CN115725708A - Method for detecting nucleotide by using biological Hermite - Google Patents

Method for detecting nucleotide by using biological Hermite Download PDF

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CN115725708A
CN115725708A CN202210784770.4A CN202210784770A CN115725708A CN 115725708 A CN115725708 A CN 115725708A CN 202210784770 A CN202210784770 A CN 202210784770A CN 115725708 A CN115725708 A CN 115725708A
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pore
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insulating film
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耿佳
包锐
陈慕天
魏于全
赵长健
李开菊
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Sichuan University
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Abstract

The invention belongs to the field of nanopore detection, and particularly relates to a method for detecting nucleotide by using a biological Hermite. The invention provides a method for detecting nucleotide in a sample, which comprises the following steps: s1 adding said sample to a hermite system, said hermite system comprising: the transistor comprises a hermite hole, an insulating film, a first medium and a second medium, wherein the hermite hole is an MscS hermite hole, the hermite hole has a heptamer structure which is radially symmetrical and is shaped like a cylinder, and the heptamer structure comprises 7 side openings and 1 bottom opening; the sample is added to the first medium; s2 applying a driving force to said first medium and said second medium, the nucleotides in said sample interacting with said pores and generating an electrical signal; s3, analyzing the electric signal, and further identifying the nucleotide in the sample.

Description

Method for detecting nucleotide by using biological Hermite
Chinese patent application No. CN2021110062606, entitled "biomicropore system for dNTPs and detection of new corona virus based on PaMscS" filed on 30.08.2021, and chinese patent application No. CN2021110042496, entitled "biomicropore system for small molecule drug detection and whole blood detection based on PaMscS", filed on 30.2021, both priority patent applications are incorporated by reference in their entirety.
Technical Field
The invention belongs to the field of nanopore detection, and particularly relates to a method for detecting nucleotide by using a biological Hermite.
Background
The nanopore single molecule detection technology is a sensing detection technology which integrates the advantages of simple operation, high sensitivity, high detection speed, no need of marking and the like, and is widely applied to the fields of protein detection, gene sequencing, marker detection and the like. At present, the cost, sensitivity and precision of gene detection are the main problems to be solved urgently in the development of the detection technology, so the development of a novel nanopore material is a key means for solving the problems.
A biological nanopore is a naturally occurring nanoscale pore whose pore size is similar to the size of many important biological molecules. Specific blocking currents and translocation events occur when molecules pass through the channels inside the nanopore. Qualitative and quantitative analysis of the target molecule can be achieved according to the blocking current and translocation frequency of the molecule. Therefore, the size of the channel pore is a dominant factor affecting the detection capability and the application range of the nanopore. Some protein nanopores with appropriate channel pore sizes have been used for nanotechnology applications, such as α -hemolysin (α -haemolysin, α -HL), mspA, csgG, aerolysin (Aerolysin), phi29 connectors, and the like. These biological nanopores are derived primarily from bacterial porins or virus phyla and have pore sizes (1.0 nm-3.6 nm) that are approximately single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) in size. Thus, they are suitable for detecting nucleic acids and have been used for DNA/RNA sequencing, nucleic acid biomarker detection, and biomolecular interaction studies. However, local modifications of biological nanopores, such as site-directed mutagenesis or modification of specific adapters, are currently required to adapt to a wider sequencing range according to specific application requirements. Taking alpha-HL as an example, the limited pore diameter is about 1.4nm, so the application range is only limited in the analysis of ssDNA, RNA or other molecules, and the dNMPs can be directly detected by using cyclodextrin (cyclodextrin) modification without fluorescent labeling. However, modification of the pore size of biological nanopores by modification means requires a great deal of bioengineering technical assistance, and furthermore, protein pores are much less flexible in terms of size adjustment than solid state nanopores. In this sense, there is an urgent need to find a nanopore with a flexible structure to efficiently detect molecules of various sizes.
The arrangement of mononucleotide dNTPs as letters of life characters contains a huge amount of information. These versatility of dNTPs not only make them an integral part of genetic material, but also make them possible in terms of DNA probes, species identification by DNA barcodes (DNAbarcoding), and efficient application of DNA computing (DNA computing). For detection of dNTPs, traditional techniques including HPLC, fluorescence detection and qPCR often require fluorescent labels or expensive equipment.
In summary, the present invention provides a method for detecting multiple nucleotides based on a small conductance mechanical force sensitive channel, an ehrlichore, a pore size protein smaller than a nanopore, to ameliorate the deficiencies of the prior art.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting a nucleotide in a sample, and the specific technical scheme is as follows.
A method for detecting a nucleotide in a sample, comprising the steps of:
s1 adding the sample to a rice bore system, the rice bore system comprising: an angstrom pore, an insulating film, a first dielectric, a second dielectric, wherein said angstrom pore is embedded in said insulating film, said insulating film separates said first dielectric from said second dielectric, said angstrom pore provides a channel that connects said first dielectric with said second dielectric, said angstrom pore is an MscS angstrom pore, said angstrom pore has a radially symmetric and cylindrically shaped heptamer structure, said heptamer structure comprises 7 side openings and 1 bottom opening; the sample is added to the first medium;
s2 applying a driving force to said first medium and said second medium, the nucleotides in said sample interacting with said pores and generating an electrical signal;
s3, analyzing the electric signal, and further identifying the nucleotide in the sample.
Further, the charge properties and/or pore size of the openings are adjustable.
Further, the adjustment of the opening includes subjecting the insulating film to a mechanical force and/or changing the physical state of the insulating film.
Further, the mechanical force stimulus comprises one or more of a change in osmotic pressure difference of a medium on both sides of the insulating film, a direct physical stimulus of a microneedle to the insulating film, and a stimulus of a negative pressure of air to the insulating film.
Further, the aperture of the opening may be adjusted according to the following:
(1) Selecting a type of the first medium and the second medium; and/or
(2) A difference in osmotic pressure between the first medium and the second medium.
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.
Further, the difference in concentration between the first medium and the second medium is about 0-270mM.
Further, the hermite pore is an MscS variant hermite pore.
Further, the MscS variants comprise a side-hole volume variant and/or a side-hole charge variant.
Further, the E.oryzae is derived from a Bacillus.
Further, the hermite pores include one or more of pseudomonas aeruginosa, escherichia coli, thermophilic anaerobes tengchongensis, and helicobacter pylori.
Further, the hermite pore is a pamsccs variant hermite pore. The mutation site of the PaMscS variant emmetropore is located at the side opening of the cytoplasmic region of PaMscS.
Further, the PaMscS variant emmer pore includes one or more of 130A, 130H, 180R, 271I, 130S, and 130P.
