CN115541680A - Biomolecule detection method based on tunneling electrode - Google Patents

Abstract

The invention discloses a method for detecting biomolecules based on a tunneling electrode, which belongs to the technical field of nanometer devices and biosensing. The detection method includes: (1) preparing a pair of tunneling electrodes with a nano-gap on the tip of a nanopipette or a silicon chip, and modifying the modifier interacting with the biomolecules to be tested to obtain a functionalized tunneling electrode; ( 2) Combine the biomolecules to be tested on the functionalized tunneling electrodes, and then place them in the solution that reacts with the biomolecules to be tested, or directly place the functionalized tunneling electrodes in the solution containing the biomolecules to be tested, using The galvanometer detects the tunneling current signal in real time, and then analyzes the molecular structure or molecular behavior of the biomolecules to be measured by analyzing the current signal. The present invention provides a novel single-molecule sensor that combines functionalized tunneling electrodes with biomolecules, providing unlimited opportunities for understanding biomolecular omics at the molecular level, and can be used in various chemical and biochemical applications .

Description

A Biomolecular Detection Method Based on Tunneling Electrode

technical field

The invention belongs to the technical field of nano devices and biosensing, and in particular relates to a method for detecting biomolecules based on a tunneling electrode.

Background technique

At the heart of molecular bioelectronics research is the construction of nanoscale biomolecular experimental platforms that allow electronic signaling within biomolecules. Biomolecules such as proteins and nucleic acids have diverse molecular recognition capabilities, and under certain circumstances, electron tunneling is prone to occur. Therefore, the integration of biomolecules such as proteins into nanoelectronic devices has always been the pursuit of many biosensing applications. In particular, understanding and manipulating protein-mediated charge transport is of fundamental importance in electrochemical processes, tunneling detection, and ultimately in the rational design of next-generation bioelectronic devices.

Generally, biomolecule-mediated tunneling electron transport relies on the capture of biomolecules between a pair of closely spaced electrodes with a gap of less than 5 nm, which can be monitored by measuring the current response on the connected tunneling electrodes as a characterization of biomolecular behavior. . Early studies have demonstrated that redox-active protein molecules can control the charge transport process through proteins and can serve as active components of micro/nanomolecular electronic circuits. Azurin and cytochrome C (Cyt C) have been widely exploited and exhibit unique molecular bioelectronic behaviors by modulating their redox properties. However, the properties of electronic charge transport are influenced by biomolecular structures, electrode-biomolecule interfaces, and energy level alignment, all of which affect device performance.

The key to single-molecule high-precision biomolecular monitoring lies in the design and fabrication of stable, reproducible, and controllable probes. In this regard, many researchers have made great efforts. The most commonly used methods employ proximal probe techniques, including scanning probe microscopy, mechanically formed break junctions, and lithographically formed nanogap. For example, the most common scanning tunneling microscope fracture junction (STM-BJ) is capable of forming a gap between an atomically sharp tip and a conductive surface. Advanced photolithographic methods, such as mechanically controllable break junctions (MCBJ), can be fabricated on a larger scale, and the size of the electrode gap can be easily adjusted. However, the above two methods are applied to detect the conductance of biomolecules, which are subject to many limitations. For example, the reproducibility and stability of single biomolecular connections are still challenging. Taking proteins as an example, most probe technologies mainly rely on the use of Monolayers of redox-active proteins in a substantially dry state have rarely been studied in solution, which hinders further applications in real samples. Second, technologies such as STM-BJ cannot be used independently in real environments. At the same time, the excessive leakage current generated by nanoelectrodes fabricated by traditional methods usually causes molecular signals to be submerged in the background current.

Contents of the invention

The purpose of the present invention is to provide a biomolecular detection method based on a tunneling electrode, which combines electron tunneling and biomolecular modification technologies to prepare a tunneling electrode with excellent performance, which is applied to DNA, RNA, protein, sugar, etc. detection of various biomolecules.

