WO2023123292A1 - Preparation method for nanopore biosensor and sequencing method - Google Patents

Preparation method for nanopore biosensor and sequencing method Download PDF

Info

Publication number
WO2023123292A1
WO2023123292A1 PCT/CN2021/143407 CN2021143407W WO2023123292A1 WO 2023123292 A1 WO2023123292 A1 WO 2023123292A1 CN 2021143407 W CN2021143407 W CN 2021143407W WO 2023123292 A1 WO2023123292 A1 WO 2023123292A1
Authority
WO
WIPO (PCT)
Prior art keywords
voltage
membrane
nanopore
preparation
protein
Prior art date
Application number
PCT/CN2021/143407
Other languages
French (fr)
Chinese (zh)
Inventor
王佩如
董宇亮
郑荣荣
季州翔
黎宇翔
曾涛
章文蔚
徐讯
Original Assignee
深圳华大生命科学研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳华大生命科学研究院 filed Critical 深圳华大生命科学研究院
Priority to PCT/CN2021/143407 priority Critical patent/WO2023123292A1/en
Publication of WO2023123292A1 publication Critical patent/WO2023123292A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention relates to the field of nanopore sensor preparation, in particular to a preparation method and a sequencing method of a nanopore biosensor.
  • Nanopore detection technology originated from the invention of the Coulter counter and the recording technology of single-channel current. Neher and Sakamann used the patch clamp technique to measure membrane potential in 1976 to study protein channels. In 1996, Kasianowicz et al. proposed the use of ⁇ -hemolysin ( ⁇ -hemolysin) to detect DNA, which opened a new field of single-molecule nanopore detection.
  • ⁇ -hemolysin ⁇ -hemolysin
  • Nanopore sequencing also known as single-molecule sequencing technology, can sequence nucleic acids without polymerase amplification; it has the characteristics of long read length, fast speed, high throughput, and low cost.
  • the commonly used nanopores in the world can be divided into solid pores and biological pores; biological pores are nanochannel proteins, which are mainly composed of membrane proteins or viral motor proteins.
  • biological pores can be prepared in large quantities through protein expression and purification, and have good uniformity; based on protein structure, mutations can be carried out purposefully, so as to realize the detection of different analytes.
  • the principle of biological nanopore detection a voltage is applied to both ends of the nanochannel protein inserted into the membrane layer, driven by an electric field, so that the molecules of the analyte are electrophoresed from one end of the hole through the nanopore, and the ion current collected by the external circuit will change, and the passing current will change.
  • the size, time and other characteristics of the test object can be realized.
  • nanochannel proteins are inserted into membrane materials under the action of a constant voltage to form nanopore biosensors.
  • the existing method for preparing nanopore biosensors includes: after forming a phospholipid membrane at the central hole of the electrolytic cell, adding the prepared nanochannel protein to one side of the membrane, and applying an appropriate constant voltage to promote the fusion of the channel protein and the phospholipid bilayer membrane To form a nanopore channel, use a patch clamp amplifier or other electrical signal amplifiers to collect current signals, and judge whether the porin is successfully inserted into the phospholipid membrane by observing the change of the current.
  • the use of a constant voltage can enable the nanochannel protein to be efficiently inserted into the membrane material for detection, the nanochannel protein is easy to slip out of the membrane material during the detection process, resulting in interruption of detection, and the usable time of the nanopore biosensor is short, thereby reducing Sequencing throughput.
  • the main purpose of the present invention is to provide a preparation method and a sequencing method of a nanopore biosensor, so as to solve the problem in the prior art that the nanochannel protein is easy to slip out of the membrane material during the detection process.
  • a method for preparing a nanopore biosensor includes: under the action of the first voltage, inserting the nanochannel protein into the membrane material to form a biosensor with a nanopore In the nanopore biosensor of the channel, the first voltage is an increasing voltage.
  • the membrane material includes a phospholipid bimolecular membrane or a polymer membrane; preferably, the membrane rupture voltage of the membrane material is greater than 0.8V, and the membrane rupture voltage is the minimum voltage for breaking the membrane material; preferably, the composition of the phospholipid bimolecular membrane Including DPHPC (4ME: 16PC);
  • the high molecular polymer membrane comprises the high molecular polymer membrane of diblock or triblock;
  • the nanochannel protein comprises the channel protein with pore size greater than 0.5nm, less than 10nm;
  • the nanochannel protein includes a membrane protein or a viral motor channel protein; preferably, the membrane protein includes one or more of the following: ⁇ -hemolysin, Mycobacterium smegmatis porin A, cytolysin A, Escherichia coli CsgG, Aerolysin, PA63; preferably, the viral motor channel protein includes one or more of the following: phage phi29, phage SPP
  • the initial voltage of the first voltage is less than the rupture voltage; preferably, the initial voltage of the first voltage is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the end voltage of the first voltage is less than the rupture voltage Membrane voltage; preferably, the cut-off voltage is 0.18V.
  • the first voltage is a step-up or continuously-rising voltage; preferably, the step-up includes an increase of 0.01-0.8V every 10 minutes, more preferably 0.01-0.4V; preferably, the first voltage starts from the initial voltage, according to Increase the incremental speed of 0.04V every 10min to the end voltage; preferably, when the conductance value of the nanopore biosensor is greater than 1nS, stop applying the first voltage.
  • the preparation method before adding the nanochannel protein and applying the first voltage, also includes applying a second voltage to the membrane material, and the second voltage includes the first periodic voltage and the first constant voltage; preferably, the first periodic voltage Including the first square wave voltage or the first triangular wave voltage; preferably, the high level of the first square wave voltage is less than or equal to the membrane rupture voltage; preferably, the high level of the first square wave voltage is 0.01-0.8V, further Preferably, it is 0.02-0.05V; preferably, the low level of the first square wave voltage is negative; preferably, the frequency of the first square wave voltage is 0.1-100 Hz, more preferably 0.5 Hz.
  • the first constant voltage is 0.01-0.8V, preferably 0.02-0.05V; preferably, applying the second voltage to the membrane material includes: first applying the first periodic voltage to the membrane material, and then applying the first constant voltage , preferably, the first periodic voltage is applied for 1 to 100 cycles, and the first constant voltage lasts for 1 to 100 s; preferably, the second voltage is repeatedly applied to the membrane material, preferably 1 to 100 times.
  • the preparation method further includes: applying a third voltage to the nanopore biosensor, the third voltage includes a second periodic voltage and a second constant voltage; preferably, the nanopore
  • a third voltage is applied to the nanopore biosensor
  • the second periodic voltage includes a second square wave voltage or a second triangular wave voltage; preferably, the high voltage of the second square wave voltage
  • the level is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the low level of the second square wave voltage is negative; preferably, the frequency of the second square wave voltage is 0.1-100Hz, more preferably 0.5 Hz; preferably, the second constant voltage is 0.02-0.05V;
  • applying the third voltage to the nanopore biosensor includes: first applying the second periodic voltage to the nanopore biosensor, and then applying the second constant voltage;
  • the second periodic voltage is repeated for 1-100 times, and the second constant voltage last
  • the preparation method further includes: continuously applying a fourth voltage to the nanopore biosensor, and the fourth voltage is 0.01-0.8V, preferably 0.02-0.05V.
  • the nanopore biosensor prepared by any one of the above preparation methods is provided. After the nanopore biosensor is subjected to a constant voltage of 0.18V for 1 hour, the slippage rate of the nanochannel protein Less than ⁇ 10%.
  • a sequencing method includes: using any one of the above preparation methods to prepare a nanopore biosensor; using the nanopore biosensor to perform single-molecule sequencing.
  • the substance to be tested is selected from small molecule, DNA, RNA, polypeptide or protein.
  • a preparation method of liposome drug includes: under the action of increasing voltage, the drug is inserted into the lipid membrane to form liposome drug , the drug is a protein drug.
  • the drug is an antibody drug.
  • the lipid membrane is a phospholipid membrane, more preferably a vesicle formed by a phospholipid membrane.
  • the incremental voltage is a step-up voltage or a continuously rising voltage.
  • the preparation method also includes: performing flexible treatment on the lipid membrane; preferably, using a method of alternately applying periodic voltage and constant voltage to the lipid membrane Be flexible.
  • a drug delivery method includes: preparing the drug into a liposome drug by the above preparation method; delivering the drug in the form of a liposome drug into in the cell.
  • the drug is delivered into the cell in the form of liposome drug based on voltage stimulation.
  • the first voltage is used to promote the fusion of the nanochannel protein and the membrane material, thereby improving the stability of the nanopore biosensor and reducing the loss of the nanochannel protein from the membrane material. probability of slippage.
  • Fig. 1 shows the construction flowchart of constructing the nanopore biosensor according to the embodiment of the present invention 1 and 2;
  • Fig. 2 shows the current schematic diagram of the nanochannel protein slipping from the phospholipid bilayer according to Examples 1, 2 and Comparative Example 1 of the present invention; the figure shows the current value of a single nanopore channel, which is about 190pA; about 250s , the current decreases from 190pA to around 0pA;
  • FIG. 3 shows a schematic diagram of the slippage rate of nanochannel proteins according to Examples 1 and 2 of the present invention and Comparative Example 1.
  • Nanopore biosensors As mentioned in the background technology, although the nanopore biosensor prepared by the existing technology can efficiently insert the nanochannel protein into the membrane material for detection, the nanochannel protein is easy to slip out of the membrane material during the detection process. cause the detection to be interrupted. Nanopore biosensors have low stability and short usable time, which reduces sequencing throughput.
  • the inventors tried to insert nanochannel proteins into membrane materials to form nanopore biosensors under the action of increasing voltage, and found that the nanopore biosensors prepared by this preparation method had good stability and the slippage of nanochannel proteins low rate. Therefore, a series of protection schemes of the present application are proposed.
  • a method for preparing a nanopore biosensor includes: under the action of a first voltage, inserting a nanochannel protein into a membrane material to form a biosensor with a nanopore In the nanopore biosensor of the channel, the first voltage is an increasing voltage.
  • the nanochannel protein under the action of the first voltage, can be inserted into the membrane material, and the nanochannel protein and the membrane material together constitute a nanopore biosensor with a nanopore channel.
  • the first voltage is different from the constant voltage in the prior art, and is an increasing voltage, including but not limited to step-up or continuous-rise voltage.
  • the membrane material includes a phospholipid bimolecular membrane or a polymer membrane; preferably, the membrane rupture voltage of the membrane material is greater than 0.8V, and the membrane rupture voltage includes but is not limited to 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7 or 0.8V, the membrane breaking voltage is the minimum voltage to break down the membrane material; preferably, the composition of the phospholipid bimolecular membrane includes DPHPC (4ME: 16PC); preferably, the polymer membrane includes diblock or triblock Block polymer membranes; preferably, nanochannel proteins include channel proteins with a pore size greater than 0.5 nm and less than 10 nm; preferably, nanochannel proteins include but are not limited to membrane proteins or viral motor channel proteins; preferably, membrane proteins Including but not limited to ⁇ -hemolysin (alpha-hemolysin), Mycobacterium smegmatis porin A (MspA), cytolysin A (ClyA), Escherichia coli C
  • the above membrane rupture voltage is the voltage that exceeds the membrane's ability to withstand, and different membrane materials have different membrane rupture voltages.
  • the membrane rupture voltage of phospholipid bimolecular membrane is about 0.3V, and the membrane rupture voltage of some polymer membranes can reach 0.8V.
  • the nanochannel protein in the nanopore biosensor has a pore size greater than 0.5 nm, allowing ions and analytes to pass through; the pore size needs to be larger than the size of the analyte to be measured.
  • Membrane proteins such as alpha-hemolysin, MspA, ClyA, CsgG, aerolysin or PA63, and viral motor channels such as phage phi29, phage SPP1, phage T3, phage T4 or phage T7
  • the proteins are all proteins with pore diameters, and can be used as nanochannel proteins to prepare nanopore biosensors.
  • Single-stranded DNA is about 1 nm in diameter, and double-stranded DNA is about 2 nm in diameter.
  • the smaller pore size porins MspA and ⁇ -hemolysin (with a pore size between 1-1.5 nm) only allow single-stranded DNA to pass through the channel and do not allow double-stranded DNA to perforate.
  • Phospholipid membranes may comprise or consist of phospholipids, for example, selected from the group consisting of Diphytanyl-phosphatidylcholine (DPhPC), 1,2-Diphytanyl-sn-glycero-3-phosphocholine, 1,2- Di-O-phytanoyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), di Palmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanoyl- sn-glycerol, 1,2-dipalmitoy
  • the phospholipid bimolecular membrane DPHPC (4ME: 16PC) utilized in this application was purchased from vani polar lipids company, article number: 850356, chemical name: 1,2-diphytanoyl-sn-glycero-3-phosphocholine.
  • the polymer film may comprise a polymer selected from the group consisting of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene , polyisoprene, polybutadiene, polyvinyl chloride, polyalkylacrylate (PAA), polyalkylmethacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylic acid Esters, polysulfones, polyvinyl ethers, poly(propylene oxide) and their copolymers, radical-substituted C1-C6 alkyl acrylates and methacrylates, acrylamides, methacrylamides, (C1-C6 alkyl ) acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylated acrylates and methacryl
  • the initial voltage of the first voltage is lower than the rupture voltage; preferably, the initial voltage of the first voltage is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the first The end voltage of the voltage is less than the rupture voltage; preferably, the end voltage includes 0.15-0.8V, including 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8V, more preferably 0.18V.
  • the first voltage in this application is an increasing voltage, and its initial voltage and termination voltage should be less than the membrane rupture voltage, so as to prevent the applied voltage from exceeding the maximum voltage that the membrane can withstand, causing the membrane to be electrically broken down, resulting in nanopores
  • the biosensor is destroyed.
  • the initial voltage of the first voltage is 0.02-0.05V, and the end voltage is 0.18V.
  • Increasing the first voltage within this range can make the nanochannel protein insert into the membrane material under the action of the first voltage, and prepare nanopores. biological sensor.