Further, the nucleotide comprises one or more of dGTP, dATP, dTTP, dCTP, dUTP, GTP, ATP, TTP, CTP, UTP.
Further, the insulating film includes a phospholipid film and/or a polymer film.
Further, the first medium and/or the second medium comprises one or more of a sodium chloride solution, a lithium chloride solution, a cesium chloride solution, a potassium chloride solution, and a sodium bromide solution.
In another aspect, the present invention further provides a kit for rapid detection of nucleotides, wherein the kit comprises:
(1) MscS Ammi pore;
(2) An insulating film;
(3) And (4) electrically conducting liquid.
Further, the MscS emmer pore comprises a side-pore volume variant and/or a side-pore charge variant of MscS.
Further, the insulating film includes a phospholipid film and/or a polymer film.
Further, the electrically conductive liquid includes one or more of a sodium chloride solution, a lithium chloride solution, a cesium chloride solution, a potassium chloride solution, and a sodium bromide solution.
Further, the MscS angstrom pore comprises a PaMscS variant angstrom pore.
Further, the PaMscS variant emmer pore includes one or more of 130A, 130H, 180R, 271I, 130S, and 130P.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for detecting a nucleotide in a sample using an angstrom pore system, wherein the angstrom pore system comprises an MscS angstrom pore. The invention creatively utilizes the characteristics of a small-conductance mechanical force sensitivity channel (MscS) to detect the nucleotides in a sample, and the characteristics are specifically represented as follows:
1) The MscS emmetropore has a narrow pore size. It is estimated that MscS angstrom pores have a pore size in the range of-6-16 angstrom, which is much smaller than that commonly used in the prior art (e.g., alpha-hemolysin nanopores have a pore size of about 1.4-2.4nm, i.e., 14-24 angstrom).
2) The MscS Hermitian pore size is tunable (also can be understood as structurally flexible). The MscS emmetropore can convert a mechanical stimulus to an electrical or biochemical signal within milliseconds, thereby initiating modulation of the tunnel configuration. By utilizing the sensitivity of the MscS angstrom pores to the mechanical force stimulation on the insulating film and/or the change of the physical state of the insulating film, the adjustment of the MscS angstrom pores can be realized by influencing the insulating film without complicated chemical modification. For example, the concentrations 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 across the insulating film and thus the pore size, to achieve optimization of the selectivity for dNTPs and to improve the discrimination of dNTPs. Protein nanopores of the prior art typically have a fixed channel structure, and direct detection of nucleotides (e.g., dntps and the like) typically requires additional protein engineering modifications or introduction of chemical modifications. The MscS angstrom pore diameter related by the invention can realize reversible in-situ adjustment only by changing external conditions, and is suitable for direct monomolecular sensing and identification of nucleotides (also can be understood as direct detection of nucleotides).
3) The methods provided by the invention can directly detect and distinguish one or more nucleotides, and can also be used in combination with other strategies to further detect the presence of a target nucleic acid in a sample. In addition, mutations can be introduced into the side hole of the MscS emmer pore, adjusting the volume (e.g., replacing W with a, S, P) and charge (e.g., replacing W with H, replacing K with R) of the amino acids at the side hole to achieve better detection of specific charged molecules and molecules of specific size.
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 a host organism containing such DNA sequences.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.
FIG. 1 shows electrophysiological testing and dNTP detection based on a PaMscS Ammi well;
FIG. 2 shows the transport frequency of dNTPs through a PaMscS1 pore under different osmotic pressure conditions;
figure 3 shows the detection of AFP aptamers and miR21 by PaMscS1 through dNTPs depletion;
FIG. 4 shows the SDS-PAGE results for PaMscS proteins (including: 1: wild-type PaMscS;2: W130A mutant; 3: K180R mutant; 4: marker);
fig. 5 shows the current signal or current profile for either wild-type or mutant pamsccs;
FIG. 6 shows the current trajectory through a single PaMscS1 angstrom pore at a ramp voltage of 0mV to +100 mV;
fig. 7 shows the transport capacity of different ions through the PaMscS1 pore;
fig. 8 shows the translocation frequency statistics of dNTPs through a PaMscS1 emm pore at different voltages (n = 3);
fig. 9 shows the current trajectory and residence time distribution for PaMscS1 detection of single nucleotides;
figure 10 shows that single-stranded DNA cannot be translocated through the PaMscS1 pore;
FIG. 11 shows the results of native PAGE electrophoresis of PCR reagents mixed with miR21 and AFP aptamers;
FIG. 12 shows dNTP detection based on a wild-type PaMscS emittor pore;
figure 13 shows a single channel embedded current trace for wild-type EcMscS (voltage +100mV, conductivity 30mm;
FIG. 14 shows the channel scan voltage (-100 mV to 100 mV) for wild-type EcMscS;
figure 15 shows conductance profiles of wild-type EcMscS;
figure 16 shows a sequence alignment of PaMscS with MscS of other bacteria.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrases "comprising a component of' 8230; \8230;" does not exclude the presence of another like element in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.
In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "in a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed range. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range of 1 to 6 should be read as having specifically disclosed sub-ranges 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., as well as individual numbers within that range, e.g., 1,2,3,4,5, and 6. The above rules apply regardless of the breadth of the range.
Detailed description of the drawings
FIG. 1: A. electrophysiological measurement chamber schematic. B. Single pore insertions of PaMscS1 and PaMscS2 Am pores at +50mv voltage. I-V relationships for PaMscS1 and PaMscS2 Am pores over a voltage range of-50 mv to +50mv. Conductance profiles for PaMscS s1 and PaMscS2 angstrom wells (N =18, respectively) (buffer conditions-cis: 300mM NaCl, -trans: 30mM NaCl). E. dNTPs were detected by a PaMscS mutant Ammi well (buffer conditions-cis: 300mM NaCl, -trans: 30mM NaCl, and voltage +50 mv).
FIG. 2: the translocation frequencies of dCTP (expressed in orange) and dGTP (expressed in blue) were tested under different osmotic pressure differences, symmetric (a, 300mM NaCl. Four sets of dNTPs concentrations, 0.5mM, 1.0mM, 1.5mM and 2.0mM, were tested for dCTP and dGTP translocation. D. At the symmetrical and low positionsRelationship between translocation frequency and dCTP/dGTP concentration under osmotic pressure and high osmotic pressure conditions (each data point n = 3). E. Under 3 different osmotic pressure conditions, f dCTP And f dGTP The rate of increase of (c).
