocking current peak was observed.
图28：MscS家族的序列比对。图28d中红色突出显示的残基在4条序列中是相同的；序列上方的圆柱指定为α螺旋和β链。Figure 28: Sequence alignment of the MscS family. Residues highlighted in red in Figure 28D are identical across the 4 sequences; columns above the sequences designate α-helices and β-strands.
埃米孔emikon
本发明所使用的埃米孔是小电导机械力敏感性通道(Mechanosensitive channel of small conductance,MscS)，优选的是PaMscS(铜绿假单胞菌小电导机械力敏感性通道)或其变体。所述变体(也可以理解为“突变体”)可以是由生物体(例如铜绿假单胞菌)表达的天然存在变体。变体还包括由重组技术产生的非天然存在变体。在本发明中，“PaMscS变体”、“突变型PaMscS”、“突变体PaMscS”、“PaMscS突变体”表示相同含义，除非另有说明。The angstrom pore used in the present invention is a small conductance mechanosensitive channel (Mechanosensitive channel of small conductance, MscS), preferably PaMscS (Pseudomonas aeruginosa small conductance mechanosensitive channel) or a variant thereof. The variant (also understood as "mutant") may be a naturally occurring variant expressed by an organism such as Pseudomonas aeruginosa. Variants also include non-naturally occurring variants produced by recombinant techniques. In the present invention, "PaMscS variant", "mutant PaMscS", "mutant PaMscS", and "PaMscS mutant" have the same meaning unless otherwise specified.
在本发明的一个实施例中，所述埃米孔可以为MscS变体。可以对SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4的氨基酸序列进行氨基酸取代，例如对单个或多个氨基酸取代。取代可以是保守取代或非保守取代。作为优选，可以对SEQ ID NO:1或SEQ ID  NO:2或SEQ ID NO:3或SEQ ID NO:4的氨基酸序列的一个或多个位置进行非保守取代，其中被取代的氨基酸残基被化学性质和/或物理尺寸明显不同的氨基酸所替换。进一步地，所述MscS变体可分为侧孔体积变体和侧孔电荷变体。侧孔体积变体是指突变位点位于胞质端的侧面开口(也可以理解为“侧孔”)处且通过改变该位点的氨基酸，使得其侧孔体积改变的变体。侧孔电荷变体是指突变位点位于胞质端的侧面开口处且通过改变该位点的氨基酸，使得其侧孔电荷改变的变体。例如，所述侧孔体积变体可以是将较大体积的氨基酸(例如，色氨酸(W))替换成较小体积的氨基酸(例如，丙氨酸(A)、丝氨酸(S)或脯氨酸(P))，或反之。侧孔电荷变体可以是将带某种电荷的氨基酸替换成带相反电荷或中性的氨基酸，也可以是将中性的氨基酸替换成带电荷的氨基酸。一般来说，带正电荷的氨基酸的非限定性实例包括组氨酸、精氨酸和赖氨酸；带负电荷的非限定性实例包括天冬氨酸和谷氨酸；中性的非限定性实例包括甘氨酸、丙氨酸、苯丙氨酸、缬氨酸、亮氨酸、异亮氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、脯氨酸和色氨酸。氨基酸的保守取代或非保守取代，以及对氨基酸的许多不同类型(删除、置换、添加)等修饰都是本领域公知的，本领域技术人员可根据实际情况来对MscS进行修饰，以得到相应MscS变体。修饰的手段包括对相应的DNA序列进行修改(例如，修改DNA序列信息后直接合成相应蛋白质或者利用PCR对DNA序列进行定点突变)，进而得到相应变体(及其对应的DNA序列)。In one embodiment of the present invention, the angstrompore may be a MscS variant. Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, for example single or multiple amino acid substitutions. Substitutions may be conservative or non-conservative. Preferably, non-conservative substitutions can be made to one or more positions of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, wherein the substituted amino acid residue is replaced by Amino acids that differ significantly in chemical properties and/or physical size are substituted. Further, the MscS variants can be divided into side hole volume variants and side hole charge variants. The side pore volume variant refers to a variant in which the mutation site is located at the side opening (also known as "side pore") at the cytoplasmic end and the volume of the side pore is changed by changing the amino acid at this site. The side hole charge variant refers to a variant in which the mutation site is located at the side opening of the cytoplasmic end and the side hole charge is changed by changing the amino acid at this site. For example, the side pore volume variant may be the substitution of a larger amino acid (e.g. tryptophan (W)) for a smaller amino acid (e.g. alanine (A), serine (S) or proline amino acid (P)), or vice versa. Side hole charge variants can be the substitution of a certain charged amino acid for an oppositely charged or neutral amino acid, or the substitution of a neutral amino acid for a charged amino acid. In general, non-limiting examples of positively charged amino acids include histidine, arginine, and lysine; non-limiting examples of negatively charged amino acids include aspartic acid and glutamic acid; neutral, non-limiting Examples include glycine, alanine, phenylalanine, valine, leucine, isoleucine, cysteine, asparagine, glutamine, serine, threonine, tyrosine, Methionine, Proline and Tryptophan. Amino acid conservative substitutions or non-conservative substitutions, as well as many different types of amino acid modifications (deletion, substitution, addition) and other modifications are well known in the art, and those skilled in the art can modify MscS according to the actual situation to obtain the corresponding MscS Variants. The means of modification include modifying the corresponding DNA sequence (for example, directly synthesizing the corresponding protein after modifying the DNA sequence information or using PCR to perform site-directed mutation on the DNA sequence), and then obtain the corresponding variant (and its corresponding DNA sequence).
在一个具体实施例中，所述MscS变体可以是PaMscS变体。所述PaMscS变体例如包括130A、130H、180R、271I、130S和130P中的一种或多种。PaMscS的侧孔体积突变体例如包括130A、130S、130P，PaMscS的侧孔电荷变体例如包括130H、180R、271I。这样的修饰可以改变被修饰的侧孔的孔径(也可以理解为“孔道尺寸”)，进而提高对特定分子体积的分析物的检测能力；还可以改变被修饰的侧孔通道的局部电荷特性，进而提高对特定带电荷分析物的检测能力；还可以增强所述PaMscS变体的蛋白通道电流的稳定性。In a specific embodiment, said MscS variant may be a PaMscS variant. The PaMscS variants include, for example, one or more of 130A, 130H, 180R, 271I, 130S and 130P. The side pore volume mutants of PaMscS include, for example, 130A, 130S, and 130P, and the side pore charge variants of PaMscS, for example, include 130H, 180R, and 271I. Such modification can change the pore diameter of the modified side hole (also can be understood as "pore size"), thereby improving the detection ability of the analyte with a specific molecular volume; it can also change the local charge characteristics of the modified side hole channel, Further, the detection ability of specific charged analytes is improved; the stability of the protein channel current of the PaMscS variant can also be enhanced.
在本发明的一个实施例中，所述埃米孔可以为野生型PaMscS，其虽背景噪声较高，但仍具备检测分析物的能力。In one embodiment of the present invention, the angstrompore may be wild-type PaMscS, which has the ability to detect analytes despite its high background noise.
在本发明的一个实施例中，所述埃米孔可以为野生型EcMscS(大肠杆菌小电导机械力敏感性通道)或其变体。EcMscS与PaMscS的结构高度相似，且同样能够形成稳定的通道电流，具备检测分析物的能力。PaMscS与EcMscS的序列相似性为60％。而对氨基酸的保守取代或非保守取代，以及对氨基酸的许多不同类型(删除、置换、添加)等修饰都是本领域公知的，本领域技术人员可根据实际情况来对EcMscS进行修饰，以得到相应EcMscS变体。In one embodiment of the present invention, the angstrom pore may be wild-type EcMscS (Escherichia coli small conductance mechanosensitive channel) or a variant thereof. The structures of EcMscS and PaMscS are highly similar, and they can also form a stable channel current and have the ability to detect analytes. The sequence similarity between PaMscS and EcMscS is 60%. Conservative substitutions or non-conservative substitutions of amino acids, as well as many different types of modifications (deletion, substitution, addition) to amino acids are well known in the art, and those skilled in the art can modify EcMscS according to actual conditions to obtain Corresponding EcMscS variant.
在本发明另一个实施例中，除大肠杆菌(Escherichia coli)和铜绿假单胞菌(Pseudomonas aeruginosa)外，所述埃米孔还可以源自其他杆菌，例如腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis)和幽门螺杆菌(Helicobacterpylori)。PaMscS与TtMscS、HpMscS的结构同样高度相似，而序列相似性分别为55％、44％。结合PaMscS与EcMscS的实际电生理检测结果可以看出，MscS能够作为埃米孔检测分析物的原因在于其高度相似的结构和类似的功能。而对氨基酸的保守取代或非保守取代，以及对氨基酸的许多不同类型(删除、置换、添加)等修饰都是本领域公知的，本领域技术人员可根据实际情况来对MscS进行修饰，以得到相应MscS变体。In another embodiment of the present invention, in addition to Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa (Pseudomonas aeruginosa), the emipore can also be derived from other bacilli, such as Tengchong thermophilic anaerobic bacteria (Thermoanaerobacter tengcongensis ) and Helicobacter pylori. The structures of PaMscS and TtMscS and HpMscS are also highly similar, and the sequence similarities are 55% and 44%, respectively. Combining the actual electrophysiological detection results of PaMscS and EcMscS, it can be seen that the reason why MscS can be used as an analyte in the angmipore is due to its highly similar structure and similar functions. Conservative substitutions or non-conservative substitutions of amino acids, as well as many different types of modifications (deletion, substitution, addition) to amino acids are well known in the art, and those skilled in the art can modify MscS according to actual conditions to obtain Corresponding MscS variant.
分析物Analyte
所述分析物是荷电物质。如果分析物带有净电荷则它是荷电的。所述分析物可以荷负电也可以荷正电。如果分析物带有净负电荷则它是荷负电的。如果分析物带有净正电荷则它是荷正电的。合适的分析物应为尺寸小于或等于所述埃米孔孔径的物质，优选为核苷酸、氨基酸、肽、药物分子。The analyte is a charged species. An analyte is charged if it has a net charge. The analyte can be negatively or positively charged. An analyte is negatively charged if it has a net negative charge. An analyte is positively charged if it has a net positive charge. Suitable analytes should be substances with a size smaller than or equal to the angstrom pore diameter, preferably nucleotides, amino acids, peptides, drug molecules.
在本发明的一个实施例中，所述分析物可以为核苷酸。“核苷酸”是指由杂环碱基、糖和磷酸基团组成的单体单元。应当理解，杂环碱基包括天然存在的碱基(鸟嘌呤(G)、腺嘌呤(A)、胞嘧啶(C)、胸腺嘧啶(T)和尿嘧啶(U))及非天然存在的碱基类似物。糖包括天然存在的 糖(脱氧核糖和核糖)及非天然存在的糖类似物。所述核苷酸包括脱氧核糖核苷酸和核糖核苷酸，例如ATP、dATP、CTP、dCTP、GTP、dGTP、UTP、TTP、dUTP、GMP、UMP、TMP、CMP、dGMP、dAMP、dTMP、dCMP、dUMP、ADP、GDP、TDP、UDP、CDP、dADP、dGDP、dTDP、dUDP、dCDP。所述核苷酸包括天然存在的核苷酸和以天然存在的核苷酸相似的方式与核酸杂交的非天然存在的核苷酸类似物。所述核苷酸为游离的(或者，可以理解为“单个”)。作为优选，核苷酸为ATP、dATP、CTP、dCTP、GTP、dGTP、UTP、TTP、dUTP。In one embodiment of the present invention, the analyte may be a nucleotide. "Nucleotide" refers to a monomeric unit consisting of a heterocyclic base, a sugar and a phosphate group. It is to be understood that heterocyclic bases include naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T) and uracil (U)) as well as non-naturally occurring bases base analogs. Sugars include naturally occurring sugars (deoxyribose and ribose) and non-naturally occurring sugar analogs. The nucleotides include deoxyribonucleotides and ribonucleotides such as ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, GMP, UMP, TMP, CMP, dGMP, dAMP, dTMP, dCMP, dUMP, ADP, GDP, TDP, UDP, CDP, dADP, dGDP, dTDP, dUDP, dCDP. The nucleotides include naturally occurring nucleotides and non-naturally occurring nucleotide analogs that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. The nucleotides are free (or, it may be understood as "single"). Preferably, the nucleotides are ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP.
在本发明的一个实施例中，所述分析物可以为氨基酸。“氨基酸”是指蛋白质中发现的20种天然存在的氨基酸中的任何氨基酸、天然存在的氨基酸的D-立体异构体(例如，D-苏氨酸)、非天然氨基酸以及化学修饰的氨基酸。这些氨基酸类型中的每一种类型不是相互排斥的。下列缩写用于20种天然存在的氨基酸：丙氨酸(Ala；A)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、精氨酸(Arg；R)、半胱氨酸(Cys；C)、谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、甲硫氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。氨基酸的具体性质(例如，极性、带电荷、亲水性、平均体积)对本领域技术人员是已知的。在本发明另一个实施例中，所述分析物可以为短肽，例如二肽。In one embodiment of the present invention, the analyte may be an amino acid. "Amino acid" refers to any of the 20 naturally occurring amino acids found in proteins, the D-stereoisomers of naturally occurring amino acids (eg, D-threonine), unnatural amino acids, and chemically modified amino acids. Each of these amino acid types are not mutually exclusive. The following abbreviations are used for the 20 naturally occurring amino acids: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine amino acid (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I ), Leucine (Leu; L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F), Proline (Pro; P), Serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). The specific properties (eg, polarity, charge, hydrophilicity, average volume) of amino acids are known to those skilled in the art. In another embodiment of the present invention, the analyte may be a short peptide, such as a dipeptide.
在本发明的一个实施例中，所述分析物可以为药物分子。药物分子可以是一种化合物。更具体地，“药物分子”可以是具有1000g/mol或更低分子量的药物(例如，低于800、700、600、500、400、300或200g/mol)。作为优选，药物分子可以是氨基糖苷类抗生素。在本发明的另一个实施例中，药物分子包括氨基酸及其盐(包括不能成药的氨基酸)和肽。In one embodiment of the present invention, the analyte may be a drug molecule. A drug molecule can be a compound. More specifically, a "drug molecule" may be a drug having a molecular weight of 1000 g/mol or lower (eg, lower than 800, 700, 600, 500, 400, 300 or 200 g/mol). Preferably, the drug molecule can be an aminoglycoside antibiotic. In another embodiment of the present invention, the drug molecules include amino acids and their salts (including non-druggable amino acids) and peptides.
埃米孔***Emikon system
“埃米孔***”包括具有埃米级尺寸的孔(简称为“埃米孔”)、绝缘膜、第一介质和第二介质。在本发明的一个实施例中，所述具有埃米级尺寸的孔为小电导机械力敏感性通道(MscS)埃米孔。所述具有埃米级尺寸的孔优选为具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口。在本发明的一个实施例中，所述具有埃米级尺寸的孔具有一个径向对称且形状似圆柱体的典型七聚体结构，所述七聚体结构包含8个开口，其中7个相等的开口分布在侧面，第8个开口分布于底部且由7个亚基形成；上述8个开口孔径大小均可调节。所述具有埃米级尺寸的孔允许所述分析物从所述绝缘膜的一侧易位到另一侧。The "angstrom hole system" includes a hole having a size in the angstrom order (abbreviated as "angstrom hole"), an insulating film, a first medium, and a second medium. In one embodiment of the present invention, the pore with a size in the Angstrom order is a small conductance mechanosensitive channel (MscS) Angstrom pore. The pores with angstrom size are preferably heptamer structures with radial symmetry and cylindrical shape, and the heptamer structures include 7 side openings and 1 bottom opening. In one embodiment of the present invention, the pore with Angstrom size has a typical heptamer structure with radial symmetry and cylindrical shape, and the heptamer structure contains 8 openings, 7 of which are equal The openings are distributed on the side, and the eighth opening is distributed on the bottom and formed by 7 subunits; the pore size of the above 8 openings can be adjusted. The pores having Angstrom dimensions allow translocation of the analyte from one side of the insulating membrane to the other.