To achieve the above object, the present invention adopts the following technical solutions:

The invention provides a biomolecule detection method based on a tunneling electrode, comprising the following steps:

(1) Prepare a pair of tunneling electrodes with a nano-gap on the tip of a nanopipette or a silicon wafer, and then modify the surface of the pair of tunneling electrodes with modifiers that can interact with the biomolecules to be tested to obtain a functionalized tunneling electrode ;

(2) The biomolecule to be tested interacts with the modification on the functionalized tunneling electrode, and then placed in a solution containing a substance that reacts with the biomolecule to be tested, or the functionalized tunneling electrode is placed directly on the In the solution containing the biomolecules to be tested, an ammeter is used to detect the tunneling current signal in real time to obtain the corresponding tunneling current signal of the biomolecules, and then analyze the molecular structure or molecular behavior of the biomolecules to be tested by analyzing the current signal.

In the present invention, the surface of the tunneling electrode is functionally modified, and the modified substance interacts with the biomolecules to be tested, so that the biomolecules to be tested are combined with the tunneling electrode pair, and the tunneling current signal can be continuously monitored under different bias conditions. Analyze the bioelectronic behavior or molecular structure of individual biomolecules.

The biomolecules to be tested include protein, DNA, RNA, sugar.

In step (1), the nano-gap tunneling electrode pair is prepared on the tip of the nano-pipette or the silicon wafer by means of electrochemical deposition, chemical etching, mechanically controllable cleavage or electro-etching.

The preparation method of the nano-pipette comprises: inserting a metal wire into the multi-channel thin tube, and drawing the multi-channel thin tube into a nano-pipette with a tip at one end by applying external force.

Preferably, the conductive metal wire in the channel of the nanopipette is made of gold wire, and one side of the multi-channel thin tube is drawn to form a nanopipette with a tip, which is electrochemically deposited on the carbon nanoelectrode to prepare a nanogap gold electrode.

During the electrochemical deposition process, an AC bias voltage is applied between the two nanometer electrode pairs, and the current flowing through the external resistor in the loop is detected by a lock-in amplifier. conductance. The conductance value is negatively correlated with the size of the nano-gap between the electrode pairs. Therefore, through the feedback circuit, different conductance values are used as the termination marks of the electrochemical deposition to prepare the nano-gap electrode pairs with different spacings.

The method for preparing the nano-gap tunneling electrode pair on the silicon wafer comprises: spin-coating photoresist after the silicon wafer is cleaned, and then exposing the silicon wafer, developing the exposed silicon wafer in a developer, and then developing the developed silicon wafer Carry out metal sputtering or evaporation coating, and finally put the silicon wafer into the stripping solution to remove the photoresist and excess metal. Preferably, metal sputtering or evaporation coating is used to deposit gold electrodes with nanometer gaps on the silicon wafer.

Preferably, the positive photoresist is AZ5214e, the developing solution is rzx3038 positive developing solution, and the stripping solution is acetone.

Preferably, the photoresist with a thickness of 1-3 μm is spin-coated on the silicon wafer and then pre-baked. The temperature of the oven is 90-110° C. Baking for 2 minutes, the exposed silicon wafer is immersed in the developer solution, the exposed area is dissolved as the subsequent electrode deposition area, and then an Au layer with a thickness of 200nm is deposited by metal sputtering or evaporation coating to form a pair of nano-gap tunneling electrodes.

The tunneling electrode has one or more pairs of nano-gap electrodes; the range of the nano-gap is 0.1nm-10nm; the nano-gap is the minimum distance between two electrodes in the electrode pair. Preferably, the nano-gap ranges from 1.5 nm to 3.1 nm.

Further, in step (1), the modification method includes electrostatic adsorption, hydrogen bonding, and chemical adsorption.

Preferably, the modifier may be, but not limited to, a biotin-streptavidin complex, protein or nucleic acid molecule with thiol modification. The modification is connected to the electrode through a sulfhydryl group.

Further, in step (2), the current signal analysis method includes: calculating the real-time conductance according to the bias voltage and the tunneling current signal, performing frequency distribution statistics on the conductance, and screening the waveform of the conductance change in a short period of time.

