A biomolecule detection method based on tunneling electrodes

Abstract

The present invention discloses a method for detecting biomolecules based on tunneling electrodes, which belongs to the field of nanodevices and biosensor technology. The detection method comprises: (1) preparing a tunneling electrode pair with a nanogap on the tip of a nanopipette or a silicon wafer, modifying a modifier that interacts with the biomolecule to be detected, and preparing a functionalized tunneling electrode; (2) binding the biomolecule to be detected to the functionalized tunneling electrode, and then placing it in a solution that reacts with the biomolecule to be detected, or directly placing the functionalized tunneling electrode in a solution containing the biomolecule to be detected, using an ammeter to detect the tunneling current signal in real time, and analyzing the current signal to further analyze the molecular structure or molecular behavior of the biomolecule to be detected. The present invention provides a new single-molecule sensor that combines the functionalized tunneling electrode with the biomolecule, providing unlimited opportunities for understanding biomolecular omics at the molecular level, and can be used in various chemical and biochemical applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano devices and biosensors, and in particular relates to a method for detecting biomolecules based on tunneling electrodes. Background Art

[0002] The core of molecular bioelectronics research is the construction of nanoscale biomolecular experimental platforms that allow for electronic signaling within biomolecules. Biomolecules such as proteins and nucleic acids possess diverse molecular recognition capabilities and, under certain circumstances, are susceptible to electron tunneling. Therefore, integrating biomolecules such as proteins into nanoelectronic devices has been a long-standing goal for numerous biosensing applications. In particular, understanding and manipulating protein-mediated charge transport is fundamentally important in electrochemical processes, tunneling detection, and ultimately, the rational design of next-generation bioelectronic devices.

[0003] Typically, 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. The current response at the connected tunneling electrodes can be measured as a proxy for monitoring biomolecule 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 / nano-molecular electronic circuits. Azurin and cytochrome C (Cyt C) have been widely utilized and exhibit unique molecular bioelectronic behaviors by regulating their redox properties. However, the characteristics of electronic charge transport are affected by the biomolecular structure, the electrode-biomolecule interface, and the energy level arrangement, all of which affect the performance of the device.

[0004] The key to high-precision single-molecule biomolecular monitoring lies in the design and fabrication of stable, reproducible, and controllable probes. Numerous researchers have devoted significant effort to this endeavor. The most commonly used approaches employ proximal probe techniques, including scanning probe microscopy (SPM), mechanically formed break junctions (JNs), and photolithographically formed nanogaps. For example, the most common scanning tunneling microscopy break junction (STM-BJ) creates a gap between an atomically sharp tip and a conductive surface. Advanced photolithographic methods, such as mechanically controlled break junctions (MCBJs), enable larger-scale fabrication and easily tunable electrode gap sizes. However, both of these approaches face numerous limitations when applied to biomolecular conductance monitoring. For example, the reproducibility and stability of single biomolecule junctions remain challenging. For example, most probe techniques rely on monolayers of redox-active proteins in a largely dry state, with few studies conducted in solution. This hinders their application to real-world samples. Furthermore, techniques like STM-BJs cannot be used independently in real-world environments. Furthermore, the excessive leakage current generated by conventionally fabricated nanoelectrodes often causes the molecular signal to be submerged in the background current. Summary of the Invention

[0005] The purpose of the present invention is to provide a biomolecule detection method based on tunneling electrodes, which combines electron tunneling and biomolecule modification technologies to prepare tunneling electrodes with excellent performance, and is applied to the detection of various biomolecules including DNA, RNA, proteins, sugars, etc.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a biomolecule detection method based on a tunneling electrode, comprising the following steps:

[0008] (1) Preparing a tunneling electrode pair with a nanogap on a nanopipette tip or a silicon wafer, and then modifying the surface of the tunneling electrode pair with a modifier that can interact with the biomolecule to be detected to obtain a functionalized tunneling electrode;

[0009] (2) The biomolecule to be tested is interacted with the modified substance and bound to the functionalized tunneling electrode, which is then placed in a solution containing a substance that reacts with the biomolecule to be tested, or the functionalized tunneling electrode is directly placed in a solution containing the biomolecule to be tested, and an ammeter is used to detect the tunneling current signal in real time to obtain the tunneling current signal corresponding to the biomolecule. The molecular structure or molecular behavior of the biomolecule to be tested is analyzed by analyzing the current signal.

