Single molecule detection device and method based on electrochemical gating technology in quantum tunneling region

Abstract

The invention provides a single molecule detection device and method based on an electrochemical gating technology in a quantum tunneling region, and relates to a detection technology for regulating molecular energy level alignment and regulating molecular conductance through gate voltage, a functionalized tunneling electrode is prepared at the tip of a nano pipettor and is placed in a solution containing single molecules, and the electrochemical gating technology in the quantum tunneling region is used for detecting the single molecules in the nano pipettor. The gate potential and the bias voltage are dynamically adjusted, tunneling current signals flowing through the single molecule are recorded and analyzed in real time, the structure, the charge state and the energy state of the single molecule can be actively regulated and controlled, and dynamic detection of the single molecule is achieved according to the conductivity change spectrum of the single molecule under different potential conditions. And the single molecule detection precision based on the tunneling electrode is obviously improved.

Description

Technical Field

[0001] The present invention relates to the field of single-molecule detection and nano-sensing technology. Specifically, it relates to a single-molecule detection method based on an electrochemically gated technology within the quantum tunneling region. Background Art

[0002] Single-molecule detection technology is an important breakthrough in contemporary analytical science. Its core lies in getting rid of the limitation of averaging the behaviors of a large number of molecules in traditional ensemble measurements, directly observing the real-time structure, dynamic behaviors, and interactions of individual molecules, so as to reveal the heterogeneity and complexity of the behaviors of individual molecules. This technology captures the unique signals of individual molecules in chemical reactions, biological processes, or physical effects through ultra-high-sensitivity instruments, enabling scientists to trace transient intermediates, low-probability reaction paths, and dynamic fluctuations of intermolecular interactions that cannot be resolved by traditional methods.

[0003] However, traditional single-molecule detection essentially still belongs to a passive recording mode. Its observation process is limited by the occurrence probability of spontaneous molecular behaviors, unable to actively regulate the physical and chemical states of the research object, and difficult to synchronously analyze the correlations among molecular structure, charge state, and energy state, with significant bottlenecks in dynamic control accuracy and information dimension.

[0004] For example, when studying voltage-dependent ion channel proteins, conventional single-molecule fluorescence technology can only record the conformational fluctuations of the protein at a fixed membrane potential, but cannot apply electrochemical stimuli in real time to simulate dynamic regulation under physiological conditions; in catalytic reaction monitoring, although scanning probe technology can locate individual active sites, it is difficult to precisely control the adsorption strength of reaction intermediates while applying a potential.

[0005] This passivity has led to many key scientific problems (such as the microscopic mechanism of electric-field-driven molecular conformational transitions, the charge separation dynamics of excited-state molecules) remaining at the theoretical speculation stage, and there is an urgent need to develop a new generation of single-molecule research paradigms that combine active manipulation and high-precision detection.

[0006] Different biomolecules have diverse molecular recognition capabilities, and in specific cases, are prone to electron tunneling and can be used for single-molecule detection. The charge transport of single molecules is fundamentally important in electrochemical processes, tunneling detection, and ultimately in the rational design of next-generation bioelectronic devices. Single-molecule-mediated tunneling electron transport depends on the capture of biomolecules between a pair of electrodes with a closely spaced gap of less than 100 nm, and the current response on the connected tunneling electrodes can be measured as a characterization for monitoring the behaviors of biomolecules. However, the tunneling current generated by single molecules will change with the movement or position of single molecules, the signal is easily interfered, and the solution environment usually contains various different single molecules, resulting in unstable and rather messy detection signals for single molecules, and the precision for detecting single molecules is not ideal.

[0007] In the early stage, our research group developed a method for detecting single molecules based on tunneling electrodes (CN115541680A). However, it requires functional modification on the tunneling electrodes to form a single-molecule junction with the target molecule, stabilize the detection signal, and enable the target molecule to undergo redox reactions for detection. The detection process is more complex, and it can only detect substances that can bind to the functional modification. For example, in this patent, peroxidase was used to modify the tunneling electrode, and only hydrogen peroxide that can be catalyzed by peroxidase can be detected. At the same time, this method only performs static detection, which belongs to passive monitoring and cannot actively regulate the physical and chemical states of the research object. The detection sensitivity is still limited, and it is difficult to detect some single molecules with small current changes during static detection.

[0008] Therefore, there is an urgent need to find a method for detecting target molecules with higher precision based on quantum tunneling. Summary of the Invention

[0009] Aiming at the problems existing in the prior art, the present invention provides a single-molecule detection device and method based on electrochemically gated technology in the quantum tunneling region. It is a detection technology that regulates the molecular energy level alignment and adjusts the molecular conductance through the gate voltage. By preparing a functionalized tunneling electrode at the tip of a nanopipette, placing it in a solution containing single molecules, dynamically adjusting the gate potential and bias voltage, and real-time recording and analyzing the tunneling current signal flowing through the single molecule, it can actively regulate the structure, charge state, and energy state of the single molecule. According to the conductance change map of the single molecule under different potential conditions, the dynamic detection of the single molecule is realized, and the detection accuracy of detecting single molecules based on tunneling electrodes is significantly improved.

[0010] On the one hand, the present invention provides a device for detecting single molecules. The device includes a tunneling electrode. The molecule to be detected is located between the nano-gaps of the tunneling electrode and is under different potential conditions to detect the conductance change of the molecule to be detected, thereby judging the type of the molecule to be detected.

[0011] The device for detecting single molecules provided by the present invention is a quantum tunneling probe detection device. During the detection using the tunneling electrode, this device can adjust the potential in real time to achieve the dynamic detection of single molecules. Its main principle is: based on the different conductance changes of different molecules to be detected under different potential conditions, the conductance change map of each target molecule under different potential conditions is constructed as the fingerprint map of the target molecule to form a database. Then, by detecting the conductance change of the molecule to be detected in the sample and comparing it with the conductance change map of the known target molecule in the database, it can be judged which specific target molecule the molecule to be detected is. Compared with the existing single-molecule static detection methods, the detection device provided by the present invention for detecting single molecules is not only more convenient to operate but also has higher detection accuracy.

[0012] Existing quantum tunneling probe detection devices can only detect single molecules statically and passively. They need to add specific reagents to promote redox reactions and perform specific modifications on the tunneling electrodes to stabilize the detection signal. Especially for some single molecules with no obvious change in current before and after the redox reaction during static detection, it is often difficult to detect them or the detection sensitivity is low, making it difficult to meet the accuracy requirements.