Specifically, when the biomolecule to be tested is a protein with enzymatic catalytic activity, in step (1), the functional modification includes: first modifying the surface of the tunneling electrode with thiol biotin, and then combining with streptavidin to obtain a functional In step (2), the protein to be tested is biotin-modified and bound to the functionalized tunneling electrode, and then the tunneling electrode is placed in a solution containing a catalytic substrate, and an ampere meter is used to detect the tunneling electrode in real time. Current: Calculate the real-time conductance according to the bias voltage and tunneling current signal, the conductance G=I/V, conduct frequency distribution statistics on the conductance, screen the waveform of the conductance change in a short period of time, and analyze the instantaneous state change process of the catalytic reaction from the instantaneous waveform change.

When the biomolecule to be tested is DNA, in step (1), the functional modification includes: respectively modifying the probe DNA I and the probe DNA II on both ends of the tunnel electrode pair to prepare a functional tunnel electrode; The needle DNA I includes a complementary sequence I complementary to the second half of the target DNA chain, the probe DNA II includes a complementary sequence II complementary to the first half of the target DNA chain, and one end of the probe DNA I and the probe DNA II is modified with Sulfhydryl group; in step (2), place the functionalized tunneling electrode in the solution containing the DNA molecule to be tested, and use an ammeter to detect the tunneling current in real time. If the current value increases abruptly, and maintains an oscillating signal , it is judged that the DNA molecule to be tested is the target DNA.

Preferably, the thiol group is modified at the 5' end of the probe DNA I, and the 5' end of the sequence has a poly(T) sequence; the 3' end of the probe DNA II is modified with a thiol group, and the 3' end of the sequence has a poly(T) sequence.

When the biomolecule to be tested is a DNA mutation chain, in step (1), the functional modification includes: first modifying dithiobis(succinimidyl propionate) on one end of the tunneling electrode pair, and then combining with the probe DNA reaction, so that the two ends of the probe DNA are respectively connected to the two ends of the tunneling electrode pair to obtain a functionalized tunneling electrode; the probe DNA contains a complementary sequence to the template DNA, and its two ends are respectively modified with amino and sulfhydryl groups ; In step (2), first place the functionalized tunneling electrode in a solution containing template DNA, use an ammeter to detect the tunneling current, and obtain a tunneling current signal corresponding to the template DNA; then take out the tunneling electrode, and remove the template The DNA and the probe DNA are unwound; then the untwisted functionalized tunneling electrode is placed in the solution containing the DNA mutation strand to be tested, and the tunneling current is detected by an ammeter to obtain the tunneling current signal corresponding to the DNA mutation strand to be tested ; Finally, by comparing the tunneling current signal corresponding to the template DNA and the tunneling current signal corresponding to the DNA mutation chain, the degree of base mutation in the DNA mutation chain is judged.

Wherein the amino terminal of the probe DNA is connected with dithiobis(succinimidyl propionate) (DSP), and the sulfhydryl terminal is connected with one side electrode of the unmodified DSP.

Preferably, the probe DNA also includes a poly(T) sequence at the 5' end and a poly(T) sequence at the 3' end in addition to the complementary sequence.

In step (2), the condition for unwinding the template DNA and the probe DNA is to place in ultrapure water at 93-98°C.

Terminology Explanation:

When the term "nano-pipette" is used herein, it generally refers to a pipette-like shape with a tip on one side and a multi-channel thin tube with a tip size of nanometer scale.

The term "interstices" as used herein generally refers to pores, channels or passages formed or otherwise provided in a material. The material may be a solid material, such as a substrate. The gap may be disposed adjacent or proximate to the sensing circuit or an electrode coupled to the sensing circuit. In some examples, the gaps have a characteristic width or diameter on the order of 0.1 nanometers to about 100 nm. A gap having a nanoscale width may be referred to as a "nanogap". In some cases, the width of the nanogap can be smaller than the diameter of a biomolecule or a subunit (eg, monomer) of a biomolecule.

