A method of protecting a nanopore protein and uses thereof

Abstract

This application provides a method and application for protecting nanoporins. The protection method includes: embedding the nanoporin onto a membrane, the membrane dividing a solution into two parts, which are connected through the nanoporin; and applying a negative voltage to one side of the solution containing the nanoporin before membrane crosslinking. This application enables an initiator to approach the membrane and undergo membrane crosslinking while remaining away from the nanoporin, thus protecting the nanoporin and preventing damage that could cause abnormalities in the opening current or even subsequent sequencing current.

Description

Technical Field

[0001] This application relates to the field of nanopore sequencing technology, specifically to a method for protecting nanopore proteins and its application. Background Technology

[0002] Gene sequencing technology uses nanopores as biosensors, which are embedded in an insulating amphiphilic membrane. When a single-stranded DNA molecule passes through the nanopore, the different current changes caused by different base groups are read by the signal receiver in the nanopore, and then the corresponding base recognition is performed, thereby realizing the detection of gene sequences.

[0003] When preparing molecular membranes for gene sequencing chips, a first layer of electrolyte, a second layer of organic nonpolar film-forming solution, and a third layer of electrolyte polar solvent are typically introduced sequentially into the film-forming region of the gene sequencing chip, so that the nonpolar solvent of the amphiphilic material is sandwiched between the two polar solvent layers to form a molecular membrane.

[0004] Existing molecular membrane support devices suffer from technical problems such as uneven film formation, low film stability, and significant differences in film formation between various microsupport structures due to unreasonable structural design. Therefore, cross-linking the molecular membrane is employed to improve membrane stability. However, during cross-linking, free radicals generated by the initiator can enter the confinement regions of nanoporous proteins, disrupting their structure and causing the nanopore opening current to deviate significantly from normal values. Summary of the Invention

[0005] To solve the above problems, this application adopts the following technical solution:

[0006] The inventive point of this application is to provide a method for protecting a nanoporin, wherein the nanoporin is embedded in a membrane that divides a solution into two parts, and the two parts of the solution are connected through the nanoporin, comprising: applying a negative voltage to one side of the solution of the nanoporin before the membrane crosslinks.

[0007] Optionally, the negative voltage range is (-180)mV to (-300)mV.

[0008] Optionally, the method further includes adding nanoparticles to the solution on the other side of the nanoporous protein; the nanoparticles being non-negative or having a density greater than that of water.

[0009] Optionally, the nanoparticles include one or more of the following: gold nanoparticles, SiO2 nanoparticles, Fe3O4 nanoparticles, silver nanoparticles, and platinum nanoparticles.

[0010] Optionally, the size of the nanoparticles is not smaller than the pore size of the nanoporin.

[0011] Optionally, the size of the nanoparticles is 1 to 5 times the pore size of the nanoporin, preferably 1 to 2 times.

[0012] Optionally, the membrane is a copolymer membrane containing unsaturated bonds; the initiator for membrane crosslinking includes a negatively charged initiator.

[0013] Optionally, it includes: (1) applying a negative voltage to one side of the nanoporin solution; and (2) adding a solution containing membrane crosslinking initiation to the other side of the nanoporin to perform membrane crosslinking.

[0014] Optionally, the method includes: (1) adding nanoparticles to the solution on the other side of the nanoporin; (2) applying a negative voltage to the solution on one side of the nanoporin; and (3) adding a solution containing membrane crosslinking initiator to the other side of the nanoporin to perform membrane crosslinking.

[0015] Another inventive point of this application is to provide an application of the protection method described above in nanopore sequencing.