[0013] The device provided by the present invention can perform dynamic detection of single molecules based on the electrochemical gating technology within the quantum tunneling region. Through the double-electrode quantum tunneling probe in the quantum tunneling probe detection device, combined with the weak current detection device of the three-electrode system, it actively regulates the matching degree between the molecular energy level and the electrode, breaks through the passive observation limitation of traditional single-molecule detection, and realizes the dynamic control and high-dimensional analysis of single-molecule conductance.

[0014] The potential refers to the voltage applied to the single molecule to be detected during the detection using the tunneling electrode, including the gate potential and the bias voltage.

[0015] The conductance refers to the resistance value obtained by the molecule to be detected under specific current and voltage conditions.

[0016] The type of the molecule to be detected refers to determining what specific molecule the molecule to be detected is.

[0017] Furthermore, the different potentials, including different gate potentials and bias voltages, record and analyze the tunneling current signals flowing through the target molecule and / or the molecule to be detected in real time, and obtain the conductance change map of the target molecule and / or the molecule to be detected.

[0018] The dynamic detection described in the present invention refers to the real-time adjustment of the gate potential and the bias voltage during the detection using the tunneling electrode; the static detection refers to the gate potential and the bias voltage remaining unchanged during the detection using the tunneling electrode.

[0019] The gate potential refers to the voltage applied to the independent gate electrode (i.e., the overall offset of the voltages of the two working electrodes relative to the voltage of the reference electrode), which is used to regulate the energy level structure of the system. The bias voltage refers to the voltage applied between the two tunneling electrodes (that is, the voltage applied across the single molecule), which is used to drive electron tunneling. For example, taking the reference electrode voltage as the reference voltage, its voltage can be regarded as 0. The potential of working electrode 1 is Vr, and the potential of working electrode 2 is Vr + Vb. Among them, Vr is called the gate voltage, that is, the overall offset of the voltages of the two working electrodes relative to the voltage of the reference electrode, and Vb is called the bias voltage between the two electrodes.

[0020] The conductance change spectrum refers to a current signal spectrum obtained when, as the gate potential and bias voltage change, both the current flowing through a single molecule and its voltage change, and its conductance (equivalent to the resistance value obtained at a specific voltage and current) also continuously changes. Subsequently, based on the voltage and current, a conductance distribution histogram for conductance is prepared, and the type of the molecule to be detected is determined by observing the position and height of the conductance peaks in the conductance distribution histogram. The type mentioned here refers to determining which specific target molecule the molecule to be detected is.

[0021] Further, the tunneling electrode has a nano-gap tunneling electrode pair, which is fabricated on the tip of a nanopipette, and the nano-gap of the nano-gap tunneling electrode pair is 0.1 - 100 nm.

[0022] Further, the tunneling electrode is fabricated by heating and softening and pulling a double-channel glass pipette; the double-channel glass pipette is filled with a conductive material, a metal wire is inserted as the tail end, and a nano-gap tunneling electrode pair is deposited at the tip; the material of the nano-gap tunneling electrode pair is a single metal or a metal mixture.

[0023] Based on a quantum tunneling probe detection device with controllable spacing, the present invention actively adjusts the gate voltage and the bias voltage applied across the target molecule, changes the matching degree between the molecular energy level and the electrode, enhances or suppresses the tunneling signal of a specific molecule, thereby obtaining the dynamic change information of the conductance of the target molecule with respect to the gate potential and the bias voltage, constructing a "potential - bias voltage" response matrix of conductance, and analyzing the dependence of its conductance on the electrode potential.

[0024] The controllable-spacing quantum tunneling probe detection device includes a double-electrode quantum tunneling probe (abbreviation: tunneling electrode), a weak current detection device, and a liquid environment containing the target molecule. The double-electrode quantum tunneling probe is a double-hole glass pipette with one end pulled into a tip (nanometer to micrometer scale), filled with a conductive substance inside, and the tip is an electrode pair with a nano-gap; the tip of the quantum tunneling probe is placed in the liquid environment containing the target molecule; the tail end of the quantum tunneling probe is connected to the weak current detection device, and this device can simultaneously control the potential of the tunneling probe electrode relative to the reference electrode.

[0025] The present invention actively controls the matching degree between the molecular energy level and the electrode through a double-electrode quantum tunneling probe in combination with a weak current detection device of a three-electrode system, and realizes the dynamic manipulation and high-dimensional analysis of single-molecule conductance.

[0026] The nano-gap between the nano-gap tunneling electrode pairs directly affects the sensitivity, resolution, and signal stability of single-molecule detection. The smaller the gap, the stronger the tunneling current, the higher the spatial resolution, the easier the signal is to detect, the clearer the fingerprint spectrum, and the higher the detection sensitivity. However, the smaller the gap, the greater the difficulty in fabricating the tunneling electrode.

[0027] The tunneling electrode provided by the present invention has a nano-gap of 0.1 to 100 nm and can be used for efficient dynamic detection of single molecules.

[0028] In some embodiments, the nano-gap is preferably 0.1 to 10 nm.

[0029] In some embodiments, the nano-gap is preferably 1.5 to 3.1 nm.

[0030] Furthermore, the tunneling electrode is fabricated by heating and softening a double-channel glass pipette and then pulling it; the double-channel glass pipette is filled with a conductive material, a metal wire is inserted as the tail end, and a pair of nano-gap tunneling electrodes is deposited at the tip.

[0031] In some embodiments, the method for preparing the double-electrode quantum tunneling probe is as follows: the double-channel glass pipette is pulled into a probe with a tip at one end by heating and softening while applying an external force; a conductive material is filled into the double-channel glass pipette, and at the same time, a conductive metal wire is inserted as the tail end; a pair of electrodes with a nano-gap is deposited at the tip.

[0032] The heating methods for heating and softening include: microwave heating, resistance furnace heating, laser heating, etc.

[0033] The conductive material in the nano-pipette channel is selected as pyrolytic carbon, and the conductive metal wire is made of copper wire.

[0034] The pair of electrodes with a nano-gap is made of a metal or a metal mixture and has a nano-gap. Preferably, the pair of electrodes with a nano-gap uses gold, and the gold electrodes with a nano-gap are prepared at the tip of the glass tube by electrochemical deposition; the gap is the minimum of the gaps between the two electrodes in the pair of electrodes, and the nano-gap refers to the range of this gap being 0.1 nm to 100 nm. Preferably, the nano-gap range is 1.5 nm to 3.1 nm.

[0035] In some embodiments, the deposition of the pair of nano-gap tunneling electrodes at the tip is prepared by electrochemical deposition. The specific process of preparing the nano-gap electrodes by electrochemical deposition is as follows: an uncontacted pair of electrodes is deposited at the tip of the nano-pipette by an electrochemical method, and gold is deposited by a constant current and a self-terminating voltage feedback method. The termination criterion of the method is: when the voltage between the pair of electrodes approaches 0 volts, it indicates that the distance between the pair of electrodes is in the nano-scale, and the electrochemical deposition is terminated.