[0010] The present invention functionalizes the surface of the tunneling electrode pair, and the modified substance interacts with the biomolecule to be tested, so that the biomolecule to be tested is combined with the tunneling electrode pair. By continuously monitoring the tunneling current signal under different bias conditions, the bioelectronic behavior or molecular structure of a single biomolecule can be analyzed.

[0011] The biomolecules to be detected include proteins, DNA, RNA, and sugars.

[0012] In step (1), a nanogap tunneling electrode pair is prepared on a nanopipette tip or a silicon wafer by electrochemical deposition, chemical etching, mechanically controllable cracking or electrical etching.

[0013] The preparation method of the nanopipette comprises: inserting a metal wire into a multi-channel capillary tube, and drawing the multi-channel capillary tube into a nanopipette with a pointed tip at one end by applying external force.

[0014] Preferably, the conductive metal wire in the nanopipette channel is gold wire, a nanopipette with a tip is formed by drawing a single side of a multi-channel capillary tube, and a gold electrode with a nanogap is prepared by electrochemical deposition on the carbon nanoelectrode.

[0015] During electrochemical deposition, an AC bias is applied between two nanoelectrode pairs. A lock-in amplifier measures the current flowing through an external resistor in this loop. The conductance between the nanoelectrode pairs is calculated based on the current magnitude and the voltage divider between the nanoelectrode pairs. The conductance is negatively correlated with the size of the nanogap between the electrodes. Therefore, using a feedback circuit, different conductance values ​​serve as the termination marker for electrochemical deposition, allowing the fabrication of nanogap electrode pairs with varying spacing.

[0016] A method for fabricating a nanogap tunneling electrode pair on a silicon wafer includes: cleaning the silicon wafer, spin-coating a photoresist, exposing the wafer to light, developing the exposed wafer in a developer, then sputtering or evaporating a metal film on the developed wafer, and finally placing the wafer in a stripping solution to remove the photoresist and excess metal. Preferably, gold electrodes with nanogap are deposited on the silicon wafer by metal sputtering or evaporation.

[0017] Preferably, the photoresist is AZ5214e positive photoresist, the developer is rzx3038 positive photoresist, and the stripping solution is acetone.

[0018] Preferably, a photoresist with a thickness of 1 to 3 μm is spin-coated on the silicon wafer and then pre-baked at an oven temperature of 90 to 110°C for 5 to 15 minutes. The wafer is then exposed to UV light for 5 seconds and then baked at 120°C for 2 minutes. The exposed silicon wafer is immersed in a developer, and the exposed area is dissolved as a subsequent electrode deposition area. Then, a 200 nm thick Au layer is deposited by metal sputtering or evaporation coating to form a nanogap tunneling electrode pair.

[0019] The tunneling electrode comprises one or more nanogap electrode pairs; the nanogap ranges from 0.1 nm to 10 nm; the nanogap is the minimum distance between two electrodes in an electrode pair. Preferably, the nanogap ranges from 1.5 nm to 3.1 nm.

[0020] Furthermore, in step (1), the modification method includes electrostatic adsorption, hydrogen bonding, and chemical adsorption.

[0021] Preferably, the modified substance may be, but is not limited to, a biotin-streptavidin complex, a protein, or a nucleic acid molecule with thiol modification, and the modified substance is connected to the electrode via the thiol group.