[0016] Compared with the prior art, this application has the following advantages:

[0017] This application applies a negative voltage to the bottom of the nanoporin. When a negative voltage is applied to the bottom of the nanoporin, the negatively charged initiator is subjected to an electric field force away from the nanoporin, preventing it from entering the confinement region of the nanoporin and damaging its structure. Consequently, the nanopore opening current does not deviate significantly from the normal value. Furthermore, because the electric field force on the membrane surface is significantly smaller than that of the nanoporin, the initiator can still approach the copolymer membrane and initiate cross-linking. In other words, this application enables the initiator to approach the membrane and perform membrane cross-linking while remaining away from the nanoporin, thus protecting the nanoporin and preventing damage that could cause abnormalities in the opening current or even subsequent sequencing current. Attached Figure Description

[0018] Figure 1 This is a structural diagram of a gene sequencing chip provided in one embodiment of this application.

[0019] Figure 2 This is a schematic diagram of the voltage distribution at the inlet of a nanoporous protein provided in an embodiment of this application.

[0020] Figure 3 This is a diagram showing the positional relationship between nanopore channels and nanoparticles provided in an embodiment of this application.

[0021] Figure 4 This is a current diagram for detecting the breakdown voltage of a membrane provided in one embodiment of this application.

[0022] Figure 5(a) is a comparison diagram of the opening current provided in an embodiment of this application.

[0023] Figure 5(b) is a diagram illustrating the physical meaning of the opening current provided in an embodiment of this application.

[0024] Figure 6 This is a schematic diagram of a pre-crosslinking membrane provided in an embodiment of this application.

[0025] Figure 7 This is a schematic diagram of a cross-linked membrane provided in one embodiment of this application.

[0026] Figure 8 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0027] Figure 9 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0028] Figure 10 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0029] Figure 11 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0030] Figure 12 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0031] Figure 13 This is an example of an embodiment of the cross-linked porous protein provided in this application.

[0032] Figure 14 This is an example of an embodiment of the cross-linked porous protein provided in this application. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0035] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0036] In preparing the molecular membrane for gene sequencing chips, the following is first obtained: Figure 1 The chip shown has a Pt electrode at its bottom, and a microwell structure is formed by photolithography using Su-8 photoresist. Then, a first layer of electrolyte (an aqueous solution of 200 mM KCl, 150 mM K3Fe(CN)6, and 100 mM K4Fe(CN)6) is sequentially introduced into this microwell structure to wet the entire film-forming area. Next, a second layer of organic nonpolar film-forming solution is introduced to displace some of the first layer of electrolyte. Finally, a third layer of electrolyte (an aqueous solution of 200 mM KCl, 150 mM K3Fe(CN)6, and 100 mM K4Fe(CN)6) is introduced, causing the nonpolar solvent of the amphiphilic material to be sandwiched between two polar solvent layers to form a molecular film.

[0037] The nanoporous protein solution was diluted to 1 / 1000 with an electrolyte consisting of 200 mM KCl, 150 mM K3Fe(CN)6, and 100 mM K4Fe(CN)6, and added to the chip. A voltage of 180 mV was applied, and the applied voltage was removed when the sequencing unit current abruptly changed from 0 nA to around 0.3 nA. At this point, the nanoporous protein had been embedded in the membrane.

[0038] The number of nanoporous proteins corresponds one-to-one with the number of microwells, meaning that only one nanoporous protein is embedded in each microwell. When the chip contains multiple microwells, the entire chip also contains a corresponding number of nanoporous proteins.

[0039] A method for protecting a nanoporin is provided, wherein the nanoporin is embedded in a membrane that divides a solution into two parts, which are connected by the nanoporin. The method includes applying a negative voltage to one side of the solution containing the nanoporin before the membrane is cross-linked.

[0040] When a negative voltage is applied, an electric field is generated through the nanopore channels, such as... Figure 2 As shown, a relatively strong negative voltage is generated, especially at the edges of the nanoporous channels; and the initiator for membrane crosslinking also carries a negative charge, thus keeping the initiator away from the nanoporous channels, thereby preventing the negatively charged initiator from damaging the nanopores during crosslinking. At the same time, since the electric field force on the membrane surface is significantly smaller than that of the nanoporous protein, persulfate radicals can still approach the copolymer membrane and initiate crosslinking.

[0041] The voltage range of negative voltage is (-180)mV to (-300)mV.