[0036] In addition, the smaller the nano-gap, the higher the detection sensitivity, but it is also more susceptible to environmental perturbations. Therefore, if modifications are added to the tunneling electrode, it also helps to improve the effect of resisting environmental perturbations.

[0037] Further, the tail end of the tunneling electrode is connected to a weak current detection device, and the weak current detection device includes two working electrodes and a reference electrode; the reference electrode is used to provide a stable potential reference; the two working electrodes respectively regulate the voltage relative to the reference electrode and detect the weak current flowing through this electrode.

[0038] The weak current detection device is a three-electrode system device. One of the two working electrodes is connected to one of the conductive metal wires at the tail end of the double-hole pipette, and the other electrode is connected to the reference electrode. The two working electrodes can respectively regulate the voltage relative to the reference electrode and simultaneously detect the weak current flowing through this electrode. The tip of the double-hole pipette and the reference electrode are placed in a liquid environment containing the target molecule.

[0039] Further, the reference electrode includes any one or more of a standard hydrogen electrode, a saturated calomel electrode, a mercuric oxide electrode, a mercurous sulfate electrode, a copper sulfate reference electrode, a silver-silver chloride electrode, and a solid-state reference electrode.

[0040] Further, the nano-gap tunneling electrode pair is modified, and the modification can form a single-molecule junction with the molecule to be detected.

[0041] Theoretically, as long as a molecule passes through the tunneling region during tunneling detection, a signal can be observed. However, the conductance of each single molecule also varies in different directions. For example, for dopamine molecules, the detection results are different when measuring the conductance from the left and right sides and from the top and bottom sides.

[0042] Therefore, if the tunneling electrode is modified with a molecule, the molecule to be detected can be captured through the modified molecule, which can, to a certain extent, fix the posture of the molecule to be detected (thereby fixing the tilt angle of the molecule and making the molecular structure basically fixed). For example, hydrogen bonds are modified on the nano-gap tunneling electrode pair to form a single-molecule junction with dopamine. One hydrogen bond of a nano-electrode connects the hydroxyl group of dopamine, and one end of the hydrogen bond connects the amino group of dopamine. In this way, the measured conductance signal will be more stable and it is easier to accurately detect the molecule to be detected.

[0043] Of course, there can be various modifications. For example, some modifications can form strong chemical bonds to fix the molecule to be detected, thus forming a path of electrode-molecule-electrode, which can be measured for a long time. However, this method is only applicable to detecting a single type of single molecule. For example, CN115541680A can only detect hydrogen peroxide that can react with peroxidase and cannot detect multiple different single molecules simultaneously.

[0044] In order to detect multiple different single molecules simultaneously, it is preferred to modify the tunneling electrode with relatively weak hydrogen bonds. This modification method does not fix a molecule for a long time. Instead, after capturing a certain single molecule for testing, the single molecule will fall off, and then the next single molecule will be captured. In this way, multiple different single molecules can be detected in a complex solution environment, realizing the simultaneous detection of multiple single molecules.

[0045] On the other hand, the present invention provides a method for detecting a target molecule, and the method includes the following steps:

[0046] (1) Placing the tunneling electrode in a solution containing the molecule to be detected;

[0047] (2) Dynamically detecting the change in the conductance of the molecule to be detected under different potential conditions, obtaining a conductance change map of the molecule to be detected, and thereby judging the type of the molecule to be detected.

[0048] Further, the method includes the following steps:

[0049] (1) Placing the tunneling electrode in a solution containing the target molecule;

[0050] (2) Dynamically adjusting the gate potential and the bias voltage, recording and analyzing in real time the tunneling current signal flowing through the target molecule, obtaining a conductance change map of the target molecule, and constructing a database containing the conductance change maps of different target molecules;

[0051] (3) Placing the tunneling electrode in a solution containing the molecule to be detected;

[0052] (4) Dynamically adjusting the gate potential and the bias voltage, recording and analyzing in real time the tunneling current signal flowing through the molecule to be detected, obtaining a conductance change map of the molecule to be detected, and comparing it with the database to judge which target molecule the molecule to be detected is;

[0053] Both the target molecule and the molecule to be detected are single molecules.

[0054] Further, under the condition of dynamically adjusting the gate potential and the bias voltage, the target molecule and / or the molecule to be detected undergoes any one or more of redox reactions, molecular conformation transformation reactions, and molecular conjugation mode switching reactions, thereby causing a change in its conductance.

[0055] Different single molecules may undergo different reactions under the condition of dynamically adjusting the gate potential and bias voltage of the tunneling electrode. For example, single molecules with redox activity will undergo redox reactions. According to the different oxidation potentials of different single molecules or the different conductances of different molecular redox states, they can be distinguished. For example, single molecules with molecular conformational reaction activity will undergo conformational reactions. According to the different conformational reaction potentials of different single molecules, they can be distinguished. Another example is single molecules with molecular conjugate mode switching reaction activity, which will undergo molecular conjugate mode switching reactions. According to the different molecular conjugate mode switching reaction potentials of different single molecules, they can be distinguished.

[0056] Furthermore, under the condition of dynamically adjusting the gate potential and bias voltage, the target molecule and / or the molecule to be detected undergo redox reactions.

[0057] Since most single molecules have redox activity, and the process of spontaneous redox reactions of different single molecules is more specific, it is more suitable to be used as a fingerprint to detect single molecules.

[0058] During the process of dynamically adjusting the gate potential and bias voltage, the spontaneous redox reaction of single molecules usually includes the following three processes: 1. Being in a stable reduced state; 2. Starting to be oxidized as the bias voltage increases; 3. Continuing to increase the bias voltage, and the molecule is continuously oxidized. The potentials required for the three processes of different single molecules are different, so their conductance change spectra are also completely different.

[0059] In addition, although the existing static detection methods also use redox reactions, they must add components that promote redox reactions or other reactions in the reaction solution. However, the method provided by the present invention does not require adding any components that promote redox reactions or other reactions. Only by changing the potential, the conductance of the single molecule changes under different potential conditions, so that it spontaneously undergoes redox reactions, conformational change reactions, molecular conjugate mode switching reactions, etc. Since the conductance change situations of different single molecules are different at specific potentials, the detection of single molecules is achieved by detecting this change process. Such a detection method can significantly improve the detection accuracy compared with static detection, enabling single molecules that could not be detected by static detection to be successfully detected in dynamic detection.

[0060] Furthermore, the way for the molecule to be detected to enter the nano-gap of the nano-gap tunneling electrode pair is any one or more of the following: free diffusion, electrostatic adsorption, hydrogen bond adsorption, covalent bond adsorption, specific binding, optical tweezers, π-π stacking.