FIG. 3: A. schematic diagram of detection strategy. B. Current traces for the no-target control group, miR21 group, AFP aptamer group, and both miR21 and AFP aptamer groups. Current distribution for c.4 detection 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 depletion and a relatively lower frequency of dATP translocation; samples with AFP aptamers will result in more dGTP consumption and a relatively lower dGTP translocation frequency; samples with both miR21 and AFP aptamers will result in more dATP and dGTP consumption and relatively lower dATP and dGTP translocation frequencies (buffer conditions-cis end: 300mM NaCl, -trans end: 100mM NaCl, voltage +50mV, n =3 per experiment).
FIG. 5 is a schematic view of: A. background signal frequencies of wild-type PaMscS and mutant PaMscS1, paMscS2, background noise frequencies of PaMscS1 and PaMscS2 are lower than wild-type PaMscS (voltage +50mv, n.gtoreq.3). Time of insertion of PaMscS1 and PaMscS2 angstrom-rice pores, paMscS2 angstrom-rice pores have higher membrane fusion efficiency (n.gtoreq.3) than PaMscS 1. dNTPs for PaMscS s1 and PaMscS2 angstrom wells blocked the current distribution.
FIG. 6: current traces through a single PaMscS1 angstrom pore at a ramp voltage of 0mV to +100 mV: when the voltage was raised above +90mV, voltage gating was observed (buffer conditions-cis: 300mM NaCl, -trans: 30mM NaCl, sampling frequency: 4999 hz).
FIG. 7: the buffering conditions were: 300mM NaCl on the cis side and 30mM NaCl on the trans side, with n.gtoreq.3 for each data point, mean. + -. SD.
FIG. 9: residence time distribution: dGTP (A), dATP (B), dTTP (C) and dCTP (D); the concentration of each nucleotide was 2mM and the buffer conditions were-cis terminal: 300mM NaCl, -trans end: 30mM NaCl, voltage +50mV.
FIG. 10: voltage: +50mV; buffer conditions are as follows: cis-30 mM NaCl, trans-30 mM NaCl. The final concentration of ssDNA was 5. Mu.M, and the sequence was 5'TAGCTTATCAGACTGATGTTGA 3' (SEQ ID NO: 5).
FIG. 11:1: 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 aptamer).
Aminodorum pratense (L.) pers
The emittor pore used in the invention is a small-conductance channel of small conductance product, mscS, preferably PaMscS (Pseudomonas aeruginosa small-conductance mechanical force sensitive channel) or a variant thereof. The variant (which may also be understood as a "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, "pamsccs variant", "mutant pamsccs", "pamsccs mutant" mean the same unless otherwise specified.
In one embodiment of the invention, the emmetropic pore may be an MscS variant. Amino acid substitutions, for example single or multiple amino acid substitutions, may be made to the amino acid sequence of SEQ ID NO 1 or 2 or 3 or 4. Substitutions may be conservative or non-conservative. Preferably, one or more positions of the amino acid sequence of SEQ ID NO 1 or 2 or 3 or 4 are non-conservatively substituted, wherein the amino acid residue to be substituted is replaced by an amino acid of significantly different chemical and/or physical size. Further, the MscS variants can be divided into side hole volume variants and side hole charge variants. A side hole volume variant refers to a variant in which the mutation site is located at the lateral opening (also understood as "side hole") of the cytoplasmic end and the side hole volume is changed by changing the amino acid at that site. The side hole charge variant refers to a variant in which a mutation site is located at the side opening of the cytoplasmic end and the side hole charge thereof is changed by changing the amino acid of the site. For example, the side-hole volume variant can be a substitution of a larger volume of an amino acid (e.g., tryptophan (W)) for a smaller volume of an amino acid (e.g., alanine (a), serine (S), or proline (P)), or vice versa. The side hole charge variant can be obtained by replacing an amino acid with a certain charge with an amino acid with an opposite charge or with a neutral charge, or by replacing an amino acid with a neutral charge. In general, non-limiting examples of positively charged amino acids include histidine, arginine, and lysine; non-limiting examples of negatively charged include aspartic acid and glutamic acid; non-limiting examples of neutrality include glycine, alanine, phenylalanine, valine, leucine, isoleucine, cysteine, asparagine, glutamine, serine, threonine, tyrosine, methionine, proline, and tryptophan. Conservative or non-conservative substitutions of amino acids, as well as many different types of modifications (deletions, substitutions, additions) of amino acids, etc., are well known in the art, and one skilled in the art can modify MscS as appropriate to obtain the corresponding MscS variant. Means for modification include modification of the corresponding DNA sequence (e.g., direct synthesis of the corresponding protein after modification of DNA sequence information or site-directed mutagenesis of the DNA sequence by PCR) to obtain the corresponding variant (and its corresponding DNA sequence).
In a particular embodiment, the MscS variant can be a PaMscS variant. The PaMscS variants include, for example, one or more of 130A, 130H, 180R, 271I, 130S, and 130P. Side-hole volume mutants of PaMscS include, for example, 130A, 130S, 130P, side-hole charge variants of PaMscS include, for example, 130H, 180R, 271I. Such modifications can alter the pore size (also understood as "pore size") of the modified side pore, thereby improving the detection capability for analytes of a particular molecular volume; the local charge characteristics of the modified side-hole channel can also be changed, so that the detection capability of a specific charged analyte is improved; the stability of the protein channel current of the PaMscS variants can also be enhanced.
In one embodiment of the invention, the hermite pore may be a wild-type pamsccs that has a high background noise but still has the ability to detect an analyte.
In one embodiment of the invention, the hermite pore may be a wild-type EcMscS (e.coli small conductance mechanical force sensitive tunnel) or a variant thereof. EcMscS are highly similar in structure to pammscs and are also capable of forming stable tunnel currents with the ability to detect analytes. Sequence similarity of PaMscS to EcMscS was 60%. Conservative substitutions or non-conservative substitutions for amino acids, as well as many different types of modifications (deletions, substitutions, additions) to amino acids, are well known in the art, and one skilled in the art can modify EcMscS to obtain corresponding EcMscS variants, as appropriate.
In another embodiment of the invention, the herminium pores may be derived from other bacilli, such as anaerobe tengcnatus (Thermoanaerobacter tengconsis) and helicobacter pylori (helicobacter pylori), in addition to Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa. PaMscS has the same high similarity with TtMscS and HpMscS, and the sequence similarity is 55% and 44% respectively. In combination with the results of actual electrophysiological measurements of PaMscS and EcMscS, it can be seen that MscS is able to detect analytes as hermite pores because of its highly similar structure and similar function. Conservative or non-conservative substitutions for amino acids, as well as many different types of modifications (deletions, substitutions, additions) to amino acids, are known in the art, and one skilled in the art can modify MscS to obtain corresponding MscS variants, as the case may be.
