Electrochemical buffer solution for biological nanopores and preparation method therefor

Abstract

The present invention relates to the field of biochemical detection, and specifically relates to an electrochemical buffer solution for biological nanopores and a preparation method therefor. Provided is an electrochemical buffer solution which has high cost performance and is capable of reducing the loss of biological nanopores. The buffer solution comprises potassium ferricyanide, potassium ferrocyanide, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, EDTA and mannitol. When the electrochemical buffer solution is used for biological nanometer detection, the loss rate of biological nanopores can be significantly reduced, the stability of biological nanopores is improved, the flux of detection is increased, and the problem of biological nanopore loss is effectively overcome. Moreover, the electrochemical buffer solution can also be used in monomolecular sensing technology and / or ultra-trace element detection technology, and can be used as a buffer reagent for improving the stability of detection.

Description

Electrochemical buffer for biological nanopore and preparation method thereof Technical Field

[0001] The present invention relates to the field of biochemical detection, and in particular to an electrochemical buffer solution for biological nanopores and a preparation method thereof. Background Art

[0002] Nanopores are holes with a nanometer diameter. They can be produced by pore-forming proteins or holes in synthetic materials such as silicon and graphene. The two are called biological nanopores and solid-state nanopores, respectively. When a nanopore exists in an electrically insulating membrane, it can be used as a single-molecule sensor with the characteristics of being label-free, rapid, and ultra-highly sensitive. The detection principle is based on detecting the ionic current passing through the nanopore under the action of a voltage. For example, when RNA or single-stranded DNA molecules in a 1M KCl solution pass through a nanopore with a diameter of 1.5 nanometers, the current is blocked by about 90%. Solid-state nanopores have significant advantages in stability, current noise, and process integration, but are limited by the level of semiconductor process manufacturing and have the problems of complex manufacturing and high cost.

[0003] Electrochemical sensors based on biological nanopores have the advantages of relatively low cost, high portability, simple sample processing, and instant result analysis. They have broad application prospects in DNA sequencing, nucleic acid analysis, protein and peptide analysis, organic small molecule detection, and metal ion detection.

[0004] In products or technologies that use biological nanopores for single-molecule sensing, the nanopores gradually lose activity over time (hereinafter referred to as pore loss), rendering them incapable of completing their sensing tasks. Improving analytical efficiency requires maintaining throughput, which means maintaining a sufficient number of active nanopores. The stability of biological nanopores depends primarily on three factors: the applied electrical load, the buffer system, and the stability of the pore protein itself. Reducing the applied electrical load directly reduces analysis speed and is generally not considered an optimization strategy.

[0005] Currently, there are two main approaches to ensuring analytical throughput. One is to directly increase the number of nanopores, which will correspondingly increase the number of effective nanopores at each time period. The other is to screen and synthesize more stable porins to fundamentally overcome the problem of effective nanopore loss. However, increasing analytical throughput by directly increasing the number of nanopores significantly increases the production process difficulty and cost, resulting in a low cost-effectiveness. Screening and synthesizing more stable porins is technically difficult, has a long development cycle, and carries a high risk of failure, making it generally considered a preferred option rather than a mandatory one.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide an electrochemical buffer solution for biological nanopores and a preparation method thereof.

[0008] The present invention provides an electrochemical buffer comprising water, an electron buffer pair, a metal chelating agent, a protein protective agent and an ion buffer;

[0009] The electronic buffer pair includes potassium ferrocyanide and potassium ferrocyanide;

[0010] The metal chelating agent includes EDTA;

[0011] The protein protective agent includes at least one of trehalose, sorbitol, sucrose, polyethylene glycol, gelatin, glycine and / or mannitol;

[0012] The ion buffer includes at least one of a PB buffer, a HEPES buffer, or a Tris buffer.

[0013] In the present invention, the protective agent of the electrochemical buffer solution is screened;

[0014] In some embodiments of the present invention, the protective agent is trehalose;

[0015] In other embodiments of the present invention, the protective agent is glycine;

[0016] In other embodiments of the present invention, the protective agent is mannitol;

[0017] The experimental results show that the addition of mannitol and glycine is beneficial to reducing the damage rate of biological nanopores; the addition of mannitol is optimal, and the damage rate of biological nanopores is lowest when the mannitol concentration is 10mM, so that the electrochemical buffer of the present invention has a good effect of reducing biological nanopore damage.

[0018] Furthermore, the present invention screens the ion buffers that are matched with the protective agent;

[0019] In some embodiments of the present invention, the ion buffer is a PB buffer;

[0020] In other embodiments of the present invention, the ion buffer is a HEPES buffer;

[0021] In other embodiments of the present invention, the ion buffer is a Tris buffer;

[0022] The experimental results may indicate that the loss rate of the bio-nanopore of the electrochemical buffer solution containing the PB ion buffer and the protective agent is further reduced, and the reduction in the loss rate of the bio-nanopore is beneficial to the improvement of the detection flux of the stabilizing agent.