[0022] Furthermore, in step (2), the current signal analysis method includes: calculating the real-time conductance based on 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.

[0023] Specifically, when the biological molecule to be tested is a protein with enzyme catalytic activity, in step (1), the functional modification includes: first, modifying the surface of the tunneling electrode with thiol biotin, and then binding it with streptavidin to obtain a functionalized tunneling electrode; in step (2), the protein to be tested is modified with biotin and then bound to the functionalized tunneling electrode, and then the tunneling electrode is placed in a solution containing a catalytic substrate, and the tunneling current is detected in real time using an ammeter; the real-time conductance is calculated based on the bias voltage and the tunneling current signal, the conductance G=I / V, the conductance is subjected to frequency distribution statistics, the waveform of the conductance change in a short period of time is screened, and the transient state change process of the catalytic reaction is analyzed from the transient waveform change.

[0024] When the biological molecule to be detected is DNA, in step (1), the functional modification includes: modifying probe DNA I and probe DNA II at the two ends of the tunneling electrode pair respectively to obtain a functionalized tunneling electrode; the probe DNA I contains a complementary sequence I that is complementary to the second half of the target DNA chain, and the probe DNA II contains a complementary sequence II that is complementary to the first half of the target DNA chain, and one end of the probe DNA I and probe DNA II is modified with a thiol group; in step (2), the functionalized tunneling electrode is placed in a solution containing the DNA molecule to be detected, and the tunneling current is detected in real time using an ammeter. If the current value increases suddenly and maintains an oscillating signal, it is determined that the DNA molecule to be detected is the target DNA.

[0025] 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.

[0026] When the biological molecule to be detected is a DNA mutation chain, in step (1), the functional modification includes: first modifying one end of the tunneling electrode pair with disulfide bis(succinimidyl propionate), and then reacting with the probe DNA so that the two ends of the probe DNA are respectively connected to the two ends of the tunneling electrode pair to prepare a functionalized tunneling electrode; the probe DNA contains a complementary sequence complementary to the template DNA, and its two ends are respectively modified with an amino group and a thiol group; in step (2), first placing the functionalized tunneling electrode in a solution containing the template DNA, using an ammeter to detect the tunneling current, and obtaining a tunneling current signal corresponding to the template DNA; then taking out the tunneling electrode, and unwinding the template DNA and the probe DNA; then placing the unwound functionalized tunneling electrode in a solution containing the DNA mutation chain to be detected, using an ammeter to detect the tunneling current, and obtaining a tunneling current signal corresponding to the DNA mutation chain to be detected; 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 determined.

[0027] The amino end of the probe DNA is connected to dithiobis(succinimidyl propionate) (DSP), and the sulfhydryl end is connected to one side electrode of unmodified DSP.

[0028] Preferably, the probe DNA includes, in addition to the complementary sequence, a poly(T) sequence at the 5' end and a poly(T) sequence at the 3' end.

[0029] In step (2), the template DNA and the probe DNA are unwound in ultrapure water at 93-98°C.

[0030] Terminology Notes:

[0031] The term "nanopipette" as used herein generally refers to a multi-channel tube that is similar in shape to a pipette and has a tip on one side with a tip size of nanometers.

[0032] The term "gap" as used herein generally refers to a pore, channel, or passage formed or otherwise provided in a material. The material may be a solid material, such as a substrate. The gap may be arranged adjacent to or in proximity to a sensing circuit or an electrode coupled to the sensing circuit. In some examples, the gap has a characteristic width or diameter on the order of 0.1 nanometers to about 100 nm. A gap having a nanometer-scale width may be referred to as a "nanogap." In some cases, the width of a nanogap may be smaller than the diameter of a biomolecule or a subunit (e.g., a monomer) of a biomolecule.