The term "electrode" as used herein generally refers to a material or component that can be used to measure electrical current. An electrode (or electrode assembly) can be used to measure current flow into and out of another electrode. In some cases, electrodes can be disposed in a channel (eg, nanogap) and used to measure the current across the channel. The current may be a tunneling current. Such currents can be detected as biomolecules, such as proteins, flow through the nanogap. In some cases, a sensing circuit coupled to an electrode provides an applied voltage across the electrode to generate a current. Alternatively or in addition, the electrodes may be used to measure and/or identify conductance associated with biomolecules such as amino acid subunits or protein monomers. In this case, tunneling current can be related to conductance.

In some examples, the pair of nanoelectrodes includes individual nanoelectrodes separated by a gap, the nanogap having a length of 0.1 nm to 100 nm. Nanoelectrodes can be of any convenient shape or size, and can comprise any conductive material. Each electrode disclosed herein may be made of a different material or a mixture of materials such as an alloy.

Nanoelectrodes are used to measure electrical currents that can travel through and/or across molecules. The current may be a tunneling current. Measuring the current can be used to determine the sequence of a biopolymer, eg a nucleic acid molecule (eg DNA or RNA) or a protein. For mass measurement, the gap spacing between the electrodes of one or more nanoelectrode pairs can be stable and controllable.

The beneficial effect that the present invention possesses:

The present invention takes a single biomolecule as the monitoring object and provides a novel single-molecule sensor. By functionalizing the tunneling electrode with a fixed gap, the functionalized tunneling electrode is combined with the biomolecule, which is useful for the study of a single biomolecule. The electronic/electrochemical properties of the molecule are critical. By utilizing good device stability, label-free capability, and high-speed data acquisition, it is possible to directly study stochastic fluctuations in environmental conditions and reveal in real time the conductance pathways and further conformational states of individual biomolecules relevant to biological processes, such as protein folding and DNA sequencing. Therefore, this approach combined with biomolecular engineering such as DNA, protein, etc., opens up unlimited opportunities for understanding biomolecular omics at the molecular level, which can be used in various chemical and biochemical applications.

Description of drawings

Figure 1 is a pair of tunneling electrodes with nano-gap prepared on a silicon wafer.

Fig. 2 is the real-time tunneling current signal (I-t) of the hydrogen peroxide reaction catalyzed by peroxidase in Example 1.

Figure 3 shows the instantaneous waveform response of peroxidase catalyzed hydrogen peroxide reaction. Four representative waveform changes were selected, representing different conductance changes in the process of peroxidase catalyzing hydrogen peroxide reaction.

Fig. 4 is a histogram of frequency statistics of peroxidase-catalyzed hydrogen peroxide reaction.

Figure 5 is a pair of tunneling electrodes with a nanogap at the tip of a nanopipette.

FIG. 6 is a schematic flow chart of segmentally modified template DNA recognizing complementary DNA strands in Example 2. FIG.

Fig. 7 is the real-time tunneling current signal (I-t) after recognizing the complementary DNA strand.

FIG. 8 is a schematic diagram of DNA template strands connected to both ends of the tunneling electrode in Example 3. FIG.

Figure 9 is a statistical histogram of the frequency of binding complementary DNA strands (upper) and mutant DNA strands (lower).

detailed description

The present invention will be further described below in conjunction with specific embodiments. The following examples are only used to illustrate the present invention, and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention belong to the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

Example 1

1. The tunneling electrode is prepared by the chip method. The preparation process includes:

Step 1: Clean the silicon wafer in the cleaning solution;

Specifically: put the silicon wafer in acetone for 5 minutes, take it out, put it in isopropanol for 5 minutes, and rinse it with 18.2 MΩ·cm ultrapure water.

Step 2: Spin-coat photoresist on the cleaned silicon wafer; expose the silicon wafer; develop the exposed silicon wafer in a developer; perform metal sputtering or evaporation coating on the developed silicon wafer; Place in a stripper to remove photoresist and excess metal.