[0023] In some specific embodiments, in the electrochemical buffer,

[0024] The protein protective agent is mannitol;

[0025] The ion buffer is a PB buffer;

[0026] The PB buffer includes potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0027] In a specific embodiment, the electrochemical buffer comprises: 90-200 mM potassium ferrocyanide, 90-200 mM potassium ferrocyanide, 3.42 mM potassium dihydrogen phosphate, 21.58 mM dipotassium hydrogen phosphate, 0.01-10 mM EDTA, and 6-20 mM mannitol; the pH of the electrochemical buffer is 6.0-9.0.

[0028] Preferably, the electrochemical buffer comprises: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 3.42 mM potassium dihydrogen phosphate, 21.58 mM dipotassium hydrogen phosphate, 1 mM EDTA, and 10 mM mannitol; the pH of the buffer is 8.0.

[0029] The electrochemical buffer described in the present invention is improved on the basis of the electrochemical buffer components commonly used in traditional bio-nanomaterials. A protective agent is added to the traditional electrochemical buffer. The addition of the protective agent significantly reduces the loss rate of the bio-nanopore during detection. Furthermore, the present invention screens the ion buffer that matches the protective agent, further reducing the loss rate of the bio-nanopore during the detection process, improving the stability of the bio-nanopore during application, and increasing the detection flux. The results show that compared with the traditional electrochemical buffer, the electrochemical buffer described in the present invention reduces the bio-nanopore pore loss rate from 61.3% to 11.6% after 4 hours.

[0030] The present invention provides a method for preparing the electrochemical buffer solution, which comprises dissolving an electronic buffer pair, a metal chelating agent, a protein protective agent and an ion buffer solution in water, adjusting the pH, and filtering to obtain the electronic buffer solution.

[0031] Furthermore, in the preparation method, the pH is adjusted to 8.0; and the reagent for adjusting the pH is KOH solution.

[0032] The present invention also provides the application of the electrochemical buffer solution and / or the electrochemical buffer solution prepared by the preparation method in biological nanopore sensing technology, single molecule sensing technology and / or ultratrace element detection technology.

[0033] The present invention provides a kit comprising:

[0034] Detection probe and electrochemical buffer of the present invention;

[0035] Or it includes a detection probe and an electrochemical buffer solution prepared by the present invention and the preparation method.

[0036] In the present invention, the detection probes include biological probes and nanomaterial probes. The biological probes are molecules that label properties or physical states of nucleic acids, proteins, cell structures, etc., and include nucleic acid probes, protein probes, and cell structure probes. The nucleic acid probes include, but are not limited to, DNA probes, cDNA probes, RNA probes, and / or oligonucleotide probes, etc., which are not limited in the present invention. The nanomaterial probes include silver nanoprobes and / or magnetic nanoprobes, etc.

[0037] Furthermore, the kit of the present invention may also include other reagents or equipment that are compatible with the detection instrument or detection means, but the present invention does not limit this.

[0038] The present invention provides a detection method, which utilizes at least one of the following I) to III) to detect tumors, microorganisms, nucleic acids and / or trace elements using biological nanopore technology:

[0039] 1), electrochemical buffer solution of the present invention;

[0040] II), an electrochemical buffer solution prepared by the preparation method of the present invention;

[0041] III), the kit of the present invention.

[0042] In the present invention, the tumor includes primary tumors and metastatic tumors thereof; the microorganisms include fungi, bacteria, viruses or their respective propagules, etc.; the nucleic acids include DNA, cDNA, RNA, PNA, etc., which are not limited in the present invention.

[0043] The present invention provides a cost-effective electrochemical buffer solution that can reduce bionanopore loss. The buffer solution comprises potassium ferrocyanide, potassium ferrocyanide, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, EDTA, and mannitol. When used in bionanopore detection, the electrochemical buffer solution can significantly reduce the bionanopore loss rate, improve bionanopore stability, and increase detection throughput, effectively overcoming the problem of bionanopore loss. The electrochemical buffer solution can also be used in single-molecule sensing technology and / or ultratrace element detection technology, serving as a buffering agent to improve detection stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 shows the pore damage under different protective agents;

[0046] Figure 2 shows the pore damage under different protective agent concentrations (mannitol);

[0047] Figure 3 shows pore damage at different protectant concentrations (glycine);

[0048] Figure 4 shows the pore damage under different buffers;

[0049] Figure 5 shows the comparative data of buffer test. DETAILED DESCRIPTION

[0050] The present invention provides electrochemical buffers for biological nanopores and methods for preparing the same. Those skilled in the art can refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described using preferred embodiments. It is apparent that those skilled in the art can modify, adapt, and combine the methods and applications herein to implement and apply the present invention without departing from the disclosure, spirit, and scope of the present invention.