在本发明的一个实施例中，所述具有埃米级尺寸的孔被嵌入所述绝缘膜中，所述绝缘膜(也可以理解为，所述埃米级尺寸的孔和所述绝缘膜的复合体)将所述第一介质与所述第二介质分隔开，所述具有埃米级尺寸的孔的孔道提供连通所述第一介质与所述第二介质的通道；向所述第一介质和所述第二介质之间施加驱动力后，位于所述第一介质的分析物与所述MscS埃米孔相互作用以形成电流(即电信号)。在本发明中，“第一介质”是指所述分析物被加入所述埃米孔***时位于的介质；“第二介质”则是指被所述绝缘膜分隔开的两部分介质中，“第一介质”的另一侧。在本发明中，驱动力是指通过电势、电渗流、浓度梯度等方式驱动分析物与所述埃米孔相互作用的力。In one embodiment of the present invention, the hole with the angstrom-scale size is embedded in the insulating film, and the insulating film (also can be understood as, the hole with the angstrom-scale size and the insulating film Composite) separates the first medium from the second medium, and the channels with pores in the Angstrom order provide passages connecting the first medium and the second medium; After a driving force is applied between the first medium and the second medium, the analyte located in the first medium interacts with the MscS pore to form a current (ie, an electrical signal). In the present invention, "first medium" refers to the medium in which the analyte is added to the angstrom pore system; "second medium" refers to the two parts of the medium separated by the insulating film. , the other side of the "first medium". In the present invention, the driving force refers to the force driving the interaction between the analyte and the angstrom pore by means of potential, electroosmotic flow, concentration gradient and the like.
所述第一介质和所述第二介质可以相同或不同，并且所述第一介质和所述第二介质可以包括电导液。所述电导液为碱金属卤化物水溶液，具体为氯化钠(NaCl)、氯化锂(LiCl)、氯化铯(CsCl)、氯化钾(KCl)、溴化钠(NaBr)。在本发明一个实施例中，所述第一介质和所述第二介质含有的电导液的浓度是不同的，换句话说，所述第一介质和所述第二介质中电导液的浓度存在差值，进而使得所述绝缘膜两侧的渗透压存在差值。所述第一介质和/或所述第二介质还可以包括缓冲液，例如HEPES。所述第一介质和/或所述第二介质的浓度范围可以是30mM-3M。The first medium and the second medium may be the same or different, and the first medium and the second medium may comprise electrically conductive fluids. The conductive liquid is an aqueous alkali metal halide solution, specifically sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl), and sodium bromide (NaBr). In one embodiment of the present invention, the concentrations of the conductive liquid contained in the first medium and the second medium are different, in other words, the concentrations of the conductive liquid in the first medium and the second medium exist The difference, and then there is a difference in the osmotic pressure on both sides of the insulating film. The first medium and/or the second medium may also comprise a buffer, such as HEPES. The concentration range of the first medium and/or the second medium may be 30mM-3M.
绝缘膜是指具有搭载埃米孔(或纳米孔)并阻塞非埃米孔(或纳米孔)通过的离子电流 的能力的膜。所述绝缘膜可以包括磷脂膜和/或高分子膜。示例性的磷脂膜包括DPHPC、DOPC、E.coli lipid，示例性的高分子膜包括三嵌段共聚物高分子膜。An insulating film refers to a film that has the ability to host angstrompores (or nanopores) and block ionic currents passing through non-angstrompores (or nanopores). The insulating film may include a phospholipid film and/or a polymer film. Exemplary phospholipid membranes include DPHPC, DOPC, E.coli lipid, and exemplary polymer membranes include triblock copolymer polymer membranes.
本埃米孔***可包含本文所描述的任何小电导机械力敏感性通道，例如野生型PaMscS(SEQ ID NO:1)、野生型EcMscS(SEQ ID NO:2)、野生型TtMscS(SEQ ID NO:3)和野生型HpMscS(SEQ ID NO:4)及其相应变体，上述四种MscS的具体序列信息如表4所示。例如，所述小电导机械力敏感性通道可以为突变体PaMscS1(W130A)、突变体PaMscS2(K180R)、突变体PaMscS3(V271I)。The present pore system can comprise any of the small conductance mechanosensitive channels described herein, such as wild-type PaMscS (SEQ ID NO: 1), wild-type EcMscS (SEQ ID NO: 2), wild-type TtMscS (SEQ ID NO :3) and wild-type HpMscS (SEQ ID NO: 4) and its corresponding variants, the specific sequence information of the above four MscSs is shown in Table 4. For example, the small conductance mechanosensitive channel can be mutant PaMscS1 (W130A), mutant PaMscS2 (K180R), mutant PaMscS3 (V271I).
表4：四种MscS的氨基酸序列信息Table 4: Amino acid sequence information of four MscS
Figure PCTCN2022115022-appb-000001
Figure PCTCN2022115022-appb-000001
在本发明的一个具体实施例中，所述埃米孔***包括两个电解液室，其被绝缘膜分隔开而形成反式(-trans)隔室和顺式(-cis)隔室，所述埃米孔的孔嵌入绝缘膜中，绝缘膜上只有小电导机械力敏感性通道埃米孔来连通上述两个电解液室。当向上述两个电解液室施加电势时，电解液室中溶液中的电解质离子通过电泳移动并穿过所述埃米孔。In a specific embodiment of the present invention, the Angstrom pore system includes two electrolyte chambers, which are separated by an insulating membrane to form a trans (-trans) compartment and a cis (-cis) compartment, so The holes of the above-mentioned angstrom holes are embedded in the insulating film, and there are only small conductance mechanical force-sensitive channel angmometer holes on the insulating film to communicate with the above-mentioned two electrolyte chambers. When a potential is applied to the above two electrolyte chambers, electrolyte ions in solution in the electrolyte chambers move by electrophoresis and pass through the Angstrom pores.
在本发明的一个实施例中，所述小电导机械力敏感性通道(MscS)埃米孔可以被嵌入绝缘膜中，但其保留了响应绝缘膜受到的机械力刺激和绝缘膜的物理状态变化而改变蛋白质结构的能力。具体地，机械力刺激包括所述绝缘膜两侧的渗透压变化、微针对所述绝缘膜的直接物理刺激、气压负压对所述绝缘膜的刺激等。绝缘膜的物理变化包括所述绝缘膜的厚度变化、所述绝缘膜的组成成分变化、所述绝缘膜的表面曲率变化。所述改变蛋白质结构包括改变MscS的开口的电荷性质和/或孔径大小。进一步地，可利用MscS埃米孔改变的开口的电荷性质和/或孔径大小来检测不同的分析物。本发明所涉及的埃米孔的孔径的可调节范围可以是5–15埃米。In one embodiment of the present invention, the small conductance mechanosensitive channel (MscS) angstrom hole can be embedded in the insulating film, but it retains the response to the mechanical stimulation of the insulating film and the change of the physical state of the insulating film The ability to change the structure of a protein. Specifically, mechanical force stimulation includes osmotic pressure changes on both sides of the insulating membrane, direct physical stimulation of micro-targeting on the insulating membrane, stimulation of the insulating membrane by negative air pressure, and the like. The physical change of the insulating film includes the change of the thickness of the insulating film, the change of the composition of the insulating film, and the change of the surface curvature of the insulating film. Said altering the protein structure comprises altering the charge properties and/or pore size of the openings of MscS. Further, the charge properties and/or pore size of the altered opening of the MscS angstrompore can be utilized to detect different analytes. The pore diameter of the angstrom hole involved in the present invention can be adjusted in the range of 5-15 angstrom.
所述埃米孔和分析物之间的相互作用The interaction between the pore and the analyte
所述分析物可与所述埃米孔在所述绝缘膜两侧的任一侧接触。所述分析物可以与所述绝缘膜两侧中的任一侧相接触，使得所述分析物穿过所述埃米孔的通道以到达所述绝缘膜的另一侧。在这种情况下，所述分析物在其经由所述孔的通道穿过所述绝缘膜时，与所述埃米孔 相互作用。或者，所述分析物可与所述绝缘膜的侧面接触，所述绝缘膜的侧面可使所述分析物与所述埃米孔相互作用，使其与所述埃米孔分离并停留在所述绝缘膜的同一侧。所述分析物可以以任何方式并在任何位点与所述埃米孔相互作用。所述分析物还可以撞击到所述埃米孔，与所述埃米孔相互作用，使其与所述埃米孔分离并停留在所述绝缘膜的同一侧。The analyte may be in contact with the Angstrom pore on either side of the insulating film. The analyte may be in contact with either side of the insulating film such that the analyte passes through the passage of the Angstrom pore to the other side of the insulating film. In this case, the analyte interacts with the Angstrom pore as it passes through the insulating membrane via the passage of the pore. Alternatively, the analyte may be in contact with the side of the insulating film that allows the analyte to interact with the Angstrom pore, separate it from the Angstrom pore, and reside in the Angstrom pore. on the same side as the insulating film. The analyte can interact with the pore in any manner and at any site. The analyte may also impinge on the Angstrompore, interact with the Angstrompore, separate it from the Angstrompore and reside on the same side of the insulating membrane.
在所述分析物与所述埃米孔相互作用的过程中，所述分析物会以该分析物特异性的方式影响流过所述埃米孔的电流，即流经所述埃米孔的电流对特定分析物是特征性的。可进行对照实验以测定特定分析物对流过所述埃米孔的电流的效应，然后以鉴定样本中的特定分析物或测定样本中是否存在特定分析物。更具体地，可以根据通过检测分析物所获得的电流模式与在相同的条件下使用已知的分析物获得的已知的电流模式进行比较，以鉴定分析物的存在与否或浓度等。During the interaction of the analyte with the pore, the analyte affects the current flowing through the pore in a manner specific to the analyte, i.e. the current flowing through the pore The current is characteristic of a particular analyte. Control experiments can be performed to determine the effect of a particular analyte on the current flowing through the angstrom pore, and then to identify the particular analyte in the sample or to determine the presence or absence of the particular analyte in the sample. More specifically, the presence or absence or concentration of the analyte can be identified based on the comparison of the current pattern obtained by detecting the analyte with the known current pattern obtained using the known analyte under the same conditions.
本发明的埃米孔***还可以包括一个或多个测量流过所述埃米孔的电流的测量装置，例如膜片钳放大器或数据采集设备。The angstrompore system of the present invention may also include one or more measuring devices that measure the current flowing through the angstrompore, such as patch clamp amplifiers or data acquisition devices.
样本sample
所述分析物可存在于任何合适的样本中。本发明通常在已知含有或怀疑含有所述分析物的样本上进行。本发明可以在含有一种或多种种类未知的分析物的样本上进行。或者，本发明可以确认所述一种或多种已知存在或预计存在于所述样本中的分析物的种类。The analyte can be present in any suitable sample. The invention is generally performed on samples known to contain or suspected to contain the analyte. The invention can be performed on samples containing one or more analytes of unknown type. Alternatively, the present invention may identify the species of said one or more analytes known to be present or predicted to be present in said sample.
所述样本可以是生物样本。本发明可以在获自或提取自任何生物或微生物的样本上在体外进行。本发明还可以在获自或提取自任何病毒的样本上在体外进行。优选地，所述样本为流体样本。所述样本通常包括体液。所述样本可以是体液样本，例如尿液、血液、血清、血浆、淋巴液、囊肿液、胸膜液、腹水液、腹膜液、羊水、***、脑脊液、支气管肺泡灌洗液、母乳、泪液、唾液、痰或其组合。所述样本可以源自人类，也可以源自其他哺乳动物。所述样本可以是非生物样本。所述非生物样本优选地为流体样本，例如饮用水、海水、河水以及用于实验室试验的试剂。The sample can be a biological sample. The invention may be performed in vitro on a sample obtained or extracted from any organism or microorganism. The invention can also be performed in vitro on samples obtained or extracted from any virus. Preferably, the sample is a fluid sample. The sample typically includes bodily fluids. The sample may be a body fluid sample, such as urine, blood, serum, plasma, lymph fluid, cyst fluid, pleural fluid, ascitic fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tear fluid, Saliva, sputum, or a combination thereof. The sample can be derived from humans or from other mammals. The sample can be a non-biological sample. The non-biological samples are preferably fluid samples such as drinking water, sea water, river water and reagents for laboratory tests.
所述样本在分析之前可以不经过处理，例如直接在全血中检测所述分析物。所述样本在分析之前也可以经过处理，例如通过离心、过滤、稀释、沉淀或其他本领域已知的物理手段或化学手段。The sample may not be processed prior to analysis, eg, the analyte is detected directly in whole blood. The sample may also be treated prior to analysis, eg, by centrifugation, filtration, dilution, sedimentation, or other physical or chemical means known in the art.
在本发明的一个实施例中，所述样本为全血样本。In one embodiment of the present invention, the sample is a whole blood sample.
在本发明一个实施例中，所述样本为核酸扩增产物。In one embodiment of the present invention, the sample is a nucleic acid amplification product.
检测样本中核酸的存在的方法Method for detecting the presence of nucleic acid in a sample
本发明还提供一种检测样本中核酸的存在的方法。所述方法包括：S1将样本置于核酸扩增体系并进行核酸扩增，确定所述核酸扩增体系中底物核苷酸的数量，获得所述样本的核酸扩增产物；S2将所述样本的核酸扩增产物加入埃米孔***，所述埃米孔***包括：埃米孔、绝缘膜、第一介质、第二介质，其中所述蛋白质埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口，所述样本的核酸扩增产物被加入到所述第一介质；S3向所述第一介质和所述第二介质之间施加驱动力，所述样本的核酸扩增产物中剩余核苷酸与所述埃米孔相互作用并产生电信号(电流)；S4对所述电信号进行量化，获得所述剩余核苷酸的数量；S5将所述剩余核苷酸的数量与所述底物核苷酸的数量进行对比，确定所述样本中所述目标核酸是否存在。在一些实施例中，S1可以和S2同时进行或在同一体系中进行。在检测前，还可以设置阈值，例如，只有剩余核苷酸中的至少一种的数量低于该阈值时，视为样本中目标核酸存在；或者，只有全部种类的剩余核苷酸的数量高该阈值时，视为样本中目标核酸不存在。The invention also provides a method of detecting the presence of nucleic acid in a sample. The method includes: S1 placing the sample in a nucleic acid amplification system and performing nucleic acid amplification, determining the number of substrate nucleotides in the nucleic acid amplification system, and obtaining a nucleic acid amplification product of the sample; S2 placing the The nucleic acid amplification product of the sample is added to the angstrom hole system, and the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the protein angstrom hole is embedded in the insulating film, The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, and the angstrom hole is MscS angstrom A hole, the Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, the heptamer structure includes 7 side openings and 1 bottom opening, and the nucleic acid amplification product of the sample is added to the The first medium; S3 applies a driving force between the first medium and the second medium, and the remaining nucleotides in the nucleic acid amplification product of the sample interact with the angstrom pores and generate electrical signals (current); S4 quantifies the electrical signal to obtain the quantity of the remaining nucleotides; S5 compares the quantity of the remaining nucleotides with the quantity of the substrate nucleotides to determine the sample Whether the target nucleic acid described in is present. In some embodiments, S1 and S2 can be performed simultaneously or in the same system. Before detection, a threshold can also be set, for example, only when the quantity of at least one of the remaining nucleotides is lower than the threshold, it is considered that the target nucleic acid exists in the sample; or, only the quantity of all kinds of remaining nucleotides is high When this threshold is reached, it is considered that the target nucleic acid does not exist in the sample.
本发明的“消耗策略”实际上适用于多种跨膜孔，而并不局限于本发明的埃米孔。跨膜孔是在某种程度上穿过膜的结构。它允许由施加的驱动力驱动的分析物流过膜或膜内。跨膜 孔通常穿过整个膜，使得分析物可以从膜的一侧流到膜的另一侧。然而，跨膜孔不是必须得穿过膜。它的一端可以封闭。例如，孔可以是膜中的阱、间隙、通道、沟槽或狭缝，分析物可以沿着它流动或流入其中。可以使用任何跨膜孔来实施本发明“消耗策略”。跨膜孔可以是生物的或人造的。合适的孔可以是蛋白质孔、多核苷酸孔和固态孔。本发明中的跨膜孔至少应具备检测和区分多种核苷酸的能力，优选为跨膜蛋白孔。换句话说，本发明涉及的跨膜蛋白孔能够允许由驱动力驱动的分析物从膜的一侧流到另一侧。The "depletion strategy" of the present invention is actually applicable to a variety of transmembrane pores, and is not limited to the angstrom pore of the present invention. A transmembrane pore is a structure that passes through a membrane to some extent. It allows the analyte to flow through or within the membrane driven by an applied driving force. Transmembrane pores typically span the entire length of the membrane, allowing analytes to flow from one side of the membrane to the other. However, the transmembrane pore does not necessarily have to pass through the membrane. It can be closed at one end. For example, a pore may be a well, gap, channel, groove or slit in a membrane along which or into which analyte may flow. Any transmembrane pore can be used to implement the "depletion strategy" of the present invention. Transmembrane pores can be biological or artificial. Suitable pores may be protein pores, polynucleotide pores and solid state pores. The transmembrane pore in the present invention should at least have the ability to detect and distinguish multiple nucleotides, preferably a transmembrane protein pore. In other words, the present invention relates to transmembrane protein pores that allow the flow of analytes from one side of the membrane to the other, driven by a driving force.