[0042] Above this voltage, the membrane is prone to rupture or the porins may pop out, which is detrimental to the stability of the entire nanopore system; below this voltage, the effectiveness is greatly reduced due to factors such as the medium, and the porins cannot be protected.

[0043] Optionally, the method further includes adding nanoparticles to the solution on the other side of the nanoporous protein; the nanoparticles being non-negative or having a density greater than that of water.

[0044] Nanoparticles are added to the solution above the nanoporin, and these nanoparticles block the entrance of the nanoporin; at this time, adding an over-negatively charged initiator will prevent it from entering the nanopore.

[0045] Nanoparticles can only sink and come into contact with proteins when their density is greater than that of water.

[0046] When these particles are non-negatively charged and approach the membrane surface, the negatively charged electric field near the nanoporin attracts them, causing them to be located at the nanoporin inlet. Therefore, the density of the nanoparticles is not critical. These particles are preferably positively charged.

[0047] Nanoparticles include one or more of the following: gold nanoparticles, SiO2 nanoparticles, Fe3O4 nanoparticles, silver nanoparticles, and platinum nanoparticles.

[0048] The size of the nanoparticles is not smaller than the pore size of the nanoporin inlet.

[0049] The nanoparticles have amino-modified functional groups on their surface. When the solution is acidic, the nanoparticles are positively charged and move towards the openings of the porin more quickly and precisely.

[0050] When the size of the nanoparticle is greater than or equal to that of the nanoporin, it can block the entrance to the nanoporin; if the nanoparticle is smaller than the pore size of the nanoporin entrance, a similar effect will occur, except that the nanoparticle will be located inside the nanoporin. Figure 3 As shown, the channel structure generated by the nanoporin decreases progressively from top to bottom until it reaches its shortest point (generally referred to as the contraction zone). When the nanoparticle is smaller than the pore size of the nanoporin inlet, the nanoparticle can be located at any position from the inlet to the contraction zone. That is, the size of the nanoparticle is not smaller than the pore size of the nanoporin contraction zone. However, since the nanoporous channel will continue to be used subsequently, to avoid the nanoparticle entering the nanoporous channel too deeply and causing adverse effects, it is preferable that the size of the nanoparticle is not smaller than the pore size of the nanoporin inlet. More preferably, the size of the nanoparticle is 1 to 5 times the pore size of the nanoporin inlet, which can be 1, 2, 3, 4, 5 times, or any multiple of this value; preferably 1 to 2 times.

[0051] Since the inlet size of most nanoporous proteins is currently around 5 nm, the size of nanoparticles is preferably not less than 5 nm.

[0052] For example, the MSPA ion channel protein has an opening size of 4.8 nm [Perera, Ayomi S, et al. Nanoscopic surfactant behavior of the porin MspA in aqueous media. Beilstein Journal of Nanotechnology 4.1(2013):278-284]; the CsgG protein has an opening size of 5.2 nm [Cao B, et al. Structure of the nonameric bacterial amyloid secretion channel. ProcNatl Acad Sci USA. 2014 Dec 16; 111(50):E5439-44].

[0053] Specifically, the size of the nanoparticles can be adjusted according to the opening size of the corresponding protein.

[0054] The amount of nanoparticles used is positively correlated with the number of nanopores; the number of nanoparticles should not be less than the number of nanopores.

[0055] The preferred number of nanoparticles is 1 to 10,000 times the number of nanopores embedded in the membrane; more preferably 100 to 10,000 times; even more preferably 1,000 to 10,000 times; it can be 1,000 times, 2,000 times, 3,000 times, 4,000 times, 5,000 times, 6,000 times, 7,000 times, 8,000 times, 9,000 times, 10,000 times, or any multiple of this range. This avoids excessive nanoparticles, which could affect membrane cross-linking; it also reduces the risk of insufficient nanoparticles, which would fail to provide protection.

[0056] The amount of nanoparticles is generally (5 × 10⁻⁶). 10 )~(5×10 15 The number of nanoparticles can be approximately determined by adjusting their volume (e.g., nanoparticles per ml).