[0061] Different ways for the molecule to be detected to enter the nano-gap can also be determined by different modification methods of the tunneling electrode.

[0062] Of course, there can be various modifications. For example, some modifications can form strong chemical bonds to fix the analyte molecule, thus forming an electrode-molecule-electrode pathway for long-term measurement. However, this method is only applicable to detecting a single type of single molecule. For instance, CN115541680A can only detect hydrogen peroxide that can react with peroxidase and cannot detect multiple different single molecules simultaneously.

[0063] To detect multiple different single molecules simultaneously, it is preferred to modify the tunneling electrode with relatively weak hydrogen bonds. This modification method does not fix a molecule for a long time. Instead, after capturing a single molecule for testing, the single molecule will fall off, and then the next single molecule will be captured. In this way, multiple different single molecules can be detected in a complex solution environment, achieving the simultaneous detection of multiple single molecules.

[0064] In some methods, there are many connection methods that can be compatible with most analytes, such as electrostatic adsorption, hydrogen bond adsorption, covalent bond adsorption, optical tweezers, π-π stacking, etc. Hydrogen bond is one of the better choices. Because for the modification method using hydrogen bond connection, its binding strength is neither too strong nor too weak, and this modification method does not fix a molecule for a long time. Instead, after capturing a single molecule for testing, the single molecule will fall off, and then the next single molecule will be captured. In this way, multiple different single molecules can be detected in a complex solution environment, achieving the simultaneous detection of multiple single molecules, and the universality is very high. While using other modification methods, such as aptamers, peroxidases, etc., can only detect one target substance and cannot detect multiple different single molecules simultaneously.

[0065] Furthermore, the analysis method of the tunneling current signal includes: analyzing the baseline current, analyzing the high-conductance state current, analyzing the low-conductance state current, analyzing the difference between each current, analyzing the proportion of each state and the transition frequency, either one or more of them.

[0066] Furthermore, the adjustment range of the gate potential is -1V to 1V; the adjustment range of the bias voltage is 1nV to 600mV.

[0067] Since the conductance of the single molecule changes immediately with the change of the gate potential and the bias voltage, and the reaction occurs instantaneously, the time interval of adjusting different gate potentials and bias voltages has no impact. It can be adjusted every 1S, or every 1min, or even every 1 hour, and the obtained conductance change diagrams are almost no different.

[0068] Furthermore, the analyte molecule or target molecule is a substance whose conductance will change under different potentials.

[0069] Theoretically, for any single molecule, as long as its conductance changes during the processes of adjusting the gate potential and the bias voltage, it can be detected by dynamically detecting this change process, thereby detecting the single molecule.

[0070] Further, the molecule to be detected or the target molecule includes any one or more of proteins, nucleic acids, sugars, neurotransmitter molecules, and small chemical molecules.

[0071] Further, the molecule to be detected or the target molecule is a substance that undergoes redox reactions under different potential conditions.

[0072] During the processes of adjusting the gate potential and the bias voltage, there are many reasons for the change in conductance. For example, redox reactions, molecular conformation transformation reactions, molecular conjugation mode switching reactions, etc. Among them, the most common one is the redox reaction. All single molecules with different redox activities under different potential conditions can be accurately detected by the method provided by the present invention.

[0073] Further, the molecule to be detected or the target molecule includes molecules that are difficult to accurately detect by static detection using tunneling electrodes. The static detection refers to that during the detection process, the gate potential and the bias voltage remain unchanged; the molecules that are difficult to accurately detect by static detection using tunneling electrodes include those in which the conductance peak distributions of the molecules to be detected are very close during static detection, making it difficult to distinguish which single molecule it is.

[0074] Static detection can only detect through the current change before and after the redox reaction of a single molecule. The static detection spectra of some single molecules are very close, so it is impossible to distinguish which single molecule it is. For example, dopamine and norepinephrine have no obvious difference in conductance distribution during static detection, and it is impossible to distinguish whether it is dopamine or norepinephrine. However, through the dynamic detection method of tunneling electrodes provided by the present invention, it is possible to accurately distinguish dopamine and norepinephrine in the solution through their conductance change processes.

[0075] Further, the molecules that are difficult to accurately detect by static detection using tunneling electrodes include any one or more of dopamine, serotonin, and norepinephrine.

[0076] Further, the solution containing the molecule to be detected also contains ultrapure water or a buffer solution.

[0077] In some cases, the solution needs to be pure and free of impurities, such as ultrapure water.

[0078] In some cases, in order to simulate the body fluids of humans or animals to a certain extent, a buffer solution such as PBS that mimics body fluids as much as possible can be used.

[0079] On the other hand, the present invention provides a detection method for detecting target molecules that are difficult to detect or distinguish by static detection. The method includes the following steps:

[0080] (1) Place the tunneling electrode in a solution containing the target molecule and / or the molecule to be detected;

[0081] (2) Dynamically adjust the gate potential and the bias voltage, record and analyze in real time the tunneling current signal flowing through the target molecule and / or the molecule to be detected, and obtain the conductance change map of the target molecule and / or the molecule to be detected.

[0082] Furthermore, the target molecules that are difficult to detect or distinguish by static detection include any one or more of dopamine, serotonin, and norepinephrine.

[0083] The beneficial effects of the present invention are as follows:

[0084] 1. Provide a brand-new single-molecule dynamic detection method. By dynamically adjusting the gate potential and the bias voltage, record and analyze in real time the tunneling current signal flowing through the single molecule, and according to the conductance change map of the single molecule under different potential conditions, realize the dynamic detection of the single molecule, and significantly improve the detection accuracy of detecting single molecules based on the tunneling electrode.

[0085] 2. Adopt a nano-gap electrode pair, use a single molecule to construct a conductive channel, and combine with an electrochemical three-electrode system to apply a gate voltage (Vg) to directly control the energy level arrangement, redox state or protonation degree of the molecule, realize the precise control of the single molecule structure, charge state, and energy state, and obtain the molecular conductance fingerprint map (high and low conductance state statistical distribution) by regulating the gate potential and the bias voltage.

[0086] 3. Compared with the traditional method, the breakthrough advantage of this technology lies in the innovation of the dynamic regulation ability.

[0087] 4. Through dynamic detection, it is possible to accurately distinguish and detect single molecules that are difficult to distinguish by static detection through the dynamic conductance change situation.

[0088] 5. By selecting a suitable modification method for the tunneling electrode, it can be made to be compatible with most molecules to be detected at the same time, and realize the simultaneous detection of multiple single molecules.

[0089] 6. Not only make up for the technical shortcomings of traditional single-molecule detection, but also show unique potential in the fields of molecular electronic devices, precision catalytic design, and biomolecular dynamic analysis.