本领域技术人员可以在理解本发明的基础上，选择其他能够检测(和区分)不同核苷酸的跨膜孔来检测目标核酸的存在与否。Those skilled in the art can select other transmembrane pores capable of detecting (and distinguishing) different nucleotides to detect the presence or absence of the target nucleic acid on the basis of understanding the present invention.
“核酸”是指以单链或双链形式存在的脱氧核糖核苷酸或核糖核苷酸聚合物。目标核酸的“核酸扩增”是指体外构建与目标核酸序列至少一部分相同或互补的核酸链的过程，仅当该目标核酸存在于样本中时才可能发生核酸扩增过程。在核酸扩增过程，通常利用酶(例如核酸聚合酶、转录酶)来产生目标核酸或者其片段的多个拷贝，或者与该目标核酸或者其片段互补的序列的多个拷贝。发生核酸扩增过程后，核酸扩增体系的底物核苷酸会随拷贝数的增加而相应地减少。图3和图4为核酸扩增的示例。"Nucleic acid" refers to a polymer of deoxyribonucleotides or ribonucleotides in single- or double-stranded form. "Nucleic acid amplification" of a target nucleic acid refers to the process of constructing a nucleic acid chain in vitro that is at least partially identical or complementary to a target nucleic acid sequence, and the nucleic acid amplification process can only occur when the target nucleic acid is present in a sample. During nucleic acid amplification, enzymes (such as nucleic acid polymerase, transcriptase) are usually used to generate multiple copies of a target nucleic acid or a fragment thereof, or multiple copies of a sequence complementary to the target nucleic acid or a fragment thereof. After the nucleic acid amplification process occurs, the substrate nucleotides of the nucleic acid amplification system will decrease correspondingly with the increase of the copy number. Figure 3 and Figure 4 are examples of nucleic acid amplification.
本发明方法的原理基于体外核酸扩增技术，以消耗核酸扩增体系中的底物核苷酸，因此常见的体外核酸扩增技术，例如聚合酶链反应(PCR)、连接酶链反应(LCR)、链置换扩增技术(SDA)、转录介导的扩增技术(TMA)、环介导等温扩增技术(LAMP)都可以配合本发明提供的方法所使用。The principle of the method of the present invention is based on in vitro nucleic acid amplification technology, to consume the substrate nucleotide in the nucleic acid amplification system, so common in vitro nucleic acid amplification technology, such as polymerase chain reaction (PCR), ligase chain reaction (LCR) ), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Loop-Mediated Isothermal Amplification (LAMP) can all be used in conjunction with the method provided by the invention.
在一个具体实施例中，本发明提供的方法可以检测样本中是否存在新冠病毒核酸。In a specific embodiment, the method provided by the present invention can detect whether there is a new coronavirus nucleic acid in a sample.
在一个具体实施例中，本发明提供的方法可以检测样本中是否存在单链核酸。In a specific embodiment, the method provided by the present invention can detect whether there is a single-stranded nucleic acid in a sample.
在本发明的一个实施例中，本发明通过构建合适的引物(例如，SARS-CoV-2核酸的特异性引物)并将所述引物引入到核酸扩增体系(包括底物dNTPs、聚合酶、反转录酶)。如果样本中存在该新冠病毒核酸，则所述新冠病毒核酸在合适的条件下核酸扩增，消耗底物dNTPs并生成所述核酸的多个拷贝。而将所述样本的核酸扩增产物加入本发明提供的埃米孔***后，所述核酸扩增体系中的大分子物质(例如，酶、多核苷酸等)无法穿过埃米孔，也就是说，所述核酸扩增体系中只有游离的单核苷酸能够穿过所述埃米孔并产生特异性电流，进而确定剩余核苷酸的数量，以判断样本中是否存在目标新冠病毒核酸(即如果样本中无目标新冠病毒核酸，则剩余核苷酸的数量更接近核酸扩增前的底物核苷酸数量；如果样本中存在新冠病毒核酸，则剩余核苷酸的数量显著低于核酸扩增前的底物核苷酸数量，更具体地，底物核苷酸中可能至少一种核苷酸被完全消耗)。In one embodiment of the present invention, the present invention constructs suitable primers (for example, specific primers for SARS-CoV-2 nucleic acid) and introduces the primers into the nucleic acid amplification system (including substrate dNTPs, polymerase, reverse transcriptase). If the new coronavirus nucleic acid exists in the sample, the new coronavirus nucleic acid is amplified under suitable conditions to consume substrate dNTPs and generate multiple copies of the nucleic acid. After the nucleic acid amplification product of the sample is added to the angstrom pore system provided by the present invention, macromolecular substances (such as enzymes, polynucleotides, etc.) in the nucleic acid amplification system cannot pass through the angstrom pore, nor That is to say, only free single nucleotides in the nucleic acid amplification system can pass through the angstrompore and generate a specific current, and then determine the number of remaining nucleotides to determine whether there is a target new coronavirus nucleic acid in the sample (That is, if there is no target new coronavirus nucleic acid in the sample, the number of remaining nucleotides is closer to the number of substrate nucleotides before nucleic acid amplification; if there is new coronavirus nucleic acid in the sample, the number of remaining nucleotides is significantly lower than The number of substrate nucleotides before nucleic acid amplification, more specifically, at least one of the substrate nucleotides may be completely consumed).
在本发明的一个实施例中，本发明通过构建合适的探针(例如，所述探针包括与目标核酸序列互补配对的序列和多聚核苷酸序列)并将所述探针引入到核酸扩增体系(包括底物dNTPs、聚合酶)。如果样本中存在该目标核酸序列，则所述目标核酸序列在合适的条件下核酸扩增，消耗底物dNTPs并生成所述目标核酸序列的多个拷贝；更具体地，由于探针还具有多聚核苷酸序列(例如，Poly T、Poly A、Poly C、Poly G)，因此，所述目标核酸序列与所述探针结合后，还会大量消耗与多聚核苷酸序列相对应的底物核苷酸，因此可以从某种底物核苷酸的特异性消耗来判断样本中是否存在目标核酸序列。将所述样本的核酸扩增产物加入本发明提供的埃米孔***后，所述核酸扩增体系中的大分子物质(例如，酶、多核苷酸等)无法穿过埃米孔，也就是说，所述核酸扩增体系中只有游离的单核苷酸能够穿过所述埃米孔并产生特异性电流，进而确定剩余核苷酸的数量，以判断样本中是否存在目标核酸序列(即如果样本中无目标核酸序列，则剩余核苷酸的数量更接近核酸扩增前的底物核苷酸数量；如果样本中存在目标核酸序列，则剩余核苷酸的数量显著低于核酸扩增前的底物核苷酸数量且探针中所对应的多聚核苷酸被大量消耗。基于此，可设计不同的探针在同一个核酸扩增体系中同时检测不同的目标核酸序列。In one embodiment of the present invention, the present invention constructs a suitable probe (for example, the probe includes a sequence complementary to the target nucleic acid sequence and a polynucleotide sequence) and introduces the probe into the nucleic acid Amplification system (including substrate dNTPs, polymerase). If the target nucleic acid sequence exists in the sample, the target nucleic acid sequence is amplified under suitable conditions, consumes substrate dNTPs and generates multiple copies of the target nucleic acid sequence; more specifically, since the probe also has multiple Polynucleotide sequence (for example, Poly T, Poly A, Poly C, Poly G), therefore, after described target nucleic acid sequence is combined with described probe, also can consume a large amount of corresponding to polynucleotide sequence Substrate nucleotides, therefore, the presence or absence of target nucleic acid sequences in samples can be judged from the specific consumption of certain substrate nucleotides. After the nucleic acid amplification product of the sample is added to the angstrom pore system provided by the present invention, macromolecular substances (such as enzymes, polynucleotides, etc.) in the nucleic acid amplification system cannot pass through the angstrom pore, that is, Said, in the nucleic acid amplification system, only free single nucleotides can pass through the angstrom hole and generate a specific current, and then determine the number of remaining nucleotides to judge whether there is a target nucleic acid sequence in the sample (i.e. If there is no target nucleic acid sequence in the sample, the number of remaining nucleotides is closer to the number of substrate nucleotides before nucleic acid amplification; if there is a target nucleic acid sequence in the sample, the number of remaining nucleotides is significantly lower than that of nucleic acid amplification The number of substrate nucleotides in the front and the corresponding polynucleotides in the probe are consumed in large quantities. Based on this, different probes can be designed to simultaneously detect different target nucleic acid sequences in the same nucleic acid amplification system.
检测样本中的药物分子的方法Method for detecting drug molecules in a sample
本发明还提供一种检测样本中的药物分子的方法，所述方法包括：S1将所述样本加入埃米孔***，所述埃米孔***包括：埃米孔、绝缘膜、第一介质、第二介质，其中所述埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口；所述样本被加入到所述第一介质；S2向所述第一介质和所述第二介质施加驱动力，所述样本中的药物分子与所述埃米孔相互作用并产生电信号；S3分析所述电信号，进而识别所述样本中的药物分子。The present invention also provides a method for detecting drug molecules in a sample, the method comprising: S1 adding the sample to an angstrom hole system, the angstrom hole system comprising: an angstrom hole, an insulating film, a first medium, a second medium, wherein the Angstrom hole is embedded in the insulating film separating the first medium from the second medium, the Angstrom hole providing communication with the first medium With the channel of the second medium, the angstrom hole is a MscS angstrom hole, the angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 sides Opening and 1 bottom opening; the sample is added to the first medium; S2 applies a driving force to the first medium and the second medium, and the drug molecule in the sample interacts with the angstrom hole function and generate an electrical signal; S3 analyzes the electrical signal, and then identifies the drug molecule in the sample.
在本发明的一个实施例中，所述样本为体液样本。所述体液样本可以是尿液、血液、血清、血浆、淋巴液、囊肿液、胸膜液、腹水液、腹膜液、羊水、***、脑脊液、支气管肺泡灌洗液、母乳、泪液、唾液、痰或其组合。所述样本在分析之前可以不经过处理，例如直接在全血中检测所述分析物。当然，所述样本在分析之前也可以经过处理，例如通过离心、过滤、稀释、沉淀或其他本领域已知的物理手段或化学手段。本发明所指的样本包括不经过处理的样本和经过处理的样本。In one embodiment of the present invention, the sample is a body fluid sample. The body fluid sample can be urine, blood, serum, plasma, lymph fluid, cystic fluid, pleural fluid, ascitic fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tear fluid, saliva, sputum or a combination thereof. The sample may not be processed prior to analysis, eg, the analyte is detected directly in whole blood. Of course, the sample can also be processed before analysis, such as by centrifugation, filtration, dilution, precipitation or other physical or chemical means known in the art. The samples referred to in the present invention include untreated samples and processed samples.
在本发明的一个实施例中，所述药物分子的可检测范围可以是大于10nM(也可以理解为检测下限为10nM)。作为优选，所述药物分子的可检测范围可以是10nM～1mM。如果药物分子的浓度远大于10nM(例如10mM)，可以将其浓度稀释至10nM～1mM。In one embodiment of the present invention, the detectable range of the drug molecule may be greater than 10 nM (it can also be understood that the detection limit is 10 nM). Preferably, the detectable range of the drug molecule may be 10nM-1mM. If the concentration of the drug molecule is much greater than 10 nM (eg 10 mM), its concentration can be diluted to 10 nM-1 mM.
在本发明的一个实施例中，所述药物分子是一种化合物。更具体地，“药物分子”可以是具有1000g/mol或更低分子量的药物(例如，低于800、700、600、500、400、300或200g/mol)。作为优选，药物分子可以是氨基糖苷类抗生素，例如硫酸庆大霉素、硫酸新霉素、西索米星等。在本发明的另一个实施例中，药物分子包括氨基酸及其盐(包括不能成药的氨基酸)和肽。In one embodiment of the invention, said drug molecule is a compound. More specifically, a "drug molecule" may be a drug having a molecular weight of 1000 g/mol or lower (eg, lower than 800, 700, 600, 500, 400, 300 or 200 g/mol). Preferably, the drug molecule can be an aminoglycoside antibiotic, such as gentamicin sulfate, neomycin sulfate, sisomicin and the like. In another embodiment of the present invention, the drug molecules include amino acids and their salts (including non-druggable amino acids) and peptides.
在本发明的一个实施例中，本发明检测体液样本中的药物分子，其中药物分子的检测限为10nM。换句话说，体液样本中的所述药物分子的可检测范围可以是大于10nM。In one embodiment of the present invention, the present invention detects drug molecules in body fluid samples, wherein the detection limit of drug molecules is 10 nM. In other words, the detectable range of the drug molecule in the bodily fluid sample may be greater than 10 nM.
在本发明的一个实施例中，本发明检测全血样本(也可以理解为“血液样本”)中的药物分子。将所述全血样本加入本发明提供的埃米孔***后，所述全血样本中的细胞(例如，红细胞、白细胞、血小板)和大分子物质(例如蛋白质)无法穿过埃米孔，也就是说，所述全血样本中的细胞和大分子物质不会使本发明提供的埃米孔堵塞。而存在于所述全血样本中的药物分子能够穿过所述埃米孔并产生特异性电流，本发明提供的埃米孔可以灵敏地识别较低浓度的所述药物分子，进而判断全血样本中所述药物分子的存在及浓度。In one embodiment of the present invention, the present invention detects drug molecules in whole blood samples (also known as "blood samples"). After the whole blood sample is added to the angstrom pore system provided by the present invention, the cells (for example, red blood cells, white blood cells, platelets) and macromolecular substances (such as proteins) in the whole blood sample cannot pass through the angstrom pore, nor can they pass through the angstrom pore system. That is to say, cells and macromolecular substances in the whole blood sample will not block the angstrom pore provided by the present invention. However, the drug molecules present in the whole blood sample can pass through the angstrom pore and generate a specific current. The angstrom hole provided by the present invention can sensitively recognize the drug molecule at a lower concentration, and then judge the whole blood The presence and concentration of the drug molecule in the sample.
在本发明的一个实施例中，本发明提供的方法可以用于对受试者体内的药物分子的血药浓度的连续监测。In one embodiment of the present invention, the method provided by the present invention can be used for continuous monitoring of the blood drug concentration of the drug molecule in the subject.
在本发明的一个实施例中，采用PaMscS2(K180R)和PaMscS3(V271I)埃米孔来检测全血样本中的药物分子，但其他MscS及其相应变体也包含在本发明的保护范围之中，依据是上述变体均有对药物分子进行传感检测的能力。In one embodiment of the present invention, PaMscS2 (K180R) and PaMscS3 (V271I) angmipores are used to detect drug molecules in whole blood samples, but other MscS and their corresponding variants are also included in the protection scope of the present invention , based on the fact that the above variants all have the ability to sense and detect drug molecules.
具体实施举例：Specific implementation examples:
实施例一Embodiment one
材料与方法：Materials and Methods:
氯化钠(NaCl，>99.0％，CAS#7647-14-5)、dNTP混合物(>99.0％)、dATP(>97％，CAS#1927-31-7)、dCTP(>98％，CAS#102783-51-7)、dGTP(>98％，CAS#93919-41-6)、dTTP(>98％，CAS#18423-43-3)购自Sangon Biotech。酵母提取物(CAS#8013-01-2)、胰蛋白酶(CAS#73049-73-7)、氨苄青霉素钠盐(≥98.5％，CAS#69-52-3)、Tris(≥99.9％，CAS#77-86-1)、咪唑(Imidazole)(≥99％，CAS#288-32-4)、十二烷基-β-D-麦芽糖苷(n-Dodecyl-β-D-Maltopyranoside，DDM)(≥99％，CAS#69227-93-6)、异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside，IPTG)(≥99％，CAS#367-93-1)、苯甲磺酰氟 (phenylmethylsulfonyl fluoride，PMSF)(≥99.％，CAS#329-98-6)、4-(2-羟乙基)哌嗪-1-乙磺酸(4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid，HEPES)(>99.5％，CAS#7365-45-9)购自Sigma-Aldrich。大肠杆菌提取磷脂购自Avanti。PrimeSTAR HS DNA聚合酶购自TaKaRa。pUC57载体质粒、DNA模板、miRNA-21、AFP适配体由Sangon Biotech合成，其序列信息于表1列出。Sodium Chloride (NaCl, >99.0%, CAS#7647-14-5), dNTP Mixture (>99.0%), dATP (>97%, CAS#1927-31-7), dCTP (>98%, CAS# 102783-51-7), dGTP (>98%, CAS#93919-41-6), dTTP (>98%, CAS#18423-43-3) were purchased from Sangon Biotech. Yeast Extract (CAS#8013-01-2), Trypsin (CAS#73049-73-7), Ampicillin Sodium Salt (≥98.5%, CAS#69-52-3), Tris (≥99.9%, CAS #77-86-1), Imidazole (≥99%, CAS#288-32-4), Dodecyl-β-D-maltoside (n-Dodecyl-β-D-Maltopyranoside, DDM) (≥99%, CAS#69227-93-6), isopropyl-β-D-thiogalactoside (IPTG) (≥99%, CAS#367-93-1 ), phenylmethylsulfonyl fluoride (PMSF) (≥99.%, CAS#329-98-6), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (4-(2 -Hydroxyethyl)piperazine-1-ethanesulfonic acid, HEPES) (>99.5%, CAS#7365-45-9) was purchased from Sigma-Aldrich. Phospholipids extracted from Escherichia coli were purchased from Avanti. PrimeSTAR HS DNA polymerase was purchased from TaKaRa. The pUC57 vector plasmid, DNA template, miRNA-21, and AFP aptamer were synthesized by Sangon Biotech, and their sequence information is listed in Table 1.