Analyte
The analyte is a charged species. An analyte is charged if it carries a net charge. The analyte may 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 pore size of the hermite pores, preferably nucleotides, amino acids, peptides, drug molecules.
In one embodiment of the 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 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 base analogs. Sugars include naturally occurring sugars (deoxyribose and ribose) as well as 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, dTDDP, 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, alternatively, can be understood as "single"). Preferably, the nucleotide is ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP.
Angstrom pore system
The "angstrom pore system" includes pores having angstrom-scale dimensions (simply referred to as "angstrom pores"), an insulating film, a first dielectric, and a second dielectric. In one embodiment of the invention, the pores having angstrom-scale dimensions are small conductance mechanical force sensitive channel (MscS) angstrom pores. The pores having angstrom-scale dimensions are preferably heptameric structures with radial symmetry and cylinder-like shapes, comprising 7 side openings and 1 bottom opening. In one embodiment of the invention, the pores with angstrom-scale dimensions have a typical heptameric structure radially symmetric and shaped like a cylinder, comprising 8 openings, with 7 equal openings distributed on the sides, the 8 th opening distributed on the bottom and formed by 7 subunits; the aperture sizes of the 8 openings can be adjusted. The pores having angstrom-scale dimensions allow the analyte to translocate from one side of the insulating film to the other.
In one embodiment of the present invention, the pores with angstrom scale size are embedded in the insulating film, the insulating film (it can also be understood that the composite of the angstrom scale size pores and the insulating film) separates the first medium from the second medium, the pore channel of the angstrom scale size pores provides a channel for communicating the first medium and the second medium; upon application of a driving force between the first medium and the second medium, an analyte located in the first medium interacts with the MscS Eimeria pores to form an electrical current (i.e., an electrical signal). In the present invention, "first medium" refers to the medium in which the analyte is located when added to the hermite system; the "second dielectric" refers to the other side of the "first dielectric" in the two parts of the dielectric separated by the insulating film. In the present invention, the driving force refers to the force that drives the analyte to interact with the pores by means of electrical 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 an electrically conductive liquid. The electric conduction liquid is an alkali metal halide aqueous solution, and specifically is sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl) and sodium bromide (NaCl). In one embodiment of the present invention, the first medium and the second medium contain electrically conductive liquids having different concentrations, in other words, there is a difference in the concentrations of the electrically conductive liquids in the first medium and the second medium, so that there is a difference in osmotic pressure across the insulating film. The first medium and/or the second medium may also include a buffer, such as HEPES. The concentration of the first medium and/or the second medium may range from 30mM to 3M.
The insulating film refers to a film having the ability to carry angstrom pores (or nanopores) and block an ion current passing through non-angstrom pores (or nanopores). The insulating film may include a phospholipid film and/or a polymer film. Exemplary insulating films include DPHPC, DOPC, e.
The present angstrom pore system may comprise any of the small conductance mechanical force sensitive 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 their corresponding variants, with specific sequence information for the four MscS listed in Table 3. For example, the small conductance mechanical force-sensitive channel can be a mutant PaMscS1 (W130A), a mutant PaMscS2 (K180R), a mutant PaMscS3 (V271I) (which has the ability to detect small molecule drugs, experimental data not shown).
In one embodiment of the invention, the angstrom pore system includes two electrolyte chambers separated by an insulating film to form a trans (-trans) compartment and a cis (-cis) compartment, the pores of the angstrom pore being embedded in the insulating film, there being only small conductance mechanical force sensitive channels on the insulating film to communicate the two electrolyte chambers. When an electric potential is applied to the above two electrolyte chambers, electrolyte ions in the solution in the electrolyte chamber move through the angstrom pore by electrophoresis.
In one embodiment of the invention, the small conductance mechanical force sensitive channel (MscS) hermite pores may be embedded in the insulator film, but retain the ability to alter the protein structure in response to mechanical stimuli to which the insulator film is subjected and changes in the physical state of the insulator film. Specifically, the mechanical force stimulation includes osmotic pressure change on both sides of the insulating film, direct physical stimulation of the insulating film by micro-needles, stimulation of the insulating film by air pressure negative pressure, and the like. The physical change of the insulating film includes a change in thickness of the insulating film, a change in composition of the insulating film, and a change in curvature of a surface of the insulating film. The altering the protein structure comprises altering a charge property and/or pore size of the opening of the MscS. Further, the charge properties and/or pore size of the openings altered by the MscS-angstrom pores can be used to detect different analytes. The adjustable range of the aperture of the hermite hole related by the invention can be 5-15 hermite.
Interaction between the ehmitic pore and an analyte
The analyte may be in contact with the hermite hole 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 hermite to the other side of the insulating film. In this case, the analyte interacts with the angstrom pore as it passes through the insulating film via the passage of the pore. Alternatively, the analyte may be in contact with a side of the insulating film, which may allow the analyte to interact with the angstrom pore, causing it to separate from the angstrom pore and stay on the same side of the insulating film. The analyte may interact with the pores in any manner and at any site. The analyte may also impinge on the angstrom pore, interacting with the angstrom pore, causing it to separate from the angstrom pore and reside on the same side of the insulating film.
During the interaction of the analyte with the angstrom pore, the analyte affects the current flowing through the angstrom pore in a manner specific to the analyte, i.e., the current flowing through the angstrom pore 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 hermite pores, and then to identify the particular analyte in the sample or to determine whether the particular analyte is present in the sample. More specifically, the presence, absence, concentration, or the like of an analyte can be identified based on a comparison of a current pattern obtained by detecting the analyte with a known current pattern obtained using a known analyte under the same conditions.
The angstrom well system of this invention may also include one or more measuring devices, such as patch clamp amplifiers or data acquisition equipment, that measure the current flowing through the angstrom well.
Sample(s)
The analyte may be present in any suitable sample. The invention is typically performed on samples known to contain, or suspected of containing, the analyte. The invention may be performed on samples containing one or more species of unknown analyte. Alternatively, the invention may identify the one or more species of analyte known to be present or expected to be present in the sample.
The sample may be a biological sample. The invention may be carried out in vitro on a sample obtained or extracted from any organism or microorganism. The invention may also be carried out in vitro on a sample obtained or extracted from any virus. Preferably, the sample is a fluid sample. The sample typically comprises a bodily fluid. The sample may be a bodily fluid sample, such as urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, sputum, or a combination thereof. The sample may be derived from a human or other mammal. The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample, such as drinking water, sea water, river water, and reagents for laboratory testing.
The sample may be untreated prior to analysis, for example by detecting the analyte directly in whole blood. The sample may also be processed prior to analysis, for example by centrifugation, filtration, dilution, precipitation or other physical or chemical means known in the art.