Specifically:

1. Spin coating, spin coating photoresist on the cleaned silicon wafer, the thickness of the glue is 3 μm;

2. Pre-baking, the photoresist is pre-baked to improve the adhesion to the substrate. The pre-baked treatment is specifically placed in an oven with a temperature of 90°C for 10 minutes;

3. Exposure, exposure for 5 seconds on a UV exposure machine (Karl SUSS MA/BA6);

4. Post-bake, bake at 120°C for 2 minutes to generate cross-linking reaction in the exposed area;

5. Development, after flood exposure, the grid lines can be dissolved in the developer to deposit Au in the subsequent steps;

6. Evaporation, depositing an Au layer with a thickness of 200nm by a thermal evaporator (VNANO Vacuum Technology Co., Ltd);

7. Stripping, washing away unnecessary photoresist and metal to obtain the metal electrode arrangement on the silicon wafer.

The photoresist used is positive resist AZ5214e, the developer is rzx3038 positive resist developer, and the stripping solution is acetone.

The resulting scanning electron microscope image of the tunneling electrode pair is shown in Fig. 1 .

2. Enzyme-catalyzed reaction of modified protein

Step 1: Put the prepared tunneling electrode into an ethanol solution containing mercaptobiotin (concentration: 100nM), soak it for 2 hours and take it out;

Step 2: Soak the tunneling electrode treated in step 1 in a streptavidin solution (concentration: 10 μM, PBS buffer: 10 mM, pH 7.4) for 2 hours and take it out;

Step 3: Soak the tunneling electrode treated in step 2 in a biotin-modified peroxidase solution (concentration: 10 μM, PBS buffer: 10 mM, pH 7.4) for 2 hours and take it out;

Step 4: Connect the processed tunneling electrodes to the test system;

Step 5: Add 10 μL of 30% hydrogen peroxide to the sample cell.

The test system described in step 4 is specifically composed of Amplifier (Molecular Devices, model no. MultiClamp 700B), Digitizer (Molecular Devices, model no. Axon Digidata1550B), and Headstage (Molecular Devices, model no. CV 7B).

The real-time recording image of the obtained current is shown in Fig. 2 .

Signal processing is performed on the obtained data to filter out instantaneous waveform changes, as shown in Figure 3. The instantaneous state change process of the catalytic reaction can be studied from the instantaneous waveform change.

Calculate the real-time conductance according to the bias voltage and tunneling current signals, conductance G=I/V, make statistics on different conductances, and make a frequency histogram, as shown in Figure 4. It can be seen from the frequency histogram that there are two state distributions in the overall catalytic reaction.

Example 2

1. Prepare the tunneling electrode, the process is as follows:

Step 1. Place the metal wire in the thin glass tube;

Specifically, the thin glass tube is a double channel with an outer diameter of 1.2 mm, an inner diameter of 0.90 mm, and a length of 100 mm. Two identical metal wires are respectively inserted from the tail. The metal wires are gold wires, the length is 2 cm, and the outer diameter is 25 μm. ;

Step 2, drawing a thin glass tube containing a wire inside;

Specifically, first, the thin glass tube containing the gold wire is cleaned, and the cleaning steps are as follows: rinse with 18.2 MΩ·cm ultrapure water, put it into a plasma cleaning machine to clean the surface debris, and clean for 30 minutes.

The cleaned thin glass tube containing gold wire inside is drawn by P-2000 laser drawing machine, and drawn by a three-step method: the first step is to apply a laser beam to heat the middle part of the thin glass tube, and draw it at the same time. The parameters are set as Heat: 350, Filament: 4, Velocity: 15, Delay: 120, Pull: 0, and the middle part of the thin glass tube is heated and melted to form an hourglass-shaped structure; the second step is to connect both ends of the thin glass tube with tetrafluoroethylene The hose is sealed and connected, and the inside of the thin glass tube is evacuated for 6 minutes. Then heat seal the glass tube and the wire. The heat sealing steps are: heat the glass tube for 4 seconds, cool for 1 minute, and reheat for 7 seconds; the drawing parameters are set as Heat: 390, Filament: 3, Velocity: 12. Delay: 120, Pull: 0; the third step, heat the thin glass tube again after the wire is sealed, and apply pulling force to both ends of the thin glass tube at the same time, the drawing parameters are set as Heat: 520, Filament: 3, Velocity: 36, Delay: 170, Pull: 50. At the end of the drawing procedure, a wire-containing nanopipette with a tip on one side is formed.