表1 miR21和AFP适配体的序列Table 1 Sequences of miR21 and AFP aptamers
Figure PCTCN2022115022-appb-000002
Figure PCTCN2022115022-appb-000002
野生型和突变体PaMscS的表达和纯化：Expression and purification of wild-type and mutant PaMscS:
使用基因特异性引物通过聚合酶链反应(PCR)扩增出来自铜绿假单胞菌基因组DNA中PaMscS的基因。使用ClonExpress II One Step Cloning Kit(Vazyme)将基因***质粒中。将包含PaMscS基因的质粒的的大肠杆菌BL21(DE3)细胞在37℃、存在50μg/mL氨苄青霉素的Luria-Bertani(LB)培养基中培养并表达纯化。通过SDS-PAGE分析确定峰值。特别说明，本发明中野生型和突变型蛋白的表达纯化步骤一样，只是由于野生型蛋白与突变型蛋白存在序列差异，因此在质粒合成阶段有差异。The gene from PaMscS in Pseudomonas aeruginosa genomic DNA was amplified by polymerase chain reaction (PCR) using gene-specific primers. Genes were inserted into plasmids using the ClonExpress II One Step Cloning Kit (Vazyme). The Escherichia coli BL21 (DE3) cells containing the plasmid of the PaMscS gene were cultured at 37° C. in Luria-Bertani (LB) medium in the presence of 50 μg/mL ampicillin, and expressed and purified. Peaks were identified by SDS-PAGE analysis. In particular, the expression and purification steps of the wild-type and mutant proteins in the present invention are the same, but there are differences in the plasmid synthesis stage due to sequence differences between the wild-type protein and the mutant protein.
本发明实验团队通过建模揭示了该蛋白质埃米孔的结构，其为径向对称且类似圆柱体的典型七聚体。它包含8个开口，7个在侧面，1个在底部。N-末端残基1-13太过灵活，无法在模型中解析。在拓扑结构上，PaMscS可分为2个部分，跨膜区和大的胞质部分。每个单体产生三个N-末端跨膜螺旋，包括TM1(残基17～52)、TM2(残基58～83)和TM3(残基90～122)。C-末端胞质区可分为中间β结构域和COOH末端结构域。每个亚单位中的TM1和TM2以反平行方向排列在一起，TM1穿过通道外侧的双层膜并且TM2形成中心层，从而形成围绕通道轴的渗透路径。TM3螺旋可以描述为两个螺旋片段，TM3a和TM3b，在Gly108处被一个明显的扭结(kink)～53°分开，其在同源性是保守的残基。TM3a像TM1一样，以不同的偏转穿过该膜，而TM3b则返回细胞质，与胞质区域相互作用。此外，7个亚基形成一个半径为
Figure PCTCN2022115022-appb-000003
的中心孔，其感受张力并与构象变化有关。比较铜绿假单胞菌和大肠杆菌的MscS结构，前者的TM1和TM2的倾斜角度小于后者，这导致TM区域的大偏转，尤其是TM1和TM2之间的环。在胞质区，中间β结构域(残基123～172)包含5条β链，其与其他不同亚基的β链紧密相连。并且C末端结构域(177～273残基)以5条β链和2条α螺旋组成，其为混合结构。在相邻单体的这两个结构域之间，在侧面有7个相等的开口，清晰可见，半径约为
Figure PCTCN2022115022-appb-000004
其被提出认为是EcMscS中的离子渗透的原因。除这些入口外，第8个开口存在于蛋白质底部，其通过7条β链表现，最窄半径为
Figure PCTCN2022115022-appb-000005
在所有尺寸中，延伸至
Figure PCTCN2022115022-appb-000006
的PaMscS2平行于七倍轴，并在垂直方向宽度为
Figure PCTCN2022115022-appb-000007
PaMscS的结构类似于关闭状态(PDB:2OAU)下的EcMscS，TM结构域有超过101个rmsd为
Figure PCTCN2022115022-appb-000008
的C α原子，但在开放状态(PDB:2VV5)下，TM区域存在较大差异，rmsd为
Figure PCTCN2022115022-appb-000009
这些结果表明PaMscS在结构上的构象反映了关闭状态。总的来说，PaMscS埃米孔独特而精细的结构显示出用于检测的巨大潜力。 The experimental team of the present invention revealed the structure of the protein angstrom pore through modeling, which is a typical heptamer that is radially symmetrical and resembles a cylinder. It contains 8 openings, 7 on the sides and 1 on the bottom. N-terminal residues 1-13 are too flexible to be resolved in the model. Topologically, PaMscS can be divided into 2 parts, the transmembrane region and the large cytoplasmic part. Each monomer generates three N-terminal transmembrane helices, including TM1 (residues 17-52), TM2 (residues 58-83) and TM3 (residues 90-122). The C-terminal cytoplasmic region can be divided into an intermediate β domain and a COOH terminal domain. TM1 and TM2 in each subunit are aligned together in an antiparallel orientation, with TM1 passing through the bilayer membrane on the outside of the channel and TM2 forming the central layer, thus forming a permeation pathway around the channel axis. The TM3 helix can be described as two helical segments, TM3a and TM3b, separated by a distinct kink ~53° at Gly108, residues that are conserved in homology. TM3a, like TM1, crosses the membrane with a different deflection, while TM3b returns to the cytoplasm, where it interacts with the cytoplasmic domain. Furthermore, 7 subunits form a radius of
Figure PCTCN2022115022-appb-000003
The central pore of , which senses tension and is involved in conformational changes. Comparing the MscS structures of Pseudomonas aeruginosa and Escherichia coli, the slant angles of TM1 and TM2 of the former are smaller than those of the latter, which leads to a large deflection of the TM region, especially the loop between TM1 and TM2. In the cytoplasmic region, the middle β-domain (residues 123-172) contains 5 β-strands that are tightly connected to β-strands of other different subunits. And the C-terminal domain (177-273 residues) is composed of 5 β-strands and 2 α-helices, which is a mixed structure. Between these two domains of adjacent monomers, there are seven equal openings on the sides, clearly visible, with a radius of approx.
Figure PCTCN2022115022-appb-000004
It has been proposed to be responsible for ion permeation in EcMscS. In addition to these entrances, an 8th opening exists at the bottom of the protein, which is expressed by 7 β-strands with a narrowest radius of
Figure PCTCN2022115022-appb-000005
In all sizes, extends to
Figure PCTCN2022115022-appb-000006
PaMscS2 is parallel to the heptad axis and has a vertical width of
Figure PCTCN2022115022-appb-000007
The structure of PaMscS is similar to that of EcMscS in the closed state (PDB:2OAU), and the TM domain has more than 101 rmsd as
Figure PCTCN2022115022-appb-000008
C α atoms, but in the open state (PDB:2VV5), there is a large difference in the TM region, the rmsd is
Figure PCTCN2022115022-appb-000009
These results suggest that the structural conformation of PaMscS reflects the closed state. Overall, the unique and fine-grained structure of PaMscS emipores shows great potential for detection.
位点突变：Site mutation:
对相应的氨基酸位点所对应的DNA序列进行修改(包括DNA序列修改后直接送合成或者PCR突变)，将目标突变位点所在的DNA序列修改为突变蛋白所对应的DNA序列。突变型PaMscS蛋白埃米孔的突变蛋白可以包括130A、130H、180R、271I、130S或130P。Modify the DNA sequence corresponding to the corresponding amino acid site (including direct synthesis or PCR mutation after DNA sequence modification), and modify the DNA sequence where the target mutation site is located to the DNA sequence corresponding to the mutant protein. Mutant PaMscS protein Amypore muteins may include 130A, 130H, 180R, 271I, 130S or 130P.
膜的合并和单通道记录：Merging and single-channel recording of membranes:
实验在由华纳仪器(Warner instrument)提供的垂直样品池中进行，如果没有特别提到，所有的电流痕迹都由采样频率为9900Hz的HEKA EPC 10USB膜片钳放大器记录。1μL的25mg/mL大肠杆菌膜被预涂在杯子的150μm孔口上，然后将1mL的电解质溶液(-trans端： 100mM NaCl，10mM HEPES，pH7.0；-cis端：300mM NaCl，10mM HEPES，pH7.0)分别加入到样品池的两面。然后用1mL吸管从-cis端吸出样品池的大约2/3的电解质溶液。当平均电流接近0pA时，电解质溶液被驱动到样品池的-cis侧，以形成一个平面磷脂双分子膜。磷脂膜形成后，PaMscS1蛋白溶液被加入到-cis端中。当PaMscS1被嵌入到平面磷脂双分子膜中时，电流有明显的变化。蛋白质被嵌入后，更换1mL溶液，然后进行后续实验。Experiments were performed in a vertical sample cell provided by Warner instrument, and all current traces were recorded by a HEKA EPC 10USB patch-clamp amplifier with a sampling frequency of 9900 Hz if not specifically mentioned. 1μL of 25mg/mL E. coli membrane was pre-coated on the 150μm orifice of the cup, and then 1mL of electrolyte solution (-trans end: 100mM NaCl, 10mM HEPES, pH7.0; -cis end: 300mM NaCl, 10mM HEPES, pH7.0 .0) were added to both sides of the sample cell. Then use a 1mL pipette to suck out about 2/3 of the electrolyte solution in the sample cell from the -cis end. When the average current is close to 0 pA, the electrolyte solution is driven to the -cis side of the sample cell to form a planar phospholipid bilayer. After the phospholipid film was formed, the PaMscS1 protein solution was added to the -cis end. When PaMscS1 was embedded in planar phospholipid bilayers, there was a clear change in the current. After the protein was embedded, 1 mL of the solution was replaced before subsequent experiments.
单核苷酸检测：Single nucleotide detection:
通过MscS埃米孔检测不同组的扩增产物。将不同的样品加入-cis端，在+50mV下记录并观察20分钟。当平面磷脂膜上形成稳定的PaMscS1埃米孔时，将待检测的单核苷酸加入样品孔的-cis端，然后施加电压，记录电流信号。Different sets of amplification products were detected by MscS angstrompores. Different samples were added to the -cis end, recorded at +50 mV and observed for 20 min. When a stable PaMscS1 angstrom hole is formed on the planar phospholipid membrane, the single nucleotide to be detected is added to the -cis end of the sample hole, and then a voltage is applied to record the current signal.
冠状病毒临床样本的收集和处理：Collection and processing of clinical samples for coronavirus:
SARS-CoV-2患者的临床样本通过咽拭子(患者编号：1～15)和血液(患者编号：16～22)采集，经反转录后获得cDNA。Clinical samples of SARS-CoV-2 patients were collected through throat swabs (patient numbers: 1-15) and blood (patient numbers: 16-22), and cDNA was obtained after reverse transcription.
RNA反转录和PCR扩增：RNA reverse transcription and PCR amplification:
本发明SARS-CoV-2 RNA反转录扩增***如下：随机六聚体(60μM)和锚poly T(23):1μL，dNTP混合物(各10mM):1μL，RNA样品:11μL。将反应在热循环仪中、于65℃孵育5分钟。立即将样品放在冰上快速冷却>1分钟。然后，在一个干净的预PCR罩中，将下列试剂与样品混合：5X SuperScript IV缓冲液：4μL，DTT(100mM)：1μL，RNaseOUT RNase抑制剂：1μL，Superscript IV逆转录酶：1μL。使用42℃ 50分钟和70℃ 10分钟的程序将样品在热循环仪中孵化后获得cDNA。本发明实验中的cDNAPCR扩增***如下：ddH2O：26.5μL，5×PrimeSTAR缓冲液(Mg2+Plus)：10μL，dNTP混合物(2.5mM)：4μL，引物1(正向引物ORF P1，10μM)：2μL，引物2(反向引物ORF P2，10μM)：2μL，PrimeSTARHS DNA聚合酶(2.5U/μL)：0.5μL，cDNA样品：5μL。扩增程序如下：95℃预热5分钟，98℃热变性10秒。55℃折射退火15秒，然后72℃延伸12秒。重复所述循环，共35次。上述引物由本研究团队自主合成，序列如下：The SARS-CoV-2 RNA reverse transcription amplification system of the present invention is as follows: random hexamer (60 μM) and anchor poly T (23): 1 μ L, dNTP mixture (each 10 mM): 1 μ L, RNA sample: 11 μ L. Reactions were incubated in a thermal cycler at 65°C for 5 minutes. Immediately place samples on ice to rapidly cool >1 min. Then, in a clean pre-PCR hood, mix the following reagents with the sample: 5X SuperScript IV Buffer: 4 µL, DTT (100 mM): 1 µL, RNaseOUT RNase Inhibitor: 1 µL, Superscript IV Reverse Transcriptase: 1 µL. cDNA was obtained after incubating the samples in a thermal cycler using a program of 42 °C for 50 min and 70 °C for 10 min. The cDNA PCR amplification system in the experiment of the present invention is as follows: ddH2O: 26.5 μL, 5×PrimeSTAR buffer (Mg2+Plus): 10 μL, dNTP mixture (2.5mM): 4 μL, primer 1 (forward primer ORF P1, 10 μM): 2 μL, primer 2 (reverse primer ORF P2, 10 μM): 2 μL, PrimeSTARHS DNA polymerase (2.5U/μL): 0.5 μL, cDNA sample: 5 μL. The amplification program was as follows: preheating at 95°C for 5 minutes, heat denaturation at 98°C for 10 seconds. Refractive annealing at 55°C for 15 seconds, followed by extension at 72°C for 12 seconds. The cycle was repeated for a total of 35 times. The above primers were independently synthesized by our research team, and the sequences are as follows:
ORF P1：TTGTTTGAATAGTAGTTGTCTGA(SEQ ID NO:7)ORF P1: TTGTTTGAATAGTAGTTGTCTGA (SEQ ID NO:7)
ORF P2：TCAACTCAATATGAGTATGGTACTG(SEQ ID NO:8)ORF P2: TCAACTCAATATGAGTATGGTACTG (SEQ ID NO:8)
逆转录酶环状介导的等温扩增(RT-LAMP)和检测：Reverse Transcriptase Loop-mediated Isothermal Amplification (RT-LAMP) and Detection:
进行包含以下体系的RT-LAMP反应：ddH 2O：4.1μL，
Figure PCTCN2022115022-appb-000010
Colorimetric LAMP 2X Master Mix(DNA&RNA)：12.5μL，F3(20μM)：0.2μL，B3(20μM)：0.2μL，FIP(20μM)：1μL，BIP(20μM)：1μL，LoopF(20μM)：0.5μL，LoopB(20μM)：0.5μL，模板：5μL。LAMP反应在60℃下运行30分钟。 Perform an RT-LAMP reaction containing the following system: ddH 2 O: 4.1 μL,
Figure PCTCN2022115022-appb-000010
Colorimetric LAMP 2X Master Mix (DNA&RNA): 12.5 μL, F3 (20 μM): 0.2 μL, B3 (20 μM): 0.2 μL, FIP (20 μM): 1 μL, BIP (20 μM): 1 μL, LoopF (20 μM): 0.5 μL, LoopB (20 μM): 0.5 μL, Template: 5 μL. The LAMP reaction was run at 60°C for 30 minutes.