In one embodiment of the invention, the sample is a nucleic acid amplification product.
Method for detecting the presence of nucleic acids in a sample
The invention also provides a method of detecting the presence of a nucleic acid in a sample. The method comprises the following steps: s1, placing a sample in a nucleic acid amplification system, carrying out 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, adding a nucleic acid amplification product of the sample to an E.coli well system comprising: an emittor pore, an insulating film, a first medium, a second medium, wherein said protein emittor pore is embedded in said insulating film, said insulating film separates said first medium from said second medium, said emittor pore provides a passageway that communicates said first medium with said second medium, said emittor pore is an MscS emittor pore having a radially symmetric and cylindrically shaped heptameric structure comprising 7 side openings and 1 bottom opening, said nucleic acid amplification product of said sample being added to said first medium; s3 applying a driving force between said first medium and said second medium, remaining nucleotides in nucleic acid amplification products of said sample interacting with said pores and generating an electrical signal (current); s4, quantifying the electric signal to obtain the number of the residual nucleotides; s5, comparing the number of the residual nucleotides with the number of the substrate nucleotides to determine whether the target nucleic acid exists in the sample. In some embodiments, S1 may be performed simultaneously with S2 or in the same system. Before detection, a threshold value may also be set, e.g., the presence of target nucleic acid in the sample is considered to be present only if the amount of at least one of the remaining nucleotides is below the threshold value; alternatively, only if the number of remaining nucleotides of all species is above the threshold is the target nucleic acid considered to be absent from the sample.
The "depletion strategy" of the present invention is applicable to a wide variety of transmembrane pores in nature and is not limited to the angstrom pores of the present invention. One skilled in the art will appreciate that other transmembrane pores capable of detecting (and distinguishing) different nucleotides may be selected to detect the presence or absence of a target nucleic acid based on an understanding of the present invention.
"nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form. "nucleic acid amplification" of a target nucleic acid refers to a process of constructing in vitro a nucleic acid strand that is identical or complementary to at least a portion of the target nucleic acid sequence, the nucleic acid amplification process only being possible when the target nucleic acid is present in a sample. In nucleic acid amplification processes, enzymes (e.g., nucleic acid polymerases, transcriptases) are typically utilized to generate multiple copies of a target nucleic acid or fragment thereof, or multiple copies of a sequence complementary to the target nucleic acid or fragment thereof. After the nucleic acid amplification process occurs, the number of nucleotides in the substrate of the nucleic acid amplification system decreases correspondingly with the increase of the copy number. For example, FIG. 3A shows one type of nucleic acid amplification system.
The principle of the method is based on the in vitro nucleic acid amplification technology to consume the substrate nucleotides in the nucleic acid amplification system, so that common in vitro nucleic acid amplification technologies, such as Polymerase Chain Reaction (PCR), ligase Chain Reaction (LCR), strand displacement amplification technology (SDA), transcription-mediated amplification Technology (TMA) and loop-mediated isothermal amplification technology (LAMP), can be used with the method provided by the invention.
In one embodiment, the methods provided herein allow for the detection of the presence of a novel coronavirus nucleic acid in a sample.
In one embodiment, the methods provided herein can detect the presence or absence of single-stranded nucleic acid in a sample.
In one embodiment of the invention, the invention provides for nucleic acid amplification of the new coronavirus nucleic acid under suitable conditions, consuming the substrates dNTPs and generating multiple copies of the nucleic acid, by constructing suitable primers (e.g., primers specific for the SARS-CoV-2 nucleic acid) and introducing the primers into a nucleic acid amplification system (including the substrates dNTPs, polymerase, reverse transcriptase) if present in the sample. After the nucleic acid amplification product of the sample is added to the hermite system provided by the present invention, macromolecular substances (e.g., enzymes, polynucleotides, etc.) in the nucleic acid amplification system cannot pass through the hermite, that is, only free mononucleotides in the nucleic acid amplification system can pass through the hermite and generate specific current, and then the number of remaining nucleotides is determined to determine whether the target neocoronavirus nucleic acid exists in the sample (i.e., if no target neocoronavirus nucleic acid exists in the sample, the number of remaining nucleotides is closer to the number of substrate nucleotides before nucleic acid amplification; if the neocoronavirus nucleic acid exists in the sample, the number of remaining nucleotides is significantly lower than the number of substrate nucleotides before nucleic acid amplification, and more specifically, at least one of the substrate nucleotides may be completely consumed).
In one embodiment of the invention, the invention is performed by constructing a suitable probe (e.g., the probe comprises a sequence that complementarily pairs with a target nucleic acid sequence and a polynucleotide sequence) and introducing the probe into a nucleic acid amplification system (including substrates dNTPs, a polymerase), and if the target nucleic acid sequence is present in the sample, nucleic acid amplifying the target nucleic acid sequence under suitable conditions, consuming the substrates dNTPs and generating multiple copies of the target nucleic acid sequence; more specifically, since the probe also has a polynucleotide sequence (e.g., poly T, polyA, poly C, poly G), the presence or absence of the target nucleic acid sequence in the sample can be determined from the specific consumption of a certain substrate nucleotide because the substrate nucleotide corresponding to the polynucleotide sequence is consumed in a large amount after the target nucleic acid sequence is bound to the probe. After the nucleic acid amplification product of the sample is added to the hermite system provided by the present invention, macromolecular substances (e.g., enzymes, polynucleotides, etc.) in the nucleic acid amplification system cannot pass through the hermite, that is, only free mononucleotides in the nucleic acid amplification system can pass through the hermite and generate specific current, so as to determine the number of remaining nucleotides, so as to determine whether a target nucleic acid sequence exists in the sample (i.e., if no target nucleic acid sequence exists in the sample, the number of remaining nucleotides is closer to the number of substrate nucleotides before nucleic acid amplification, and if a target nucleic acid sequence exists in the sample, the number of remaining nucleotides is significantly lower than the number of substrate nucleotides before nucleic acid amplification and the corresponding polynucleotides in the probe are greatly consumed).
Specific examples are as follows:
example one
The material and the method are as follows:
sodium chloride (NaCl, >99.0%, CAS # 7647-14-5), dNTP mix (> 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 (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), phenylmethylsulfonylfluoride (phenylmethylsulfonylfluoroxyl, PMSF) (≧ 99%, CAS # 329-98-6), 4- (2-Hydroxyethyl) piperazine-1- (2-ethylsulfonic acid) 4- (2-ethylsulfonic acid), sigma # 739-99%, sigma # 9-99-9-4, sigma # 9-4), and isopropyl-D-thiogalactoside (IPTG) (≧ 99%, CAS # 329-93-1). Coli extracted phospholipids were purchased from Avanti. PrimeSTAR HS DNA polymerase was purchased from TaKaRa. pUC57 vector plasmids, DNA templates, miRNA-21, AFP aptamers were synthesized by Sangon Biotech, and the sequence information is listed in Table 1.