[0090] 7. In the field of molecular electronics, the voltage regulation capability of this technology provides a realistic path for the development of single-molecule switches and storage devices; in catalysis science, this technology can optimize the potential response characteristics of active sites at the single-molecule level and guide the design of efficient catalysts; in the biomedical field, this technology can analyze the conformational changes of proteins and the regulation mechanism of enzyme activity under the action of electric fields, providing a new perspective for disease mechanism research and drug development.

[0091] 8. With the deep involvement of machine learning algorithms, this technology is expected to further break through the limitations of time and space resolution, promote single-molecule science from the "observation era" to the "control era", and become a core tool for revealing the dynamic nature of the microscopic world. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Schematic diagram of the overall structure of a quantum tunneling probe device (tunneling electrode) based on a double-hole glass pipette in the quantum tunneling probe detection device in Example 1;

[0093] Figure 2 Schematic diagram of the pyrolytic carbon deposition process in Example 1;

[0094] Figure 3 This is a schematic diagram of the structure in which the thiol group of the molecule in Example 3 can form a gold-sulfur bond with the gold electrode, and the carbonyl group and the amino group on the other side of the molecule can form a hydrogen bond with the hydroxyl group and the amino group on the dopamine molecule to form a single molecule junction;

[0095] Figure 4 This is an overall configuration diagram of the quantum tunneling detection device in Example 3;

[0096] Figure 5 It is a characteristic current diagram when the gate voltage WE1 is -200 mV to 300 mV and the voltage difference Bias is 100 mv in Example 3;

[0097] Figure 6 This is a characteristic current diagram when the gate voltage WE1 is -200 mV to 300 mV and the voltage difference Bias is 200 mV in Example 3;

[0098] Figure 7 The conductance diagram of Example 3 in which the gate voltage WE1 is -200, -100, 0, 100 mV, the voltage difference Bias is 100 mV, and WE2 is WE1+Bias;

[0099] Figure 8 The conductance diagrams of Example 3 are as follows: WE1 is 200, 300, 400, 500 mV, the voltage difference Bias is 100 mv, and WE2 is WE1+Bias;

[0100] Figure 9 In Example 3,Figure 7 and Figure 8 Molecular conductance fingerprint spectra integrated on one graph (where G represents the gate voltage, for example, G: -200 mV means the gate voltage is -200 mV);

[0101] Figure 10 Are the conductance distribution diagrams of DA and NE during static detection in Example 4;

[0102] Figures 11 to 17 Are the conductance distribution diagrams of DA and NE during dynamic detection in Example 4, where Figures 11 to 17 The gate voltages WE1 in are -200, -100, 0, 100, 200, 300, 400 mV respectively, the voltage difference Bias is 100 mv, and WE2 is WE1 + Bias;

[0103] Figure 18 Is the circuit schematic diagram in Example 4;

[0104] Figure 19 Is the molecular conductance fingerprint spectrum of norepinephrine (NE) in Example 4 (where G represents the gate voltage, for example, G: -200 mV means the gate voltage is -200 mV).

[0105] Detailed description

[0106] Tunneling electrode

[0107] When the term "electrode" is used in this article, it generally refers to a material or component that can be used to measure current. An electrode (or electrode component) can be used to measure the current flowing into and out of another electrode. In some cases, an electrode can be arranged in a channel (such as a nanogap) and used to measure the current across the channel. The current can be a tunneling current. Such a current can be detected when a biomolecule (such as a protein) flows through the nanogap. In some cases, a sensing circuit coupled to the electrode provides an applied voltage across the electrode to generate a current. As an alternative or supplement, the electrode can be used to measure and / or identify the conductance associated with a biomolecule (such as an amino acid subunit or a protein monomer). In this case, the tunneling current can be related to the conductance.

[0108] A tunneling electrode is an electrode structure that utilizes the quantum tunneling effect to achieve electron transport. Quantum tunneling refers to the phenomenon where electrons penetrate a certain probability in a region prohibited by classical mechanics (such as an insulating layer or a potential barrier), which is one of the fundamental characteristics of quantum mechanics. When the potential barrier between the electrode and the target material (or another electrode) is very thin (usually at the nanoscale), electrons can pass through the barrier to form a tunneling current, which is the quantum tunneling effect. The tunneling current is extremely sensitive to the thickness and height of the potential barrier and usually follows an exponential decay relationship. Different from traditional electrodes, tunneling electrodes do not rely on Ohmic contacts (i.e., electrons do not need to cross the barrier through thermal excitation), but directly achieve electron transport through quantum tunneling.

[0109] Tunneling electrodes usually consist of a metal-insulator-metal (MIM) or metal-insulator-semiconductor (MIS) structure. Common insulating layer materials include aluminum oxide (Al2O3), silicon dioxide (SiO2), silicon nitride (Si3N4), etc., and the thickness is usually below a few nanometers.

[0110] Tunneling electrodes include independent nanoelectrodes separated by a nano-gap, and the length of the nano-gap is from 0.1 nm to 100 nm. Tunneling electrodes can have any convenient shape or size and can contain any conductive material. Each tunneling electrode disclosed in the present invention can be made of different materials or a mixture of materials such as alloys.

[0111] Tunneling electrodes are used to measure the current that can pass through and / or cross molecules, and the current can be a tunneling current. Measuring the current can be used to determine the sequence of biopolymers, such as nucleic acid molecules (such as DNA or RNA) or proteins. For mass measurement, the gap spacing between the electrodes of one or more pairs of tunneling electrodes can be stable and controllable.

[0112] Quantum tunneling probe detection device

[0113] A quantum tunneling probe detection device is an instrument that utilizes the quantum tunneling effect to achieve high-sensitivity detection. Its core principle is to monitor the change in the tunneling current in the nanoscale gap of the tunneling electrode to reflect the physical or chemical characteristics of the sample to be measured. Such devices have important applications in the fields of nanotechnology, surface science, biosensing, etc. The most typical representative is the scanning tunneling microscope (STM), but it also includes other dedicated sensors.

[0114] Gate potential

[0115] The gate potential refers to the voltage applied to an independent gate electrode (i.e., the overall offset of the voltages of two working electrodes relative to the voltage of the reference electrode), which is used to regulate the energy level structure of the system and control the charge distribution inside the device through an electrostatic field. It is the voltage applied through a gate (Gate) in electronic devices such as field-effect transistors, quantum dots, single-electron devices, etc., and is used to regulate the distribution of charge carriers, the energy band structure, or the conduction state of the conduction channel inside the device. Its core function is to control the behavior of current or quantum states through the electric field effect.