  • the nanopore biosensor prepared by the above preparation method has high stability, and the nanochannel protein is not easy to slip off from the membrane material during use. It can be used for a long time, which helps to increase the sequencing throughput.
  • the first voltage is a step-up or continuously rising voltage; preferably, the step-up includes increasing 0.01-0.8V every 10 minutes, more preferably 0.01-0.4V, including but not limited to 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4V, 0.45V, 0.5V, 0.55V, 0.6V, 0.65V, 0.7V, 0.75V or 0.8V; preferably, the first The voltage starts from the initial voltage and increases to the final voltage at an increasing rate of 0.04V every 10min; preferably, when the conductance value of the nanopore biosensor is greater than 1nS, the application of the first voltage is stopped.
  • the first voltage includes, but is not limited to, a step-up voltage (ie, a stepwise increasing voltage) or a continuously increasing voltage (ie, a linearly increasing voltage). If the first voltage is increased in steps, the specific increase speed is not particularly limited. In the above preferred embodiment, the step is increased by 0.04V every 10 minutes, from the initial voltage to the end voltage.
  • the preparation method before adding the nanochannel protein and applying the first voltage, also includes applying a second voltage to the membrane material, and the second voltage includes the first periodic voltage and the first constant voltage; preferably , the first periodic voltage includes a first square wave voltage or a first triangular wave voltage; preferably, the high level of the first square wave voltage is less than or equal to the membrane rupture voltage; preferably, the high level of the first square wave voltage is 0.01 to 0.8V, more preferably 0.02 to 0.05V (specifically 0.01V, 0.02V, 0.04V, 0.06V, 0.08V, 0.1V, 0.15V, 0.2V, 0.25V, 0.3V, 0.35V, 0.4V V, 0.45V, 0.5V, 0.55V, 0.6V, 0.65V, 0.7V, 0.75V or 0.8V); preferably, the low level of the first square wave voltage is negative; preferably, the first square wave voltage The frequency is 0.1-100 Hz, more preferably 0.5 Hz.
  • the first constant voltage is 0.01-0.8V (specifically, it can be 0.01V, 0.02V, 0.04V, 0.06V, 0.08V, 0.1V, 0.15V, 0.2V, 0.25V, 0.3 V, 0.35V, 0.4V, 0.45V, 0.5V, 0.55V, 0.6V, 0.65V, 0.7V, 0.75V or 0.8V), more preferably 0.02-0.05V; preferably, apply a second The voltage includes: first applying the first periodic voltage to the membrane material, and then applying the first constant voltage.
  • the first periodic voltage is performed for 1 to 100 cycles, including but not limited to 1, 2, 3, 5, 10 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 cycles
  • the first constant voltage lasts for 1-100s, including but not limited to 1, 2, 5, 10, 14, 20, 30, 40, 50, 60, 70, 80, 90 or 100s
  • the second voltage is repeatedly applied to the membrane material, preferably 1 to 100 times, including but not limited to 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times.
  • the preparation method Before adding the nanochannel protein and applying the first voltage, the preparation method includes applying a second voltage to the membrane material that has been formed to stimulate the membrane material to make it more flexible, which is beneficial for the subsequent insertion of the nanochannel protein into the membrane material, so that The stability of the finally prepared nanopore biosensor is improved, and the nanochannel protein is not easy to slip from the membrane material during use.
  • the above-mentioned second voltage includes a first periodic voltage and a first constant voltage.
  • the first periodic voltage includes but is not limited to a first square wave voltage or a first triangular wave voltage.
  • the periodic voltage is used to stimulate the membrane material to make it more flexible, which is more conducive to the insertion of nanochannel proteins.
  • the high level (i.e. the maximum voltage) of the first square wave voltage is controlled to be less than the membrane rupture voltage, i.e. the high level is within the range of 0.01-0.8V, further Preferably, it may be in the range of 0.02 to 0.05V.
  • the low level of the first square wave voltage can be negative, that is, the low level voltage is a negative number of the high level voltage.
  • the first square wave voltage includes but is not limited to 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100 Hz.
  • the frequency of the first square wave voltage is 0.5 Hz, that is, one period of the first square wave voltage is 2s, and within one period, the high level lasts for 1s, and the low level lasts for 1s.
  • the second voltage also includes a first constant voltage, which is 0.02-0.05V.
  • the second voltage includes: first applying the first cycle voltage to the membrane material reagent, and then applying a constant voltage, and the two voltages are cycled for many times, so that the membrane material is more flexible.
  • the preparation method further includes: applying a third voltage to the nanopore biosensor, the third voltage includes the second periodic voltage and the second Constant voltage; preferably, when the conductance value of the nanopore biosensor is greater than 1nS, a third voltage is applied to the nanopore biosensor; preferably, the second periodic voltage includes a second square wave voltage or a second triangular wave voltage; preferably, The high level of the second square wave voltage is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the low level of the second square wave voltage is negative; preferably, the frequency of the second square wave voltage is 0.1 ⁇ 100Hz, the second square wave voltage includes but not limited to 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100Hz, more preferably 0.5Hz; preferably, the second constant voltage is 0.02-0.05V; preferably, applying the third voltage to the nanopore biosensor includes: first applying a second periodic voltage to the nanopore biosensor
  • the third voltage includes a second periodic voltage and a second constant voltage.
  • the second periodic voltage includes but is not limited to a second square wave voltage or a second triangular wave voltage. The combination of periodic voltage and constant voltage can stabilize the nanochannel protein inserted into the membrane material.
  • the high level (ie, the maximum voltage) of the second square wave voltage needs to be lower than the membrane rupture voltage, and the high level is 0.01-0.8V, preferably 0.02-0.05V.
  • the low level of the second square wave voltage can be negative, that is, the low level voltage is a negative number of the high level voltage.
  • the second square wave voltage includes but is not limited to 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100 Hz.
  • the frequency of the second square wave voltage is 0.5 Hz, that is, one period of the second square wave voltage is 2s, and within one period, the high level lasts for 1s, and the low level lasts for 1s.
  • the third cycle voltage also includes a second constant voltage, the second constant voltage is 0.01-0.8V, preferably 0.02-0.05V, and the combination of the cycle voltage and the constant voltage makes the nanopore biosensing more stable.
  • the preparation method further includes: continuously applying a fourth voltage to the nanopore biosensor, the fourth voltage is 0.01-0.8V, preferably 0.02 ⁇ 0.05V.
  • the two effects of the third and fourth voltages include: (1) preventing more nanochannel proteins from inserting into the same phospholipid membrane, resulting in porosity. If multiple nanochannel proteins appear on a phospholipid membrane, it cannot be used for detection; (2) it can stabilize the constructed nanopore biosensor and reduce the slippage rate. Before applying the third voltage, the nanopore biosensor has been prepared.
  • a nanopore biosensor prepared by the above preparation method is provided. After the nanopore biosensor is subjected to a constant voltage of 0.18V for 1 hour, the slippage rate of the nanochannel protein is less than ⁇ 10%.
  • the nanopore biosensor prepared by the above preparation method has good stability, and the nanochannel protein inserted into the membrane material is not easy to slip from the membrane material during use, that is, under the action of a constant voltage, and can continue to function for a long average time. After the nanopore biosensor acts on a constant voltage of 0.18V for 1 hour, the slipping rate of the nanochannel protein is less than ⁇ 10%. However, in the nanopore biosensor prepared by the existing constant voltage technology, the slippage rate of the nanochannel protein is greater than 30% under the same conditions.
  • a sequencing method comprising: using the above-mentioned preparation method to prepare a nanopore biosensor; using the nanopore biosensor to perform single-molecule sequencing of a substance to be tested.
  • the substance to be tested is selected from small molecules, DNA, RNA, polypeptides or proteins.
  • the nanopore biosensor prepared by the above preparation method can be applied in a corresponding device to perform single-molecule sequencing of the substance to be tested.
  • Species to be tested include small molecules, DNA, RNA, peptides or proteins.
  • the step of inserting the nanopore channel protein into the membrane material in the aforementioned preparation method of the nanopore biosensor of the present application is similar to the principle of encapsulating the protein drug in the liposome carrier to form the liposome drug, thus , in the fourth typical embodiment of the present application, a preparation method of a liposome drug is provided, the preparation method comprising: under the action of increasing voltage, inserting the drug into the lipid membrane to form a liposome drug,
  • the drug is a protein drug. Under the action of increasing voltage, it is more conducive to the stable insertion of protein drugs into the lipid membrane and not easy to slip out, which in turn helps to improve the reliability of preparing protein drugs into liposome drugs, thereby facilitating the improvement of delivery efficiency.
  • the specific protein drug can be protein drug or polypeptide drug in any structural form.
  • the drug is an antibody drug.
  • Lipid membranes used as lipid delivery carriers are suitable for this application, such as phospholipid membranes, phospholipid monomolecular membranes, or phospholipid bimolecular membranes, more preferably vesicles formed by phospholipid membranes.
  • Specific classes of phospholipid membranes are exemplified as follows: selected from diphytanyl-phosphatidylcholine (DPhPC), 1,2-diphytanyl-sn-glycero-3-phosphocholine, 1,2-di-O- Phytanoyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), dipalmitoylphosphatidyl Choline (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatid
  • the phospholipid bimolecular membrane DPHPC (4ME: 16PC) utilized in this application was purchased from vani polar lipids company, article number: 850356, chemical name: 1,2-diphytanoyl-sn-glycero-3-phosphocholine.
  • the initial voltage and termination voltage of the above-mentioned incremental voltage should be less than the membrane rupture voltage of the lipid membrane, so as to prevent the applied voltage from exceeding the maximum voltage that the lipid membrane can withstand, causing the lipid membrane to be electrically broken down, causing liposome drug destroyed.
  • the initial voltage of the incremental voltage is 0.02-0.1V, preferably 0.02-0.05V, and the end voltage is 0.18V. Increasing within this range can make it easier for the protein drug to be inserted into the lipid membrane and not easy to slip off, so that the package better drug stability.
  • the aforementioned increasing voltage may be a step-up voltage (ie, a stepwise increasing voltage), or a continuously increasing voltage (ie, a linearly increasing voltage). If the voltage is increased incrementally in the form of a step increase, there is no particular limitation on the specific increase speed. In the above preferred embodiment, the voltage is increased by 0.04V every 10 minutes during the step increase, from the initial voltage step to the end voltage.
  • the preparation method further includes: performing a flexible treatment on the lipid film;
  • the flexible treatment is performed by alternately applying periodic voltage (such as square wave voltage or triangular wave voltage) and constant voltage to the lipid membrane.
  • a periodic voltage (such as square wave voltage or triangular wave voltage) is first applied to the lipid membrane, and then a constant voltage is applied.
  • the periodic voltage is carried out for 1 to 100 cycles, including but not limited to 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cycles, constant voltage for 1-100s, including but not limited to 1, 2, 5, 10, 14 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 s; preferably, repeatedly apply periodic voltage and constant voltage to the lipid membrane repeatedly, preferably repeating 1 to 100 times, including but not limited to repeated 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times.
  • the high level (i.e. the maximum voltage) of the square wave voltage is controlled to be less than the membrane rupture voltage, i.e. the high level is 0.01 ⁇ It is within the range of 0.8V, more preferably within the range of 0.02 to 0.05V.
  • the low level of the square wave voltage can be negative, that is, the low level voltage is the negative number of the high level voltage.
  • Square wave voltages include, but are not limited to, 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100 Hz.
  • the frequency of the square wave voltage is 0.5 Hz, that is, one period of the first square wave voltage is 2s, and within one period, the high level lasts for 1s, and the low level lasts for 1s.
  • a drug delivery method includes: preparing the drug into a liposome drug by the above preparation method; and delivering the drug into cells in the form of liposome drug.
  • the protein drug can be delivered into the cell in the form of a conventional liposome (for example, with Cell membrane fusion, endocytosis into the cell, etc.).
  • the drug is delivered into the cell in the form of liposome drug based on voltage stimulation.
  • the way of voltage stimulation helps to improve the efficiency of cell entry.
  • the phospholipid membrane DPHPC (4ME: 16PC) was stimulated by the second voltage to make it more flexible to promote the insertion.
  • a voltage of ⁇ 0.02V was applied for 1s each, for a total of three cycles; then a constant voltage of 0.02V was applied for 14s. This above-mentioned step was repeated 30 times.
  • the nanochannel protein CsgG mutant (amino acid sequence shown by SEQ ID NO: 1) to one side of the membrane, and apply the first voltage.
  • the first voltage is: an initial voltage of 0.02V, an end voltage of 0.18V, and an increase of 0.04V every 10 minutes.
  • amino acid sequence of the CsgG transmembrane protein mutant is SEQ ID NO: 1:
  • the phospholipid membrane DPHPC (4ME: 16PC) was stimulated by the second voltage to make it more flexible to promote the insertion.
  • a voltage of ⁇ 0.05V was applied for 1s each, for a total of three cycles; then a constant voltage of 0.05V was applied for 14s. This above-mentioned step was repeated 30 times.
  • the nanochannel protein CsgG mutant is added to one side of the membrane and a first voltage is applied.
  • the first voltage is: an initial voltage of 0.05V, an end voltage of 0.18V, and an increase of 0.04V every 10 minutes.
  • FIG. 1 shows a flow chart of constructing a nanopore biosensor according to Embodiment 1 or 2 of the present invention.
  • the nanochannel proteins of 2 nanopore biosensors slipped out within 1 hour, and the slipping ratio was 1.7%, as shown in FIG. 3 .
  • the prepared nanochannel protein CsgG mutant was added to one side of the membrane, and a constant voltage of 0.18V was applied to promote the bilayer of the channel protein and phospholipid.
  • Molecular layer membrane fusion to form nanoporous biosensors.
  • Figure 2 shows that a nanochannel protein slides out from the phospholipid membrane, causing detection failure, and the current is less than 10pA (voltage: 0.18V) after the slide out.
  • the present invention utilizes the first, second and third voltages to prepare nanopore biosensors, which can significantly increase the stability of nanopore biosensors , reducing the slippage rate of nanochannel proteins on it.