TABLE 1 sequences of miR21 and AFP aptamers
Figure BDA0003720073860000111
Expression and purification of wild-type and mutant PaMscS:
the gene for PaMscS from Pseudomonas aeruginosa genomic DNA was amplified by Polymerase Chain Reaction (PCR) using gene-specific primers. The gene was inserted into a plasmid using the Clonexpress II One Step Cloning Kit (Vazyme). Coli BL21 (DE 3) cells containing a plasmid of PaMscS gene were cultured at 37 ℃ in Luria-Bertani (LB) medium in the presence of 50. Mu.g/mL ampicillin and purified by expression. The peak was determined by SDS-PAGE analysis. In particular, the expression and purification steps of the wild-type protein and the mutant protein are the same in the invention, but the wild-type protein and the mutant protein have sequence difference, so that the wild-type protein and the mutant protein have difference in plasmid synthesis stage.
The experimental team of the invention reveals the structure of the protein Hermite pore through modeling, and the protein Hermite pore is a typical heptamer which is radially symmetrical and similar to a cylinder. It contains 8 openings, 7 on the side and 1 on the bottom. The N-terminal residues 1-13 are too flexible to be resolved in the model. Topologically, paMscS can be divided into 2 parts, a transmembrane region and a large cytoplasmic part. Each monomer produces three N-terminal transmembrane helices, including TM1 (residues 17-52), TM2 (residues 58-83) and TM3 (residues 90-122). The C-terminal cytoplasmic domain can be divided into an intermediate beta domain and a COOH terminal domain. The TM1 and TM2 in each subunit are aligned together in an anti-parallel orientation, with TM1 passing through the bilayer membrane outside the channel and TM2 forming the central layer, thereby forming a permeation pathway around the channel axis. The TM3 helix can be described as two helix segments, TM3a and TM3b, separated by a distinct kink (kink) at Gly108 to 53 °, which are residues conserved in homology. TM3a passes through the membrane with different deflections like TM1, while TM3b returns to the cytoplasm and interacts with the cytoplasmic region. In addition, 7 subunits form a E-radius
Figure BDA0003720073860000118
Which senses tension and is associated with a conformational change. Comparing the MscS structures of the pseudomonas aeruginosa and the Escherichia coli, the inclination angle of the TM1 and the TM2 of the pseudomonas aeruginosa and the Escherichia coli is smaller than that of the MscS structures of the Escherichia coliThis results in a large deflection of the TM region, especially the loop between TM1 and TM 2. In the cytoplasmic region, the intermediate beta domain (residues 123-172) contains 5 beta strands, which are tightly linked to the beta strands of other different subunits. And the C-terminal domain (residues 177-273) consists of 5 beta strands and 2 alpha helices, which are mixed structures. Between these two domains of adjacent monomers, there were 7 equal openings on the sides, clearly visible, with a radius of about
Figure BDA0003720073860000112
It is proposed to be the cause of ion permeation in EcMscS. In addition to these entries, the 8 th opening is present in the bottom of the protein, expressed by 7 beta strands with a minimum radius of E.E.E.E.
Figure BDA0003720073860000113
In all sizes, extend to &
Figure BDA0003720073860000114
Is parallel to the seven-fold axis and has a width in the vertical direction of
Figure BDA0003720073860000115
The structure of PaMscS is similar to EcMscS in the off state (PDB: 2 OAU), with over 101 rmsd of the TM domain
Figure BDA0003720073860000116
C of (A) α Atomic, but in the open state (PDB: 2VV 5), there is a large difference in the TM region, rmsd is
Figure BDA0003720073860000117
These results indicate that the conformation of PaMscS in structure reflects the off state. Overall, the unique and fine structure of PaMscS hermite pores shows great potential for detection.
Site mutation:
modifying the DNA sequence corresponding to the corresponding amino acid site (including direct synthesis or PCR mutation after DNA sequence modification), and modifying the DNA sequence of the target mutation site into the DNA sequence corresponding to the mutant protein. Mutant proteins of mutant PaMscS proteins emmer pores may include 130A, 130H, 180R, 271I, 130S, or 130P.
Merging of membranes and single-channel recording:
the experiment was performed in a vertical cuvette supplied by the Warner instrument (Warner Instrument) and all traces of current were recorded by a HEKAEPC 10USB patch clamp amplifier with a sampling frequency of 9900Hz, if not otherwise specified. mu.L of a 25mg/mL E.coli membrane was precoated on the 150 μm orifice of the cup, and then 1mL of an electrolyte solution (-trans end: 100mM NaCl,10mM HEPES, pH7.0; cis end: 300mM NaCl,10mM HEPES, pH 7.0) was added to each side of the sample cell. Approximately 2/3 of the electrolyte solution in the cell was then aspirated from the-cis end using a 1mL pipette. When the average current approaches 0pA, the electrolyte solution is driven to the-cis side of the sample cell to form a planar phospholipid bilayer membrane. After phospholipid membrane formation, a solution of PaMscS1 protein was added to the-cis terminus. When PaMscS1 was embedded in planar phospholipid bilayer membranes, there was a significant change in current. After the protein was inserted, 1mL of the solution was replaced, and then the subsequent experiment was performed.
Detecting single nucleotide:
different sets of amplification products were detected by MscS Ammi well. Different samples were added to the-cis end, recorded at +50mV and observed for 20 min. When a stable PaMscS1 angstrom pore is formed on the planar phospholipid membrane, the mononucleotide to be detected is added to the-cis end of the sample pore, then a voltage is applied and a current signal is recorded.
Example two
Electrophysiological detection and dNTPs detection based on PaMscS ehmits pores:
the basic function of MscS is a fast on/off switch in response to mechanical stimuli, such as changes in membrane tension during osmotic pressure. The cytoplasmic domain of MscS functions as a molecular sieve that balances osmotic agent loss during osmoadaptation (osmoadaptation). In the protein of the present invention, in the hermite pore, 7 side pores from the cytoplasmic region play a key role in the translocation (translocation) of ions and solutes. Therefore, the side-hole mutants pamsccs 1 (W130A) and pamsccs 2 (K180R) were selected for subsequent studies due to their low background noise (fig. 4, fig. 5A-C). In electrophysiological experiments, purified proteins were added to the-cis end of the electrophysiological device (FIG. 1A). When the PaMscS mutant channel is embedded in a bilayer lipid membrane (BLM, a kind of insulating membrane), stable channel current hopping can be observed at a voltage of +50mV (fig. 1B). At voltages ranging from-50 mV to +50mV, the channel conductance of PaMscS1 remained stable (FIG. 1C), and when the voltage was higher than +90mV, the gating probability of PaMscS1 increased (FIG. 6). The conductance of the PaMscS1 angstrom wells was 0.64 ± 0.02nS (n =91, gaussian-fit peak ± SD, -cis end: 300mM NaCl, -trans end: 30mM NaCl), and the conductance profile of the PaMscS2 angstrom wells was 34.9 ± 7.0pA (mean ± SD from 18 independent insertion events) (fig. 1D). Ion transport results for PaMscS1 indicated that PaMscS1 had better selectivity for Br "(fig. 7).