[0057] The membrane is a copolymer membrane containing unsaturated bonds; the initiators for membrane crosslinking include negatively charged initiators.

[0058] The membrane is preferably a diblock copolymer or triblock copolymer containing unsaturated bonds. Among them, unsaturated bonds include carbon-carbon double bonds (alkenyl), carbon-oxygen double bonds (carboxyl, carbonyl, aldehyde), carbon-nitrogen double bonds, carbon-carbon triple bonds (alkynyl), carbon-nitrogen triple bonds (cyano), etc.

[0059] Copolymers containing unsaturated bonds can be diblock copolymers or triblock copolymers containing alkenyl groups; specifically, they can be as shown in the following formula:

[0060]

[0061] Where n ranges from 5 to 40; m ranges from 5 to 40; and p ranges from 5 to 40.

[0062] The concentration of the copolymer is 2 mg / ml to 100 mg / ml; the solvent is generally a hydrocarbon group; preferably n-decane, n-tetradecane, n-hexadecane, etc.

[0063] The type of membrane crosslinking can be crosslinking caused by an initiator.

[0064] Negatively charged initiators are those that carry a negative charge during a reaction. These initiators can generate negatively charged free radicals or ions, thereby initiating the reaction.

[0065] Negatively charged initiators include organic peroxides and inorganic peroxides.

[0066] Inorganic peroxides include persulfates, specifically potassium persulfate, sodium persulfate, and ammonium persulfate.

[0067] Organic peroxides include one or more of the following: ketones, phenyl esters, phenyl ketones, azo compounds, and phenylphosphides.

[0068] Phenyl esters include one or more of the following: tert-butyl peroxybenzoate (TBPB), tert-butyl peroxy(2-ethylhexanoate) (TBPO), and 2-ethylhexyl 4-dimethylaminobenzoate.

[0069] Ketones include cyclohexanone peroxide (CHP) and / or methyl ethyl ketone peroxide (MEKPO).

[0070] Phenyl ketones include one or more of the following: 1-hydroxycyclohexylphenyl ketone, methyl benzoylformate, 2,2-methoxy-phenylacetophenone, benzoyl peroxide (BPO), and Irgacure 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetone).

[0071] Azo compounds include: 2,2-azobis(2-methylphenylimidazolium)-dihydrochloride.

[0072] Phenylphosphorus compounds include one or more of the following: (2,4,6-trimethylbenzoyl)diphenylphosphine oxide, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, and phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite.

[0073] The amount of organic initiator added was 10 mg / ml.

[0074] Preferably, the above-mentioned initiator can be used for aqueous phase initiation; more preferably, Irgacure 2959 is preferred.

[0075] The method for protecting nanoporin includes: (1) applying a negative voltage to a solution on one side of the nanoporin; and (2) adding a solution containing membrane crosslinking initiator to the other side of the nanoporin to perform membrane crosslinking.

[0076] Specifically:

[0077] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 In the chip shown;

[0078] 2) Add a copolymer film containing unsaturated bonds;

[0079] 3) Add electrolyte; apply a negative voltage after embedding the nanoporous protein;

[0080] 4) Rinse the electrolyte above the copolymer membrane with a KCl solution that is isotonic with the electrolyte, add a solution containing an initiator, and then let it stand to carry out the crosslinking reaction.

[0081] Prepare a solution containing the initiator: Take the initiator, adjust the osmotic pressure of the solution to be the same as that of the electrolyte using KCl, and remove oxygen by vacuum to obtain a solution containing the initiator.

[0082] The method for protecting nanoporin includes: (1) adding nanoparticles to a solution on the other side of the nanoporin; (2) applying a negative voltage to a solution on one side of the nanoporin; and (3) adding a solution containing membrane crosslinking initiator to the other side of the nanoporin to perform membrane crosslinking.