Abstract

A preparation method for a nanopore biosensor and a sequencing method. The preparation method comprises: under the action of a first voltage, inserting a nanochannel protein into a membrane material to form a nanopore biosensor having a nanopore channel, wherein the first voltage is an incremental voltage. By means of the incremental voltage, the fusion of the nanochannel protein and the membrane material is promoted, thereby improving the stability of the nanopore biosensor, and reducing the probability of the nanochannel protein slipping out of the membrane material.

Description

纳米孔生物传感器的制备方法及测序方法Preparation method and sequencing method of nanopore biosensor 技术领域technical field
本发明涉及纳米孔传感器制备领域，具体而言，涉及一种纳米孔生物传感器的制备方法及测序方法。The invention relates to the field of nanopore sensor preparation, in particular to a preparation method and a sequencing method of a nanopore biosensor.
背景技术Background technique
纳米孔检测技术起源于Coulter计数器的发明以及单通道电流的记录技术。Neher和Sakamann在1976年利用膜片钳技术测量膜电势，研究蛋白通道。1996年，Kasianowicz等提出了利用α-溶血素(α-hemolysin)检测DNA，开启了单分子纳米孔检测的新领域。Nanopore detection technology originated from the invention of the Coulter counter and the recording technology of single-channel current. Neher and Sakamann used the patch clamp technique to measure membrane potential in 1976 to study protein channels. In 1996, Kasianowicz et al. proposed the use of α-hemolysin (α-hemolysin) to detect DNA, which opened a new field of single-molecule nanopore detection.
纳米孔测序，又被称为单分子测序技术，无需聚合酶扩增可对核酸进行测序；具有长读长、速度快、通量高、成本低等特点。目前国际上常用的纳米孔可以分为固态孔和生物孔；生物孔即纳米通道蛋白，主要由膜蛋白或病毒马达蛋白构成。相比固态孔材料，生物孔可以通过蛋白表达纯化大量制备，且均一性好；基于蛋白结构可有目的的进行突变，从而实现不同待测物的检测。生物纳米孔检测的原理：***膜层的纳米通道蛋白两端加一个电压，通过电场驱动，使待测物分子从孔一端电泳通过纳米孔，在外电路收集的离子电流会发生改变，通过电流改变的大小、时间等特征从而实现对待测物的表征。Nanopore sequencing, also known as single-molecule sequencing technology, can sequence nucleic acids without polymerase amplification; it has the characteristics of long read length, fast speed, high throughput, and low cost. At present, the commonly used nanopores in the world can be divided into solid pores and biological pores; biological pores are nanochannel proteins, which are mainly composed of membrane proteins or viral motor proteins. Compared with solid-state pore materials, biological pores can be prepared in large quantities through protein expression and purification, and have good uniformity; based on protein structure, mutations can be carried out purposefully, so as to realize the detection of different analytes. The principle of biological nanopore detection: a voltage is applied to both ends of the nanochannel protein inserted into the membrane layer, driven by an electric field, so that the molecules of the analyte are electrophoresed from one end of the hole through the nanopore, and the ion current collected by the external circuit will change, and the passing current will change. The size, time and other characteristics of the test object can be realized.
目前，纳米通道蛋白在恒定电压的作用下***到膜材内，从而形成纳米孔生物传感器。现有的制备纳米孔生物传感器的方法包括：电解池中央小孔处形成磷脂膜后，在膜的一侧加入制备好的纳米通道蛋白，施加适当恒定电压促进通道蛋白与磷脂双分子层膜融合形成纳米孔通道，利用膜片钳放大器或其他电信号放大器用于采集电流信号，通过观察电流的变化判断是否由孔蛋白成功***到磷脂膜内。使用恒定电压尽管能够使纳米通道蛋白高效***到膜材内实现检测，但纳米通道蛋白易在检测过程中从膜材内滑出，导致检测中断，纳米孔生物传感器可使用的时间短，从而降低测序通量。At present, nanochannel proteins are inserted into membrane materials under the action of a constant voltage to form nanopore biosensors. The existing method for preparing nanopore biosensors includes: after forming a phospholipid membrane at the central hole of the electrolytic cell, adding the prepared nanochannel protein to one side of the membrane, and applying an appropriate constant voltage to promote the fusion of the channel protein and the phospholipid bilayer membrane To form a nanopore channel, use a patch clamp amplifier or other electrical signal amplifiers to collect current signals, and judge whether the porin is successfully inserted into the phospholipid membrane by observing the change of the current. Although the use of a constant voltage can enable the nanochannel protein to be efficiently inserted into the membrane material for detection, the nanochannel protein is easy to slip out of the membrane material during the detection process, resulting in interruption of detection, and the usable time of the nanopore biosensor is short, thereby reducing Sequencing throughput.
发明内容Contents of the invention
本发明的主要目的在于提供一种纳米孔生物传感器的制备方法及测序方法，以解决现有技术中纳米通道蛋白易在检测过程中从膜材内滑出的问题。The main purpose of the present invention is to provide a preparation method and a sequencing method of a nanopore biosensor, so as to solve the problem in the prior art that the nanochannel protein is easy to slip out of the membrane material during the detection process.
为了实现上述目的，根据本发明的一个方面，提供了一种纳米孔生物传感器的制备方法，该制备方法包括：在第一电压的作用下，使纳米通道蛋白***膜材内，形成具有纳米孔通道的纳米孔生物传感器，第一电压为递增电压。In order to achieve the above object, according to one aspect of the present invention, a method for preparing a nanopore biosensor is provided. The preparation method includes: under the action of the first voltage, inserting the nanochannel protein into the membrane material to form a biosensor with a nanopore In the nanopore biosensor of the channel, the first voltage is an increasing voltage.
进一步地，膜材包括磷脂双分子膜或高分子聚合物膜；优选地，膜材的破膜电压大于0.8V，破膜电压为击破膜材的最小电压；优选地，磷脂双分子膜的组成包括DPHPC(4ME：16PC)；优选地，高分子聚合物膜包括两嵌段或三嵌段的高分子聚合物膜；优选地，纳米通道蛋白包 括孔径大于0.5nm、小于10nm的通道蛋白；优选地，纳米通道蛋白包括膜蛋白或病毒马达通道蛋白；优选地，膜蛋白包括以下一种或多种：α-溶血素、耻垢分枝杆菌孔蛋白A、溶细胞素A、大肠杆菌CsgG、气单胞菌溶素、PA63；优选地，病毒马达通道蛋白包括以下一种或多种：噬菌体phi29、噬菌体SPP1、噬菌体T3、噬菌体T4、噬菌体T7。Further, the membrane material includes a phospholipid bimolecular membrane or a polymer membrane; preferably, the membrane rupture voltage of the membrane material is greater than 0.8V, and the membrane rupture voltage is the minimum voltage for breaking the membrane material; preferably, the composition of the phospholipid bimolecular membrane Including DPHPC (4ME: 16PC); Preferably, the high molecular polymer membrane comprises the high molecular polymer membrane of diblock or triblock; Preferably, the nanochannel protein comprises the channel protein with pore size greater than 0.5nm, less than 10nm; Preferably Preferably, the nanochannel protein includes a membrane protein or a viral motor channel protein; preferably, the membrane protein includes one or more of the following: α-hemolysin, Mycobacterium smegmatis porin A, cytolysin A, Escherichia coli CsgG, Aerolysin, PA63; preferably, the viral motor channel protein includes one or more of the following: phage phi29, phage SPP1, phage T3, phage T4, and phage T7.
进一步地，第一电压的起始电压小于破膜电压；优选地，第一电压的起始电压为0.01～0.8V，进一步优选为0.02-0.05V；优选地，第一电压的终止电压小于破膜电压；优选地，终止电压为0.18V。Further, the initial voltage of the first voltage is less than the rupture voltage; preferably, the initial voltage of the first voltage is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the end voltage of the first voltage is less than the rupture voltage Membrane voltage; preferably, the cut-off voltage is 0.18V.
进一步地，第一电压为阶梯上升或持续上升的电压；优选地，阶梯上升包括每10min增加0.01～0.8V，更优选0.01～0.4V；优选地，第一电压为从起始电压开始，按照每10min增加0.04V的递增速度递增至终止电压；优选地，纳米孔生物传感器的电导值大于1nS时，停止施加第一电压。Further, the first voltage is a step-up or continuously-rising voltage; preferably, the step-up includes an increase of 0.01-0.8V every 10 minutes, more preferably 0.01-0.4V; preferably, the first voltage starts from the initial voltage, according to Increase the incremental speed of 0.04V every 10min to the end voltage; preferably, when the conductance value of the nanopore biosensor is greater than 1nS, stop applying the first voltage.
进一步地，在加入纳米通道蛋白、施加第一电压前，制备方法还包括对膜材施加第二电压，第二电压包括第一周期性电压和第一恒定电压；优选地，第一周期性电压包括第一方波电压或第一三角波电压；优选地，第一方波电压的高电平小于或等于破膜电压；优选地，第一方波电压的高电平为0.01～0.8V，进一步优选为0.02～0.05V；优选地，第一方波电压的低电平为负；优选地，第一方波电压的频率为0.1～100Hz，进一步优选为0.5Hz。Further, before adding the nanochannel protein and applying the first voltage, the preparation method also includes applying a second voltage to the membrane material, and the second voltage includes the first periodic voltage and the first constant voltage; preferably, the first periodic voltage Including the first square wave voltage or the first triangular wave voltage; preferably, the high level of the first square wave voltage is less than or equal to the membrane rupture voltage; preferably, the high level of the first square wave voltage is 0.01-0.8V, further Preferably, it is 0.02-0.05V; preferably, the low level of the first square wave voltage is negative; preferably, the frequency of the first square wave voltage is 0.1-100 Hz, more preferably 0.5 Hz.
进一步地，第一恒定电压为0.01～0.8V，优选为0.02～0.05V；优选地，对膜材施加第二电压包括：对膜材先施加第一周期性电压，然后再施加第一恒定电压，优选地，第一周期性电压进行1～100个周期，第一恒定电压持续1～100s；优选地，对膜材重复多次施加第二电压，优选重复1～100次。Further, the first constant voltage is 0.01-0.8V, preferably 0.02-0.05V; preferably, applying the second voltage to the membrane material includes: first applying the first periodic voltage to the membrane material, and then applying the first constant voltage , preferably, the first periodic voltage is applied for 1 to 100 cycles, and the first constant voltage lasts for 1 to 100 s; preferably, the second voltage is repeatedly applied to the membrane material, preferably 1 to 100 times.
进一步地，在形成具有纳米孔通道的纳米孔生物传感器之后，制备方法进一步包括：对纳米孔生物传感器施加第三电压，第三电压包括第二周期性电压和第二恒定电压；优选地，纳米孔生物传感器的电导值大于1nS时，对纳米孔生物传感器施加第三电压；优选地，第二周期性电压包括第二方波电压或第二三角波电压；优选地，第二方波电压的高电平为0.01～0.8V，进一步优选为0.02～0.05V；优选地，第二方波电压的低电平为负；优选地，第二方波电压的频率为0.1～100Hz，进一步优选为0.5Hz；优选地，第二恒定电压为0.02～0.05V；优选地，对纳米孔生物传感器施加第三电压包括：对纳米孔生物传感器先施加第二周期性电压，然后再施加第二恒定电压；优选地，第二周期性电压重复1～100次，第二恒定电压持续1～100s；优选地，对纳米孔生物传感器重复多次施加第三电压，优选地，重复1～100次。Further, after forming the nanopore biosensor with a nanopore channel, the preparation method further includes: applying a third voltage to the nanopore biosensor, the third voltage includes a second periodic voltage and a second constant voltage; preferably, the nanopore When the conductance value of the pore biosensor is greater than 1nS, a third voltage is applied to the nanopore biosensor; preferably, the second periodic voltage includes a second square wave voltage or a second triangular wave voltage; preferably, the high voltage of the second square wave voltage The level is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the low level of the second square wave voltage is negative; preferably, the frequency of the second square wave voltage is 0.1-100Hz, more preferably 0.5 Hz; preferably, the second constant voltage is 0.02-0.05V; preferably, applying the third voltage to the nanopore biosensor includes: first applying the second periodic voltage to the nanopore biosensor, and then applying the second constant voltage; Preferably, the second periodic voltage is repeated for 1-100 times, and the second constant voltage lasts for 1-100 s; preferably, the third voltage is repeatedly applied to the nanopore biosensor, preferably, repeated for 1-100 times.
进一步地，在对纳米孔生物传感器重复多次施加第三电压之后，制备方法还包括：对纳米孔生物传感器持续施加第四电压，第四电压为0.01～0.8V，优选为0.02～0.05V。Further, after repeatedly applying the third voltage to the nanopore biosensor, the preparation method further includes: continuously applying a fourth voltage to the nanopore biosensor, and the fourth voltage is 0.01-0.8V, preferably 0.02-0.05V.
为了实现上述目的，根据本发明的第二个方面，提供了上述任一种制备方法所制备的纳米孔生物传感器，纳米孔生物传感器在0.18V恒定电压作用1小时后，纳米通道蛋白的滑脱率小于<10％。In order to achieve the above object, according to the second aspect of the present invention, the nanopore biosensor prepared by any one of the above preparation methods is provided. After the nanopore biosensor is subjected to a constant voltage of 0.18V for 1 hour, the slippage rate of the nanochannel protein Less than <10%.
为了实现上述目的，根据本发明的第三个方面，提供了一种测序方法，该测序方法包括：利用上述任一种制备方法制备纳米孔生物传感器；利用纳米孔生物传感器对待测物质进行单分子测序。In order to achieve the above object, according to the third aspect of the present invention, a sequencing method is provided, the sequencing method includes: using any one of the above preparation methods to prepare a nanopore biosensor; using the nanopore biosensor to perform single-molecule sequencing.
进一步地，待测物质选自小分子、DNA、RNA、多肽或蛋白质。Further, the substance to be tested is selected from small molecule, DNA, RNA, polypeptide or protein.
为了实现上述目的，根据本发明的第四个方面，提供了一种脂质体药物的制备方法，该制备方法包括：在递增电压的作用下，使药物***脂质膜内形成脂质体药物，药物为蛋白药物。In order to achieve the above object, according to the fourth aspect of the present invention, a preparation method of liposome drug is provided, the preparation method includes: under the action of increasing voltage, the drug is inserted into the lipid membrane to form liposome drug , the drug is a protein drug.
进一步地，药物为抗体药物。Further, the drug is an antibody drug.
进一步地，脂质膜为磷脂膜，更优选为磷脂膜形成的囊泡。Further, the lipid membrane is a phospholipid membrane, more preferably a vesicle formed by a phospholipid membrane.
进一步地，递增电压为阶梯上升的电压或持续上升的电压。Further, the incremental voltage is a step-up voltage or a continuously rising voltage.
进一步地，在使药物***脂质膜内形成脂质体药物前，该制备方法还包括：对脂质膜进行柔性处理；优选地，采用对脂质膜交替施加周期性电压与恒定电压的方式进行柔性处理。Further, before the drug is inserted into the lipid membrane to form a liposome drug, the preparation method also includes: performing flexible treatment on the lipid membrane; preferably, using a method of alternately applying periodic voltage and constant voltage to the lipid membrane Be flexible.
为了实现上述目的，根据本发明的第五个方面，提供了一种药物的递送方法，递送方法包括：将药物上述制备方法制备成脂质体药物；将药物以脂质体药物的形式递送入细胞内。In order to achieve the above object, according to the fifth aspect of the present invention, a drug delivery method is provided. The delivery method includes: preparing the drug into a liposome drug by the above preparation method; delivering the drug in the form of a liposome drug into in the cell.
进一步地，基于电压刺激的方式将药物以脂质体药物的形式递送入细胞内。Further, the drug is delivered into the cell in the form of liposome drug based on voltage stimulation.