In the case of insertion of a PaMscS1 Am pore in BLM and the presence of dNTPs in the-cis terminus, translocation signals of the dNTPs can be observed at positive voltages. The translocation frequency of dNTPs increased with increasing voltage (figure 8). Both PaMscS1 and PaMscS2 were analyzed for dNTP mix detection (dNTPs concentration: 0.2mM, voltage: +50mV, -cis end: 300mM NaCl, -trans end: 30mM NaCl). A significant difference in the blocking current distribution can be observed between the two mutants, indicating that translocation of dNTPs is associated with the side hole of the PaMscS hermite pore (fig. 1E). That is, under the same detection conditions, the PaMscS1 and PaMscS2 angstrom pores appear to be different for dNTPs blocking current distribution. In particular, the PaMscS1 angstrom pore exhibited 3 peaks for the four dNTPs cocktail blocking rates, while the PaMscS2 angstrom pore exhibited 2 peaks for the four dNTPs cocktail blocking rates. Since the difference between the pamsccs 1 and pamsccs 2 mutations is in the side hole amino acid difference, it is presumed that the detection signal of dNTPs correlates with the side hole. Since PaMscS1 has a better discriminating effect on dNTPs mixtures, it is more suitable for discriminating dNTPs mixtures. Whereas for pamsccs 2 it shows more stable channel conductance and relatively higher membrane fusion efficiency, and thus it is more suitable for subsequent rapid diagnosis (fig. 5A-C). The wild-type PaMscS emmer pore exhibited 2 peaks for the four dNTPs cocktail blocking rates (fig. 12). The current trajectory and residence time distribution of pamsccs 1 for single nucleotide detection are shown in fig. 9.
Compared with the currently reported composition containing am7 beta CD and MoS 2 Compared to the nanoporous α -hemolysin of α -Hederin, although the detection accuracy of the PaMscS s1 angstrom pore was lower than the reported optimal biological nanopore containing am7 β CD conjugate (i.e., constructed using an α -hemolysin mutein and a 6-amino-6 deoxy- β -cyclodextrin aptamer), the translocation speed was comparable to the solid state nanopore. In a single stranded DNA (ssDNA) detection experiment, 50. Mu.M ssDNA was detected in a buffer of 30mM NaCl/300mM NaCl at a bias voltage of +50mV, whereas no translocation event was observed due to its narrow channel size (FIG. 10). Thus, the PaMscS emmer pore has the potential to be a useful small molecule sensor.
EXAMPLE III
Selective adjustment of PaMscS1 angstrom wells was used for optimized dNTPs detection:
given the mechanical force sensitivity of the PaMscS1 angstrom pores, the experimenter adjusted the selectivity of the PaMscS1 angstrom pores by applying different osmotic pressure differentials. To maintain constant charge characteristics of dCTP and dGTP at different osmotic pressure differences, the experimental personnel maintained the conductivity buffer concentration at the-cis end at 300mM and varied the conductivity buffer concentration at the-trans end to vary the osmotic pressure difference. The detection capacity of PaMscS1 angstrom pores for large and small molecule dGTP and dCTP was tested under 3 conditions of differential osmotic pressure, including symmetric (symmetric) conditions (FIG. 2A,300mM NaCl/300mM NaCl, +50mV bias), low differential osmotic pressure conditions (FIG. 2B,100mM NaCl/300mM NaCl, +50mV bias) and high differential osmotic pressure conditions (FIG. 2C,30mM NaCl/300mM NaCl, +50mV bias). Under the condition of symmetric osmotic pressure, the translocation frequency of dCTP is from 0.16 +/-0.03 s -1 Increase to 0.22 + -0.07 s -1 And dGTP is from 0.09 + -0.02 s -1 Change to 0.07. + -. 0.003s -1 . The translocation frequency of dCTP was from 0.34. + -. 0.1s under conditions of low differential permeability (LOD) -1 Increase to 0.67 + -0.14 s -1 And dGTP is from 0.06 + -0.01 s -1 Increased to 0.3 + -0.04 s -1 . Under high osmotic pressure (HOD) conditions, the translocation frequency of dCTP is from 0.12 + -0.04 s -1 Increase to 0.22 + -0.07 s -1 And dGTP is from 0.37 +/-0.08 s -1 Increased to 1.12 + -0.12 s -1 (fig. 2D, n =3, mean ± s.e.m for each experiment). Figure 2E summarizes the detection of dGTP and dCTP, and it is summarized that low differential osmotic pressure conditions show the highest increase in translocation events for dCTP, while high differential osmotic pressure conditions show the highest increase in translocation events for dGTP (figure 2E). The low osmotic pressure conditions show a balanced capture capacity for both dCTP and dGTP compared to the reduced capture efficiency of dCTP under high osmotic pressure conditions. Therefore, different dNTPs can be captured more accurately by adjusting different osmotic pressure differences.
In summary, given that the size and charge characteristics of dGTP and dCTP remain constant under given experimental conditions, and that the channel size of the MscS family (e.g., ecMscS, hpMscS, atMsL1 proteins, etc.) can vary under different pressure, osmolarity conditions, or membrane potential, the experimenter can conclude that: the differences in selectivity of the PaMscS 1-angstrom pore for dNTPs are caused by changes in the size of the tunnel under different osmotic pressure conditions.
Example four:
detection of various biomarkers through PaMscS1 angstrom pores:
experimenters designed a strategy for discriminating dNTP barcode probes and verified the application of the dNTP barcode probes in the detection of various biomarkers through a PaMscS1 angstrom pore. The specific operation is as follows: two probes were designed to bind to the target DNA sequence and cause specific dNTPs depletion in the chain polymerization reaction, the principle of which is: the probe A and the probe B have different base distributions, when amplification occurs, the amplification of the probe A or the probe B can cause a large amount of base-rich consumption, and the information of which probe is obtained by judging the main consumption type of the base. And (3) probe A: the miR21 probe: comprises a sequence which is complementarily paired with miR21 and a bar code sequence of polyT; and a probe B: AFP aptamer probe: comprising a sequence complementary paired to an AFP aptamer and a barcode sequence of poly C. When either probe A or probe B is present in the sample, the polymerase chain reaction can be activated, consuming either dATP or dGTP in the reaction system (FIG. 3A). The results of native PAGE electrophoresis of miR21 and AFP aptamers are shown in FIG. 11.