[0083] Specifically:

[0084] 1) Add the electrolyte as follows Figure 1 In the chip shown;

[0085] 2) Add a copolymer film containing unsaturated bonds;

[0086] 3) Add electrolyte; embed nanoporous proteins;

[0087] 4) Add amino-modified nanoparticles with osmotic pressure adjusted using KCl solution;

[0088] 5) Apply a negative voltage;

[0089] 6) Prepare a solution containing the initiator: Take the initiator, adjust the osmotic pressure of the solution to be the same as that of the electrolyte using KCl, and remove oxygen by vacuum to obtain a solution containing the initiator.

[0090] 7. Rinse the copolymer membrane with a KCl solution isotonic with the electrolyte to remove the electrolyte, add a solution containing an initiator, and let it stand for 20 minutes to carry out the crosslinking reaction; then add electrolyte to quench the crosslinking reaction.

[0091] The method for testing membrane breakdown voltage is as follows: An initial voltage of 140 mV is applied, with the voltage increased by 10 mV every 3 seconds. Because the membrane contains nanoporous proteins, when the membrane is not yet broken down, the current detected by a single sequencing unit is approximately 1.0 nA. When the membrane breaks down, the current detected by a single sequencing unit increases by two orders of magnitude, reaching approximately 100 nA. The voltage applied across the membrane at this point is recorded; this voltage is the breakdown voltage.

[0092] For example Figure 4 After 14 pressurization cycles, the current rapidly increased (in the red box), indicating that the membrane ruptured. The breakdown voltage of this porous membrane was 480mV.

[0093] At a sampling rate of 5 kHz, 10,000 consecutive sampling points were selected, and the standard deviation σ was calculated. If σ < 16 / 6.6 pA, the pore opening current of the pore protein is considered normal; see Figures 5(a) and 5(b) for details.

[0094] Calculation formula:

[0095]

[0096] Where, x i The i-th sampling point; x (the horizontal line above) is the mean; N is the total number of sampling points, n = 6.6 × standard deviation σ.

[0097] According to the above formula, the state of the hole can be determined from the current signal of the hole in Figure 5. The smaller the value, the less noise, and the better the state of the hole.

[0098] Preparation of amino-modified gold nanoparticles: 20 ml of 0.01% chloroauric acid solution and 2 ml of 1% histidine solution were mixed and stirred at room temperature in the dark for 3 hours. Then, 5 ml of 0.1% amino-polyethylene glycol-thiol (average relative molecular mass 500) was added, and the mixture was stirred at room temperature in the dark for 8 hours. The solution was dialyzed using a dialysis band with a molecular weight of 2000 to obtain amino-modified gold nanoparticles with a diameter of 5 nm.

[0099] Nanoparticles can be prepared using the methods described above, or commercially available products can be used directly.

[0100] Example 1

[0101] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1In the chip shown.

[0102] 2) Add 10 mg / ml of the copolymer in n-decane solution; Copolymer: hydrophobic end length of 10 1,2-butadiene (n = 10) and hydrophilic end length of 5 polyacrylamide (m = 5).

[0103] 3) Add electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6); after embedding the nanoporous protein (MSPA ion channel protein), apply a voltage of -200mV;

[0104] 4) Rinse off the electrolyte above the copolymer membrane with 600mM KCl solution, and add a solution containing the initiator (prepare the initiator solution: take initiator: 20mmol / L K2S2O8 and 10mmol / L Na2S2O5 aqueous solution, adjust the osmotic pressure of the solution to be the same as the osmotic pressure of the electrolyte with KCl, and remove oxygen by vacuum to obtain a solution containing the initiator: 20mmol / L K2S2O8, 10mmol / L Na2S2O5, 600mM KCl); let stand for 20min to carry out the crosslinking reaction; then add electrolyte to quench the crosslinking reaction.

[0105] The obtained membrane and nanoporous protein system were tested; the breakdown voltage was 600mV, indicating that the cross-linked membrane was more stable; and the states of the membrane before and after cross-linking were compared. Figure 6 and Figure 7 As shown, the membrane boundary becomes clearer after crosslinking, and the membrane size no longer changes over time.