核酸扩增和检测：Nucleic acid amplification and detection:
本发明的核酸扩增体系如下：ddH2O：64.6μL，5×PrimeSTAR缓冲液(Mg2+Plus)：20μL，dNTP混合物(各10mM)：6μL，引物1(miRNA21，100μM)：0.4μL，引物2(AFP适配体，10μM)：4μL，模板1(提取，poly T)：2μL，模板2(poly C，1μM)：2μL，PrimeSTAR HS DNA聚合酶(2.5U/μL)：1μL。扩增程序如下：95℃预热5min且98℃热变性10s。60℃折射退火15秒，然后68℃延伸23秒。共重复循环30次，核酸扩增结果如图13所示。The nucleic acid amplification system of the present invention is as follows: ddH2O: 64.6 μL, 5×PrimeSTAR buffer (Mg2+Plus): 20 μL, dNTP mixture (each 10 mM): 6 μL, primer 1 (miRNA21, 100 μM): 0.4 μL, primer 2 ( AFP aptamer, 10 μM): 4 μL, template 1 (extracted, poly T): 2 μL, template 2 (poly C, 1 μM): 2 μL, PrimeSTAR HS DNA polymerase (2.5U/μL): 1 μL. The amplification procedure was as follows: preheating at 95°C for 5 minutes and thermal denaturation at 98°C for 10s. Refractive annealing at 60°C for 15 seconds, followed by extension at 68°C for 23 seconds. A total of 30 cycles were repeated, and the nucleic acid amplification results are shown in FIG. 13 .
电生物学数据分析：Electrobiological data analysis:
在本发明中，电生物学数据由Clampfit软件处理，并用Origin软件绘图。In the present invention, electrobiological data were processed by Clampfit software and plotted by Origin software.
实施例二Embodiment two
基于PaMscS埃米孔的电生理检测和dNTP检测：Electrophysiological detection and dNTP detection based on PaMscS pore:
MscS的基本功能是对机械刺激(如渗透压期间的膜张力变化)反应的快速开/关的开关。MscS的胞质结构域功能为在渗透适应(osmoadaptation)过程中平衡渗透剂的损失的分子筛。本发明的蛋白质埃米孔中，来自胞质区的7个侧孔在离子和溶质的易位(translocation)过程中起着关键作用。因此，侧孔突变体PaMscS1(W130A)和PaMscS2(K180R)由于低背景 噪音而被选择用于后续研究(图5、图6A-C)。在电生理实验中，将纯化的蛋白质加入到电生理装置的-cis端中(图1A)。当PaMscS突变体通道嵌入双层脂质膜(BLM，绝缘膜的一种)时，在+50mV的电压下可以观察到稳定的通道电流跳跃(图1B)。在范围从-50mV至+50mV的电压下，PaMscS1的通道电导保持稳定(图1C)，并且当电压高于+90mV时，PaMscS1的门控概率增加(图7)。PaMscS1埃米孔的电导为0.64±0.02nS(n＝91，高斯拟合的峰值±SD，-cis端：300mM NaCl，-trans端：30mM NaCl)，PaMscS2埃米孔的电导分布为34.9±7.0pA(平均值±SD，来自18个独立***事件)(图1D)。PaMscS1的离子转运结果表明，PaMscS1对Br-具有更好的选择性(图8)。The basic function of MscS is a rapid on/off switch in response to mechanical stimuli such as changes in membrane tension during osmolarity. The cytoplasmic domain of MscS functions as a molecular sieve that balances the loss of osmolytes during osmoadaptation. In the protein angstrompore of the present invention, the seven side pores from the cytoplasmic region play a key role in the translocation of ions and solutes. Therefore, side hole mutants PaMscS1(W130A) and PaMscS2(K180R) were selected for subsequent studies due to low background noise (Fig. 5, Fig. 6A-C). For electrophysiological experiments, the purified protein was added to the -cis end of the electrophysiological device (Figure 1A). When the PaMscS mutant channel was embedded in a bilayer lipid membrane (BLM, a type of insulating membrane), a steady channel current jump could be observed at a voltage of +50 mV (Fig. 1B). The channel conductance of PaMscS1 remained stable at voltages ranging from -50 mV to +50 mV (Fig. 1C), and the gating probability of PaMscS1 increased when the voltage was higher than +90 mV (Fig. 7). The conductance of PaMscS1 angstrompore is 0.64±0.02nS (n=91, the peak value of Gaussian fitting±SD, -cis end: 300mM NaCl, -trans end: 30mM NaCl), and the conductance distribution of PaMscS2 angstrompore is 34.9±7.0 pA (mean ± SD, from 18 independent insertion events) (Fig. 1D). The ion transport results of PaMscS1 showed that PaMscS1 had better selectivity for Br- (Fig. 8).
在BLM中***PaMscS1埃米孔，并在-cis端中存在dNTPs的情况下，在正电压下可以观察到dNTPs的易位信号。dNTPs的易位频率随着电压的增加而增加(图9)。PaMscS1和PaMscS2都被分析用于dNTP混合物检测(dNTPs浓度：0.2mM，电压：+50mV，-cis端：300mM NaCl，-trans端：30mM NaCl)。在两个突变体之间可以观察到阻塞电流分布的明显差异，表明dNTPs的易位与PaMscS埃米孔的侧孔有关(图1E)，即，在同样的检测条件下，PaMscS1和PaMscS2埃米孔对于dNTPs阻塞电流的分布呈现出不同的情况。具体表现为PaMscS1埃米孔对于四种dNTPs混合物阻塞率呈现3个峰，而PaMscS2埃米孔对于四种dNTPs混合物阻塞率呈现2个峰。因为PaMscS1和PaMscS2突变的差异在于侧孔氨基酸的差异，因此推测dNTPs的检测信号与侧孔相关。由于PaMscS1对dNTP混合物有更好的区分效果，它更适合用于dNTPs混合物的区分。而对于PaMscS2，它表现出更稳定的通道电导和相对更高的膜融合效率，因此它更适合用于接着的快速诊断(图6A-C)。野生型PaMscS埃米孔对于四种dNTPs混合物阻塞率呈现2个峰(图29)。PaMscS1检测单核苷酸的电流轨迹和停留时间分布如图10A-D所示。In the presence of PaMscS1 angstrompores in the BLM and the presence of dNTPs in the -cis end, translocation signals of dNTPs could be observed at positive voltages. The translocation frequency of dNTPs increased with increasing voltage (Fig. 9). Both PaMscS1 and PaMscS2 were analyzed for dNTP mixture detection (dNTPs concentration: 0.2mM, voltage: +50mV, -cis end: 300mM NaCl, -trans end: 30mM NaCl). A clear difference in blocking current distribution could be observed between the two mutants, indicating that the translocation of dNTPs is associated with the side holes of the PaMscS angstrompore (Fig. 1E), i.e., PaMscS1 and PaMscS2 angstrom Pores present a different profile for the distribution of dNTPs blocking currents. Specifically, PaMscS1 angstrompores showed three peaks for the blocking rate of the four dNTPs mixtures, while PaMscS2 angstrompores showed two peaks for the blocking rate of the four dNTPs mixtures. Because the difference between PaMscS1 and PaMscS2 mutations lies in the amino acid difference of the side hole, it is speculated that the detection signal of dNTPs is related to the side hole. Since PaMscS1 has a better discrimination effect on dNTP mixtures, it is more suitable for the discrimination of dNTPs mixtures. As for PaMscS2, it exhibited more stable channel conductance and relatively higher membrane fusion efficiency, so it was more suitable for subsequent rapid diagnosis (Fig. 6A-C). The blocking rate of the wild-type PaMscS pore for the four dNTPs mixtures showed two peaks (Fig. 29). The current traces and residence time distributions of PaMscS1 detecting single nucleotides are shown in Fig. 10A-D.
与目前报道的含有am7βCD、MoS 2和α-Hederin的纳米孔α-溶血素相比，虽然PaMscS1埃米孔的检测准确度低于报道的含有am7βCD结合物(即利用α溶血素突变蛋白与6-氨基-6脱氧-β-环糊精适配体构建)的最佳生物纳米孔，但易位速度与固态纳米孔相当。在单链DNA(ssDNA)检测实验中，50μM的ssDNA在30mM NaCl/300mM NaCl的缓冲条件下和+50mV偏置电压下进行检测，而由于其通道尺寸较窄，因此未观察到易位事件(图11)。因此，PaMscS突变体埃米孔有潜力成为一种有用的小分子传感器。 Compared with the currently reported nanopore α-hemolysin containing am7βCD, MoS 2 and α-Hederin, although the detection accuracy of PaMscS1 angiopore is lower than that of the reported conjugate containing am7βCD (i.e., using α-hemolysin mutein with 6 -Amino-6-deoxy-β-cyclodextrin aptamer construct), but the translocation rate is comparable to that of solid-state nanopores. In single-stranded DNA (ssDNA) detection experiments, 50 μM ssDNA was detected under buffer conditions of 30 mM NaCl/300 mM NaCl and a bias voltage of +50 mV, and no translocation events were observed due to its narrow channel size ( Figure 11). Therefore, the PaMscS mutant emipore has the potential to be a useful small molecule sensor.
实施例三Embodiment three
PaMscS1埃米孔的选择性调整用于优化的dNTPs检测：Selective tuning of the PaMscS1 Angstrom pore for optimized dNTPs detection:
考虑到PaMscS1埃米孔的机械力敏感性，实验人员通过应用不同的渗透压差来调节PaMscS1埃米孔的选择性。为了在不同的渗透压差下保持dCTP和dGTP的恒定电荷特性，实验人员将-cis端的电导缓冲液浓度保持在300mM并改变-trans端的电导液浓度来改变渗透压差。在3种渗透压差条件下测试了PaMscS1埃米孔对大分子dGTP和小分子dCTP的检测能力，包括对称(symmetric)条件(图2A，300mM NaCl/300mM NaCl，+50mV偏置电压)，低渗透压差条件(图2B，100mM NaCl/300mM NaCl，+50mV偏置电压)和高渗透压差条件(图2C，30mM NaCl/300mM NaCl，+50mV偏置电压)。在对称渗透压条件下，dCTP的易位频率从0.16±0.03s -1增加到0.22±0.07s -1，而dGTP从0.09±0.02s -1变化到0.07±0.003s -1。在低渗透压差(LOD)条件下，dCTP的易位频率从0.34±0.1s -1增加到0.67±0.14s -1，而dGTP从0.06±0.01s -1增加到0.3±0.04s -1。在高渗透压差(HOD)条件下，dCTP的易位频率从0.12±0.04s -1增加到0.22±0.07s -1，而dGTP从0.37±0.08s -1增加到1.12±0.12s -1(图2D，每个实验n＝3，平均值±S.E.M)。图2E总结了dGTP和dCTP的检测，并且它总结为低渗透压差条件显示dCTP的最高的易位事件增加，而高渗透压差条件显示最高的dGTP的易位事件增加(图2E)。与高渗透压条件的dCTP的降低的捕获效率相比，低渗透压差条件显示对dCTP和dGTP二者的平衡的捕获能力。鉴于dGTP和dCTP的大小和电荷特性在给定的实验条件下保持恒定，且MscS家族(例如EcMscS、HpMscS、AtMsL1蛋白等)的通道大小在不同的压力、渗 透压条件或膜电位下可以变化，实验人员可以得出结论：PaMscS1埃米孔对dNTPs的选择性差异是由不同渗透压差条件下的通道大小的变化引起的。 Considering the mechanical sensitivity of PaMscS1 angstrompores, the experimenters tuned the selectivity of PaMscS1 angstrompores by applying different osmotic pressure differences. In order to maintain the constant charge characteristics of dCTP and dGTP under different osmotic pressure differences, the experimenters kept the conductivity buffer concentration at the -cis end at 300 mM and changed the conductivity buffer concentration at the -trans end to change the osmotic pressure difference. The PaMscS1 angstrompore was tested for its ability to detect macromolecular dGTP and small molecular dCTP under three osmotic pressure differential conditions, including symmetric conditions (Fig. 2A, 300mM NaCl/300mM NaCl, +50mV bias voltage), low Osmotic pressure differential conditions (FIG. 2B, 100 mM NaCl/300 mM NaCl, +50 mV bias voltage) and high osmotic pressure differential conditions (FIG. 2C, 30 mM NaCl/300 mM NaCl, +50 mV bias voltage). Under symmetric osmotic pressure conditions, the translocation frequency of dCTP increased from 0.16±0.03s -1 to 0.22±0.07s -1 , while that of dGTP changed from 0.09±0.02s -1 to 0.07±0.003s -1 . Under low osmotic pressure difference (LOD) conditions, the translocation frequency of dCTP increased from 0.34±0.1s -1 to 0.67±0.14s -1 , while that of dGTP increased from 0.06±0.01s -1 to 0.3±0.04s -1 . Under high osmotic pressure difference (HOD) conditions, the translocation frequency of dCTP increased from 0.12±0.04s -1 to 0.22±0.07s -1 , while that of dGTP increased from 0.37±0.08s -1 to 1.12±0.12s -1 ( Figure 2D, n=3 per experiment, mean ± SEM). Figure 2E summarizes the detection of dGTP and dCTP, and it concludes that low osmolality conditions showed the highest increase in dCTP translocation events, while high osmolality conditions showed the highest increase in dGTP translocation events (Figure 2E). Low osmolarity differential conditions showed a balanced capture capacity for both dCTP and dGTP compared to the reduced capture efficiency of dCTP for high osmolarity conditions. Given that the size and charge properties of dGTP and dCTP remain constant under given experimental conditions, and the channel size of the MscS family (such as EcMscS, HpMscS, AtMsL1 proteins, etc.) can vary under different pressure, osmotic pressure conditions or membrane potentials, The experimenters can conclude that the difference in the selectivity of the PaMscS1 angstrompore to dNTPs is caused by the variation of the channel size under different osmotic pressure difference conditions.
实施例四：Embodiment four:
通过PaMscS2检测SARS-CoV-2临床样本：Detection of SARS-CoV-2 clinical samples by PaMscS2:
对SARS-CoV-2核酸进行快速而简单的检测是对SARS-CoV-2流行的重要预防方法。在此，实验人员使用SARS-CoV-2Orf1ab(cDNA)的特异性引物进行SARS-CoV-2的检测(图3A)。由于PaMscS2埃米孔具有较高的膜融合效率，因此被应用于检测。目标序列为172bp，且扩增产物的片段分析表明引物可用于目标基因的扩增(表2)。合成了SARS-CoV-2 Orf1ab基因并检测了SARS-CoV-2Orf1ab基因的梯度浓度。在10^3拷贝数/mL至10^11拷贝数/mL的浓度范围内，合成的SARS-CoV-2 Orf1ab基因可以被检测到(图3B)。然后，对22个临床样本进行了检测，包括15个确诊病人的样本和7个健康对照组的样本。通过埃米孔检测的15个阳性样本和6个阴性样本(病人编号：1～15，17～22)都显示出与临床检测一致的结果(表3)，1个阴性样本被诊断为假阳性结果(病人编号：16)。本方法的特异性为86％，灵敏度为100％(图3C)。作为核酸扩增监测的纳米设备，PaMscS突变体埃米孔***可以与如聚合酶链式反应和链式置换扩增等各种NAAT(NucleicAcidAmplification Tests，核酸扩增检测)(图12)相结合，进行目标基因的检测(图3D)，该孔还具有监测逆转录过程的潜力，其可以实现对目标RNA的快速且无扩增的检测。Rapid and simple detection of SARS-CoV-2 nucleic acid is an important preventive method against the SARS-CoV-2 epidemic. Here, experimenters used specific primers for SARS-CoV-2 Orf1ab (cDNA) for the detection of SARS-CoV-2 (Fig. 3A). Due to the high membrane fusion efficiency of PaMscS2 pore, it was used for detection. The target sequence is 172bp, and the fragment analysis of the amplified product shows that the primers can be used for the amplification of the target gene (Table 2). The SARS-CoV-2 Orf1ab gene was synthesized and the gradient concentration of the SARS-CoV-2 Orf1ab gene was detected. The synthetic SARS-CoV-2 Orf1ab gene could be detected in the concentration range from 10^3 copies/mL to 10^11 copies/mL (Fig. 3B). Then, 22 clinical samples were tested, including 15 samples from confirmed patients and 7 samples from healthy controls. All 15 positive samples and 6 negative samples (patient numbers: 1-15, 17-22) tested by Amipore showed results consistent with clinical testing (Table 3), and 1 negative sample was diagnosed as a false positive Results (patient number: 16). The method had a specificity of 86% and a sensitivity of 100% (Fig. 3C). As a nano-device for nucleic acid amplification monitoring, the PaMscS mutant angstrom system can be combined with various NAAT (Nucleic Acid Amplification Tests, nucleic acid amplification detection) such as polymerase chain reaction and chain displacement amplification (Figure 12), For the detection of the target gene (Fig. 3D), the well also has the potential to monitor the reverse transcription process, which enables rapid and amplification-free detection of the target RNA.