[0033] The term "electrode" as used herein generally refers to a material or component that can be used to measure electric current. An electrode (or electrode component) can be used to measure the current flowing into or out of another electrode. In some cases, an electrode can be arranged in a channel (e.g., a nanogap) and used to measure the current flowing across the channel. The current can be a tunneling current. Such a current can be detected when a biomolecule (e.g., a protein) flows through a nanogap. In some cases, a sensing circuit coupled to an electrode provides an applied voltage across the electrode to generate an electric current. Alternatively or in addition, the electrode can be used to measure and / or identify the conductance associated with a biomolecule (e.g., an amino acid subunit or a protein monomer). In this case, the tunneling current can be related to the conductance.

[0034] In some examples, the nanoelectrode pair includes independent nanoelectrodes separated by a gap, wherein the nanogap length is 0.1 nm to 100 nm. The nanoelectrodes can have any convenient shape or size and can be composed of any conductive material. Each electrode disclosed herein can be made of a different material or a mixture of materials, such as an alloy.

[0035] Nanoelectrodes are used to measure the current that can pass through and / or across molecules. The current can be a tunneling current. Measuring the current can be used to determine the sequence of biopolymers, such as nucleic acid molecules (e.g., DNA or RNA), or proteins. For mass measurement, the gap spacing between the electrodes of one or more nanoelectrode pairs can be stable and controllable.

[0036] The present invention has the following beneficial effects:

[0037] This invention uses single biomolecules as monitoring targets, providing a novel single-molecule sensor. By functionalizing a fixed-gap tunneling electrode and then combining it with the biomolecule, this sensor is crucial for studying the electronic and electrochemical properties of single biomolecules. By leveraging excellent device stability, label-free capabilities, and high-speed data acquisition, it is possible to directly study random fluctuations under environmental conditions and reveal in real time the conductance pathways of single biomolecules and further conformational states relevant to biological processes, such as protein folding and DNA sequencing. Therefore, this approach, combined with engineering of biomolecules such as DNA and proteins, offers unlimited opportunities for understanding biomolecular omics at the molecular level and can be used in a variety of chemical and biochemical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A pair of tunneling electrodes with nanogaps fabricated on a silicon wafer.

[0039] Figure 2 It is the real-time tunneling current signal (It) of the peroxidase-catalyzed hydrogen peroxide reaction in Example 1.

[0040] Figure 3 Four representative waveform changes were selected to represent the transient waveform response of peroxidase-catalyzed hydrogen peroxide reaction, representing the different conductivity changes of peroxidase in the process of catalyzing hydrogen peroxide reaction.

[0041] Figure 4 This is the frequency statistical histogram of peroxidase-catalyzed hydrogen peroxide reaction.

[0042] Figure 5 is a tunneling electrode pair with a nanogap located at the tip of the nanopipette.

[0043] Figure 6 Schematic diagram of the process of segmented modified template DNA recognizing complementary DNA chains in Example 2.

[0044] Figure 7 It is the real-time tunneling current signal (It) after recognizing the complementary DNA chain.

[0045] Figure 8Schematic diagram of the DNA template chain connected to both ends of the tunneling electrode in Example 3.

[0046] Figure 9 The frequency statistical histograms of binding complementary DNA chains (top) and mutant DNA chains (bottom). DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific examples. 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, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0049] Example 1

[0050] 1. Preparation of tunneling electrodes by chip method. The preparation process includes:

[0051] Step 1: Place the silicon wafer in the cleaning solution for cleaning;

[0052] Specifically, the silicon wafer was placed in acetone and ultrasonicated for 5 minutes, then taken out and placed in isopropanol and ultrasonicated for 5 minutes, and then rinsed with 18.2 MΩ·cm ultrapure water.

[0053] 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 the silicon wafer in a stripping solution to remove the photoresist and excess metal.