应用本发明的技术方案，在制备纳米孔生物传感器时，利用递增的第一电压，促进纳米通道蛋白与膜材的融合，从而提高纳米孔生物传感器的稳定性，减小纳米通道蛋白从膜材中滑脱的概率。Applying the technical scheme of the present invention, when preparing the nanopore biosensor, the first voltage is used to promote the fusion of the nanochannel protein and the membrane material, thereby improving the stability of the nanopore biosensor and reducing the loss of the nanochannel protein from the membrane material. probability of slippage.
附图说明Description of drawings
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The accompanying drawings constituting a part of the present application are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:
图1示出了根据本发明实施例1和2的构建纳米孔生物传感器的构建流程图；Fig. 1 shows the construction flowchart of constructing the nanopore biosensor according to the embodiment of the present invention 1 and 2;
图2示出了根据本发明实施例1、2和对比例1的纳米通道蛋白从磷脂双分子层滑脱的电流示意图；图中展示了单个纳米孔通道的电流值，约为190pA；在250s左右时，电流从190pA降低至0pA附近；Fig. 2 shows the current schematic diagram of the nanochannel protein slipping from the phospholipid bilayer according to Examples 1, 2 and Comparative Example 1 of the present invention; the figure shows the current value of a single nanopore channel, which is about 190pA; about 250s , the current decreases from 190pA to around 0pA;
图3示出了根据本发明实施例1、2和对比例1的纳米通道蛋白滑脱率示意图。FIG. 3 shows a schematic diagram of the slippage rate of nanochannel proteins according to Examples 1 and 2 of the present invention and Comparative Example 1.
具体实施方式Detailed ways
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with examples.
如背景技术所提到的，利用现有技术制备的纳米孔生物传感器，虽然能够使纳米通道蛋白高效***到膜材内实现检测，但纳米通道蛋白易在检测过程中从膜材内滑出，导致检测中断。纳米孔生物传感器的稳定性低，可使用的时间短，从而降低测序通量。As mentioned in the background technology, although the nanopore biosensor prepared by the existing technology can efficiently insert the nanochannel protein into the membrane material for detection, the nanochannel protein is easy to slip out of the membrane material during the detection process. cause the detection to be interrupted. Nanopore biosensors have low stability and short usable time, which reduces sequencing throughput.
因而，在本申请中发明人尝试在递增电压的作用下将纳米通道蛋白***膜材中形成纳米孔生物传感器，并发现该制备方法制备的纳米孔生物传感器的稳定性好，纳米通道蛋白的滑脱率低。因而提出了本申请的一系列保护方案。Therefore, in the present application, the inventors tried to insert nanochannel proteins into membrane materials to form nanopore biosensors under the action of increasing voltage, and found that the nanopore biosensors prepared by this preparation method had good stability and the slippage of nanochannel proteins low rate. Therefore, a series of protection schemes of the present application are proposed.
在本申请第一种典型的实施方式中，提供了一种纳米孔生物传感器的制备方法，该制备方法包括：在第一电压的作用下，使纳米通道蛋白***膜材内，形成具有纳米孔通道的纳米孔生物传感器，第一电压为递增电压。In the first typical embodiment of the present application, a method for preparing a nanopore biosensor is provided. The preparation method includes: under the action of a first voltage, inserting a nanochannel protein into a membrane material to form a biosensor with a nanopore In the nanopore biosensor of the channel, the first voltage is an increasing voltage.
在上述制备方法中，在第一电压的作用下，纳米通道蛋白能够***膜材内，纳米通道蛋白和膜材共同构成具有纳米孔通道的纳米孔生物传感器。第一电压与现有技术中的恒压不同，为递增电压，包括但不限于阶梯上升或持续上升的电压。In the above preparation method, under the action of the first voltage, the nanochannel protein can be inserted into the membrane material, and the nanochannel protein and the membrane material together constitute a nanopore biosensor with a nanopore channel. The first voltage is different from the constant voltage in the prior art, and is an increasing voltage, including but not limited to step-up or continuous-rise voltage.
在一种优选的实施例中，膜材包括磷脂双分子膜或高分子聚合物膜；优选地，膜材的破膜电压大于0.8V，破膜电压包括但不限于0.2、0.3、0.4、0.5、0.6、0.7或0.8V，破膜电压为击破膜材的最小电压；优选地，磷脂双分子膜的组成包括DPHPC(4ME：16PC)；优选地，高分子聚合物膜包括两嵌段或三嵌段的高分子聚合物膜；优选地，纳米通道蛋白包括孔径大于0.5nm、小于10nm的通道蛋白；优选地，纳米通道蛋白包括但不限于膜蛋白或病毒马达通道蛋白；优选地，膜蛋白包括但不限于α-溶血素(alpha-hemolysin)、耻垢分枝杆菌孔蛋白A(MspA)、溶细胞素A(ClyA)、大肠杆菌CsgG、气单胞菌溶素(aerolysin)、PA63；优选地，病毒马达通道蛋白包括但不限于噬菌体phi29、噬菌体SPP1、噬菌体T3、噬菌体T4或噬菌体T7中的一种或多种。In a preferred embodiment, the membrane material includes a phospholipid bimolecular membrane or a polymer membrane; preferably, the membrane rupture voltage of the membrane material is greater than 0.8V, and the membrane rupture voltage includes but is not limited to 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7 or 0.8V, the membrane breaking voltage is the minimum voltage to break down the membrane material; preferably, the composition of the phospholipid bimolecular membrane includes DPHPC (4ME: 16PC); preferably, the polymer membrane includes diblock or triblock Block polymer membranes; preferably, nanochannel proteins include channel proteins with a pore size greater than 0.5 nm and less than 10 nm; preferably, nanochannel proteins include but are not limited to membrane proteins or viral motor channel proteins; preferably, membrane proteins Including but not limited to α-hemolysin (alpha-hemolysin), Mycobacterium smegmatis porin A (MspA), cytolysin A (ClyA), Escherichia coli CsgG, aerolysin (aerolysin), PA63; Preferably, the viral motor channel protein includes, but is not limited to, one or more of phage phi29, phage SPP1, phage T3, phage T4 or phage T7.
上述破膜电压就是超过膜所能承受的电压，不同的膜材具有不同的破膜电压。磷脂双分子膜的破膜电压在0.3V左右，部分高分子聚合物膜的破膜电压能够达到0.8V。纳米孔生物传感器中的纳米通道蛋白的孔径大于0.5nm，允许离子和待测分析物通过；孔径需大于待测分析物尺寸。α-溶血素(alpha-hemolysin)、MspA、ClyA、CsgG、气单胞菌溶素(aerolysin)或PA63等膜蛋白，以及噬菌体phi29、噬菌体SPP1、噬菌体T3、噬菌体T4或噬菌体T7等病毒马达通道蛋白，均为具有孔径的蛋白，能够作为纳米通道蛋白，制备纳米孔生物传感器。单链DNA直径约为1nm，双链DNA直径约为2nm。因此，孔径较小的孔蛋白MspA和α-溶血素(孔径在1-1.5nm之间)只允许单链DNA穿过通道，不允许双链DNA穿孔。The above membrane rupture voltage is the voltage that exceeds the membrane's ability to withstand, and different membrane materials have different membrane rupture voltages. The membrane rupture voltage of phospholipid bimolecular membrane is about 0.3V, and the membrane rupture voltage of some polymer membranes can reach 0.8V. The nanochannel protein in the nanopore biosensor has a pore size greater than 0.5 nm, allowing ions and analytes to pass through; the pore size needs to be larger than the size of the analyte to be measured. Membrane proteins such as alpha-hemolysin, MspA, ClyA, CsgG, aerolysin or PA63, and viral motor channels such as phage phi29, phage SPP1, phage T3, phage T4 or phage T7 The proteins are all proteins with pore diameters, and can be used as nanochannel proteins to prepare nanopore biosensors. Single-stranded DNA is about 1 nm in diameter, and double-stranded DNA is about 2 nm in diameter. Therefore, the smaller pore size porins MspA and α-hemolysin (with a pore size between 1-1.5 nm) only allow single-stranded DNA to pass through the channel and do not allow double-stranded DNA to perforate.
磷脂膜可以包括磷脂或由其组成，例如，选自二植烷酰基-磷脂酰胆碱(DPhPC)、1,2-二植烷酰基-sn-甘油-3-磷酸胆碱、1,2-二-O-植烷酰基-sn-甘油-3-磷酸胆碱(DoPhPC)、棕榈酰基-油酰基-磷脂酰胆碱(POPC)、二油酰基-磷脂酰-甲基酯(DOPME)、二棕榈酰基磷脂酰胆碱(DPPC)、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酸、磷脂酰肌醇、磷脂酰甘油、鞘磷脂、1,2-二-O-植烷酰基-sn-甘油、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-350]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-550]、1,2- 二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-750]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-1000]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-2000]、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-乳糖酰基、GM1神经节苷脂、溶血磷脂酰胆碱(LPC)或其任意组合。本申请中利用的磷脂双分子膜DPHPC(4ME：16PC)购买于avanti polar lipids公司，货号：850356，化学名称：1,2-diphytanoyl-sn-glycero-3-phosphocholine。Phospholipid membranes may comprise or consist of phospholipids, for example, selected from the group consisting of Diphytanyl-phosphatidylcholine (DPhPC), 1,2-Diphytanyl-sn-glycero-3-phosphocholine, 1,2- Di-O-phytanoyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), di Palmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanoyl- sn-glycerol, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl-sn-glycerol-3 -Phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 750], 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sn-glycerol-3- Phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-lactoyl, GM1 ganglioside, lysophosphatidyl Choline (LPC) or any combination thereof. The phospholipid bimolecular membrane DPHPC (4ME: 16PC) utilized in this application was purchased from avanti polar lipids company, article number: 850356, chemical name: 1,2-diphytanoyl-sn-glycero-3-phosphocholine.
高分子聚合物膜可以包括选自下列的聚合物：聚硅氧烷、聚烯烃、全氟聚醚、全氟烃基聚醚、聚苯乙烯、聚氧丙烯、聚乙酸乙烯酯、聚氧丁烯、聚异戊二烯、聚丁二烯、聚氯乙烯、聚烷基丙烯酸酯(PAA)、聚烷基甲基丙烯酸酯、聚丙烯腈、聚丙烯、PTHF、聚甲基丙烯酸酯、聚丙烯酸酯、聚砜、聚乙烯醚、聚(环氧丙烷)及其共聚物、基取代的C1-C6烷基丙烯酸酯和甲基丙烯酸酯、丙烯酰胺、甲基丙烯酰胺、(C1-C6烷基)丙烯酰胺和甲基丙烯酰胺、N,N-二烷基-丙烯酰胺、乙氧基丙烯酸酯和甲基丙烯酸酯、聚乙二醇单甲基丙烯酸酯和聚乙二醇单甲基醚甲基丙烯酸酯、羟基取代的(C1-C6烷基)丙烯酰胺和甲基丙烯酰胺、羟基取代的C1-C6烷基乙烯基醚、乙烯基磺酸钠、苯乙烯基磺酸钠、2-丙烯酰胺-2-甲基丙磺酸、N-乙烯基吡咯、N-乙烯基-2-吡咯烷酮、2-乙烯基恶唑啉、2-乙烯基-4,4'–双烷基恶唑啉基-5-酮、2,4-乙烯基吡啶、总共具有3-5个碳原子的乙烯化不饱和羧酸，氨基(C1-C6烷基)-、单(C1-C6烷氨基)(C1-C6烷基)-和双(C1-C6烷氨基)(C1-C6烷基)-丙烯酸酯和甲基丙烯酸酯、烯丙醇、3-三甲基铵甲基丙烯酸2-羟丙基酯氯化物、二甲基氨乙基甲基丙烯酸酯(DMAEMA)、二甲基氨乙基甲基丙烯酰胺、甘油甲基丙烯酸酯、N-(1,1-二甲基-3-氧代丁基)丙烯酰胺、环亚氨基醚、乙烯基醚、包含环氧衍生物的环醚、环不饱和醚、N-取代环乙亚胺、β-内酯和β-内酰胺、乙烯酮缩醛、乙烯基缩醛和正膦。The polymer film may comprise a polymer selected from the group consisting of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene , polyisoprene, polybutadiene, polyvinyl chloride, polyalkylacrylate (PAA), polyalkylmethacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylic acid Esters, polysulfones, polyvinyl ethers, poly(propylene oxide) and their copolymers, radical-substituted C1-C6 alkyl acrylates and methacrylates, acrylamides, methacrylamides, (C1-C6 alkyl ) acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylated acrylates and methacrylates, polyethylene glycol monomethacrylate and polyethylene glycol monomethyl ether Hydroxy-substituted (C1-C6 alkyl) acrylamides and methacrylamides, hydroxyl-substituted C1-C6 alkyl vinyl ethers, sodium vinyl sulfonate, sodium styryl sulfonate, 2-propene Amide-2-methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4'-dialkyloxazolinyl -5-keto, 2,4-vinylpyridine, ethylenically unsaturated carboxylic acid with a total of 3-5 carbon atoms, amino(C1-C6 alkyl)-, mono(C1-C6 alkylamino)(C1- C6 alkyl)- and bis(C1-C6 alkylamino)(C1-C6 alkyl)-acrylates and methacrylates, allyl alcohol, 3-trimethylammonium methacrylate, 2-hydroxypropyl chloride compound, dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl ) acrylamides, cyclic imino ethers, vinyl ethers, cyclic ethers containing epoxy derivatives, cyclounsaturated ethers, N-substituted ethyleneimines, β-lactones and β-lactams, ketene acetals, Vinyl acetal and phosphorane.
在一种优选的实施例中，第一电压的起始电压小于破膜电压；优选地，第一电压的起始电压为0.01～0.8V，进一步优选为0.02-0.05V；优选地，第一电压的终止电压小于破膜电压；优选地，终止电压包括0.15～0.8V，包括0.15、0.2、0.25、0.3、0.4、0.5、0.6、0.7或0.8V，进一步优选为0.18V。In a preferred embodiment, the initial voltage of the first voltage is lower than the rupture voltage; preferably, the initial voltage of the first voltage is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the first The end voltage of the voltage is less than the rupture voltage; preferably, the end voltage includes 0.15-0.8V, including 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8V, more preferably 0.18V.
本申请中的第一电压为递增电压，其起始电压和终止电压均应小于破膜电压，防止施加的电压超过膜材所能承受的最大电压，导致膜材被电击穿，造成纳米孔生物传感器被破坏。第一电压的起始电压为0.02-0.05V，终止电压为0.18V，在第一电压此范围内进行递增，能够使纳米通道蛋白在第一电压的作用下***膜材中，制备获得纳米孔生物传感器。利用上述制备方法制备的纳米孔生物传感器的稳定性高，在使用中纳米通道蛋白不易从膜材中滑脱。可使用的时间长，有助于提高测序通量。The first voltage in this application is an increasing voltage, and its initial voltage and termination voltage should be less than the membrane rupture voltage, so as to prevent the applied voltage from exceeding the maximum voltage that the membrane can withstand, causing the membrane to be electrically broken down, resulting in nanopores The biosensor is destroyed. The initial voltage of the first voltage is 0.02-0.05V, and the end voltage is 0.18V. Increasing the first voltage within this range can make the nanochannel protein insert into the membrane material under the action of the first voltage, and prepare nanopores. biological sensor. The nanopore biosensor prepared by the above preparation method has high stability, and the nanochannel protein is not easy to slip off from the membrane material during use. It can be used for a long time, which helps to increase the sequencing throughput.
在一种优选的实施例中，第一电压为阶梯上升或持续上升的电压；优选地，阶梯上升包括每10min增加0.01～0.8V，更优选0.01～0.4V，包括但不限于0.01、0.02、0.04、0.06、0.08、0.1、0.15、0.2、0.25、0.3、0.35、0.4V、0.45V、0.5V、0.55V、0.6V、0.65V、0.7V、0.75V或0.8V；优选地，第一电压为从起始电压开始，按照每10min增加0.04V的递增速度递增至终止电压；优选地，纳米孔生物传感器的电导值大于1nS时，停止施加第一电压。In a preferred embodiment, the first voltage is a step-up or continuously rising voltage; preferably, the step-up includes increasing 0.01-0.8V every 10 minutes, more preferably 0.01-0.4V, including but not limited to 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4V, 0.45V, 0.5V, 0.55V, 0.6V, 0.65V, 0.7V, 0.75V or 0.8V; preferably, the first The voltage starts from the initial voltage and increases to the final voltage at an increasing rate of 0.04V every 10min; preferably, when the conductance value of the nanopore biosensor is greater than 1nS, the application of the first voltage is stopped.
第一电压包括但不限于阶梯上升的电压(即阶梯式递增的电压)或持续上升的电压(即线性递增的电压)。若第一电压以阶梯上升的形式进行电压的递增时，具体递增的速度并无特别限制，上述优选实施例中，在阶梯上升时每10min增加0.04V，从起始电压阶梯上升至终止电压。在对膜材和纳米通道蛋白施加第一电压的过程中，对通道的电流和电压利用膜片钳放大器或其他数据采集获取装置进行监测，当电导值(电导值＝电流÷电压)大于1nS时，即说明纳米通道蛋白已经成功***膜材中，停止施加第一电压，无需继续施加第一电压至终止电压。The first voltage includes, but is not limited to, a step-up voltage (ie, a stepwise increasing voltage) or a continuously increasing voltage (ie, a linearly increasing voltage). If the first voltage is increased in steps, the specific increase speed is not particularly limited. In the above preferred embodiment, the step is increased by 0.04V every 10 minutes, from the initial voltage to the end voltage. In the process of applying the first voltage to the membrane material and nanochannel protein, the current and voltage of the channel are monitored by a patch clamp amplifier or other data acquisition and acquisition devices, when the conductance value (conductance value = current ÷ voltage) is greater than 1nS , which means that the nanochannel protein has been successfully inserted into the membrane material, the application of the first voltage is stopped, and there is no need to continue to apply the first voltage to the stop voltage.