表2：片段长度和扩增产物浓度Table 2: Fragment Length and Amplified Product Concentration
Figure PCTCN2022115022-appb-000011
Figure PCTCN2022115022-appb-000011
表3：Ct值和临床样本的埃米孔检测结果Table 3: Ct values and Angstrom results of clinical samples
Figure PCTCN2022115022-appb-000012
Figure PCTCN2022115022-appb-000012
Figure PCTCN2022115022-appb-000013
Figure PCTCN2022115022-appb-000013
实施例五：Embodiment five:
通过PaMscS1埃米孔检测多种生物标记物：Detection of multiple biomarkers via the PaMscS1 angstrompore:
实验人员设计了一种dNTP条形码探针的区分策略，并通过PaMscS1埃米孔验证了其在多种生物标记物检测中的应用。设计了两个探针以结合目标DNA序列并且在链式聚合反应中引起特异性dNTPs消耗，其中miR21探针(探针A)：包括与一条与miR21互补配对的序列和一条polyT的条形码序列；AFP适配体探针(探针B)：包括一条与AFP适配体互补配对的序列和一条poly C的条形码序列。当样品中存在探针A或探针B时，聚合酶链反应可被激活，消耗反应体系中的dATP或dGTP(图4A)。检测无目标序列的对照样品、有miR21的样品、有AFP适配体的样品和既有miR21还有AFP适配体的样品。相应的电流轨迹和阻塞电流分布如图4B和4C所示。可以观察到，在对照组(没有miR21和AFP适配体的样品)中，dATP(δf dATP＝f dATP/f 背景)的易位事件频率的相对增加为1.7±0.2(平均值±S.E.M)，dGTP(δf dGTP＝f dGTP/f 背景)的易位事件频率的相对增加为1.8±0.03。在有miR21的样品中，δf dATP显著低于对照组(1.4±0.1，平均值±S.E.M)，同时有AFP适配体的样品中，δf dGTP降至1.0±0.3(平均值±S.E.M)。对于既有miR21又有AFP适配体的样品，与对照组相比，δf dATP(1.1±0.2，平均值±S.E.M)和δf dGTP(1.3±0.2，平均值±S.E.M)均降低(图4D)。这些结果表明，PaMscS1可以同时检测单个或多个生物标记物(每个实验n≥3)。 Experimentalists designed a discrimination strategy of dNTP barcoded probes and validated its application in the detection of multiple biomarkers through the PaMscS1 angstrom pore. Two probes were designed to bind to the target DNA sequence and cause specific dNTPs depletion in a chain polymerization reaction, wherein the miR21 probe (probe A): includes a barcode sequence complementary to miR21 and a polyT; AFP aptamer probe (probe B): includes a sequence complementary to the AFP aptamer and a poly C barcode sequence. When probe A or probe B exists in the sample, the polymerase chain reaction can be activated to consume dATP or dGTP in the reaction system ( FIG. 4A ). Control samples without target sequence, samples with miR21, samples with AFP aptamer, and samples with both miR21 and AFP aptamer were tested. The corresponding current trajectories and blocking current distributions are shown in Fig. 4B and 4C. It can be observed that in the control group (samples without miR21 and AFP aptamers), the relative increase in the frequency of translocation events for dATP (δf dATP = f dATP /f background ) was 1.7 ± 0.2 (mean ± SEM), The relative increase in frequency of translocation events for dGTP (δf dGTP =f dGTP / fbackground ) was 1.8 ± 0.03. In samples with miR21, δf dATP was significantly lower than that in the control group (1.4±0.1, mean±SEM), while in samples with AFP aptamer, δf dGTP decreased to 1.0±0.3 (mean±SEM). For samples with both miR21 and AFP aptamers, both δf dATP (1.1±0.2, mean±SEM) and δf dGTP (1.3±0.2, mean±SEM) were decreased compared with controls (Fig. 4D) . These results demonstrate that PaMscS1 can detect single or multiple biomarkers simultaneously (n ≥ 3 per experiment).
实施例六：Embodiment six:
一种增强信号的策略：A strategy to boost the signal:
A.通过离子调控增强对于待检测物的检测限，具体为对于dNTPs，通过在本发明涉及的埃米孔的胞质端加入镍离子、钴离子等二价阳离子，可以提高对于dNTPs的检测下限；B.通过电导液渗透差异的调节增强信噪比，具体为通过提高电导液浓度，可以提高对于结构类似药物分子的区分能力，通过提高磷脂膜两侧电导液的渗透差异，可以提高对于dGTP的检测效果。A. Enhance the detection limit of the substance to be detected by ion regulation, specifically for dNTPs, by adding divalent cations such as nickel ions and cobalt ions to the cytoplasmic end of the angstrom pores involved in the present invention, the lower limit of detection for dNTPs can be improved B. Enhance the signal-to-noise ratio by adjusting the penetration difference of the conductive solution, specifically by increasing the concentration of the conductive solution, the ability to distinguish drug molecules with similar structures can be improved, and by increasing the penetration difference of the conductive solution on both sides of the phospholipid membrane, it can be improved for dGTP detection effect.
实施例七：Embodiment seven:
药物检测drug testing
1.通过LC-MS的药物测量：1. Drug measurement by LC-MS:
LC-MS和LC-MS/MS分析是在岛津超快速液相色谱***(UFLC，岛津)和AB SCIEX Qtrap 5500质谱仪上进行的，配备了Turbo喷雾离子源(Turbo Spray ion source)。色谱和质谱数据的收集和分析由Analyst 1.6.2软件(AB SCIEX,USA)完成。LC-MS and LC-MS/MS analyzes were performed on a Shimadzu ultrafast liquid chromatography system (UFLC, Shimadzu) and an AB SCIEX Qtrap 5500 mass spectrometer equipped with a Turbo Spray ion source. The collection and analysis of chromatographic and mass spectrometric data were completed by Analyst 1.6.2 software (AB SCIEX, USA).
色谱分离是在WatersACQUITYUPLC BEH C18柱(2.1mm×100mm I.D.，1.7μm)上实现的。流动相由水(A)和乙腈(B)组成，梯度洗脱如下：0-1.0分钟，10-90％B；1-2.0分钟，90％B。流速为0.5mL/min。柱温和自动进样器的温度分别保持在35℃和15℃。注射量为1μL。Chromatographic separation was achieved on a WatersACQUITYUPLC BEH C18 column (2.1mm×100mm I.D., 1.7μm). The mobile phase consisted of water (A) and acetonitrile (B), and the gradient elution was as follows: 0-1.0 minutes, 10-90% B; 1-2.0 minutes, 90% B. The flow rate was 0.5 mL/min. The temperature of the column and the autosampler were maintained at 35°C and 15°C, respectively. The injection volume is 1 μL.
在MS/MS分析中，采用正电离模式用于样品检测，以如下的优化质谱参数：离子喷雾电压，5500V；去簇电压，100V；温度，500℃。选择MRM(多反应监测)模式定量来硫酸庆大霉素和IS(内标)，离子对分别为450.2-160.1、464.2-160.1、478.2-157.1和265.2-232.2。In MS/MS analysis, the positive ionization mode was used for sample detection, and the mass spectrometry parameters were optimized as follows: ion spray voltage, 5500V; declustering voltage, 100V; temperature, 500°C. Select the MRM (Multiple Reaction Monitoring) mode to quantify gentamicin sulfate and IS (internal standard), and the ion pairs are 450.2-160.1, 464.2-160.1, 478.2-157.1 and 265.2-232.2, respectively.
2.从连接到活体大鼠血管的透析装置中连续监测硫酸庆大霉素：2. Continuous monitoring of gentamicin sulfate from a dialysis device connected to a live rat blood vessel:
雄性Sprague-Dawley大鼠(250-300g)由成都达硕实验动物有限公司提供，本发明中的所有动物实验都得到了四川大学华西医院伦理委员会的批准(批准号2021885A)。Male Sprague-Dawley rats (250-300 g) were provided by Chengdu Dashuo Experimental Animal Co., Ltd. All animal experiments in the present invention were approved by the Ethics Committee of West China Hospital of Sichuan University (approval number 2021885A).
大鼠(n＝1)用2％戊巴比妥麻醉，且左股静脉被分离并***导管以提供一个静脉注射口。接下来，通过该导管注入最初的0.4mL肝素溶液(250U/mL)，然后每40分钟周期注射0.1mL以防止监测期间血块的形成。然后分离左股动脉，***导管，并立即通过预先设计的带透析膜的管连接到设备。设备中的空气被血流排出后，管与静脉导管相连，形成一个稳定的循环。对于连续监测，首先记录没有目标药物时的基线信号，然后通过静脉导管以缓慢的速度注入特定浓度的硫酸庆大霉素。对于血液浓度的间隔测量，建立一个类似的循环，既无设备也无透析膜。从动脉导管收集0、15、30、45和60分钟的血液样本，并用PaMscS3(V271I)埃米孔测量药物浓度。每次实验结束后，大鼠由颈椎脱臼处死。Rats (n=1) were anesthetized with 2% pentobarbital, and the left femoral vein was isolated and cannulated to provide an IV port. Next, an initial 0.4 mL of heparin solution (250 U/mL) was infused through the catheter, followed by 0.1 mL every 40 minute cycle to prevent clot formation during monitoring. The left femoral artery was then isolated, catheterized, and immediately connected to the device through a pre-designed tubing with a dialysis membrane. After the air in the device is expelled by the blood flow, the tube is connected to an IV catheter, creating a steady cycle. For continuous monitoring, the baseline signal in the absence of the drug of interest was first recorded, and then a specific concentration of gentamicin sulfate was infused at a slow rate through the venous catheter. For the interval measurement of the blood concentration, a similar cycle is set up, without the device and without the dialysis membrane. Blood samples at 0, 15, 30, 45, and 60 min were collected from the catheter arteriosus and drug concentrations were measured using a PaMscS3(V271I) angstrom. After each experiment, the rats were sacrificed by cervical dislocation.
3.全血检测：3. Whole blood test:
采大鼠血液10微升，加入到融合有埃米孔的样品槽中(-cis端)，施加偏置电压，记录加样一定时间内的信号频率，随后将该信号频率减去空白血液对照频率，根据所检测分子的标准曲线，计算获得该分子对应的浓度。Take 10 microliters of rat blood, add it to the sample tank with angstrom holes (-cis end), apply a bias voltage, record the signal frequency within a certain period of time after adding the sample, and then subtract the blank blood control from the signal frequency Frequency, according to the standard curve of the detected molecule, calculate the corresponding concentration of the molecule.
结果：result:
1.基于PaMscS埃米孔的药物单分子生物传感：1. Drug single-molecule biosensing based on PaMscS emipore:
根据PaMscS埃米孔的通道结构，选择小分子药物(摩尔质量小于1000g/mol)用于检测。硫酸庆大霉素(gentamicin sulphate；分子量MW:561.65)和硫酸新霉素(neomycin sulphate；分子量MW:712.72)被测试用于定量测定，因为它们在临床上的大规模使用，表明迫切需要便利的TDM(therapeutic drug monitoring，治疗药物监测)。硫酸庆大霉素和硫酸新霉素的检测实验是在300mMNaCl(-cis端)和30mMNaCl(-trans端)、10mM HEPES、pH 7.0的电解质条件下进行的，药物检测的电压为-50mV。药物加入检测***后出现了明显的阻塞电流信号(图14A、14B，图17、图18)。硫酸庆大霉素的标准曲线显示线性检测范围为10nM至10μM(N＝3)，典型的硫酸庆大霉素信号的2D密度图显示峰值阻塞电流为11.57±0.02pA，峰值停留时间为1.33±0.02ms(图14C，高斯拟合的峰值，878次阻塞事件)。硫酸新霉素的标准曲线显示线性检测范围为100nM至100μM(N＝3)，典型硫酸新霉素信号的2D密度图显示峰值阻塞电流为9.44±0.02pA，峰值停留时间为1.06±0.01ms(图14D，高斯拟合的峰值，883次阻塞事件)。更高的药物浓度可能导致通道的严重阻塞，并且使药物浓度计算变得困难(图19)。除硫酸庆大霉素和硫酸新霉素之外，PaMscS埃米孔还可以单分子传感其他药物，例如西索米星(MW:447.53)、焦磷酸(MW:177.975)。According to the channel structure of the PaMscS pore, small molecule drugs (molar mass less than 1000 g/mol) were selected for detection. Gentamicin sulfate (MW: 561.65) and neomycin sulfate (MW: 712.72) were tested for quantitative determination because of their large-scale clinical use, indicating an urgent need for convenient TDM (therapeutic drug monitoring, therapeutic drug monitoring). The detection experiments of gentamicin sulfate and neomycin sulfate were carried out under the electrolyte conditions of 300mM NaCl (-cis end) and 30mM NaCl (-trans end), 10mM HEPES, pH 7.0, and the voltage of drug detection was -50mV. After the drug was added to the detection system, an obvious blocking current signal appeared (Fig. 14A, 14B, Fig. 17, Fig. 18). The standard curve for gentamicin sulfate shows a linear detection range from 10nM to 10μM (N=3), and the 2D density plot of a typical gentamicin sulfate signal shows a peak blocking current of 11.57±0.02pA and a peak dwell time of 1.33± 0.02 ms (Figure 14C, peak of Gaussian fit, 878 occlusion events). The standard curve of neomycin sulfate showed a linear detection range from 100nM to 100μM (N=3), and the 2D density plot of a typical neomycin sulfate signal showed a peak blocking current of 9.44±0.02pA and a peak dwell time of 1.06±0.01ms ( Figure 14D, Gaussian fitted peaks, 883 occlusion events). Higher drug concentrations may lead to severe blockage of the channel and make drug concentration calculations difficult (Figure 19). In addition to gentamicin sulfate and neomycin sulfate, PaMscS emipores can also single-molecule sense other drugs, such as sisomicin (MW: 447.53), pyrophosphate (MW: 177.975).
为了评估PaMscS3(V271I)埃米孔对药物的检测精确度，使用LC-MS来测量硫酸庆大霉素的浓度。对于1.5μM的硫酸庆大霉素样品，PaMscS3(V271I)埃米孔和LC-MS呈现出相似的检测结果，表明PaMscS3(V271I)埃米孔的检测具有良好的精确度(图14E)。To evaluate the drug detection accuracy of the PaMscS3(V271I) emipore, LC-MS was used to measure the concentration of gentamicin sulfate. For the 1.5 μM gentamicin sulfate sample, PaMscS3(V271I) angstrompore and LC-MS showed similar detection results, indicating that the detection of PaMscS3(V271I) angstrompore had good precision ( FIG. 14E ).