[0106] Simultaneously, the pore-opening current of the cross-linked porin was measured, such as... Figure 8 As shown, after cross-linking, the porin still has a good pore current with a low range. The calculated standard deviation σ < 16 / 6.6 pA indicates that the protein is not damaged.

[0107] Example 2

[0108] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 In the chip shown;

[0109] 2) Add 20 mg / ml of the copolymer in n-dodecane solution; Copolymer: hydrophobic end length of 10 1,2-butadiene (n=10), hydrophilic end length of 5 polyethylene glycol (m=5).

[0110] 3) Add electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6); after embedding the nanoporous protein (CsgG protein), apply a voltage of -180mV;

[0111] 4) Rinse off the electrolyte above the copolymer membrane with 600mM KCl solution, and add a solution containing the initiator (preparation of the initiator solution: take initiator: 40mmol / L K2S2O8 and 20mmol / L Na2S2O5 aqueous solution, adjust the osmotic pressure of the solution to be the same as the osmotic pressure of the electrolyte with KCl, and remove oxygen by vacuum to obtain a solution containing the initiator: 40mmol / L K2S2O8, 20mmol / L Na2S2O5, 600mM KCl); let stand for 20min to carry out the crosslinking reaction; then add electrolyte to quench the crosslinking reaction.

[0112] The obtained membrane and nanoporin system were tested; the breakdown voltage was 600mV, indicating that the cross-linked membrane was more stable. The pore-opening current of the cross-linked porin was also measured. Figure 9 As shown, after cross-linking, the porin still has a good pore current with a low range. The calculated standard deviation σ < 16 / 6.6 pA indicates that the protein is not damaged.

[0113] Example 3

[0114] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 In the chip shown;

[0115] 2) Add 5 mg / ml of the copolymer in tetradecane solution; Copolymer: hydrophobic end length of 10 1,2-butadienes (n = 10), hydrophilic end length of 5 polyacrylamides (m = 5);

[0116] 3) Add electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6); embed nanoporous proteins (MSPA ion channel proteins); at this point, the number of proteins embedded in the membrane is approximately 2500.

[0117] 4) Take 1 μl of amino-modified material with a diameter of 5 nm and a concentration of 5 × 10⁻⁶. 13 Add a solution of gold nanoparticles per ml (pH=5) to 1 L of 600 mM KCl solution and mix well. Take 100 μl of the mixture and add... Figure 1 In the chip shown;

[0118] 5) Apply a voltage of -200mV;

[0119] 6) Rinse off the electrolyte above the copolymer membrane with 600mM KCl solution, and add a solution containing the initiator (preparation of the initiator solution: take initiator: 4 mmol / L K2S2O8 and 2 mmol / L Na2S2O5 aqueous solution, adjust the osmotic pressure of the solution to be the same as the osmotic pressure of the electrolyte with KCl, and remove oxygen by vacuum to obtain a solution containing the initiator: 600mM KCl, 4 mmol / L K2S2O8, 2 mmol / L Na2S2O5); let stand for 20 min to carry out the crosslinking reaction; then add electrolyte to quench the crosslinking reaction.

[0120] The pore current of the cross-linked porin was measured, such as... Figure 10 As shown, after cross-linking, the porin still has a good pore current with a low range. The calculated standard deviation σ < 16 / 6.6 pA indicates that the protein is not damaged.

[0121] Example 4

[0122] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 In the chip shown;

[0123] 2) Add a 15 mg / ml solution of the copolymer in hexadecane; Copolymer: hydrophobic end length is 5 units of 1,4-polybutadiene (n = 5), hydrophilic end length is 5 units of polyethylene glycol (m = 5);

[0124] 3) Add electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6); embed nanoporous proteins (MSPA ion channel proteins); at this point, the number of proteins embedded in the membrane is approximately 2500.