在一种优选的实施例中，在加入纳米通道蛋白、施加第一电压前，制备方法还包括对膜材施加第二电压，第二电压包括第一周期性电压和第一恒定电压；优选地，第一周期性电压包括第一方波电压或第一三角波电压；优选地，第一方波电压的高电平小于或等于破膜电压；优选地，第一方波电压的高电平为0.01～0.8V，进一步优选为0.02～0.05V(具体可以是0.01V、0.02V、0.04V、0.06V、0.08V、0.1V、0.15V、0.2V、0.25V、0.3V、0.35V、0.4V、0.45V、0.5V、0.55V、0.6V、0.65V、0.7V、0.75V或0.8V)；优选地，第一方波电压的低电平为负；优选地，第一方波电压的频率为0.1～100Hz，进一步优选为0.5Hz。In a preferred embodiment, before adding the nanochannel protein and applying the first voltage, the preparation method also includes applying a second voltage to the membrane material, and the second voltage includes the first periodic voltage and the first constant voltage; preferably , the first periodic voltage includes a first square wave voltage or a first triangular wave voltage; preferably, the high level of the first square wave voltage is less than or equal to the membrane rupture voltage; preferably, the high level of the first square wave voltage is 0.01 to 0.8V, more preferably 0.02 to 0.05V (specifically 0.01V, 0.02V, 0.04V, 0.06V, 0.08V, 0.1V, 0.15V, 0.2V, 0.25V, 0.3V, 0.35V, 0.4V V, 0.45V, 0.5V, 0.55V, 0.6V, 0.65V, 0.7V, 0.75V or 0.8V); preferably, the low level of the first square wave voltage is negative; preferably, the first square wave voltage The frequency is 0.1-100 Hz, more preferably 0.5 Hz.
在一种优选的实施例中，第一恒定电压为0.01～0.8V(具体可以是0.01V、0.02V、0.04V、0.06V、0.08V、0.1V、0.15V、0.2V、0.25V、0.3V、0.35V、0.4V、0.45V、0.5V、0.55V、0.6V、0.65V、0.7V、0.75V或0.8V)，更优选为0.02～0.05V；优选地，对膜材施加第二电压包括：对膜材先施加第一周期性电压，然后再施加第一恒定电压，优选地，第一周期性电压进行1～100个周期，包括但不限于1、2、3、5、10、20、30、40、50、60、70、80、90或100个周期，第一恒定电压持续1～100s，包括但不限于1、2、5、10、14、20、30、40、50、60、70、80、90或100s；优选地，对膜材重复多次施加第二电压，优选重复1～100次，包括但不限于重复1、2、3、5、10、20、30、40、50、60、70、80、90或100次。In a preferred embodiment, the first constant voltage is 0.01-0.8V (specifically, it can be 0.01V, 0.02V, 0.04V, 0.06V, 0.08V, 0.1V, 0.15V, 0.2V, 0.25V, 0.3 V, 0.35V, 0.4V, 0.45V, 0.5V, 0.55V, 0.6V, 0.65V, 0.7V, 0.75V or 0.8V), more preferably 0.02-0.05V; preferably, apply a second The voltage includes: first applying the first periodic voltage to the membrane material, and then applying the first constant voltage. Preferably, the first periodic voltage is performed for 1 to 100 cycles, including but not limited to 1, 2, 3, 5, 10 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 cycles, the first constant voltage lasts for 1-100s, including but not limited to 1, 2, 5, 10, 14, 20, 30, 40, 50, 60, 70, 80, 90 or 100s; preferably, the second voltage is repeatedly applied to the membrane material, preferably 1 to 100 times, including but not limited to 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times.
在加入纳米通道蛋白、施加第一电压前，该制备方法包括对已经成膜的膜材施加第二电压，刺激膜材使其更加具有柔性，利于后续将纳米通道蛋白***膜材中，从而使最终制备获得的纳米孔生物传感器的稳定性提高，在使用中纳米通道蛋白不易从膜材中滑脱。上述第二电压包括第一周期性电压和第一恒定电压。Before adding the nanochannel protein and applying the first voltage, the preparation method includes applying a second voltage to the membrane material that has been formed to stimulate the membrane material to make it more flexible, which is beneficial for the subsequent insertion of the nanochannel protein into the membrane material, so that The stability of the finally prepared nanopore biosensor is improved, and the nanochannel protein is not easy to slip from the membrane material during use. The above-mentioned second voltage includes a first periodic voltage and a first constant voltage.
第一周期性电压包括但不限于第一方波电压或第一三角波电压。利用周期性电压刺激膜材使之更有柔性，更利于纳米通道蛋白进行插孔。为了进一步提高所制备的纳米孔生物传感器的稳定性，将第一方波电压的高电平(即最大电压)控制在小于破膜电压，即高电平为0.01～0.8V的范围内，进一步优选可以为0.02～0.05V的范围内。第一方波电压的低电平可以为负，即低电平的电压为高电平电压的负数。第一方波电压包括但不限于0.1、0.3、0.5、1、2、5、10、20、30、50、70或100Hz。优选地，第一方波电压的频率为0.5Hz，即第一方波电压的一个周期为2s，在一个周期内，高电平持续1s，低电平持续1s。在第二电压中，还包括第一恒定电压，为0.02～0.05V。在对膜材施加第二电压时，第二电压包括：先对膜材试剂施加第一周期电压，再施加恒定电压，2种电压进行多次循环，从而使膜材更具有柔性。The first periodic voltage includes but is not limited to a first square wave voltage or a first triangular wave voltage. The periodic voltage is used to stimulate the membrane material to make it more flexible, which is more conducive to the insertion of nanochannel proteins. In order to further improve the stability of the prepared nanopore biosensor, the high level (i.e. the maximum voltage) of the first square wave voltage is controlled to be less than the membrane rupture voltage, i.e. the high level is within the range of 0.01-0.8V, further Preferably, it may be in the range of 0.02 to 0.05V. The low level of the first square wave voltage can be negative, that is, the low level voltage is a negative number of the high level voltage. The first square wave voltage includes but is not limited to 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100 Hz. Preferably, the frequency of the first square wave voltage is 0.5 Hz, that is, one period of the first square wave voltage is 2s, and within one period, the high level lasts for 1s, and the low level lasts for 1s. The second voltage also includes a first constant voltage, which is 0.02-0.05V. When applying the second voltage to the membrane material, the second voltage includes: first applying the first cycle voltage to the membrane material reagent, and then applying a constant voltage, and the two voltages are cycled for many times, so that the membrane material is more flexible.
在一种优选的实施例中，在形成具有纳米孔通道的纳米孔生物传感器之后，该制备方法 进一步包括：对纳米孔生物传感器施加第三电压，第三电压包括第二周期性电压和第二恒定电压；优选地，纳米孔生物传感器的电导值大于1nS时，对纳米孔生物传感器施加第三电压；优选地，第二周期性电压包括第二方波电压或第二三角波电压；优选地，第二方波电压的高电平为0.01～0.8V，进一步优选为0.02～0.05V；优选地，第二方波电压的低电平为负；优选地，第二方波电压的频率为0.1～100Hz，第二方波电压包括但不限于0.1、0.3、0.5、1、2、5、10、20、30、50、70或100Hz，进一步优选为0.5Hz；优选地，第二恒定电压为0.02～0.05V；优选地，对纳米孔生物传感器施加第三电压包括：对纳米孔生物传感器先施加第二周期性电压，然后再施加第二恒定电压；优选地，第二周期性电压重复1～100.次，包括但不限于重复1、2、3、5、10、20、30、40、50、60、70、80、90或100次，第二恒定电压持续1～100.s，包括但不限于1、2、5、10、14、20、30、40、50、60、70、80、90或100s；优选地，对纳米孔生物传感器重复多次施加第三电压，优选地，重复1～100次，包括但不限于重复1、2、3、5、10、20、30、40、50、60、70、80、90或100次。In a preferred embodiment, after forming the nanopore biosensor with the nanopore channel, the preparation method further includes: applying a third voltage to the nanopore biosensor, the third voltage includes the second periodic voltage and the second Constant voltage; preferably, when the conductance value of the nanopore biosensor is greater than 1nS, a third voltage is applied to the nanopore biosensor; preferably, the second periodic voltage includes a second square wave voltage or a second triangular wave voltage; preferably, The high level of the second square wave voltage is 0.01-0.8V, more preferably 0.02-0.05V; preferably, the low level of the second square wave voltage is negative; preferably, the frequency of the second square wave voltage is 0.1 ~100Hz, the second square wave voltage includes but not limited to 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100Hz, more preferably 0.5Hz; preferably, the second constant voltage is 0.02-0.05V; preferably, applying the third voltage to the nanopore biosensor includes: first applying a second periodic voltage to the nanopore biosensor, and then applying a second constant voltage; preferably, repeating the second periodic voltage for 1 ～100 times, including but not limited to repeating 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times, the second constant voltage lasts for 1～100.s, Including but not limited to 1, 2, 5, 10, 14, 20, 30, 40, 50, 60, 70, 80, 90 or 100s; preferably, repeatedly applying the third voltage to the nanopore biosensor, preferably , repeating 1 to 100 times, including but not limited to repeating 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times.
在纳米通道蛋白成功***膜材、形成纳米孔生物传感器后，进一步对纳米孔生物传感器施加第三电压，更有利于稳定纳米通道蛋白，使最终制备获得的纳米孔生物传感器的稳定性提高，进而在使用中纳米通道蛋白不易从膜材中滑脱，提高可使用时间及测序通量。在纳米孔生物传感器的电导值大于1nS时，即表明纳米通道蛋白成功***膜材，在此时停止对纳米孔生物传感器施加第一电压，开始施加第三电压。第三电压包括第二周期性电压和第二恒定电压。第二周期性电压包括但不限于第二方波电压或第二三角波电压。利用有周期性的电压和恒定电压共同作用，能够稳定***膜材中的纳米通道蛋白。After the nanochannel protein is successfully inserted into the membrane material to form a nanopore biosensor, further applying a third voltage to the nanopore biosensor is more conducive to stabilizing the nanochannel protein and improving the stability of the final prepared nanopore biosensor. In use, the nanochannel protein is not easy to slip out of the membrane material, which improves the usable time and sequencing throughput. When the conductance value of the nanopore biosensor is greater than 1 nS, it indicates that the nanochannel protein is successfully inserted into the membrane material. At this time, stop applying the first voltage to the nanopore biosensor and start applying the third voltage. The third voltage includes a second periodic voltage and a second constant voltage. The second periodic voltage includes but is not limited to a second square wave voltage or a second triangular wave voltage. The combination of periodic voltage and constant voltage can stabilize the nanochannel protein inserted into the membrane material.
为了更利于纳米通道蛋白进行插孔，第二方波电压的高电平(即最大电压)需小于破膜电压，高电平为0.01～0.8V，优选为0.02～0.05V。第二方波电压的低电平可以为负，即低电平的电压为高电平电压的负数。第二方波电压包括但不限于0.1、0.3、0.5、1、2、5、10、20、30、50、70或100Hz。优选地，第二方波电压的频率为0.5Hz，即第二方波电压的一个周期为2s，在一个周期内，高电平持续1s，低电平持续1s。在第三周期电压中还包括第二恒定电压，第二恒定电压为0.01～0.8V，优选为0.02～0.05V，利用周期电压和恒定电压共同作用，使纳米孔生物传感更加稳定。In order to facilitate the insertion of the nanochannel protein, the high level (ie, the maximum voltage) of the second square wave voltage needs to be lower than the membrane rupture voltage, and the high level is 0.01-0.8V, preferably 0.02-0.05V. The low level of the second square wave voltage can be negative, that is, the low level voltage is a negative number of the high level voltage. The second square wave voltage includes but is not limited to 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100 Hz. Preferably, the frequency of the second square wave voltage is 0.5 Hz, that is, one period of the second square wave voltage is 2s, and within one period, the high level lasts for 1s, and the low level lasts for 1s. The third cycle voltage also includes a second constant voltage, the second constant voltage is 0.01-0.8V, preferably 0.02-0.05V, and the combination of the cycle voltage and the constant voltage makes the nanopore biosensing more stable.
在一种优选的实施例中，在对纳米孔生物传感器重复多次施加第三电压之后，制备方法还包括：对纳米孔生物传感器持续施加第四电压，第四电压为0.01～0.8V，优选为0.02～0.05V。In a preferred embodiment, after repeatedly applying the third voltage to the nanopore biosensor, the preparation method further includes: continuously applying a fourth voltage to the nanopore biosensor, the fourth voltage is 0.01-0.8V, preferably 0.02 ~ 0.05V.
第三和第四电压的两个作用包括：(1)防止更多纳米通道蛋白***同一个磷脂膜中，导致多孔。如果一个磷脂膜上出现多个纳米通道蛋白，无法用作检测；(2)对构建好的纳米孔生物传感器起到稳定作用，降低滑脱率。在施加第三电压之前，纳米孔生物传感器已制备完成。The two effects of the third and fourth voltages include: (1) preventing more nanochannel proteins from inserting into the same phospholipid membrane, resulting in porosity. If multiple nanochannel proteins appear on a phospholipid membrane, it cannot be used for detection; (2) it can stabilize the constructed nanopore biosensor and reduce the slippage rate. Before applying the third voltage, the nanopore biosensor has been prepared.
在本申请第二种典型的实施方式中，提供了一种利用上述制备方法制备的纳米孔生物传感器，该纳米孔生物传感器在0.18V恒定电压作用1小时后，纳米通道蛋白的滑脱率小于<10％。In the second typical embodiment of the present application, a nanopore biosensor prepared by the above preparation method is provided. After the nanopore biosensor is subjected to a constant voltage of 0.18V for 1 hour, the slippage rate of the nanochannel protein is less than < 10%.
利用上述制备方法制备的纳米孔生物传感器，稳定性好，***膜材中的纳米通道蛋白在使用中、即恒定电压作用下，不易从膜材中滑脱，能够持续发挥作用的平均时间长。该纳米孔生物传感器在0.18V恒定电压作用1小时后，纳米通道蛋白的滑脱率小于<10％。而利用现有的恒定电压技术制备的纳米孔生物传感器，在同等条件下的纳米通道蛋白的滑脱率大于30％。The nanopore biosensor prepared by the above preparation method has good stability, and the nanochannel protein inserted into the membrane material is not easy to slip from the membrane material during use, that is, under the action of a constant voltage, and can continue to function for a long average time. After the nanopore biosensor acts on a constant voltage of 0.18V for 1 hour, the slipping rate of the nanochannel protein is less than <10%. However, in the nanopore biosensor prepared by the existing constant voltage technology, the slippage rate of the nanochannel protein is greater than 30% under the same conditions.
在本申请第三种典型的实施方式中，提供了一种测序方法，该测序方法包括：利用上述制备方法制备纳米孔生物传感器；利用纳米孔生物传感器对待测物质进行单分子测序。In the third typical embodiment of the present application, a sequencing method is provided, the sequencing method comprising: using the above-mentioned preparation method to prepare a nanopore biosensor; using the nanopore biosensor to perform single-molecule sequencing of a substance to be tested.
在一种优选的实施例中，待测物质选自小分子、DNA、RNA、多肽或蛋白质。In a preferred embodiment, the substance to be tested is selected from small molecules, DNA, RNA, polypeptides or proteins.
利用上述制备方法制备的纳米孔生物传感器，能够应用在相应装置中，对待测物质进行单分子测序。待测物质包括小分子、DNA、RNA、多肽或蛋白质。The nanopore biosensor prepared by the above preparation method can be applied in a corresponding device to perform single-molecule sequencing of the substance to be tested. Species to be tested include small molecules, DNA, RNA, peptides or proteins.