[0054] Specifically:

[0055] 1. Spin coating: Spin coating photoresist on the cleaned silicon wafer with a thickness of 3μm;

[0056] 2. Pre-baking: The photoresist is pre-baked to improve its adhesion to the substrate. The pre-baking treatment is specifically placed in an oven at 90°C for 10 minutes;

[0057] 3. Exposure: 5 seconds on a UV exposure machine (Karl SUSS MA / BA6);

[0058] 4. Post-bake at 120°C for 2 minutes to produce a cross-linking reaction in the exposed areas;

[0059] 5. Development: After flood exposure, the grid lines can be dissolved in the developer to deposit Au in the subsequent step;

[0060] 6. Evaporation: A 200 nm thick Au layer was deposited using a thermal evaporator (VNANO Vacuum Technology Co., Ltd);

[0061] 7. Stripping and washing away unnecessary photoresist and metal to obtain the metal electrode arrangement on the silicon wafer.

[0062] The photoresist used is positive photoresist AZ5214e, the developer is rzx3038 positive photoresist developer, and the stripping solution is acetone.

[0063] The scanning electron microscope image of the tunneling electrode pair is shown in Figure 2. Figure 1 shown.

[0064] 2. Modify protein and test enzymatic reaction

[0065] Step 1: Place the prepared tunneling electrode into an ethanol solution containing thiol biotin (concentration: 100 nM) and soak for 2 hours before taking it out;

[0066] Step 2: Place the tunneling electrode treated in step 1 into a streptavidin solution (concentration: 10 μM, PBS buffer: 10 mM, pH 7.4) and soak for 2 h before taking it out;

[0067] Step 3: Place the tunneling electrode treated in step 2 into a biotin-modified peroxidase solution (concentration: 10 μM, PBS buffer: 10 mM, pH 7.4) and soak for 2 h before taking it out;

[0068] Step 4: Connect the processed tunneling electrode to the test system;

[0069] Step 5: Add 10 μL of 30% hydrogen peroxide to the sample cell.

[0070] The test system described in step 4 specifically consists of an amplifier (Molecular Devices, model no. MultiClamp 700B), a digitizer (Molecular Devices, model no. Axon Digidata 1550B), and a headstage (Molecular Devices, model no. CV 7B).

[0071] The real-time recording image of the current is as follows Figure 2 shown.

[0072] The obtained data is processed to filter out instantaneous waveform changes, such as Figure 3 The transient state change process of the catalytic reaction can be studied from the transient waveform change.

[0073] The real-time conductivity is calculated based on the bias voltage and tunneling current signal. The conductivity G = I / V. The different conductivity sizes are statistically analyzed and a frequency histogram is drawn, such as Figure 4 As shown in Figure 2. From the frequency histogram, it can be seen that there are two state distributions in the overall catalytic reaction.

[0074] Example 2

[0075] 1. Prepare the tunneling electrode. The process is as follows:

[0076] Step 1: Place a metal wire in a thin glass tube;

[0077] Specifically, the thin glass tube has a double channel, an outer diameter of 1.2 mm, an inner diameter of 0.90 mm, and a length of 100 mm. Two identical gold wires with a length of 2 cm and an outer diameter of 25 μm are inserted from the tail.

[0078] Step 2: Drawing a thin glass tube containing a metal wire;

[0079] Specifically, the thin glass tube containing the gold wire is first cleaned, and the cleaning steps are as follows: rinse it with 18.2 MΩ·cm ultrapure water, place it in a plasma cleaning machine to clean surface debris, and clean it for 30 minutes.