[0125] 4) Take 1 μl of amino-modified material with a diameter of 5 nm and a concentration of 5 × 10⁻⁶. 13 Add a solution of gold nanoparticles per ml (pH = 8) to 1 L of 600 mM KCl solution and mix well. Take 100 μl of the mixture and add... Figure 1 In the chip shown;

[0126] 5) Apply a voltage of -200mV;

[0127] 6) Rinse off the electrolyte above the copolymer membrane with 600 mM KCl solution, then add a solution containing the initiator (prepared by using initiator: 15 mmol / L K2S2O8 and 7.5 mmol / L Na2S2O5 solution). Adjust the osmotic pressure of the solution to be the same as that of the electrolyte using KCl, and then deoxygenate under vacuum to obtain a solution containing the initiator: 600 mM / L KCl, 15 mmol / L K2S2O8, and 7.5 mmol / L Na2S2O5). Allow the mixture to stand for 20 min to allow the crosslinking reaction to proceed; then add electrolyte to quench the crosslinking reaction.

[0128] The pore current of the cross-linked porin was measured, such as... Figure 11 As shown, after cross-linking, the porin still exhibits good pore-opening current with a low range. Calculations show a standard deviation σ < 16 / 6.6 pA, indicating no protein damage; however, it is slightly inferior to Example 4. Figure 9 This is because the positive charge of the gold nanoparticles in the alkaline solution is not as strong as in Example 4, resulting in a difference in the protective effect.

[0129] Example 5

[0130] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 In the chip shown;

[0131] 2) Add a 15 mg / ml solution of the copolymer in n-tetane; Copolymer: hydrophobic end length is 5 units of 1,2-polyisoprene (n = 5), hydrophilic end length is 5 units of polyethylene glycol (m = 5);

[0132] 3) Add electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6); embed the MSPA ion channel protein; at this point, the number of proteins embedded in the membrane is approximately 2500.

[0133] 4) Take 1 μl of amino-modified material with a diameter of 5 nm and a concentration of 5.5 × 10⁻⁶. 13 A solution of platinum nanoparticles per ml with pH = 5 was added to 1 L of 600 mM KCl solution and mixed thoroughly. 100 μl of the mixed solution was then added to... Figure 1 In the chip shown;

[0134] 5) Apply a voltage of -300mV;

[0135] 6) Rinse off the electrolyte above the copolymer membrane with 600 mM KCl solution, then add a solution containing the initiator (prepared by taking an initiator solution of 30 mmol / L K2S2O8 and 15 mmol / L Na2S2O5 aqueous solution). Adjust the osmotic pressure of the solution to be the same as that of the electrolyte with KCl, and then deoxygenate under vacuum to obtain a solution containing the initiator: 600 mM KCl, 30 mmol / L K2S2O8, and 15 mmol / L Na2S2O5). Let it stand for 20 min to carry out the crosslinking reaction; then add electrolyte to quench the crosslinking reaction.

[0136] The pore current of the cross-linked porin was measured, such as... Figure 12 As shown, after cross-linking, the porin still has a good pore current with a low range. The calculated standard deviation σ < 16 / 6.6 pA indicates that the protein is not damaged.

[0137] Comparative Example 1

[0138] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 In the chip shown.

[0139] 2) Add 10 mg / ml of the copolymer in n-decane solution; Copolymer: hydrophobic end length of 10 1,2-butadiene (n = 10) and hydrophilic end length of 5 polyacrylamide (m = 5).

[0140] 3) Add electrolyte (aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6); embed MSPA ion channel protein;

[0141] 4) Rinse off the electrolyte above the copolymer membrane with 600mM KCl solution, and add a solution containing the initiator (prepare the initiator solution: take initiator: 20mmol / L K2S2O8 and 10mmol / L Na2S2O5 aqueous solution, adjust the osmotic pressure of the solution to be the same as the osmotic pressure of the electrolyte with KCl, and remove oxygen by vacuum to obtain a solution containing the initiator: 20mmol / L K2S2O8, 10mmol / L Na2S2O5, 600mM KCl); let stand for 20min to carry out the crosslinking reaction; then add electrolyte to quench the crosslinking reaction.