Due to the difference in ductility between the glass capillary and the metal wire, the tip of the metal wire is often wrapped in glass, so it needs to be mechanically polished with a needle grinder to expose the metal wire.

Step 3, using electrochemical deposition to prepare nano-gap electrode pairs on the tip of the metal wire;

Specifically, using a potentiostat, immerse the metal wire at the tip of the nanopipette into the electroplating solution (the specific components are: 4.4mM NH ₄ AuSO ₃ , 52mM (NH ₄ ) ₂ SO ₃ ), and the nanometer at the tip of the two metal wires The electrode pair is used as the working electrode, and a gold ball with a diameter of 2-3 mm immersed in the electroplating solution is used as the counter electrode, and an electrochemical deposition current of 2-3 μA is applied. During the electrochemical deposition process, a 4mV, 1Hz AC bias is applied between the two nanometer electrode pairs, and the current flowing through the external resistor (1kΩ) is detected by the lock-in amplifier. Calculate the conductance between the nanoelectrode pair using the voltage. At the same time, the conductance value is negatively correlated with the size of the nano-gap between the electrode pairs. Therefore, through the feedback circuit, the different conductance values are used as the termination marks of the electrochemical deposition to prepare the nano-gap electrode pairs with different spacings.

After the nanogap electrode pair is prepared, it is stored in 18.2 MΩ·cm ultrapure water.

The distance between the nanogap electrode pairs is 2.5 nm, which is estimated by Simmons model. The resulting scanning electron microscope image of the tunneling electrode pair is shown in FIG. 5 .

2. Segmentally modified template DNA recognizes complementary DNA strands

The DNA used was treated with TCEP (tris(2-carboxyethyl)phosphine), specifically, 100 μL DNA solution (concentration: 100 μM) to be treated was mixed with 10 μL TCEP solution (concentration: 100 mM), and reacted for 1 hour.

Step 1: Put the prepared tunneling electrode into the solution containing DNA template strand 1 (concentration: 10nM, PBS buffer: 10mM, pH 7.4), take it out after soaking for 2h, and the sequence of DNA template strand 1 is: 5` -TTTTTGTGCCCGCCGA-3`, HS-SH C6 modified at the 5` end;

Step 2: Put the tunneling electrode treated in step 1 into pure PBS buffer (concentration: 10mM, pH 7.4), use CHI600E, apply a constant potential -0.9v on one side for 30s, and interrupt the modification on one side DNA template strand 1;

Step 3: Put the tunneling electrode treated in step 2 into the solution containing DNA template strand 2 (concentration: 10nM, PBS buffer: 10mM, pH 7.4), soak for 2 hours and take it out, wherein the sequence of DNA template strand 2 is: 5`-CGAGAATTAGTCTTTTT-3`, the 3` end is modified with HS-SH C3;

Step 4: Connect the processed tunneling electrodes to the test system;

Step 5: Add 10 μL of DNA complementary strand 3 with a concentration of 10 nM to the sample pool. The sequence of DNA complementary strand 3 is the complementary sequence of the middle recognition part of DNA template strand 1+DNA template strand 2. The sequence of DNA complementary strand 3 is: GACTAATTCTCCTCGGCGGGCAC .

The flow diagram of segmentally modifying the template DNA to recognize the complementary DNA strand is shown in FIG. 6 .

The resulting real-time current recording image is shown in Figure 7. When DNA template strand 1 and DNA template strand 2 are combined with DNA complementary strand 3, the current real-time recording signal increases abruptly and maintains a stable oscillating signal.

Studies have shown that there are and only complementary DNA sequences that can produce the above two characteristics at the same time. Unfitted DNA strands cannot form bridges and cannot generate signals.