2.全血样本的药物浓度测量：2. Drug concentration measurement of whole blood samples:
血液是治疗药物监测(therapeutic drug monitoring，简称TDM)的主要介质，但全血成分复杂，其会对纳米孔检测造成严重干扰。常用的纳米孔MspA-2NN在-cis端中加入10μL全血样本后被阻塞(-cis端和-trans端的体积为1mL)，300s内+100mV下开放通道的概率仅为5％±4.7％(图20，电解质条件为-cis端：300mM NaCl，-trans端：300mM NaCl，10mM HEPES，pH7.0，N＝3)。相比之下，PaMscS2埃米孔即使在-cis端中加入20μL全血，也可以保持通道的开放，在300s内、+100mV下开放通道的概率为99％±1％(图15A、15B、15C，电解质条件为-cis端：130mM NaCl，-trans端：130mM NaCl，10mM HEPES，pH 7.0，N＝3)。考虑 到PaMscS3(V271I)埃米孔的稳健检测能力，在注射4mg/mL硫酸庆大霉素后的不同时间间隔(0分钟、15分钟、30分钟、45分钟和60分钟，图21、图22)，直接测量大鼠的硫酸庆大霉素血药浓度。硫酸庆大霉素标准曲线范围从0到3μM，用于计算药物浓度(图15D，N≥3)。在5个监测点中，15分钟的药物浓度最高，60分钟的浓度降低到注射前水平(0分钟)(图15E，N≥3)。PaMscS3(V271I)埃米孔测得的药物浓度趋势符合药物代谢动力学规律，表明PaMscS埃米孔可以准确测量活体大鼠体内药物浓度的变化。有趣的是，硫酸庆大霉素的阻塞电流分布显示，在130mM NaCl的缓冲液中、在较高的负电压下的两个峰值(图23)，而单组分的西索米星(sisomicin)在相同条件下显示一个阻塞电流峰值(图24)，这表明硫酸庆大霉素的两个阻塞电流峰值可能与其多组分有关。Blood is the main medium for therapeutic drug monitoring (TDM), but the composition of whole blood is complex, which will seriously interfere with nanopore detection. The commonly used nanopore MspA-2NN was blocked after adding 10 μL whole blood sample to the -cis end (the volume of the -cis end and -trans end was 1 mL), and the probability of opening the channel at +100 mV within 300 s was only 5% ± 4.7% ( Fig. 20, electrolyte conditions are -cis end: 300mM NaCl, -trans end: 300mM NaCl, 10mM HEPES, pH7.0, N=3). In contrast, the PaMscS2 angstrompore can keep the channel open even if 20 μL whole blood is added to the -cis end, and the probability of opening the channel within 300s at +100mV is 99%±1% (Fig. 15A, 15B, 15C, the electrolyte conditions are -cis end: 130mM NaCl, -trans end: 130mM NaCl, 10mM HEPES, pH 7.0, N=3). Considering the robust detection capability of the PaMscS3(V271I) angiopore, different time intervals (0 min, 15 min, 30 min, 45 min, and 60 min) after injection of 4 mg/mL gentamicin sulfate, Figure 21, Figure 22 ), direct measurement of blood concentration of gentamicin sulfate in rats. A standard curve of gentamicin sulfate ranging from 0 to 3 μM was used to calculate drug concentrations ( FIG. 15D , N > 3). Among the five monitoring points, the drug concentration at 15 minutes was the highest, and the concentration at 60 minutes decreased to the pre-injection level (0 minute) (Fig. 15E, N≥3). The drug concentration trend measured by the PaMscS3(V271I) angiopore conforms to the law of pharmacokinetics, indicating that the PaMscS angiopore can accurately measure the change of the drug concentration in the living rat. Interestingly, the blocking current profile of gentamicin sulfate showed two peaks at higher negative voltages in a buffer of 130 mM NaCl (Fig. 23), while the monocomponent sisomicin ) showed a blocking current peak under the same conditions (Fig. 24), which indicated that the two blocking current peaks of gentamicin sulfate may be related to its multi-component.
3.连接到活体大鼠血管的透析装置对药物的连续监测：3. Continuous monitoring of drugs by a dialysis device connected to the blood vessels of living rats:
由于不同的病人，特别是危重病人之中的药物代谢率有很大的差异，用间隔的TDM技术很难满足精准医疗的需要。因此，对药物浓度的连续监测是非常必要的。为了实现对活体动物药物浓度的长时间监测，实验人员引入了透析膜以避免血液消耗。在TDM过程中，少量的血液通过导管流向透析装置，然后再流回体内。血液中的小分子药物可以通过透析装置渗透到-cis端的导电液体中，而血液基本上没有消耗(图16A)。硫酸庆大霉素的标准曲线范围从0到30μM，用于连续监测的药物浓度计算(图16B)。在活体大鼠的可行性验证实验中，基于简单的透析装置，可以连续观察到硫酸庆大霉素的明显信号，直至注射后3小时(图16C)。对大鼠的不同剂量的硫酸庆大霉素，包括4mg/kg和20mg/kg，可以通过埃米孔监测装置区分(图16D)。这些结果表明，该***可以连续监测活体动物的药物浓度，损失极少。Due to the great difference in drug metabolism rate among different patients, especially critically ill patients, it is difficult to meet the needs of precision medicine with interval TDM technology. Therefore, continuous monitoring of drug concentration is very necessary. To achieve long-term monitoring of drug concentrations in living animals, experimenters introduced dialysis membranes to avoid blood consumption. During TDM, a small amount of blood flows through a catheter to a dialysis machine and then back into the body. Small-molecule drugs in the blood can permeate into the conductive liquid at the -cis end through the dialysis device with essentially no blood consumption (Fig. 16A). A standard curve of gentamicin sulfate ranging from 0 to 30 μM was used for drug concentration calculation for continuous monitoring ( FIG. 16B ). In a feasibility verification experiment in live rats, based on a simple dialysis device, a clear signal of gentamicin sulfate could be continuously observed until 3 hours after injection ( FIG. 16C ). Different doses of gentamicin sulfate to rats, including 4 mg/kg and 20 mg/kg, could be differentiated by the emipore monitoring device (Fig. 16D). These results demonstrate that the system can continuously monitor drug concentrations in living animals with minimal loss.
实施例八：Embodiment eight:
基于EcMscS埃米孔的电生理检测Electrophysiological Detection Based on EcMscS Angstrompore
当野生型EcMscS通道嵌入双层脂质膜(BLM)时，在+100mV的电压下可以观察到稳定的通道电流跳跃(图25)。在范围从-100mV至+100mV的电压下，野生型EcMscS通道电流保持稳定(图26)。野生型EcMscS埃米孔的电导为0.334±0.028nS(-cis端：300mM NaCl，-trans端：30mM NaCl)(图27)。图28a-c分别显示了EcMscS、TtMscS和HpMscS的结构，其与PaMscS的结构高度相似，即都为径向对称且形状似圆柱体的七聚体结构。此外，图28a-c和图28d进一步比较了PaMscS与EcMscS、TtMscS、HpMscS的序列，结果表明EcMscS、TtMscS、HpMscS与PaMscS具有一定的同源性，但这种同源性并非高度同源。EcMscS和PaMscS的相似性仅为60％，但其二者都有检测分析物的能力。因此，本领域技术人员可以理解，决定细菌的MscS能够作为埃米孔检测分析物的关键在于其径向对称且形状似圆柱体的七聚体结构和通道孔径，并非仅仅在于同源性。When the wild-type EcMscS channel was embedded in a bilayer lipid membrane (BLM), a stable channel current jump could be observed at a voltage of +100 mV ( FIG. 25 ). Wild-type EcMscS channel currents remained stable at voltages ranging from -100 mV to +100 mV (Figure 26). The conductance of the wild-type EcMscS pore is 0.334±0.028nS (-cis end: 300mM NaCl, -trans end: 30mM NaCl) (Figure 27). Figures 28a-c show the structures of EcMscS, TtMscS and HpMscS, respectively, which are highly similar to the structure of PaMscS, that is, they are all heptamers with radial symmetry and cylindrical shape. In addition, Figure 28a-c and Figure 28d further compared the sequences of PaMscS and EcMscS, TtMscS, HpMscS, and the results showed that EcMscS, TtMscS, HpMscS and PaMscS have certain homology, but this homology is not highly homologous. EcMscS and PaMscS are only 60% similar, but both have the ability to detect analytes. Therefore, those skilled in the art can understand that the key to determining that bacterial MscS can be used as an angstrom pore to detect analytes lies in its radially symmetrical and cylinder-like heptamer structure and channel pore size, not just homology.
实施例九：Embodiment nine:
基于PaMscS埃米孔的氨基酸检测Amino Acid Detection Based on PaMscS Angstrompore
本实施例以PaMscS1为例进行氨基酸的检测。谷氨酸(10mM)的检测实验是在300mMNaCl(-cis端)和30mMNaCl(-trans端)、10mM HEPES、pH 7.0的电解质条件下进行的，药物检测的电压为-50mV。谷氨酸的电流轨迹如图30所示。In this embodiment, PaMscS1 is taken as an example to detect amino acids. The detection experiment of glutamic acid (10mM) was carried out under the electrolyte conditions of 300mM NaCl (-cis end) and 30mM NaCl (-trans end), 10mM HEPES, pH 7.0, and the voltage of drug detection was -50mV. The current trajectory of glutamate is shown in Figure 30.
除单个氨基酸外，本发明涉及的埃米孔还可以检测短肽(例如，二肽)。如图31左图所示，将待测氨基酸与天冬氨酸载体脱水缩合，形成二肽，在300mMNaCl(-cis端)和30mMNaCl(-trans端)、10mM HEPES、pH 7.0的电解质条件下检测形成的二肽，并将其产生的电流信号与已测的氨基酸的特定电流信号比较，以判断待测氨基酸的种类。In addition to single amino acids, the Angstrompores contemplated by the present invention can also detect short peptides (eg, dipeptides). As shown in the left figure of Figure 31, the amino acid to be tested is dehydrated and condensed with the aspartic acid carrier to form a dipeptide, which is detected under the electrolyte conditions of 300mM NaCl (-cis end) and 30mM NaCl (-trans end), 10mM HEPES, pH 7.0 The dipeptide is formed, and the current signal generated by it is compared with the specific current signal of the detected amino acid to determine the type of amino acid to be tested.
总结：Summarize:
本发明中，实验人员研究了作为单分子传感的亚纳米通道PaMscS的新类别。野生型和突变型PaMscS埃米孔的结构信息和电生理学检验证实了直接检测dNTPs的能力。根据分析物的尺寸，可以在原位调整埃米孔的选择性以获得最佳的检测效率。与NAAT中增加的合成出的核酸产物的监测对比，PaMscS埃米孔可用于直接测定DNA扩增过程中DNA组分的消 耗，其变化量显著大于DNA产物。根据此策略，检测SARS-CoV-2临床标本，其特异性和敏感性分别为86％和100％。用此策略、通过dNTP条形码探针还同时检测到包括miR21和AFP适配体的两种靶生物标记物，表明PaMscS埃米孔可用于多种生物标记物的编码检测。这种埃米级孔还有能实现对低分子量分子(如氨基酸、药物)的直接检测。In the present invention, experimentalists investigated a new class of subnanochannels PaMscS as single-molecule sensing. Structural information and electrophysiological assays of wild-type and mutant PaMscS angiopores confirmed the ability to directly detect dNTPs. Depending on the size of the analyte, the selectivity of the Angstrom pore can be adjusted in situ for optimal detection efficiency. In contrast to the increased monitoring of synthesized nucleic acid products in NAAT, the PaMscS angstrompore can be used to directly measure the consumption of DNA components during DNA amplification, which varies significantly more than DNA products. According to this strategy, the specificity and sensitivity of detecting SARS-CoV-2 clinical specimens were 86% and 100%, respectively. Using this strategy, two target biomarkers including miR21 and AFP aptamer were also detected simultaneously by dNTP barcoded probes, indicating that the PaMscS pore can be used for the encoded detection of multiple biomarkers. This angstrom-scale pore can also realize the direct detection of low molecular weight molecules (such as amino acids and drugs).
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。Embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementations, and the above-mentioned specific implementations are only illustrative, rather than restrictive, and those of ordinary skill in the art will Under the enlightenment of the present invention, many forms can also be made without departing from the gist of the present invention and the protection scope of the claims, and these all belong to the protection of the present invention.
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Claims (45)

  1. 一种埃米孔***在检测带电荷分子中的应用，其特征在于，所述埃米孔***包含埃米孔、绝缘膜、第一介质和第二介质；所述埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，向所述第一介质和所述第二介质之间施加驱动力后，位于所述第一介质的所述带电荷分子与所述埃米孔相互作用；所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口。An application of an angstrom hole system in detecting charged molecules, characterized in that, the angmi hole system includes an angstrom hole, an insulating film, a first medium and a second medium; the angstrom hole is embedded in the In the insulating film, the insulating film separates the first medium from the second medium, and the Angstrom hole provides a channel connecting the first medium and the second medium, and the first medium is connected to the second medium. After a driving force is applied between the medium and the second medium, the charged molecules located in the first medium interact with the angstrom holes; the angstrom holes are MscS pore holes, and the angstrom holes The pores have a heptamer structure that is radially symmetrical and cylindrical in shape, and the heptamer structure contains 7 side openings and 1 bottom opening.
  2. 如权利要求1所述的应用，其特征在于，所述开口的电荷性质和/或孔径大小是可调节的；所述开口的调节方式可选地包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的物理状态变化，所述机械力刺激可选地包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。The application according to claim 1, characterized in that the charge properties and/or pore size of the opening are adjustable; the adjustment method of the opening optionally includes subjecting the insulating film to mechanical stimulation and/or Or change the physical state of the insulating film, the mechanical stimulation optionally includes the change of the osmotic pressure difference of the medium on both sides of the insulating film, the direct physical stimulation of micro-targeting the insulating film and the negative pressure of the air pressure on the insulating film. One or more of the stimulation of the insulating membrane.
  3. 如权利要求1所述的应用，其特征在于，所述开口的孔径可以根据以下方式来调节：The application according to claim 1, wherein the aperture of the opening can be adjusted in the following manner:
    (1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
    (2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
  4. 如权利要求1所述的应用，其特征在于，所述埃米孔源自杆菌，可选地包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。The application according to claim 1, wherein the emipore is derived from a bacillus, optionally including one of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori or Various.
  5. 如权利要求1所述的应用，其特征在于，所述埃米孔为MscS变体，所述MscS变体可选地包括侧孔体积变体和/或侧孔电荷变体。The application according to claim 1, wherein the Angstrom hole is a MscS variant, and the MscS variant optionally includes a side hole volume variant and/or a side hole charge variant.
  6. 如权利要求1所述的应用，其特征在于，所述带电荷分子包括核苷酸、氨基酸、肽、药物分子中的一种或多种。The application according to claim 1, wherein the charged molecules include one or more of nucleotides, amino acids, peptides, and drug molecules.
  7. 如权利要求1所述的应用，其特征在于，所述埃米孔为PaMscS变体，可选地包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。The application according to claim 1, wherein the angstrompore is a variant of PaMscS, optionally including one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
  8. 一种生物埃米孔***，其特征在于，所述生物埃米孔***包括埃米孔、绝缘膜、第一介质和第二介质，所述埃米孔被嵌入绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道；所述埃米孔为MscS变体埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口，所述埃米孔可选地为MscS的侧孔体积变体和/或侧孔电荷变体。A biological angstrom pore system, characterized in that, the biological angstrom hole system includes an angstrom hole, an insulating film, a first medium and a second medium, the angstrom hole is embedded in the insulating film, and the insulating film The first medium is separated from the second medium, and the angstrom hole provides a channel connecting the first medium and the second medium; the angstrom hole is a MscS variant angstrom hole, The angstrom hole has a heptamer structure that is radially symmetrical and cylindrical in shape, the heptamer structure includes 7 side openings and 1 bottom opening, and the angmi hole is optionally a side hole of MscS Volume variants and/or side hole charge variants.
  9. 如权利要求8所述的生物埃米孔***，其特征在于，所述埃米孔源自杆菌，可选地包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。The biological pore system according to claim 8, wherein the pore is derived from bacilli, optionally including Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori one or more of .
  10. 如权利要求8所述的生物埃米孔***，其特征在于，所述埃米孔为PaMscS变体，可选地包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。The biological angstrompore system according to claim 8, wherein the angstrompore is a PaMscS variant, optionally comprising one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
  11. 如权利要求8所述的生物埃米孔***，其特征在于，所述开口的电荷性质和/或孔径大小是可调节的；所述开口的调节方式可选地包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的物理状态变化，所述机械力刺激可选地包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。The biological angstrom pore system according to claim 8, wherein the charge properties and/or pore size of the opening are adjustable; the adjustment method of the opening optionally includes subjecting the insulating film to mechanical Force stimulation and/or change the physical state of the insulating film, the mechanical force stimulation optionally includes changes in the osmotic pressure difference of the medium on both sides of the insulating film, direct physical stimulation of the micro-targeted insulating film and air pressure One or more of stimulations of the insulating membrane by negative pressure.
  12. 如权利要求8所述的生物埃米孔***，其特征在于，所述开口的孔径可以根据以下方式来调节：The biological Angstrom pore system according to claim 8, wherein the aperture of the opening can be adjusted in the following manner:
    (1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
    (2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
  13. 权利要求8-12任一项所述的生物埃米孔***在检测带电荷分子中的应用，其特征在于，所述带电荷分子包括核苷酸、氨基酸、肽、药物分子中的一种或多种。The application of the biological Angstrom pore system described in any one of claims 8-12 in detecting charged molecules, wherein the charged molecules include one or more of nucleotides, amino acids, peptides, drug molecules Various.
  14. 一种检测样本中核苷酸的方法，其特征在于，包括如下步骤：A method for detecting nucleotides in a sample, comprising the steps of:
    S1将所述样本加入埃米孔***，所述埃米孔***包括：埃米孔、绝缘膜、第一介质、第二介质，其中所述埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧 面开口和1个底部开口；所述样本被加入到所述第一介质；S1 adding the sample to the angstrom hole system, the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the angstrom hole is embedded in the insulating film, the The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, the angstrom hole is a MscS angstrom hole, The Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening; the sample is added to the first medium;
    S2向所述第一介质和所述第二介质施加驱动力，所述样本中的核苷酸与所述埃米孔相互作用并产生电信号；S2 applies a driving force to the first medium and the second medium, and the nucleotides in the sample interact with the angstrom pores and generate electrical signals;
    S3分析所述电信号，进而识别所述样本中的核苷酸。S3 analyzes the electrical signal, and then identifies nucleotides in the sample.