[0051] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0052] The present invention will be further described below in conjunction with the embodiments:

[0053] Example 1 Electrochemical buffer for biological nanopores of the present invention

[0054] 1. Optimization of electrochemical buffer composition

[0055] Commonly used electrochemical buffer (self-prepared): potassium ferrocyanide 150mM; potassium ferrocyanide 150mM; HEPES 25mM; EDTA 0.1mM.

[0056] (1) Screening of protective agents

[0057] Different concentrations of protective agents were added to the commonly used electrochemical buffer solution, and the effects of different protective agents on reducing pore damage were measured. The results are shown in Figure 1. Screening found that the addition of mannitol and glycine was beneficial to reducing the pore loss rate.

[0058] (2) Determination of protective agent concentration

[0059] As shown in Figures 2 and 3, the test found that the pore loss rate was the lowest when 10 mM mannitol was added.

[0060] (3) Screening of compatible buffers after adding protective agent (10 mM mannitol)

[0061] As shown in Figure 4, the test found that PB buffer was more suitable for the system with the addition of mannitol protective agent.

[0062] (4) Optimize the composition and formulation of the electrochemical buffer solution

[0063] The optimized electrochemical buffer composition and formula are shown in Table 1.

[0064] Table 1. Electrochemical buffer composition and formulation

[0065] The preparation method is as follows: according to the formula, the required potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium ferrocyanide, potassium ferrocyanide, EDTA, and mannitol are weighed and placed in a beaker, an appropriate amount of deionized water is added, and the mixture is stirred until completely dissolved. The volume is adjusted to 1 L with deionized water, and the pH is adjusted to 8.0 with KOH solution. The mixture is filtered to obtain a buffer solution.

[0066] (5) Activity comparison test of the electrochemical buffer for the biological nanopore of the present invention and the biological nanopore using the conventional sequencing electrochemical buffer

[0067] Comparative testing of the same biological nanopore sensor using the electrochemical buffer optimized for the present invention, as shown in Table 1, and a commonly used sequencing electrochemical buffer is performed. The data are shown in Table 2. A line graph was plotted using Origin software, with the nanopore operating time as the horizontal axis and the nanopore loss rate as the vertical axis. The results are shown in Figure 5.

[0068] Table 2. Activity test results of the biological nanopore sensor using the electrochemical buffer for the biological nanopore of the present invention and the commonly used sequencing electrochemical buffer

[0069] From the data in Table 2 above, it can be seen that the 4-hour pore loss rate of the bionanopore using the buffer of the present invention is reduced from 61.3% to 11.6% compared with the commonly used buffer.

[0070] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An electrochemical buffer comprising water, an electron buffer pair, a metal chelator, a protein protectant and an ion buffer; The electronic buffer pair includes potassium ferrocyanide and potassium ferrocyanide; The metal chelator comprises EDTA; The protein protective agent includes at least one of trehalose, sorbitol, sucrose, polyethylene glycol, gelatin, glycine and / or mannitol; The ion buffer includes at least one of a PB buffer, a HEPES buffer or a Tris buffer.

2. The electrochemical buffer according to claim 1, characterized in that The protein protective agent is mannitol; The ion buffer is a PB buffer, and the PB buffer includes potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

3. The electrochemical buffer according to claim 1 or 2, characterized in that The electrochemical buffer comprises: 90-200 mM potassium ferrocyanide, 90-200 mM potassium ferrocyanide, 3.42 mM potassium dihydrogen phosphate, 21.58 mM dipotassium hydrogen phosphate, 0.01-10 mM EDTA, and 6-20 mM mannitol.

4. The electrochemical buffer according to claim 3, characterized in that The electronic buffer comprises: 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, 3.42 mM potassium dihydrogen phosphate, 21.58 mM potassium dihydrogen phosphate, 1 mM EDTA, and 10 mM mannitol.

5. The electrochemical buffer according to any one of claims 1 to 4, characterized in that: The pH of the buffer was 8.

0.

6. A method for preparing the electrochemical buffer according to any one of claims 1 to 5, comprising dissolving an electron buffer pair, a metal chelator, a protein protectant and an ion buffer in water, adjusting the pH, and filtering to obtain the electrochemical buffer.

7. The preparation method according to claim 6, characterized in that: The pH value is adjusted to 8.0; The reagent for adjusting pH is KOH solution.

8. Application of the electrochemical buffer according to any one of claims 1 to 5 and / or the electrochemical buffer prepared by the preparation method according to claim 6 or 7 in biological nanopore sensing technology, single molecule sensing technology and / or ultra-trace element detection technology.

9. A kit comprising: A detection probe and the electronic buffer according to any one of claims 1 to 5; or A detection probe and an electrochemical buffer prepared by the preparation method according to claim 6 or 7.

10. A detection method, which uses biological nanopore technology to detect tumors, microorganisms, nucleic acids and / or trace elements using at least one of the following I) to III): I), the electrochemical buffer according to any one of claims 1 to 5; II), an electrochemical buffer prepared by the preparation method according to claim 6 or 7; III), the kit according to claim 9.

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