需要说明的是，本申请的前述纳米孔生物传感器的制备方法中将纳米孔通道蛋白***膜材中的步骤，与将蛋白药物包裹于脂质体载体中形成脂质体药物的原理类似，因而，在本申请第四种典型的实施方式中，提供了一种脂质体药物的制备方法，该制备方法包括：在递增电压的作用下，使药物***脂质膜内形成脂质体药物，药物为蛋白药物。通过在递增电压的作用下，更有利于蛋白药物稳定***脂质膜内而不容易滑出，进而利于提高将蛋白药物制备成脂质体药物的可靠性，从而便于提高递送效率。It should be noted that the step of inserting the nanopore channel protein into the membrane material in the aforementioned preparation method of the nanopore biosensor of the present application is similar to the principle of encapsulating the protein drug in the liposome carrier to form the liposome drug, thus , in the fourth typical embodiment of the present application, a preparation method of a liposome drug is provided, the preparation method comprising: under the action of increasing voltage, inserting the drug into the lipid membrane to form a liposome drug, The drug is a protein drug. Under the action of increasing voltage, it is more conducive to the stable insertion of protein drugs into the lipid membrane and not easy to slip out, which in turn helps to improve the reliability of preparing protein drugs into liposome drugs, thereby facilitating the improvement of delivery efficiency.
具体的蛋白药物可以是任何结构形式的蛋白药物或多肽药物。优选地，该药物为抗体药物。The specific protein drug can be protein drug or polypeptide drug in any structural form. Preferably, the drug is an antibody drug.
用作脂质递送载体的脂质膜均适用于本申请，比如可以是磷脂膜，磷脂单分子膜，或磷脂双分子膜，更优选为磷脂膜形成的囊泡。磷脂膜的具体类别示例如下：选自二植烷酰基-磷脂酰胆碱(DPhPC)、1,2-二植烷酰基-sn-甘油-3-磷酸胆碱、1,2-二-O-植烷酰基-sn-甘油-3-磷酸胆碱(DoPhPC)、棕榈酰基-油酰基-磷脂酰胆碱(POPC)、二油酰基-磷脂酰-甲基酯(DOPME)、二棕榈酰基磷脂酰胆碱(DPPC)、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酸、磷脂酰肌醇、磷脂酰甘油、鞘磷脂、1,2-二-O-植烷酰基-sn-甘油、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-350]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-550]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-750]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-1000]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-2000]、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-乳糖酰基、GM1神经节苷脂、溶血磷脂酰胆碱(LPC)或其任意组合。本申请中利用的磷脂双分子膜DPHPC(4ME：16PC)购买于avanti polar lipids公司，货号：850356，化学名称：1,2-diphytanoyl-sn-glycero-3-phosphocholine。Lipid membranes used as lipid delivery carriers are suitable for this application, such as phospholipid membranes, phospholipid monomolecular membranes, or phospholipid bimolecular membranes, more preferably vesicles formed by phospholipid membranes. Specific classes of phospholipid membranes are exemplified as follows: selected from diphytanyl-phosphatidylcholine (DPhPC), 1,2-diphytanyl-sn-glycero-3-phosphocholine, 1,2-di-O- Phytanoyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), dipalmitoylphosphatidyl Choline (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanyl-sn-glycerol, 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1 ,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N -[methoxy(polyethylene glycol)-2000], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactoyl, GM1 ganglioside, lysophosphatidylcholine (LPC ) or any combination thereof. The phospholipid bimolecular membrane DPHPC (4ME: 16PC) utilized in this application was purchased from avanti polar lipids company, article number: 850356, chemical name: 1,2-diphytanoyl-sn-glycero-3-phosphocholine.
上述递增电压的起始电压和终止电压均应小于脂质膜的破膜电压，防止施加的电压超过脂质膜所能承受的最大电压，导致脂质膜被电击穿，造成脂质体药物被破坏。递增电压的起始电压为0.02-0.1V，优选为0.02-0.05V，终止电压为0.18V，在此范围内进行递增，能够使蛋白药物更容易***脂质膜中且不易滑脱，从而使包裹的药物稳定性更好。The initial voltage and termination voltage of the above-mentioned incremental voltage should be less than the membrane rupture voltage of the lipid membrane, so as to prevent the applied voltage from exceeding the maximum voltage that the lipid membrane can withstand, causing the lipid membrane to be electrically broken down, causing liposome drug destroyed. The initial voltage of the incremental voltage is 0.02-0.1V, preferably 0.02-0.05V, and the end voltage is 0.18V. Increasing within this range can make it easier for the protein drug to be inserted into the lipid membrane and not easy to slip off, so that the package better drug stability.
上述递增电压可以为阶梯上升的电压(即阶梯式递增的电压)，也可以是持续上升的电压(即线性递增的电压)。若以阶梯上升的形式进行电压的递增时，具体递增的速度并无特别限制，上述优选实施例中，在阶梯上升时每10min增加0.04V，从起始电压阶梯上升至终止电压。在施加递增电压的过程中，对脂质膜的电流和电压利用膜片钳放大器或其他数据采集获取装置进行监测，当电导值(电导值＝电流÷电压)大于1nS时，即说明蛋白药物已经成功***脂质膜中，停止施加递增电压，无需继续施加该递增电压。The aforementioned increasing voltage may be a step-up voltage (ie, a stepwise increasing voltage), or a continuously increasing voltage (ie, a linearly increasing voltage). If the voltage is increased incrementally in the form of a step increase, there is no particular limitation on the specific increase speed. In the above preferred embodiment, the voltage is increased by 0.04V every 10 minutes during the step increase, from the initial voltage step to the end voltage. In the process of applying increasing voltage, the current and voltage of the lipid membrane are monitored by a patch clamp amplifier or other data acquisition and acquisition devices. When the conductance value (conductance value=current ÷ voltage) is greater than 1nS, it means that the protein drug has been Upon successful insertion into the lipid membrane, the application of the incremental voltage is stopped and does not need to be continued.
为了进一步提高脂质膜对蛋白药物的包裹性能，在一些优选的实施例中，在使药物***脂质膜内形成脂质体药物前，该制备方法还包括：对脂质膜进行柔性处理；优选地，采用对脂质膜交替施加周期性电压(比如方波电压或三角波电压)与恒定电压的方式进行柔性处理。In order to further improve the encapsulation performance of the lipid film on the protein drug, in some preferred embodiments, before the drug is inserted into the lipid film to form a liposome drug, the preparation method further includes: performing a flexible treatment on the lipid film; Preferably, the flexible treatment is performed by alternately applying periodic voltage (such as square wave voltage or triangular wave voltage) and constant voltage to the lipid membrane.
在一些优选的实施例中，对脂质膜先施加周期性电压(比如方波电压或三角波电压)，然后再施加恒定电压，优选地，周期性电压进行1～100个周期，包括但不限于1、2、3、5、10、20、30、40、50、60、70、80、90或100个周期，恒定电压持续1～100s，包括但不限于1、2、5、10、14、20、30、40、50、60、70、80、90或100s；优选地，对脂质膜重复多次交替施加周期性电压和恒定电压，优选重复1～100次，包括但不限于重复1、2、3、5、10、20、30、40、50、60、70、80、90或100次。In some preferred embodiments, a periodic voltage (such as square wave voltage or triangular wave voltage) is first applied to the lipid membrane, and then a constant voltage is applied. Preferably, the periodic voltage is carried out for 1 to 100 cycles, including but not limited to 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cycles, constant voltage for 1-100s, including but not limited to 1, 2, 5, 10, 14 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 s; preferably, repeatedly apply periodic voltage and constant voltage to the lipid membrane repeatedly, preferably repeating 1 to 100 times, including but not limited to repeated 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times.
利用周期性电压刺激脂质膜更有柔性，更利于蛋白药物***脂质膜中。为了进一步提高所制备的脂质体药物的稳定性，在一种优选的实施例中，将方波电压的高电平(即最大电压)控制在小于破膜电压，即高电平为0.01～0.8V的范围内，进一步优选可以为0.02～0.05V的范围内。方波电压的低电平可以为负，即低电平的电压为高电平电压的负数。方波电压包括但不限于0.1、0.3、0.5、1、2、5、10、20、30、50、70或100Hz。优选地，方波电压的频率为0.5Hz，即第一方波电压的一个周期为2s，在一个周期内，高电平持续1s，低电平持续1s。Using periodic voltage to stimulate the lipid membrane to be more flexible is more conducive to the insertion of protein drugs into the lipid membrane. In order to further improve the stability of the prepared liposome drug, in a preferred embodiment, the high level (i.e. the maximum voltage) of the square wave voltage is controlled to be less than the membrane rupture voltage, i.e. the high level is 0.01～ It is within the range of 0.8V, more preferably within the range of 0.02 to 0.05V. The low level of the square wave voltage can be negative, that is, the low level voltage is the negative number of the high level voltage. Square wave voltages include, but are not limited to, 0.1, 0.3, 0.5, 1, 2, 5, 10, 20, 30, 50, 70 or 100 Hz. Preferably, the frequency of the square wave voltage is 0.5 Hz, that is, one period of the first square wave voltage is 2s, and within one period, the high level lasts for 1s, and the low level lasts for 1s.
在本发明的第五个方面，提供了一种药物的递送方法，递送方法包括：将药物上述制备方法制备成脂质体药物；将药物以脂质体药物的形式递送入细胞内。上述将蛋白类药物按照与纳米孔蛋白插孔步骤类似的方式***脂质膜上形成脂质体药物后，便可以将该蛋白类药物以常规脂质体的形式递送入细胞内(比如，与细胞膜融合，以细胞内吞的方式进入细胞内等)。In the fifth aspect of the present invention, a drug delivery method is provided. The delivery method includes: preparing the drug into a liposome drug by the above preparation method; and delivering the drug into cells in the form of liposome drug. After the above-mentioned protein drug is inserted into the lipid membrane to form a liposome drug in a manner similar to the nanopore protein insertion step, the protein drug can be delivered into the cell in the form of a conventional liposome (for example, with Cell membrane fusion, endocytosis into the cell, etc.).
在另一些优选的实施例中，基于电压刺激的方式将药物以脂质体药物的形式递送入细胞内。电压刺激的方式有助于提高入胞效率。In other preferred embodiments, the drug is delivered into the cell in the form of liposome drug based on voltage stimulation. The way of voltage stimulation helps to improve the efficiency of cell entry.
下面将结合具体的实施例来进一步详细解释本申请的有益效果。The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.
实施例1Example 1
首先通过第二电压刺激磷脂膜DPHPC(4ME：16PC)使其更加柔性以促进插孔，成膜后施加±0.02V电压各1s，共三个周期；然后施加0.02V恒定电压持续14s。重复该上述步骤30次。First, the phospholipid membrane DPHPC (4ME: 16PC) was stimulated by the second voltage to make it more flexible to promote the insertion. After the film was formed, a voltage of ±0.02V was applied for 1s each, for a total of three cycles; then a constant voltage of 0.02V was applied for 14s. This above-mentioned step was repeated 30 times.
加入纳米通道蛋白CsgG突变体(氨基酸序列由SEQ ID NO：1所示)至膜的一侧，施加第一电压。第一电压为：起始电压0.02V，终止电压0.18V，每10min增加0.04V。Add the nanochannel protein CsgG mutant (amino acid sequence shown by SEQ ID NO: 1) to one side of the membrane, and apply the first voltage. The first voltage is: an initial voltage of 0.02V, an end voltage of 0.18V, and an increase of 0.04V every 10 minutes.
当该通道的电导值(电导＝电流/电压)大于1nS，则对该通道施加第三电压：±0.02V电压各1s，共三个周期；然后施加0.02V恒定电压持续14s。重复以上步骤30次后持续给0.02V。When the conductance value of the channel (conductance=current/voltage) is greater than 1nS, a third voltage is applied to the channel: ±0.02V voltage for 1s each, for a total of three cycles; then a constant voltage of 0.02V is applied for 14s. Continue to supply 0.02V after repeating the above steps 30 times.
施加0.18V电压，持续观察纳米孔通道变化情况。34个纳米孔生物传感器中有3个纳米孔生物传感器的纳米通道蛋白在1小时内滑出，滑出比例8.8％，见图3。Apply a voltage of 0.18V, and continuously observe the change of the nanopore channel. Among the 34 nanopore biosensors, the nanochannel proteins of 3 nanopore biosensors slipped out within 1 hour, and the slipping ratio was 8.8%, as shown in FIG. 3 .
CsgG跨膜蛋白突变体氨基酸序列为SEQ ID NO：1：The amino acid sequence of the CsgG transmembrane protein mutant is SEQ ID NO: 1:
Figure PCTCN2021143407-appb-000001
Figure PCTCN2021143407-appb-000001
实施例2Example 2
首先通过第二电压刺激磷脂膜DPHPC(4ME：16PC)使其更加柔性以促进插孔，成膜后施加±0.05V电压各1s，共三个周期；然后施加0.05V恒定电压持续14s。重复该上述步骤30次。First, the phospholipid membrane DPHPC (4ME: 16PC) was stimulated by the second voltage to make it more flexible to promote the insertion. After the film was formed, a voltage of ±0.05V was applied for 1s each, for a total of three cycles; then a constant voltage of 0.05V was applied for 14s. This above-mentioned step was repeated 30 times.
加入纳米通道蛋白CsgG突变体至膜的一侧，施加第一电压。第一电压为：起始电压0.05V，终止电压0.18V，每10min增加0.04V。The nanochannel protein CsgG mutant is added to one side of the membrane and a first voltage is applied. The first voltage is: an initial voltage of 0.05V, an end voltage of 0.18V, and an increase of 0.04V every 10 minutes.
当该通道的电导值(电导＝电流/电压)大于1nS，则对该通道施加第三电压：±0.05V电压各1s，共三个周期；然后施加0.05V恒定电压持续14s；重复以上步骤30次后持续给0.02V。When the conductance value of the channel (conductance=current/voltage) is greater than 1nS, apply a third voltage to the channel: ±0.05V voltage for 1s each, for a total of three cycles; then apply a constant voltage of 0.05V for 14s; repeat the above steps for 30 Continue to give 0.02V after the first time.
施加0.18V电压，持续观察纳米孔通道变化情况。图1示出了根据本发明实施例1或2的构建纳米孔生物传感器的构建流程图。119个纳米孔生物传感器中有2个纳米孔生物传感器的纳米通道蛋白在1小时内滑出，滑出比例1.7％，见图3。Apply a voltage of 0.18V, and continuously observe the change of the nanopore channel. FIG. 1 shows a flow chart of constructing a nanopore biosensor according to Embodiment 1 or 2 of the present invention. Among the 119 nanopore biosensors, the nanochannel proteins of 2 nanopore biosensors slipped out within 1 hour, and the slipping ratio was 1.7%, as shown in FIG. 3 .
对比例1Comparative example 1
恒定电压插孔Constant voltage jack
在电解池中央的小孔处形成磷脂双分子层膜DPHPC(4ME：16PC)后，在膜的一侧加入制备好的纳米通道蛋白CsgG突变体，施加恒定电压0.18V，促进通道蛋白与磷脂双分子层膜融合形成纳米孔生物传感器。After the phospholipid bilayer membrane DPHPC (4ME: 16PC) was formed at the small hole in the center of the electrolytic cell, the prepared nanochannel protein CsgG mutant was added to one side of the membrane, and a constant voltage of 0.18V was applied to promote the bilayer of the channel protein and phospholipid. Molecular layer membrane fusion to form nanoporous biosensors.
施加0.18V电压，持续观察纳米孔通道变化情况。在恒定电压插孔的79个纳米孔生物传感器中，有24个纳米孔生物传感器的纳米通道蛋白在1小时内出现滑出，比例为30.4％，见图3。Apply a voltage of 0.18V, and continuously observe the change of the nanopore channel. Among the 79 nanopore biosensors inserted with constant voltage, the nanochannel proteins of 24 nanopore biosensors slipped out within 1 hour, the ratio was 30.4%, as shown in FIG. 3 .
图2表示一个纳米通道蛋白从磷脂膜上滑出，造成检测失败，滑出后电流小于10pA(电压:0.18V)。Figure 2 shows that a nanochannel protein slides out from the phospholipid membrane, causing detection failure, and the current is less than 10pA (voltage: 0.18V) after the slide out.
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本发明利用第一、第二和第三电压制备纳米孔生物传感器，能够明显增加纳米孔生物传感器的稳定性，降低其上纳米通道蛋白的滑脱率。From the above description, it can be seen that the above-mentioned embodiments of the present invention have achieved the following technical effects: the present invention utilizes the first, second and third voltages to prepare nanopore biosensors, which can significantly increase the stability of nanopore biosensors , reducing the slippage rate of nanochannel proteins on it.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (14)