[0116] Bias voltage

[0117] The bias voltage refers to the DC voltage (or voltage difference) applied between two electrodes of an electronic device or circuit, aiming to regulate the working state of the device (such as conduction, cutoff, amplification, etc.) or achieve specific functions (such as carrier injection, energy band regulation, etc.). It is a basic concept in electronics, semiconductor devices, and quantum technologies, that is, the voltage applied between two tunneling electrodes (i.e., the voltage applied across the two ends of a single molecule), which is used to drive electron tunneling. The bias voltage is the "cornerstone voltage" for the operation of electronic devices, and through the active regulation of the DC voltage, functions such as switching, amplification, and sensing of the device can be achieved.

[0118] Conductance

[0119] Conductance is a physical quantity that measures the conductive ability of a material or component, indicating the ease with which current passes through. It is the reciprocal of resistance (Resistance), i.e., G = 1 / R. Unit: Siemens (symbol S), 1 S = 1 A / V (ampere / volt). Physical meaning: The greater the conductance, the smaller the resistance of the material or device to current, and the stronger the conductivity. Detailed implementation manners

[0120] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not impose any limitations on it. The reagents used in this embodiment are all known products and are obtained by purchasing commercially available products.

[0121] Embodiment 1. Preparation of tunneling electrodes

[0122] The quantum tunneling probe device (tunneling electrode) based on a double-hole glass pipette in the quantum tunneling probe detection device provided in this embodiment is as a whole as Figure 1 shown, where ① is the nano-gap electrode pair, ② is the conductive material, ③ is the metal wire, and ④ is the double-channel glass pipette. The preparation method is as follows:

[0123] Step 1. Pull the double-hole glass pipette into a probe with a single-sided tip

[0124] The double-hole glass pipette treated by the plasma cleaner was placed in the P-2000 laser drawing instrument and drawn using a two-step method. The double-hole glass pipette had an outer diameter of 1.2 mm, an inner diameter of 0.9 mm, and a length of 100 mm.

[0125] The two-step method is as follows: in the first step, a laser beam is applied to heat the middle part of the thin glass tube, and the tube is pulled while being heated, and the pulling parameters are set to Heat: 850, Filament: 4, Velocity: 30, Delay: 160, Pull: 100; in the second step, the parameters are set to Heat: 860, Filament: 3, Velocity: 20, Delay: 140, Pull: 160. The pulling process is completed, and two identical double-hole glass probes with single-sided tips are formed.

[0126] Step 2: Depositing pyrolytic carbon in a double-hole glass tube with a single-sided tip

[0127] The double-hole glass probe prepared in step 1 is placed in a single-hole quartz glass tube. A constant flow of 0.2 m / s is passed from the tip of the double-hole glass tube to the tail end of the single-hole glass tube. 3 / min argon is used as the protective gas, and the tail end of the double-hole glass probe is connected to a rubber hose, which is connected to butane gas at 3 atmospheres of pressure. The butane flame preheats the front end of the quartz tube, and the protective gas in the tube brings heat to the tip of the double-hole tube. The tip of the double-hole tube glows yellow and stays for 10 seconds. At this time, the butane in the double-hole tube is pyrolyzed into carbon and deposited on the tip of the double-hole tube. Then slowly move the butane flame to the tail end of the double-hole tube until pyrolytic carbon is fully deposited about 1 cm from the tip of the double-hole glass tube. The pyrolytic carbon deposition process is as follows Figure 2 shown.

[0128] Step 3: Electrochemical deposition of a nano-gap gold electrode pair at the tip of a double-hole glass tube

[0129] Insert the metal wire from the tail end of the double-hole glass tube to ensure sufficient contact with the pyrolytic carbon, and use hot melt adhesive to fix the metal wire to ensure the stability of the device. Subsequently, place the tip of the double-hole glass tube into the gold plating solution, connect the metal wire at the tail end to the electrochemical workstation, and adopt a three-electrode mode. The working electrode is connected to one side of the metal wire, and the counter electrode and the reference electrode are connected to the other side of the metal wire, and they are exchanged regularly during the entire deposition process to achieve simultaneous gold plating at both ends. Use the constant current mode for the electrochemical deposition of gold, detect the voltage between the two electrodes. When the voltage between the electrode pairs approaches 0 volts, it indicates that the distance between the electrode pairs is on the nanometer scale, and the electrochemical deposition is terminated. At this time, take out the tunneling probe from the gold plating solution and place it in ultrapure water for storage. The metal wire is a copper wire with an outer diameter of 0.2 mm. The gold plating solution is a self-prepared gold electroplating solution, which contains 4.4 mM NH4AuSO3 and 52 mM (NH4)2SO3. The ultrapure water is ultrapure water with a resistivity of 18.2 MΩ·cm. The entire electrochemical deposition process is carried out in a Faraday cage.

[0130] Example 2. The method for dynamically detecting single molecules provided by the present invention

[0131] Construct a quantum tunneling probe detection device using the quantum tunneling probe (tunneling electrode) with a nano-gap prepared in Example 1, including a two-electrode quantum tunneling probe, a weak current detection device, and a liquid environment containing target molecules. The weak current detection device is a three-electrode system device. Two working electrodes are respectively connected to one of the conductive metal wires at the tail end of the double-hole pipette, and the other electrode is connected to the reference electrode. The two working electrodes can respectively regulate the voltage relative to the reference electrode and simultaneously detect the weak current flowing through this electrode.

[0132] The method for dynamically detecting single molecules using the quantum tunneling probe detection device is specifically as follows:

[0133] Step 1. After connecting the two-electrode quantum tunneling probe (tunneling electrode) and the reference electrode (such as an Ag / AgCl electrode) to the weak current detection device, place them in a liquid environment containing target molecules. Set a group of gate potentials Vg = [Vg0, Vg1, Vg2 … Vgn] and a group of bias voltages Vb = [Vb0, Vb1, Vb3 … Vbm], and combine to regulate the potentials of the two working electrodes to (Vgi, Vgi+Vbj), where i ∈ [0,n] and j ∈ [0,m]. Collect the tunneling current signals under different (Vgi, Vbj) combinations, calculate the real-time conductance values, and count the high and low conductance state distributions. Analyze the conductance response matrix of the target molecules to resolve the dynamic correlations of the conformational changes, charge states, and energy states of the target molecules. Prepare the conductance change fingerprint diagrams of various different target molecules and construct a database containing the conductance change fingerprint diagrams of a large number of target molecules.

[0134] Step 2: Using the same method as shown in Step 1, change the liquid environment to a solution containing the molecule to be detected, detect the conductance change graph of the molecule to be detected, and compare it with the database. If the conductance change graph of the molecule to be detected is Figure 1 consistent with that of a specific target molecule in the database, then it is determined that the molecule to be detected is the specific target molecule.