[0080] The cleaned thin glass tube containing gold wire inside is drawn using a P-2000 laser drawing instrument in a three-step method: the first step is to apply a laser beam to heat the middle part of the thin glass tube while drawing it. The drawing parameters are set as Heat: 350, Filament: 4, Velocity: 15, Delay: 120, Pull: 0. The middle part of the thin glass tube is heated and melted to form an hourglass-shaped structure; the second step is to seal the two ends of the thin glass tube with a tetrafluoroethylene hose and evacuate the inside of the thin glass tube for 6 minutes. The glass tube and the metal wire were then heat-sealed. The heat-sealing process involved heating the tube for 4 seconds, cooling for 1 minute, and then heating again for 7 seconds. The drawing parameters were set to: Heat: 390°C, Filament: 3°C, Velocity: 12°C, Delay: 120°C, and Pull: 0°C. In the third step, after the wire was sealed, the thin glass tube was heated again while simultaneously applying tension to both ends of the thin glass tube. The drawing parameters were set to: Heat: 520°C, Filament: 3°C, Velocity: 36°C, Delay: 170°C, and Pull: 50°C. This completed the drawing process, resulting in a nanopipette with a single-sided tip and a metal wire inside.

[0081] 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 using a needle grinder to expose the metal wire.

[0082] Step 3: fabricating a nanogap electrode pair on the tip of the metal wire by electrochemical deposition;

[0083] Specifically, using a potentiostat, the metal wire at the tip of the nanopipette is immersed in an electroplating solution (specifically, 4.4mM NH4AuSO3 and 52mM (NH4)2SO3). A pair of nanoelectrodes at the tips of the two wires serves as the working electrode, while a gold ball with a diameter of 2-3mm is immersed in the electroplating solution as the counter electrode. An electrochemical deposition current of 2-3μA is applied. During the electrochemical deposition process, a 4mV, 1Hz AC bias is applied between the two nanoelectrode pairs. A lock-in amplifier detects the current flowing through an external resistor (1kΩ) in this loop, and the conductance between the nanoelectrode pairs is calculated based on the current magnitude and the voltage divider between the nanoelectrode pairs. The conductance value is negatively correlated with the size of the nanogap between the electrode pairs. Therefore, through a feedback circuit, different conductance values ​​are used as the termination mark for electrochemical deposition to prepare nanogap electrode pairs with different spacings.

[0084] After the nanogap electrode pair was prepared, it was stored in 18.2 MΩ·cm ultrapure water.

[0085] The spacing between the nanogap electrodes is 2.5 nm, which is estimated using the Simmons model. Figure 5 shown.

[0086] 2. Segmented modification of template DNA to recognize complementary DNA strands

[0087] All DNAs used were treated with TCEP (tris(2-carboxyethyl)phosphine). Specifically, 100 μL of DNA solution to be treated (concentration: 100 μM) was mixed with 10 μL of TCEP solution (concentration: 100 mM) and reacted for 1 hour.

[0088] Step 1: Place the prepared tunneling electrode into a solution containing DNA template strand 1 (concentration: 10 nM, PBS buffer: 10 mM, pH 7.4) for 2 hours and then remove it. The sequence of DNA template strand 1 is: 5'-TTTTTGTGCCCGCCGA-3', with HS-SH C6 modified at the 5' end;

[0089] Step 2: Place the tunneling electrode treated in step 1 in pure PBS buffer (concentration: 10 mM, pH 7.4) and use CHI600E to apply a constant potential of -0.9 V on one side for 30 seconds to break the DNA template strand 1 on the unilateral modification.

[0090] Step 3: Place the tunneling electrode treated in step 2 into a solution containing DNA template chain 2 (concentration: 10 nM, PBS buffer: 10 mM, pH 7.4) for 2 hours and then take it out. The sequence of DNA template chain 2 is: 5'-CGAGAATTAGTCTTTTT-3', with HS-SH C3 modified at the 3' end;

[0091] Step 4: Connect the processed tunneling electrode to the test system;

[0092] Step 5: Add 10 μL of 10 nM DNA complementary chain 3 to the sample pool. The sequence of DNA complementary chain 3 is the complementary sequence of the middle recognition part of DNA template chain 1 + DNA template chain 2. The sequence of DNA complementary chain 3 is: GACTAATTCTCCTCGGCGGGCAC.

[0093] The schematic diagram of the process of segmented modification of template DNA to recognize complementary DNA chains is as follows Figure 6 shown.