[0142] The membrane and nanoporin system obtained above were tested; the breakdown voltage was 600 mV, the same as that in Example 1, indicating that the membrane crosslinking was not affected. The opening current of the crosslinked nanoporin was also measured. Figure 13As shown, after crosslinking, the range of the pore current increases. After calculation, the standard deviation σ > 16 / 6.6pA, indicating protein damage.

[0143] Comparative Example 2

[0144] 1) Add the electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6) to the solution as follows: Figure 1 In the chip shown.

[0145] 2) Add 10 mg / ml of the copolymer in n-decane solution; Copolymer: hydrophobic end length of 10 1,2-butadiene (n = 10) and hydrophilic end length of 5 polyacrylamide (m = 5).

[0146] 3) Add electrolyte (an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6, and 100mM K4Fe(CN)6); after embedding the nanoporous protein (MSPA ion channel protein), apply a voltage of 200mV;

[0147] 4) Rinse off the electrolyte above the copolymer membrane with 600mM KCl solution, and add a solution containing the initiator (prepare the initiator solution: take initiator: 20mmol / L K2S2O8 and 10mmol / L Na2S2O5 aqueous solution, adjust the osmotic pressure of the solution to be the same as the osmotic pressure of the electrolyte with KCl, and remove oxygen by vacuum to obtain a solution containing the initiator: 20mmol / L K2S2O8, 10mmol / L Na2S2O5, 600mM KCl); let stand for 20min to carry out the crosslinking reaction; then add electrolyte to quench the crosslinking reaction.

[0148] The membrane and nanoporin system obtained above were tested; the breakdown voltage was 600 mV, the same as that in Example 1, indicating that the membrane crosslinking was not affected. The opening current of the crosslinked nanoporin was also measured. Figure 14 As shown, after crosslinking, the range of the pore current increases. After calculation, the standard deviation σ > 16 / 6.6pA, indicating protein damage. Moreover, this comparative example shows a higher degree of protein damage compared to comparative example 1.

[0149] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the system described above can be found in the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0150] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the storage device and processing device described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0151] The term "comprising" or any other similar term is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus / device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent in such process, method, article, or apparatus / device.

[0152] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for protecting a nanopore protein, said nanopore protein being embedded in a membrane, said membrane dividing a solution into two parts, the two parts of the solution being in communication through the nanopore protein, characterized in that, include: Before membrane crosslinking, a negative voltage is applied to one side of the nanoporous protein solution.

2. The protection method according to claim 1, characterized in that, The voltage range of the negative voltage is (-180)mV to (-300)mV.

3. The protection method according to claim 1, characterized in that, Also includes: Nanoparticles are added to the solution on the other side of the nanoporin; the nanoparticles are either non-negative or have a density greater than that of water.

4. The protection method according to claim 3, characterized in that, The nanoparticles include one or more of the following: gold nanoparticles, SiO2 nanoparticles, Fe3O4 nanoparticles, silver nanoparticles, and platinum nanoparticles.

5. The protection method according to claim 3, characterized in that, The size of the nanoparticles is not smaller than the pore size of the nanoporin.

6. The protection method according to claim 3, characterized in that, The size of the nanoparticles is 1 to 5 times the pore size of the nanoporin.

7. The protection method according to claim 1, characterized in that, The membrane is a copolymer membrane containing unsaturated bonds; the initiator for membrane crosslinking includes a negatively charged initiator.

8. The protection method according to claim 1, characterized in that, include: (1) Apply a negative voltage to one side of the solution of the nanoporous protein; (2) Add the solution containing membrane crosslinking initiator to the other side of the nanoporin to perform membrane crosslinking.

9. The protection method according to claim 1, characterized in that, include: (1) Add nanoparticles to the solution on the other side of the nanoporous protein; (2) Apply a negative voltage to one side of the solution of the nanoporous protein; (3) Add the solution containing the initiator to the other side of the nanoporous protein to perform membrane crosslinking.

10. The application of the protection method as described in any one of claims 1 to 9 in nanopore sequencing.

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