  15. 如权利要求14所述的方法，其特征在于，所述开口的电荷性质和/或孔径大小是可调节的；所述开口的调节方式可选地包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的物理状态变化，所述机械力刺激可选地包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。The method according to claim 14, wherein the charge properties and/or pore size of the opening are adjustable; the adjustment of the opening optionally includes subjecting the insulating film to mechanical stimulation and/or Or change the physical state of the insulating film, the mechanical stimulation optionally includes the change of the osmotic pressure difference of the medium on both sides of the insulating film, the direct physical stimulation of micro-targeting the insulating film and the negative pressure on the insulating film. One or more of the stimulation of the insulating membrane.
  16. 如权利要求14所述的方法，其特征在于，所述开口的孔径可以根据以下方式来调节：The method of claim 14, wherein the aperture of the opening can be adjusted in the following manner:
    (1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
    (2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
  17. 如权利要求16所述的方法，其特征在于，所述第一介质与所述第二介质之间的渗透压差是通过所述第一介质与所述第二介质之间的浓度差来调节的，所述第一介质与所述第二介质之间的浓度差可选地为大约0-270mM。The method of claim 16, wherein the osmotic pressure difference between the first medium and the second medium is adjusted by the concentration difference between the first medium and the second medium Yes, the concentration difference between the first medium and the second medium is optionally about 0-270 mM.
  18. 如权利要求14所述的方法，其特征在于，所述埃米孔为MscS变体埃米孔，所述MscS变体可选地包括侧孔体积变体和/或侧孔电荷变体。The method according to claim 14, wherein the angstrom pore is a MscS variant pore, and the MscS variant optionally includes a side pore volume variant and/or a side pore charge variant.
  19. 如权利要求14所述的方法，其特征在于，所述埃米孔源自杆菌，可选地包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。The method according to claim 14, wherein the emipore is derived from a bacillus, optionally comprising one of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori or Various.
  20. 如权利要求14所述的方法，其特征在于，所述埃米孔为PaMscS变体埃米孔，可选地包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。The method according to claim 14, wherein the angstrompore is a PaMscS variant pore, optionally comprising one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
  21. 如权利要求14所述的方法，其特征在于，所述核苷酸包括dGTP、dATP、dTTP、dCTP、dUTP、GTP、ATP、TTP、CTP、UTP中的一种或多种。The method according to claim 14, wherein the nucleotide comprises one or more of dGTP, dATP, dTTP, dCTP, dUTP, GTP, ATP, TTP, CTP, UTP.
  22. 一种核苷酸快速检测试剂盒，其特征在于，所述试剂盒包括：A nucleotide rapid detection kit, characterized in that the kit comprises:
    (1)MscS埃米孔，所述MscS埃米孔可选地包括MscS的侧孔体积变体和/或侧孔电荷变体；(1) MscS angstrompores, which optionally include side pore volume variants and/or side pore charge variants of MscS;
    (2)绝缘膜，可选地包括磷脂膜和/或高分子膜；(2) insulating film, optionally including phospholipid film and/or polymer film;
    (3)电导液，所述电导液可选地包括氯化钠溶液、氯化锂溶液、氯化铯溶液、氯化钾溶液和溴化钠溶液中的一种或多种。(3) Conductive solution, the conductive solution optionally includes one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution.
  23. 如权利要求22所述的试剂盒，其特征在于，所述埃米孔为PaMscS变体埃米孔，可选地包括130A、130H、180R、271I、130S和130P中的一种或多种。The kit according to claim 22, wherein the angstrompore is a PaMscS variant pore, optionally including one or more of 130A, 130H, 180R, 271I, 130S and 130P.
  24. 一种检测样本中的药物分子的方法，其特征在于，包括如下步骤：A method for detecting drug molecules in a sample, comprising the steps of:
    S1将所述样本加入埃米孔***，所述埃米孔***包括：埃米孔、绝缘膜、第一介质、第二介质，其中所述埃米孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述埃米孔提供连通所述第一介质与所述第二介质的通道，所述埃米孔为MscS埃米孔，所述埃米孔具有径向对称且形状似圆柱体的七聚体结构，所述七聚体结构包含7个侧面开口和1个底部开口；所述样本被加入到所述第一介质；S1 adding the sample to the angstrom hole system, the angstrom hole system includes: a angstrom hole, an insulating film, a first medium, and a second medium, wherein the angstrom hole is embedded in the insulating film, the The insulating film separates the first medium from the second medium, the angstrom hole provides a channel connecting the first medium and the second medium, the angstrom hole is a MscS angstrom hole, The Angstrom hole has a heptamer structure that is radially symmetrical and shaped like a cylinder, and the heptamer structure includes 7 side openings and 1 bottom opening; the sample is added to the first medium;
    S2向所述第一介质和所述第二介质施加驱动力，所述样本中的药物分子与所述埃米孔相互作用并产生电信号；S2 applying a driving force to the first medium and the second medium, and drug molecules in the sample interact with the angstrom pores and generate electrical signals;
    S3分析所述电信号，进而识别所述样本中的药物分子。S3 analyzes the electrical signal, and then identifies drug molecules in the sample.
  25. 如权利要求24所述的方法，其特征在于，所述开口的电荷性质和/或孔径大小是可调节的；所述开口的调节方式可选地包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的物理状态变化，所述机械力刺激可选地包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。The method according to claim 24, wherein the charge properties and/or pore size of the opening are adjustable; the adjustment of the opening optionally includes subjecting the insulating film to mechanical stimulation and/or Or change the physical state of the insulating film, the mechanical stimulation optionally includes the change of the osmotic pressure difference of the medium on both sides of the insulating film, the direct physical stimulation of micro-targeting the insulating film and the negative pressure on the insulating film. One or more of the stimulation of the insulating membrane.
  26. 如权利要求24所述的方法，其特征在于，所述开口的孔径可以根据以下方式来调节：The method of claim 24, wherein the aperture of the opening can be adjusted in the following manner:
    (1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
    (2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
  27. 如权利要求24所述的方法，其特征在于，所述埃米孔为MscS变体埃米孔，所述MscS变体可选地包括侧孔体积变体和/或侧孔电荷变体。The method according to claim 24, wherein the angstrom pore is a MscS variant pore, and the MscS variant optionally includes a side pore volume variant and/or a side pore charge variant.
  28. 如权利要求24所述的方法，其特征在于，所述埃米孔源自杆菌，可选地包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。The method according to claim 24, wherein the emipore is derived from a bacillus, optionally including one of Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori or Various.
  29. 如权利要求24所述的方法，其特征在于，所述埃米孔为PaMscS变体埃米孔，可选地包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。The method according to claim 24, wherein the angstrompore is a PaMscS variant pore, optionally comprising one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
  30. 如权利要求24所述的方法，其特征在于，所述药物分子的分子量为小于1000g/mol，可选地为177.98～712.72g/mol；所述药物分子的浓度可选地为大于10nM。The method according to claim 24, wherein the molecular weight of the drug molecule is less than 1000 g/mol, optionally 177.98-712.72 g/mol; the concentration of the drug molecule is optionally greater than 10 nM.
  31. 如权利要求24所述的方法，其特征在于，所述样本为体液样本，所述体液样本可选地包括尿液、血液、血清、血浆、淋巴液、囊肿液、胸膜液、腹水液、腹膜液、羊水、***、脑脊液、支气管肺泡灌洗液、母乳、泪液、唾液、痰中的一种或多种。The method according to claim 24, wherein the sample is a body fluid sample, and the body fluid sample optionally includes urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascitic fluid, peritoneal One or more of amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, and sputum.
  32. 如权利要求31所述的方法，其特征在于，所述体液样本的样本量可选地为大于10μL；所述体液样本中的药物分子的浓度可选地为大于10nM。The method according to claim 31, wherein the sample volume of the body fluid sample is optionally greater than 10 μL; the concentration of the drug molecule in the body fluid sample is optionally greater than 10 nM.
  33. 如权利要求31所述的方法，其特征在于，所述方法进一步包括S4：将透析装置通过导管与所述第一介质连通，使得所述血液样本通过所述透析装置进入所述埃米孔***，其中S4先于S1。The method according to claim 31, characterized in that, the method further comprises S4: connecting a dialysis device with the first medium through a catheter, so that the blood sample enters the angstrom pore system through the dialysis device , where S4 precedes S1.
  34. 一种检测样本中目标核酸的存在的方法，其特征在于，包括如下步骤：A method for detecting the presence of a target nucleic acid in a sample, comprising the steps of:
    S1将样本置于核酸扩增体系并进行核酸扩增，确定所述核酸扩增体系中底物核苷酸的数量，获得所述样本的核酸扩增产物；S1 placing the sample in a nucleic acid amplification system and performing nucleic acid amplification, determining the number of substrate nucleotides in the nucleic acid amplification system, and obtaining a nucleic acid amplification product of the sample;
    S2将所述样本的核酸扩增产物加入单通道电生理检测***，所述单通道电生理检测***包括：跨膜孔、绝缘膜、第一介质、第二介质，其中所述跨膜孔被嵌入所述绝缘膜中，所述绝缘膜将所述第一介质与所述第二介质分隔开，所述跨膜孔提供连通所述第一介质与所述第二介质的通道，所述样本的核酸扩增产物被加入到所述第一介质；S2 Add the nucleic acid amplification product of the sample into a single-channel electrophysiological detection system, the single-channel electrophysiological detection system includes: a transmembrane pore, an insulating membrane, a first medium, and a second medium, wherein the transmembrane pore is covered Embedded in the insulating film, the insulating film separates the first medium from the second medium, the transmembrane pores provide channels for communicating the first medium and the second medium, the The nucleic acid amplification product of the sample is added to the first medium;
    S3向所述第一介质和所述第二介质之间施加驱动力，所述样本的核酸扩增产物中剩余核苷酸与所述跨膜孔相互作用并产生电信号；S3 applies a driving force between the first medium and the second medium, and the remaining nucleotides in the nucleic acid amplification product of the sample interact with the transmembrane pore and generate an electrical signal;
    S4对所述电信号进行量化，获得所述剩余核苷酸的数量；S4 quantifies the electrical signal to obtain the quantity of the remaining nucleotides;
    S5将所述剩余核苷酸的数量与所述底物核苷酸的数量进行对比，确定所述样本中所述目标核酸是否存在。S5 compares the quantity of the remaining nucleotides with the quantity of the substrate nucleotides to determine whether the target nucleic acid exists in the sample.
  35. 如权利要求34所述的方法，其特征在于，所述跨膜孔为MscS变体埃米孔，所述MscS变体可选地包括侧孔体积变体和/或侧孔电荷变体。The method according to claim 34, wherein the transmembrane pore is a MscS variant angstrom pore, and the MscS variant optionally includes a side pore volume variant and/or a side pore charge variant.
  36. 如权利要求35所述的方法，其特征在于，所述MscS变体埃米孔的开口的电荷性质和/或孔径大小是可调节的；所述开口的调节方式可选地包括使所述绝缘膜受到机械力刺激和/或使所述绝缘膜的物理状态变化，所述机械力刺激可选地包括所述绝缘膜两侧的介质的渗透压差变化、微针对所述绝缘膜的直接物理刺激和气压负压对所述绝缘膜的刺激中的一种或多种。The method of claim 35, wherein the charge properties and/or pore size of the opening of the MscS variant angstrom hole are adjustable; the adjustment of the opening optionally includes making the insulating The membrane is stimulated by mechanical force and/or the physical state of the insulating membrane is changed, and the mechanical stimulus optionally includes the change of the osmotic pressure difference of the medium on both sides of the insulating membrane, the direct physical action of micro-targeting the insulating membrane One or more of stimulation and negative air pressure stimulation of the insulating membrane.
  37. 如权利要求36所述的方法，其特征在于，所述开口的孔径可以根据以下方式来调节：The method of claim 36, wherein the aperture of the opening can be adjusted in the following manner:
    (1)所述第一介质和所述第二介质的种类选择；和/或(1) Type selection of the first medium and the second medium; and/or
    (2)所述第一介质与所述第二介质之间的渗透压差。(2) The osmotic pressure difference between the first medium and the second medium.
  38. 如权利要求37所述的方法，其特征在于，所述第一介质与所述第二介质之间的渗透压差是通过所述第一介质与所述第二介质之间的浓度差来调节的，所述第一介质与所述第二介质之间的浓度差可选地为大约0-270mM。The method of claim 37, wherein the osmotic pressure difference between the first medium and the second medium is adjusted by the concentration difference between the first medium and the second medium Yes, the concentration difference between the first medium and the second medium is optionally about 0-270 mM.
  39. 如权利要求35所述的方法，其特征在于，所述MscS变体埃米孔源自杆菌，可选地包括铜绿假单胞菌、大肠杆菌、腾冲嗜热厌氧菌和幽门螺杆菌中的一种或多种。The method of claim 35, wherein the MscS variant Emmy hole is derived from bacilli, optionally including Pseudomonas aeruginosa, Escherichia coli, Tengchong thermophilic anaerobic bacteria and Helicobacter pylori one or more.
  40. 如权利要求35所述的方法，其特征在于，所述MscS变体埃米孔为PaMscS变体埃米孔，可选地包括以下变体的一种或多种：130A、130H、180R、271I、130S和130P。The method according to claim 35, wherein the MscS variant angstrom is PaMscS variant angstrom, optionally comprising one or more of the following variants: 130A, 130H, 180R, 271I , 130S and 130P.
  41. 如权利要求34所述的方法，其特征在于，所述核苷酸包括核糖核苷酸和/或脱氧核糖核苷 酸，所述核苷酸可选地包括dGTP、dATP、dTTP、dCTP、dUTP、GTP、ATP、TTP、CTP、UTP中的一种或多种。The method of claim 34, wherein said nucleotides comprise ribonucleotides and/or deoxyribonucleotides, said nucleotides optionally comprising dGTP, dATP, dTTP, dCTP, dUTP One or more of , GTP, ATP, TTP, CTP, UTP.
  42. 如权利要求34-41任一项所述的方法，其特征在于，所述核酸扩增体系进一步包括：The method according to any one of claims 34-41, wherein the nucleic acid amplification system further comprises:
    (1)探针，所述探针包括互补区和重复区，所述互补区包括与所述目标核酸互补配对的序列，所述重复区包括重复同一个碱基的寡核苷酸序列，所述碱基包括A、T、C、G、U；或者(1) a probe, the probe includes a complementary region and a repeating region, the complementary region includes a sequence that is complementary to the target nucleic acid, and the repeating region includes an oligonucleotide sequence that repeats the same base, so The bases include A, T, C, G, U; or
    (2)所述目标核酸的特异性引物。(2) Specific primers for the target nucleic acid.
  43. 如权利要求34所述的方法，其特征在于，所述目标核酸为冠状病毒核酸，所述冠状病毒可选地包括SARS-CoV-2、HCoV-229E、HcoV-OC43、HcoV-NL63、HcoV-HKU1、SARS-CoV和MERS-CoV中的一种或多种。The method according to claim 34, wherein the target nucleic acid is a coronavirus nucleic acid, and the coronavirus optionally includes SARS-CoV-2, HCoV-229E, HcoV-OC43, HcoV-NL63, HcoV- One or more of HKU1, SARS-CoV and MERS-CoV.
  44. 一种病毒快速检测试剂盒，包括：A virus rapid detection kit, comprising:
    (1)MscS埃米孔，所述MscS埃米孔可选地包括MscS的侧孔体积变体和/或侧孔电荷变体；(1) MscS angstrompores, which optionally include side pore volume variants and/or side pore charge variants of MscS;
    (2)绝缘膜，可选地包括磷脂膜和/或高分子膜；(2) insulating film, optionally including phospholipid film and/or polymer film;
    (3)电导液，所述电导液可选地包括氯化钠溶液、氯化锂溶液、氯化铯溶液、氯化钾溶液和溴化钠溶液中的一种或多种；(3) Conductive solution, which optionally includes one or more of sodium chloride solution, lithium chloride solution, cesium chloride solution, potassium chloride solution and sodium bromide solution;
    (4)所述病毒的核酸的特异性引物或探针。(4) A specific primer or probe for nucleic acid of the virus.
  45. 如权利要求44所述的试剂盒，其特征在于，所述埃米孔为PaMscS变体埃米孔，可选地包括130A、130H、180R、271I、130S和130P中的一种或多种。The kit according to claim 44, wherein the angstrompore is a PaMscS variant pore, optionally including one or more of 130A, 130H, 180R, 271I, 130S and 130P.
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