Detection of control sample without target sequence, with miSamples of R21, samples with AFP aptamers, and samples with both miR21 and AFP aptamers. The corresponding current traces and blocking current distributions are shown in fig. 3B and 3C. It can be observed that in the control group (sample without miR21 and AFP aptamer), dATP (. Delta.f) dATP ＝f dATP /f Background ) Relative increase in frequency of translocation events of (d) 1.7. + -. 0.2 (mean. + -. S.E.M), dGTP (δ f) dGTP ＝f dGTP /f Background ) The relative increase in frequency of translocation events of (a) is 1.8 ± 0.03. In the miR 21-containing sample,. Delta.f dATP Significantly lower than control (1.4 + -0.1, mean + -S.E.M) in samples with AFP aptamer,. Delta.f dGTP Down to 1.0 ± 0.3 (mean ± s.e.m). For samples with both miR21 and AFP aptamer, δ f compared to control group dATP (1.1. + -. 0.2, mean. + -. S.E.M) and. Delta.f dGTP (1.3. + -. 0.2, mean. + -. S.E.M) decreased (FIG. 3D). These results indicate that by designing single or multiple specific probes, paMscS1 can detect single or multiple biomarkers simultaneously (n.gtoreq.3 per experiment).
Example five:
a strategy to enhance a signal:
A. the detection limit of the substance to be detected is enhanced through ion regulation, particularly for dNTPs, the detection limit of the dNTPs can be improved by adding divalent cations such as nickel ions and cobalt ions into the cytoplasmic end of the Hermite pore related by the invention; B. the signal to noise ratio is enhanced by adjusting the osmotic difference of the electric conduction liquid, particularly, the distinguishing capability of the medicine molecules with similar structures can be improved by improving the concentration of the electric conduction liquid, and the detection effect of dGTP can be improved by improving the osmotic difference of the electric conduction liquid on two sides of the phospholipid membrane.
Example six:
electrophysiological detection based on ecMscS-E-Mie pores
When the wild-type EcMscS channel is embedded in a Bilayer Lipid Membrane (BLM), stable channel current hopping can be observed at a voltage of +100mV (fig. 13). The wild-type EcMscS channel current remained stable at voltages ranging from-100 mV to +100mV (figure 14). The conductance of the wild-type EcMscS emittor was 0.334. + -. 0.028nS (-cis end: 300mM NaCl, -trans end: 30mM NaCl) (FIG. 15). Fig. 16a-c show the structures of EcMscS, ttMscS and HpMscS, respectively, which are highly similar to the structures of pammscs, i.e. heptameric structures that are both radially symmetric and shaped like cylinders. In addition, fig. 16a-c and fig. 16d further compare the sequences of PaMscS with EcMscS, ttMscS, and HpMscS (sequence information see table 3 below), and show that EcMscS, ttMscS, and HpMscS have some homology to pammscs, but that homology is not highly homologous. EcMscS and pammscs are only 60% similar, but both have the ability to detect analytes. Thus, it will be appreciated by those skilled in the art that the key to determining the ability of a bacterial MscS to serve as an emittor-pore analyte is its radially symmetric, cylindrically shaped heptamer structure and tunnel pore size, not just homology.
Table 3: amino acid sequence information for four MscS
Figure BDA0003720073860000141
Figure BDA0003720073860000151
While the present invention has been described with reference to the particular illustrative embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but is intended to cover various modifications, equivalent arrangements, and equivalents thereof, which may be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (10)

1. A method for detecting a nucleotide in a sample, comprising the steps of:
s1 adding said sample to a hermite system, said hermite system comprising: an angstrom pore, an insulating film, a first dielectric, a second dielectric, wherein said angstrom pore is embedded in said insulating film, said insulating film separates said first dielectric from said second dielectric, said angstrom pore provides a channel that connects said first dielectric with said second dielectric, said angstrom pore is an MscS angstrom pore, said angstrom pore has a radially symmetric and cylindrically shaped heptamer structure, said heptamer structure comprises 7 side openings and 1 bottom opening; the sample is added to the first medium;
s2 applying a driving force to said first medium and said second medium, the nucleotides in said sample interacting with said pores and generating an electrical signal;
s3, analyzing the electric signal, and further identifying the nucleotide in the sample.
2. The method of claim 1, wherein the charge properties and/or pore size of the openings are adjustable; the manner of adjustment of the opening optionally comprises subjecting the insulating film to a mechanical force stimulus, optionally comprising one or more of a change in the osmotic pressure difference of the medium across the insulating film, a direct physical stimulus of the insulating film by micro-needles and a stimulus of the insulating film by negative pressure of air pressure, and/or a change in the physical state of the insulating film.
3. The method of claim 1, wherein the aperture of the opening is adjustable according to:
(1) Selecting a type of the first medium and the second medium; and/or
(2) A difference in osmotic pressure between the first medium and the second medium.
4. The method of claim 3, wherein the osmotic pressure difference between the first medium and the second medium is regulated by a concentration difference between the first medium and the second medium, optionally between about 0-270mM.
5. The method of claim 1, wherein the emmetrore is an MscS variant emmetrore, and the MscS variant optionally comprises a side hole volume variant and/or a side hole charge variant.
6. The method of claim 1, wherein the pores of the Eimeria are derived from bacilli, optionally including one or more of Pseudomonas aeruginosa, escherichia coli, thermoanaerobacter tengciensis, and helicobacter pylori.
7. The method of claim 1, wherein the angstrom pore is a PaMscS variant angstrom pore, optionally including one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
8. The method of claim 1, wherein the nucleotides comprise one or more of dGTP, dATP, dTTP, dCTP, dUTP, GTP, ATP, TTP, CTP, UTP.
9. A kit for rapidly detecting nucleotides, which is characterized by comprising:
(1) An MscS angstrom pore, optionally comprising a side pore volume variant and/or a side pore charge variant of MscS;
(2) An insulating film, optionally comprising a phospholipid film and/or a polymeric film;
(3) An electrically conductive fluid, optionally comprising one or more of a sodium chloride solution, a lithium chloride solution, a cesium chloride solution, a potassium chloride solution, and a sodium bromide solution.
10. The kit of claim 9, wherein the angstrom pore is a pamsccs variant angstrom pore, optionally including one or more of 130A, 130H, 180R, 271I, 130S, and 130P.
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