  1. 一种纳米孔生物传感器的制备方法，其特征在于，所述制备方法包括：A preparation method of a nanopore biosensor, characterized in that the preparation method comprises:
    在第一电压的作用下，使纳米通道蛋白***膜材内，形成具有纳米孔通道的所述纳米孔生物传感器，所述第一电压为递增电压。Under the action of the first voltage, the nanochannel protein is inserted into the membrane material to form the nanopore biosensor with a nanopore channel, and the first voltage is an increasing voltage.
  2. 根据权利要求1所述的制备方法，其特征在于，所述膜材包括磷脂双分子膜或高分子聚合物膜；The preparation method according to claim 1, wherein the membrane material comprises a phospholipid bimolecular membrane or a polymer membrane;
    优选地，所述膜材的破膜电压大于0.8V，所述破膜电压为击破所述膜材的最小电压；Preferably, the membrane rupture voltage of the membrane material is greater than 0.8V, and the membrane rupture voltage is the minimum voltage for breaking the membrane material;
    优选地，所述磷脂双分子膜的组成包括DPHPC(4ME：16PC)；Preferably, the composition of the phospholipid bimolecular membrane includes DPHPC (4ME: 16PC);
    优选地，所述高分子聚合物膜包括两嵌段或三嵌段的高分子聚合物膜；Preferably, the high molecular polymer film comprises a two-block or three-block high molecular polymer film;
    优选地，所述纳米通道蛋白包括孔径大于0.5nm、小于10nm的通道蛋白；Preferably, the nanochannel protein includes a channel protein with a pore size greater than 0.5 nm and less than 10 nm;
    优选地，所述纳米通道蛋白包括膜蛋白或病毒马达通道蛋白；Preferably, said nanochannel protein comprises a membrane protein or a viral motor channel protein;
    优选地，所述膜蛋白包括以下一种或多种：α-溶血素、耻垢分枝杆菌孔蛋白A、溶细胞素A、大肠杆菌CsgG、气单胞菌溶素、PA63；Preferably, the membrane protein includes one or more of the following: α-hemolysin, Mycobacterium smegmatis porin A, cytolysin A, Escherichia coli CsgG, aerolysin, PA63;
    优选地，所述病毒马达通道蛋白包括以下一种或多种：噬菌体phi29、噬菌体SPP1、噬菌体T3、噬菌体T4、噬菌体T7。Preferably, the viral motor channel protein includes one or more of the following: phage phi29, phage SPP1, phage T3, phage T4, and phage T7.
  3. 根据权利要求2所述的制备方法，其特征在于，所述第一电压的起始电压小于所述破膜电压；The preparation method according to claim 2, characterized in that, the initial voltage of the first voltage is less than the membrane rupture voltage;
    优选地，所述第一电压的起始电压为0.01～0.8V，进一步优选为0.02-0.05V；Preferably, the initial voltage of the first voltage is 0.01-0.8V, more preferably 0.02-0.05V;
    优选地，所述第一电压的终止电压小于所述破膜电压；Preferably, the termination voltage of the first voltage is less than the membrane rupture voltage;
    优选地，所述终止电压为0.18V。Preferably, the termination voltage is 0.18V.
  4. 根据权利要求3所述的制备方法，其特征在于，所述第一电压为阶梯上升或持续上升的电压；The preparation method according to claim 3, wherein the first voltage is a step-up or continuously-rising voltage;
    优选地，所述阶梯上升包括每10min增加0.01～0.8V，更优选0.01～0.4V；Preferably, the step increase includes increasing 0.01-0.8V every 10 minutes, more preferably 0.01-0.4V;
    优选地，所述第一电压为从所述起始电压开始，按照每10min增加0.04V的递增速度递增至所述终止电压；Preferably, the first voltage starts from the initial voltage and increases to the final voltage at an incremental rate of 0.04V every 10 minutes;
    优选地，所述纳米孔生物传感器的电导值大于1nS时，停止施加所述第一电压。Preferably, when the conductance value of the nanopore biosensor is greater than 1 nS, the application of the first voltage is stopped.
  5. 根据权利要求2所述的制备方法，其特征在于，在加入所述纳米通道蛋白、施加所述第一电压前，所述制备方法还包括对所述膜材施加第二电压，所述第二电压包括第一周期性电压和第一恒定电压；The preparation method according to claim 2, characterized in that, before adding the nanochannel protein and applying the first voltage, the preparation method also includes applying a second voltage to the membrane material, and the second the voltage includes a first periodic voltage and a first constant voltage;
    优选地，所述第一周期性电压包括第一方波电压或第一三角波电压；Preferably, the first periodic voltage includes a first square wave voltage or a first triangular wave voltage;
    优选地，所述第一方波电压的高电平小于或等于破膜电压；Preferably, the high level of the first square wave voltage is less than or equal to the rupture voltage;
    优选地，所述第一方波电压的高电平为0.01～0.8V，进一步优选为0.02～0.05V；Preferably, the high level of the first square wave voltage is 0.01-0.8V, more preferably 0.02-0.05V;
    优选地，所述第一方波电压的低电平为负；Preferably, the low level of the first square wave voltage is negative;
    优选地，所述第一方波电压的频率为0.1～100Hz，进一步优选为0.5Hz。Preferably, the frequency of the first square wave voltage is 0.1-100 Hz, more preferably 0.5 Hz.
  6. 根据权利要求5所述的制备方法，其特征在于，所述第一恒定电压为0.01～0.8V，优选为0.02～0.05V；The preparation method according to claim 5, characterized in that, the first constant voltage is 0.01-0.8V, preferably 0.02-0.05V;
    优选地，对所述膜材施加所述第二电压包括：对所述膜材先施加所述第一周期性电压，然后再施加所述第一恒定电压，Preferably, applying the second voltage to the membrane material includes: first applying the first periodic voltage to the membrane material, and then applying the first constant voltage,
    优选地，所述第一周期性电压进行1～100个周期，所述第一恒定电压持续1～100s；Preferably, the first periodic voltage is performed for 1 to 100 cycles, and the first constant voltage lasts for 1 to 100s;
    优选地，对所述膜材重复多次施加所述第二电压，优选重复1～100次。Preferably, the second voltage is repeatedly applied to the membrane material, preferably 1 to 100 times.
  7. 根据权利要求1至6中任一项所述的制备方法，其特征在于，在形成具有纳米孔通道的所述纳米孔生物传感器之后，所述制备方法进一步包括：对所述纳米孔生物传感器施加第三电压，所述第三电压包括第二周期性电压和第二恒定电压；The preparation method according to any one of claims 1 to 6, characterized in that, after forming the nanopore biosensor with a nanopore channel, the preparation method further comprises: applying to the nanopore biosensor a third voltage comprising a second periodic voltage and a second constant voltage;
    优选地，所述纳米孔生物传感器的电导值大于1nS时，对所述纳米孔生物传感器施加所述第三电压；Preferably, when the conductance value of the nanopore biosensor is greater than 1 nS, the third voltage is applied to the nanopore biosensor;
    优选地，所述第二周期性电压包括第二方波电压或第二三角波电压；Preferably, the second periodic voltage includes a second square wave voltage or a second triangular wave voltage;
    优选地，所述第二方波电压的高电平为0.01～0.8V，进一步优选为0.02～0.05V；Preferably, the high level of the second square wave voltage is 0.01-0.8V, more preferably 0.02-0.05V;
    优选地，所述第二方波电压的低电平为负；Preferably, the low level of the second square wave voltage is negative;
    优选地，所述第二方波电压的频率为0.1～100Hz，进一步优选为0.5Hz；Preferably, the frequency of the second square wave voltage is 0.1-100 Hz, more preferably 0.5 Hz;
    优选地，所述第二恒定电压为0.02～0.05V；Preferably, the second constant voltage is 0.02-0.05V;
    优选地，对所述纳米孔生物传感器施加所述第三电压包括：对所述纳米孔生物传感器先施加所述第二周期性电压，然后再施加所述第二恒定电压；Preferably, applying the third voltage to the nanopore biosensor includes: first applying the second periodic voltage to the nanopore biosensor, and then applying the second constant voltage;
    优选地，所述第二周期性电压重复1～100次，所述第二恒定电压持续1～100s；Preferably, the second periodic voltage is repeated 1 to 100 times, and the second constant voltage lasts for 1 to 100s;
    优选地，对所述纳米孔生物传感器重复多次施加所述第三电压，优选地，重复1～100次。Preferably, the application of the third voltage to the nanopore biosensor is repeated multiple times, preferably 1 to 100 times.
  8. 根据利用权利要7所述的制备方法，其特征在于，在对所述纳米孔生物传感器重复多次施加所述第三电压之后，所述制备方法还包括：对所述纳米孔生物传感器持续施加第四电压，所述第四电压为0.01～0.8V，优选为0.02～0.05V。According to the preparation method according to claim 7, it is characterized in that, after repeatedly applying the third voltage to the nanopore biosensor, the preparation method further includes: continuously applying the third voltage to the nanopore biosensor The fourth voltage, the fourth voltage is 0.01-0.8V, preferably 0.02-0.05V.
  9. 权利要求1至8中任一项所述的制备方法所制备的纳米孔生物传感器，其特征在于，所述纳米孔生物传感器在0.18V恒定电压作用1小时后，所述纳米通道蛋白的滑脱率小于<10％。The nanopore biosensor prepared by the preparation method described in any one of claims 1 to 8, characterized in that, after the nanopore biosensor was subjected to a constant voltage of 0.18V for 1 hour, the slippage rate of the nanochannel protein Less than <10%.
  10. 一种测序方法，其特征在于，所述测序方法包括：A sequencing method, characterized in that the sequencing method comprises:
    利用权利要求1至8中任一项所述的制备方法制备纳米孔生物传感器；Using the preparation method described in any one of claims 1 to 8 to prepare a nanopore biosensor;
    利用所述纳米孔生物传感器对待测物质进行单分子测序。The nanopore biosensor is used to perform single-molecule sequencing of a substance to be tested.
  11. 根据权利要求10所述的测序方法，其特征在于，所述待测物质选自小分子、DNA、RNA、多肽或蛋白质。The sequencing method according to claim 10, wherein the substance to be tested is selected from small molecules, DNA, RNA, polypeptides or proteins.
  12. 一种脂质体药物的制备方法，其特征在于，所述制备方法包括：在递增电压的作用下，使药物***脂质膜内形成所述脂质体药物，所述药物为蛋白药物，A method for preparing a liposome drug, characterized in that the preparation method comprises: under the action of increasing voltage, inserting the drug into the lipid membrane to form the liposome drug, the drug is a protein drug,
    优选地，所述药物为抗体药物；Preferably, the drug is an antibody drug;
    优选地，所述脂质膜为磷脂膜，更优选为磷脂膜形成的囊泡；Preferably, the lipid membrane is a phospholipid membrane, more preferably a vesicle formed by a phospholipid membrane;
    优选地，所述递增电压为阶梯上升的电压或持续上升的电压；Preferably, the incremental voltage is a step-up voltage or a continuously rising voltage;
    优选地，在使所述药物***所述脂质膜内形成所述脂质体药物前，所述制备方法还包括：对所述脂质膜进行柔性处理；Preferably, before inserting the drug into the lipid membrane to form the liposome drug, the preparation method further includes: performing a flexible treatment on the lipid membrane;
    优选地，采用对所述脂质膜交替施加周期性电压与恒定电压的方式进行所述柔性处理。Preferably, the flexible treatment is performed by alternately applying periodic voltage and constant voltage to the lipid membrane.
  13. 一种药物的递送方法，其特征在于，所述递送方法包括：A drug delivery method, characterized in that the delivery method comprises:
    将药物按照权利要求12所述的制备方法制备成脂质体药物；The medicine is prepared into liposome medicine according to the preparation method described in claim 12;
    将所述药物以所述脂质体药物的形式递送入细胞内。The drug is delivered into the cell in the form of the liposomal drug.
  14. 根据权利要求13所述的递送方法，其特征在于，基于电压刺激的方式将所述药物以所述脂质体药物的形式递送入所述细胞内。The delivery method according to claim 13, characterized in that the drug is delivered into the cell in the form of the liposome drug based on voltage stimulation.
PCT/CN2021/143407 2021-12-30 2021-12-30 Preparation method for nanopore biosensor and sequencing method WO2023123292A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/143407 WO2023123292A1 (en) 2021-12-30 2021-12-30 Preparation method for nanopore biosensor and sequencing method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/143407 WO2023123292A1 (en) 2021-12-30 2021-12-30 Preparation method for nanopore biosensor and sequencing method