[0094] The real-time recording image of the current is as follows Figure 7 As shown, when the DNA template strand 1 and the DNA template strand 2 are combined with the DNA complementary strand 3, the current real-time recording signal increases suddenly and maintains a stable oscillation signal.

[0095] Research has shown that only complementary DNA sequences can produce both of these characteristics. DNA strands that don't fit together can't form a bridge and therefore can't generate a signal.

[0096] Example 3

[0097] 1. Preparation of Tunneling Electrode The preparation method is the same as that in Example 2.

[0098] 2. Modify template DNA to recognize mismatched DNA strands

[0099] Step 1: Soak the prepared tunneling electrode in a 1 μM DSP (disulfide bis(succinimidyl propionate)) solution (concentration: 1 μM) for 2 h. The DSP used was TCEP treatment. Specifically, 100 mL of the treated DSP solution (concentration: 100 μM) was mixed with 10 μL of TCEP solution (concentration: 100 mM) and reacted for 1 h.

[0100] Step 2: Place the tunneling electrode treated in step 1 into pure PBS buffer (concentration: 10 mM, pH 7.4) and use CHI600E to apply a constant potential of -0.9 V on one side for 30 s to interrupt the DSP modified on one side.

[0101] Step 3: Place the tunneling electrode treated in Step 2 into a solution containing DNA template strand 4 (concentration: 10 nM, PBS buffer: 10 mM, pH 7.4) for 2 hours, then remove it to allow DNA template strand 4 to connect to both ends of the tunneling electrode. The sequence of DNA template strand 4 is: 5'-TTTTTGTGCCCGCCGACGAGAATTAGTCTTTTT-3', with NH2 C6 modified at the 5' end and HS-SH C3 modified at the 3' end.

[0102] The schematic diagram of DNA template chain 4 connected to the two ends of the tunneling electrode is shown in Figure 8 shown.

[0103] Step 4: Connect the tunneling electrode processed in step 3 to the test system;

[0104] Step 5: Add 10 μL of 10 nM DNA complementary strand 5 to the sample pool for testing. The sequence of DNA complementary strand 5 is the complementary sequence of the middle recognition portion of DNA template strand 4, and the sequence of DNA complementary strand 5 is 5'-GACTAATTCTCGTCGGCGGGCAC-3'.

[0105] Step 6: Take out the tunneling electrode from step 5 and soak it in ultrapure water (18.2 MΩ·cm) at 93-98°C for 1 minute to allow the DNA template strand 4 and the DNA complementary strand 5 to unwind.

[0106] Step 7: Connect the tunneling electrode processed in step 6 to the test system again;

[0107] Step 8: Add 10 μL of 10 nM DNA mutant strand 6 to the sample pool for testing. The sequence of DNA mutant strand 6 is a single-base mutation of DNA complementary strand 5, and the sequence of DNA mutant strand 6 is 5'-GACTAATTCTCCTCGGCGGGCAC-3'.

[0108] Step 9: Comparing the difference between the above two test results, we can get the result of whether the DNA chain is mutated and mismatched.

[0109] Specifically, the current-time (It) data obtained in step 4 and step 7 were statistically analyzed, where conductance G = I / V. The data statistics were performed using Clampfit v.10.7 software (Molecular Devices), and frequency histograms of different conductance sizes were obtained, as shown in FIG. Figure 9 As shown in the figure, there are three distributions of DNA complementary chain 5, while there are only two distributions of DNA mutation chain 6. Therefore, it can be judged that DNA mutation chain 6 has mismatch caused by base mutation.

[0110] Through Example 3, mutations in specific DNA sequences can be directly detected without the need for additional nucleic acid amplification methods, and the detection accuracy can reach the level of a single base.