[0135] Example 3: Example of dynamic detection of a specific single molecule

[0136] Detect the neurotransmitter dopamine single molecule according to the method provided in Example 2:

[0137] First, functionalize the quantum tunneling detection device prepared in Example 1. Place the quantum tunneling probe (nano-gap is 1.5 - 3.1 nm, preferably 1.5 nm) in a 0.5 mM solution of 4(5)-(2-mercaptoethyl)1H-imidazole-2-carboxamide (ICA) dissolved in ethanol for 24 h. As Figure 3 shown, the mercapto group of this molecule can form a gold-sulfur bond with the gold electrode for stable connection, and the carbonyl group and amino group on the other side of this molecule can form hydrogen bonds with the hydroxyl group and amino group on the dopamine molecule, thus forming a single molecule junction.

[0138] Place the ICA-modified quantum tunneling probe in a solution containing dopamine. Connect the two metal wires at the tail end to the two working electrodes (WE1 and WE2) of the weak current detection device respectively, connect the Ag / AgCl electrode to the reference electrode of the weak current detection device, and also place it in the same liquid environment. The overall configuration is as Figure 4 shown, where ① is the quantum tunneling probe; ② is the Ag / AgCl electrode, and ③ is the glass microfluidic channel. The dopamine concentration in the dopamine solution is 100 nM, and the solvent is 0.1x PBS solution with pH = 7.4.

[0139] Apply different voltages to WE1 and WE2, and monitor the current flowing through the single molecule junction. The voltages applied to (WE1 and WE2) are (-200 mV, -100 mV), (-200 mV, 0 mV), (-100 mV, 0 mV), (-100 mV, 100 mV), (0 mV, 100 mV), (0 mV, 200 mV), (100 mV, 200 mV), (100 mV, 300 mV), (200 mV, 300 mV), (200 mV, 400 mV), (300 mV, 400 mV), (300 mV, 500 mV). Some characteristic current graphs after applying different voltages are as Figure 5 、Figure 6 As shown, Figure 5 WE1 is -200 mV ~300mV, the voltage difference Bias is 100mv, WE2 is WE1+Bias, and WE2 increases as WE1 increases. Figure 6 WE1 is -200 mV ~300mV, the voltage difference Bias is 200mv, WE2 is WE1+Bias, and WE2 increases with the increase of WE1. The current when no molecular junction is formed is I base When a molecular junction is formed, the current shows frequent transitions between high and low conductivity states. At this time, the current value of the high conductivity state is I high , the current value of the low conductivity state is I low The difference in current between high and low conductance states under different potentials and biases, as well as the difference in duration, are analyzed to form a molecular conductivity fingerprint, such as Figures 7 to 9 As shown, Figure 7 The conductivity diagrams of WE1 are -200, -100, 0, 100mV, the voltage difference Bias is 100mv, and WE2 is WE1+Bias; Figure 8 The conductivity diagrams are as follows: WE1 is 200, 300, 400, 500mV, the voltage difference Bias is 100mv, and WE2 is WE1+Bias; Figure 9 For the general Figure 7 and Figure 8 Molecular conductivity fingerprints integrated into one image.

[0140] Example 4: Comparison of the effects of dynamic detection and static detection

[0141] This example uses the method provided in Example 3 to detect single-molecule sample solutions, and detects two single molecules, dopamine (DA) and norepinephrine (NE). When no gate potential control is added (static detection), the conductivity fingerprints of these two single molecules are very similar (such as Figure 10 ), which makes it difficult to distinguish. As the gate potential changes, the conductivity of the two molecules changes in different trends, and the conductivity distribution peaks slowly separate (such as Figures 11 to 17 , where the gate voltage WE1 is -200, -100, 0, 100, 200, 300, 400mV respectively, the voltage difference Bias is 100mv, and WE2 is WE1+Bias). The circuit diagram is as follows Figure 18 As shown; the molecular conductivity fingerprint of norepinephrine (NE) is as follows Figure 19 shown.

[0142] For example, for molecule DA, at a gate potential of -200 mV, the conductance peaks are at 40000 pA and 55000 pA (where the highest peak is considered the baseline, i.e., the average signal of the solution, and the lower peak is considered the tunneling signal). At a gate potential of -100 mV, the conductance peaks are at 40000 pA and 47500 pA. At a gate potential of 0 mV, the conductance peaks are at 50000 pA and 58000 pA. At a gate potential of 100 mV, the conductance peaks are at 50000 pA and 55000 pA. At a gate potential of 200 mV, the conductance peaks are at 57000 pA and 65000 pA. At a gate potential of 300 mV, the conductance peaks are at 70000 pA and 75000 pA. At a gate potential of 400 mV, the conductance peaks are at 75000 pA and 82000 pA. For molecule NE, at a gate potential of -200 mV, the conductance peaks are at 60000 pA and 74000 pA. At a gate potential of -100 mV, the conductance peaks are at 55000 pA and 70000 pA. At a gate potential of 0 mV, the conductance peaks are at 45000 pA and 60000 pA. At a gate potential of 100 mV, the conductance peaks are at 47000 pA and 57000 pA. At a gate potential of 200 mV, the conductance peaks are at 47000 pA and 67000 pA. At a gate potential of 300 mV, the conductance peaks are at 55000 pA and 90000 pA. At a gate potential of 400 mV, the conductance peaks are at 55000 pA and 65000 pA.

[0143] When testing an unknown solution, in the conductance distribution map at a gate potential of -200 to 400, conductance distribution peaks at different positions as shown can be seen respectively. Figures 11 to 17 It can be known that there are two molecules, DA and NE, in the solution.

[0144] Thus, when statically detecting an unknown single molecule, since the static detection spectra of some single molecules are very similar and it is impossible to distinguish which single molecule it is (for example, it is impossible to distinguish whether it is DA or NE). When changed to dynamic detection, because there is conductance information at different potentials, by comparing with the information database, it can be known what molecule is detected. Moreover, when dynamically detecting, the smaller the step size of the gate potential Vg adjustment, the finer the molecular fingerprint information, the better the resolution effect, and in theory, the more substances can be distinguished.

[0145] Example 5. Influence of the modification method of the tunneling electrode on the detection result

[0146] 1. Comparison of different modification methods

[0147] In this example, the method provided in Example 3 was used to detect the single-molecule sample solution. The sample solution contained two single molecules, dopamine and norepinephrine, with the concentrations of both dopamine and norepinephrine being 100 nM. The solvent was 0.1x PBS solution with pH = 7.4. The quantum tunneling probes were modified in the following different ways: 1. ICA modification (the same as in Example 3), forming a single-molecule junction with dopamine through hydrogen bonds; 2. Modification with mercaptophenylboronic acid, forming a single-molecule junction with dopamine through covalent bonds; 3. Using a dopamine aptamer, forming a single-molecule junction with dopamine through specific binding; 4. Directly using an unmodified tunneling electrode for detection. The effects of the four different modification methods on the detection results of dopamine single molecules were investigated and analyzed respectively, and the results are shown in Table 1.