Publications (1)

Publication Number Publication Date
WO2023123292A1 true WO2023123292A1 (en) 2023-07-06

Family

ID=86997085

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/143407 WO2023123292A1 (en) 2021-12-30 2021-12-30 Preparation method for nanopore biosensor and sequencing method

Country Status (1)

Country Link
WO (1) WO2023123292A1 (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102203618A (en) * 2008-10-30 2011-09-28 郭培瑄 Membrane-integrated viral dna-packaging motor protein connector biosensor for DNA sequencing and other uses
US20120255862A1 (en) * 2011-03-08 2012-10-11 Electronic Biosciences, Llc Methods for voltage-induced protein incorporation into planar lipid bilayers
CN105637082A (en) * 2013-10-23 2016-06-01 吉尼亚科技公司 Process for biosensor well formation
US20200054999A1 (en) * 2017-05-17 2020-02-20 The Royal Institution For The Advancement Of Learning / Mcgill University Method and apparatus for making a nanopore in a membrane using an electric field applied via a conductive tip
CN110954445A (en) * 2019-10-31 2020-04-03 四川大学华西医院 Living cell biosensor and preparation method and application thereof
CN112119033A (en) * 2018-02-09 2020-12-22 俄亥俄州国家创新基金会 Nanopore sensor from bacteriophage
CN112345743A (en) * 2016-11-24 2021-02-09 牛津纳米孔技术公司 Apparatus and method for controlling insertion of membrane channels into membranes
CN113260449A (en) * 2018-12-11 2021-08-13 豪夫迈·罗氏有限公司 Systems and methods for self-limiting protein pore insertion in membranes

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102203618A (en) * 2008-10-30 2011-09-28 郭培瑄 Membrane-integrated viral dna-packaging motor protein connector biosensor for DNA sequencing and other uses
US20120255862A1 (en) * 2011-03-08 2012-10-11 Electronic Biosciences, Llc Methods for voltage-induced protein incorporation into planar lipid bilayers
CN105637082A (en) * 2013-10-23 2016-06-01 吉尼亚科技公司 Process for biosensor well formation
CN112345743A (en) * 2016-11-24 2021-02-09 牛津纳米孔技术公司 Apparatus and method for controlling insertion of membrane channels into membranes
US20200054999A1 (en) * 2017-05-17 2020-02-20 The Royal Institution For The Advancement Of Learning / Mcgill University Method and apparatus for making a nanopore in a membrane using an electric field applied via a conductive tip
CN112119033A (en) * 2018-02-09 2020-12-22 俄亥俄州国家创新基金会 Nanopore sensor from bacteriophage
CN113260449A (en) * 2018-12-11 2021-08-13 豪夫迈·罗氏有限公司 Systems and methods for self-limiting protein pore insertion in membranes
CN110954445A (en) * 2019-10-31 2020-04-03 四川大学华西医院 Living cell biosensor and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU, HANG: "Development and Preliminary Evaluation of Nanopore Single Molecule Sensors", INFORMATION & TECHNOLOGY, CHINA DOCTORAL DISSERTATIONS/MASTER'S THESES FULL-TEXT DATABASE (DOCTORAL), no. 01, 15 January 2020 (2020-01-15), pages 1 - 76, ISSN: 1674-022X *
NOAKES MATTHEW T.; BRINKERHOFF HENRY; LASZLO ANDREW H.; DERRINGTON IAN M.; LANGFORD KYLE W.; MOUNT JONATHAN W.; BOWMAN JASMINE L.;: "Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 37, no. 6, 22 April 2019 (2019-04-22), New York, pages 651 - 656, XP036797521, ISSN: 1087-0156, DOI: 10.1038/s41587-019-0096-0 *

Similar Documents

Publication Publication Date Title
US6746594B2 (en) Miniature support for thin films containing single channels or nanopores and methods for using the same
Akeson et al. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules
Khan et al. Durable proteo-hybrid vesicles for the extended functional lifetime of membrane proteins in bionanotechnology
Tanaka et al. Characterization of drug transport through tight-junctional pathway in Caco-2 monolayer: comparison with isolated rat jejunum and colon
CA2864125C (en) Methods for creating bilayers for use with nanopore sensors
EP2886663A1 (en) Nanopore sequencing using replicative polymerases and helicases
JP2015508896A5 (en)
US11439708B2 (en) Nanotube trans-membrane channels mimicking biological porins
Cho et al. Water-templated transmembrane nanopores from shape-persistent oligocholate macrocycles
CN112831395A (en) Cell-like membranes for nanopore sequencing
US10228347B2 (en) High contrast signal to noise ratio device components
EP3700670B1 (en) Removing and reinserting protein nanopores in a membrane using osmotic imbalance
Yu et al. Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations
WO2023123292A1 (en) Preparation method for nanopore biosensor and sequencing method
US20210302409A1 (en) Systems and methods for self-limiting protein pore insertion in a membrane
WO2021249985A1 (en) Faradaic systems and methods for self-limiting protein pore insertion in a membrane
Winterhalter et al. On the dynamics of the electric field induced breakdown in lipid membranes
WO2024055284A1 (en) Ion cross-linked biomimetic membrane, preparation method therefor, and use thereof
CN117384486A (en) Mixed bionic membrane and preparation method and application thereof
Renner et al. Voltage-controlled insertion of single α-hemolysin and Mycobacterium smegmatis nanopores into lipid bilayer membranes
WO2023123434A1 (en) Phospholipid/polymer bionic composite-formula membrane as well as preparation method therefor and use thereof
Vu et al. Investigation of compacted DNA structures induced by Na+ and K+ monovalent cations using biological nanopores
US20230266268A1 (en) Solid state nanopore formation
Tantawi et al. Electrophysiology of Phosphatidylserine Bilayer Membranes Using Electrochemical Impedance Spectroscopy
CN114371206B (en) Method for preparing stable uniform nano-pores

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21969649

Country of ref document: EP

Kind code of ref document: A1