Example 3

1. Preparation of the tunneling electrode The preparation method is the same as in Example 2.

2. Modified template DNA to identify mismatched DNA strands

Step 1: Soak the prepared tunneling electrode in 1 μM DSP (dithiobis(succinimidyl propionate)) solution (concentration: 1 μM) for 2 hours. The DSP used is treated with TCEP, specifically, the treated 100mL DSP solution (concentration 100μM) was mixed with 10μL TCEP solution (concentration 100mM) and reacted for 1 hour;

Step 2: Put the tunneling electrode treated in step 1 into pure PBS buffer (concentration: 10mM, pH 7.4), use CHI600E, apply a constant potential -0.9v on one side for 30s, and interrupt the modification on one side DSP;

Step 3: Put the tunneling electrode treated in step 2 into the solution containing DNA template strand 4 (concentration: 10nM, PBS buffer: 10mM, pH 7.4), soak for 2h and take it out, so that the DNA template strand 4 is connected to the tunneling electrode both ends of the electrodes. The sequence of DNA template strand 4 is: 5`-TTTTTGTGCCCGCCGACGAGAATTAGTCTTTTT-3`, wherein the 5` end is modified with NH2 C6, and the 3` end is modified with HS-SH C3;

A schematic diagram of the DNA template strand 4 connected to both ends of the tunneling electrode is shown in FIG. 8 .

Step 4: Connect the tunneling electrode processed in step 3 to the test system;

Step 5: Add 10 μL of DNA complementary strand 5 at a concentration of 10 nM to the sample pool for testing. Wherein the DNA complementary strand 5 sequence is the complementary sequence of the middle recognition part of the DNA template strand 4, and the sequence of the DNA complementary strand 5 is 5`-GACTAATTCTCGTCGGCGGGCAC-3`;

Step 6: Take out the tunneling electrode in step 5, and soak it in ultrapure water (18.2MΩ·cm) at 93-98°C for 1 minute, so that the DNA template strand 4 and the DNA complementary strand 5 are unwound;

Step 7: Connect the tunneling electrode processed in step 6 to the test system again;

Step 8: Add 10 μL of mutated DNA strand 6 at a concentration of 10 nM to the sample pool for testing. Wherein, the DNA mutant strand 6 sequence is a sequence in which a single base of the DNA complementary strand 5 is mutated, and the sequence of the DNA mutant strand 6 is 5`-GACTAATTCTCCTCGGCGGGCAC-3`;

Step 9: Comparing the difference between the above two test results, the result of whether the DNA strand is mutation-mismatched can be obtained.

Specifically, the current-time (I-t) data obtained in steps 4 and 7 are counted, conductance G=I/V, and the software used for the statistics of the data is Clampfit v.10.7 software (Molecular Devices), to obtain different conductances Frequency histogram, as shown in Figure 9. DNA complementary strand 5 has three distributions, while DNA mutation strand 6 has only two distributions, so it can be judged that DNA mutation strand 6 has mismatches caused by base mutations.

Through Example 3, the mutation of a specific DNA sequence can be directly detected without additional nucleic acid amplification means, and the detection accuracy can reach the level of a single base.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (10)

1. A biomolecule detection method based on a tunneling electrode is characterized by comprising the following steps:

(1) Preparing a tunneling electrode pair with a nanometer gap on the tip of a nanometer pipette or a silicon chip, and modifying a modifier capable of interacting with a biomolecule to be detected on the surface of the tunneling electrode pair to prepare a functional tunneling electrode;

(2) Combining the interaction of the biomolecule to be detected and the modifier on the functionalized tunneling electrode, then placing the functionalized tunneling electrode in a solution containing a substance which reacts with the biomolecule to be detected, or directly placing the functionalized tunneling electrode in the solution containing the biomolecule to be detected, detecting a tunneling current signal in real time by adopting an ammeter to obtain a tunneling current signal corresponding to the biomolecule, and analyzing the molecular structure or molecular behavior of the biomolecule to be detected by analyzing the current signal.