[0148] Table 1. Effects of Different Modification Methods of Tunneling Electrodes on Detection Results

[0149]

[0150] It can be seen from Table 1 that when the first modification method is used to form a single-molecule junction with the molecule to be detected through hydrogen bonds, the detected conductance change signal is stable and clear, and multiple single molecules can be detected simultaneously; while for the second and third methods, since the single molecules are connected through covalent bonds or strong chemical bonds, the connection is very firm and cannot fall off to replace other single molecules after connection, and only one single molecule can be detected; for the fourth method, since it is not modified, the detected conductance signal is unstable. Because each single molecule has a different structure (not a completely symmetric structure), when it enters the tunneling region, its posture will cause differences in the tunneling current, which will directly affect the accuracy of the detection results and it is difficult to achieve a one-to-one match of the conductance change fingerprint.

[0151] In addition, comparing the first and second methods, when covalent bond capture is used, the firmness of the covalent bond connection is higher than that of the hydrogen bond, but its flexibility in replacing different single molecules during the detection process is not as good as that of the hydrogen bond, resulting in some molecules to be detected not being captured. Sometimes, most of the single molecule signals in the solution are stable, while the detection signals of some single molecules are unstable, and the universality is not high. However, when hydrogen bond capture is used, the universality is higher. Therefore, it is preferably to use hydrogen bond capture of the molecule to be detected for dynamic detection.

[0152] 2. Comparison of the Hydrogen Bond Modification Situation

[0153] The method provided in Example 3 was used to detect the single-molecule sample solution. It was known that the sample solution contained two single molecules, dopamine and norepinephrine, with the concentrations of both dopamine and norepinephrine being 100 nM, and the solvent being 0.1x PBS solution with pH = 7.4. The quantum tunneling probe formed a single-molecule junction with the single molecule through hydrogen bonds, and the following different hydrogen bond modification methods were investigated simultaneously (see Table 2): 1. Modified with ICA (the same as Example 3); 2. Modified with mercaptobenzoic acid. The effects of the two different modification methods on the detection results of dopamine single molecules were investigated and analyzed respectively, and the results are shown in Table 2.

[0154] Table 2. Comparison of different hydrogen bond modification methods

[0155]

[0156] As can be seen from Table 2, when the tunneling electrode is modified with hydrogen bonds, the hydrogen bonds obtained by different substances for modification also have a certain impact on the dynamic detection effect. Because the hydrogen bonds constructed by different substances have structural differences, the effects of forming single-molecule junctions are also different. The most preferred is to use ICA for hydrogen bond modification. When used for dynamic detection, it has the best universality, the most stable signal, and the more accurate detection results.

[0157] All patents and publications mentioned in the specification of the present invention indicate that these are public technologies in the field and can be used by the present invention. All patents and publications cited herein are also listed in the references in the same way as each publication is specifically referenced individually. It can be understood that the described embodiments of the present invention are all some preferred embodiments and features. Any person of ordinary skill in the art can make some changes and variations based on the essence described in the present invention, and these changes and variations are also considered to be within the scope of the present invention and are limited by the independent claims and the dependent claims.

Claims

1. A device for detecting single molecules, characterized in that, It includes tunneling electrodes. The molecule to be measured is located between the nano - gaps of the tunneling electrodes and under different potential conditions, the conductance change of the molecule to be measured is detected to judge the type of the molecule to be measured.

2. The device according to claim 1, characterized in that, The different potentials include different gate potentials and biases. The tunneling current signals flowing through the target molecule and / or the molecule to be measured are recorded and analyzed in real - time to obtain the conductance change map of the target molecule and / or the molecule to be measured.

3. The device according to claim 1, characterized in that, The tunneling electrodes have a pair of nano - gap tunneling electrodes, which are fabricated on the tip of a nanopipette. The nano - gap of the pair of nano - gap tunneling electrodes is 0.1 - 100 nm.

4. The device according to claim 3, wherein The tunneling electrodes are fabricated by heating and softening and pulling a double - channel glass pipette. The double - channel glass pipette is filled with a conductive material, a metal wire is inserted as the tail end, and a pair of nano - gap tunneling electrodes is deposited on the tip. The material of the pair of nano - gap tunneling electrodes is a single metal or a metal mixture.

5. The device according to claim 4, characterized in that The pair of nano - gap tunneling electrodes is modified, and the modification can form a single - molecule junction with the molecule to be measured.

6. The device according to claim 1, characterized in that, The tail end of the tunneling electrodes is connected to a weak - current detection device. The weak - current detection device includes two working electrodes and a reference electrode. The reference electrode is used to provide a stable potential reference. The voltages of the two working electrodes relative to the reference electrode are respectively regulated, and the weak current flowing through these electrodes is detected.

7. A method for detecting a target molecule, characterized in that, It includes the following steps: (1) Place the tunneling electrodes in a solution containing the molecule to be measured; (2) Dynamically detect the conductance change of the molecule to be measured under different potential conditions, obtain the conductance change map of the molecule to be measured, and thus judge the type of the molecule to be measured.

8. The detection method according to claim 7, characterized in that, It includes the following steps: (1) Place the tunneling electrodes in a solution containing the target molecule; (2) Dynamically adjust the gate potential and bias, record and analyze the tunneling current signals flowing through the target molecule in real - time, obtain the conductance change map of the target molecule, and construct a database containing the conductance change maps of different target molecules; (3) Place the tunneling electrodes in a solution containing the molecule to be measured; (4) Dynamically adjust the gate potential and bias, record and analyze the tunneling current signals flowing through the molecule to be measured in real - time, obtain the conductance change map of the molecule to be measured, and compare it with the database to judge which target molecule the molecule to be measured is; Both the target molecule and the molecule to be measured are single molecules.

9. The detection method according to claim 7, wherein Under the condition of dynamically adjusting the gate potential and bias, the target molecule and / or the molecule to be measured undergoes any one or more of redox reactions, molecular conformation transformation reactions, and molecular conjugate mode switching reactions, so that its conductance changes.

10. The detection method according to claim 7, characterized in that, The ways for the molecule to be measured to enter the nano - gap of the pair of nano - gap tunneling electrodes are any one or more of the following: free diffusion, electrostatic adsorption, hydrogen - bond adsorption, covalent - bond adsorption, specific binding, optical tweezers, and π - π stacking.

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