[0111] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A biomolecule detection method based on a tunneling electrode, characterized in that: The following steps are involved: (1) Preparing a tunneling electrode pair with a nanogap on a nanopipette tip or a silicon wafer, and then modifying the surface of the tunneling electrode pair with a modifier that can interact with the biomolecule to be detected to obtain a functionalized tunneling electrode; (2) placing the functionalized tunneling electrode in a solution containing the biomolecule to be tested, using an ammeter to detect the tunneling current signal in real time, obtaining the tunneling current signal corresponding to the biomolecule, and analyzing the current signal to further analyze the molecular structure or molecular behavior of the biomolecule to be tested; When the biological molecule to be detected is DNA, in step (1), the functional modification includes: modifying probe DNA I and probe DNA II at the two ends of the tunneling electrode pair respectively to obtain a functionalized tunneling electrode; the probe DNA I contains a complementary sequence I complementary to the second half of the target DNA chain, and the probe DNA II contains a complementary sequence II complementary to 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 thiol group, and the modified substance is connected to the electrode via the thiol group; in step (2), the functionalized tunneling electrode is placed in a solution containing the DNA molecule to be detected, and the tunneling current is detected in real time using an ammeter. If the current value increases suddenly and maintains an oscillating signal, it is determined that the DNA molecule to be detected is the target DNA; When the biomolecule to be detected is a mutant DNA chain, in step (1), the functional modification includes: first modifying one end of the tunneling electrode pair with disulfide bis (succinimidyl propionate), and then reacting with the probe DNA so that the two ends of the probe DNA are respectively connected to the two ends of the tunneling electrode pair to prepare a functionalized tunneling electrode; the probe DNA contains a complementary sequence complementary to the template DNA, and its two ends are respectively modified with amino and sulfhydryl groups, the amino end is connected to disulfide bis (succinimidyl propionate), and the sulfhydryl end is connected to the electrode on one side of the unmodified disulfide bis (succinimidyl propionate); in step (2), first First, the functionalized tunneling electrode is placed in a solution containing template DNA, and the tunneling current is detected by an ammeter to obtain the tunneling current signal corresponding to the template DNA; then the tunneling electrode is removed, and the template DNA and probe DNA are unwound; then the unwound functionalized tunneling electrode is placed in a solution containing the DNA mutation chain to be tested, and the tunneling current is detected by an ammeter to obtain the tunneling current signal corresponding to the DNA mutation chain 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 can be determined.

2. The biomolecule detection method based on tunneling electrode according to claim 1, characterized in that: When the biomolecule to be detected is DNA, the thiol group is modified at the 5' end of the probe DNA I, and its 5' end has a poly (T) sequence; the thiol group is modified at the 3' end of the probe DNA II, and its 3' end has a poly (T) sequence.

3. The biomolecule detection method based on tunneling electrode according to claim 1, characterized in that: When the biomolecule to be detected is a mutant DNA chain, the probe DNA includes, in addition to the complementary sequence, a poly(T) sequence at the 5' end and a poly(T) sequence at the 3' end.

4. The biomolecule detection method based on tunneling electrode according to claim 1, characterized in that: In step (1), the method for preparing the nanopipette comprises: inserting a metal wire into a multi-channel capillary tube, and drawing the multi-channel capillary tube into a nanopipette having a pointed end at one end by applying an external force.

5. The biomolecule detection method based on tunneling electrode according to claim 1 or 4, characterized in that: In step (1), a nanogap tunneling electrode pair is prepared on a nanopipette tip or a silicon wafer by electrochemical deposition, chemical etching, mechanically controlled cracking or electrical etching; the nanogap ranges from 0.1 nm to 10 nm.

6. The biomolecule detection method based on tunneling electrode according to claim 1, wherein in step (2), the template DNA and the probe DNA are unwound by being placed in ultrapure water at 93-98°C.

7. The biomolecule detection method based on tunneling electrode according to claim 1, characterized in that: In step (2), the current signal analysis method includes: calculating the real-time conductance based on 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.

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