2. The tunneling electrode-based biomolecule detection method of claim 1, wherein the biomolecule to be detected comprises protein, DNA, RNA, sugar.

3. A tunneling electrode-based biomolecule detection method according to claim 1, wherein in step (1), the method for preparing a nanopipette comprises: and inserting a metal wire into the multichannel tubule, and drawing the multichannel tubule into a nanometer pipettor with a tip at one end by applying external force.

4. A tunneling electrode-based biomolecule detection method according to claim 1 or 3, wherein in step (1), the pair of nanogap tunneling electrodes is prepared on the nanopipette tip or the silicon wafer by electrochemical deposition, chemical etching, mechanical controlled junction splitting or electrical etching; the range of the nanometer gap is 0.1 nm-10 nm.

5. A tunneling electrode-based biomolecule detection method according to claim 1, wherein in step (1), the modification method includes electrostatic adsorption, hydrogen bonding, and chemical adsorption.

6. The biomolecule detection method based on tunneling electrode of claim 1, wherein the modification is a biotin-streptavidin complex, a protein or a nucleic acid molecule with thiol modification.

7. A tunneling electrode-based biomolecule detection method according to claim 1, wherein in the step (2), the current signal analysis method comprises: and calculating real-time conductance according to the bias voltage and the tunneling current signals, carrying out frequency distribution statistics on the conductance, and screening the waveform of conductance change in a short time.

8. The method for detecting biomolecules based on a tunneling electrode of claim 1, wherein when the biomolecule to be detected is a protein having enzymatic activity, the step (1) comprises the following functional modifications: firstly, carrying out sulfhydryl biotin modification on the surface of a tunneling electrode, and then combining with streptavidin to obtain a functionalized tunneling electrode; in the step (2), the protein to be detected is modified by biotin and then combined on the functionalized tunneling electrode, the tunneling electrode is placed in a solution containing a catalytic substrate, and the tunneling current is detected in real time by adopting an ammeter.

9. The method for detecting biomolecules based on tunneling electrodes according to claim 1, wherein when the biomolecule to be detected is DNA, the functionalized modification in step (1) comprises: respectively modifying the probe DNA I and the probe DNA II at two ends of a tunneling electrode pair to prepare a functional tunneling electrode; the probe DNA I comprises a complementary sequence I which is complementary with the second half of a target DNA chain, the probe DNA II comprises a complementary sequence II which is complementary with the first half of the target DNA chain, and one end of each of the probe DNA I and the probe DNA II is modified with a sulfhydryl group; in the step (2), the functionalized tunneling electrode is placed in a solution containing the DNA molecules to be detected, an amperemeter is adopted to detect the tunneling current in real time, and if the current value is suddenly increased and the oscillation signal is kept, the DNA molecules to be detected are judged to be the target DNA.

10. The method for detecting biomolecules based on tunneling electrodes as claimed in claim 1, wherein when the biomolecule to be detected is a DNA mutation chain, the functional modification in step (1) comprises: firstly, modifying dithiobis (succinimide propionate) at one end of a tunneling electrode pair, and then reacting with probe DNA to ensure that two ends of the probe DNA are respectively connected with two ends of the tunneling electrode pair to prepare a functional tunneling electrode; the probe DNA comprises a complementary sequence complementary with the template DNA, and both ends of the probe DNA are respectively modified with amino and sulfydryl; in the step (2), firstly, the functionalized tunneling electrode is placed in a solution containing template DNA, and a tunneling current is detected by adopting an ammeter to obtain a tunneling current signal corresponding to the template DNA; taking out the tunneling electrode, and unwinding the template DNA and the probe DNA; then placing the derotated functionalized tunneling electrode in a solution containing a DNA mutation chain to be detected, and detecting tunneling current by adopting an ammeter to obtain a tunneling current signal corresponding to the DNA mutation chain to be detected; and finally, judging the base mutation degree of the DNA mutation chain by comparing the tunneling current signal corresponding to the template DNA with the tunneling current signal corresponding to the DNA mutation chain.

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