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

Abstract

The present invention provides a single-molecule detection device and method based on electrochemical gating technology in the quantum tunneling region. It is a detection technology that regulates the alignment of molecular energy levels and adjusts molecular conductivity by regulating gate voltage. A functionalized tunneling electrode is prepared at the tip of a nanopipette and placed in a solution containing a single molecule. The gate potential and bias voltage are dynamically adjusted, and the tunneling current signal flowing through the single molecule is recorded and analyzed in real time. It can actively regulate the structure, charge state and energy state of the single molecule. According to the conductivity change spectrum of the single molecule under different potential conditions, dynamic detection of the single molecule is achieved, significantly improving the detection accuracy of single molecules based on tunneling electrode detection.

Description

Technical Field

[0001] The present invention relates to the field of single molecule detection and nanosensing technology, and in particular to a single molecule detection method based on electrochemical gating technology in a quantum tunneling region. Background Art

[0002] Single-molecule detection technology is a significant breakthrough in contemporary analytical science. Its core goal is to break free from the limitations of traditional ensemble measurements, which typically average the behavior of a large number of molecules, and directly observe the real-time structure, dynamic behavior, and interactions of individual molecules, thereby revealing the heterogeneity and complexity of their individual behaviors. This technology uses ultra-sensitive instruments to capture the unique signals of individual molecules in chemical reactions, biological processes, or physical interactions, enabling scientists to track transient intermediates, low-probability reaction pathways, and dynamic fluctuations in intermolecular interactions that are indistinguishable from traditional methods.

[0003] However, traditional single-molecule detection is essentially still a passive recording mode. Its observation process is limited by the probability of spontaneous molecular behavior. It cannot actively regulate the physical and chemical state of the research object, and it is difficult to simultaneously analyze the relationship between molecular structure, charge state and energy state. There are 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 proteins at a fixed membrane potential, but cannot apply electrochemical stimulation in real time to simulate dynamic regulation under physiological conditions; in catalytic reaction monitoring, although scanning probe technology can locate single active sites, it is difficult to precisely control the adsorption intensity of reaction intermediates while applying potential.

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

[0006] Different biomolecules have diverse molecular recognition capabilities and, under certain circumstances, are prone to electron tunneling, which can be used for single-molecule detection. Single-molecule charge transport is of fundamental importance in electrochemical processes, tunneling detection, and ultimately the rational design of next-generation bioelectronic devices. Single-molecule-mediated tunneling electron transport relies on the capture of biomolecules between a pair of closely spaced electrodes with a gap of less than 100 nm. The current response at the connected tunneling electrodes can be measured as a characterization of the biomolecule's behavior. However, the tunneling current generated by a single molecule varies with the movement or position of the single molecule, and the signal is easily interfered with. In addition, the solution environment usually contains a variety of different single molecules, resulting in unstable and chaotic single-molecule detection signals, and the accuracy for detecting single molecules is not ideal.

[0007] Our research team previously developed a method for detecting single molecules using a tunneling electrode (CN115541680A). However, this method requires functional modification of the tunneling electrode to enable it to form a single-molecule junction with the target molecule, stabilize the detection signal, and induce a redox reaction in the target molecule for detection. This makes the detection process more complex and can only detect substances that can bind to the functional modification. For example, the patent uses peroxidase to modify the tunneling electrode, which can only detect hydrogen peroxide, a substance that can be catalyzed by the oxidase. Furthermore, this method only performs static detection and is passive monitoring. It cannot actively regulate the physicochemical state of the research object. The detection sensitivity is still limited, and it is difficult to detect single molecules with small current changes during the static detection process.

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

[0009] In response to the problems existing in the prior art, the present invention provides a single-molecule detection device and method based on electrochemical gating technology in the quantum tunneling region. It is a detection technology that regulates the alignment of molecular energy levels and adjusts molecular conductivity by regulating gate voltage. A functionalized tunneling electrode is prepared at the tip of a nanopipette and placed in a solution containing a single molecule. The gate potential and bias voltage are dynamically adjusted, and the tunneling current signal flowing through the single molecule is recorded and analyzed in real time. It can actively regulate the structure, charge state and energy state of the single molecule, and realize dynamic detection of the single molecule according to the conductivity change spectrum of the single molecule under different potential conditions, thereby significantly improving the detection accuracy of single molecules based on tunneling electrodes.

[0010] On the one hand, the present invention provides a device for detecting single molecules, which includes a tunneling electrode. The molecule to be detected is located between the nanogaps of the tunneling electrode and is under different potentials. The conductivity change of the molecule to be detected is detected to determine 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. This device can adjust the potential in real time during the process of tunneling electrode detection, thereby achieving dynamic detection of single molecules. Its main principle is: based on the different conductivity changes of different target molecules under different potential conditions, a conductivity change spectrum of each target molecule under different potential conditions is constructed as a fingerprint spectrum of the target molecule, forming a database. Then, by detecting the conductivity change of the target molecule in the sample and comparing it with the conductivity change spectrum of known target molecules in the database, the specific target molecule of the target molecule to be detected is determined. Compared with existing single-molecule static detection methods, the detection device provided by the present invention is used for detecting single molecules, which 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 specifically modify the tunneling electrodes to stabilize the detection signal. In particular, for some single molecules that do not show obvious changes in current before and after the redox reaction during static detection, they are often difficult to detect or have low detection sensitivity, making it difficult to meet accuracy requirements.

[0013] The device provided by the present invention can perform dynamic detection of single molecules based on electrochemical gating technology in the quantum tunneling region. Through the dual-electrode quantum tunneling probe in the quantum tunneling probe detection device, combined with the weak current detection equipment of the three-electrode system, it can actively regulate the matching degree between the molecular energy level and the electrode, breaking through the passive observation limitations of traditional single-molecule detection and realizing dynamic manipulation and high-dimensional analysis of single-molecule conductivity.

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

[0015] The conductance refers to the resistance value obtained by the molecule to be measured 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 in real time the tunneling current signal flowing through the target molecule and / or the molecule to be tested, and obtain a conductivity change spectrum of the target molecule and / or the molecule to be tested.

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

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

[0020] The conductance change spectrum refers to the current signal spectrum obtained by changing the gate potential and bias voltage, as the current flowing through a single molecule and its voltage change. Its conductance (equivalent to the resistance value obtained at a specific voltage and current) also changes continuously. Subsequently, a conductance distribution histogram for the conductance is prepared based on the voltage and current. The position and height of the conductance peak in the conductance distribution histogram are observed to determine the type of molecule being tested. The type mentioned here refers to determining the specific target molecule being tested.

[0021] Furthermore, the tunneling electrode comprises a nanogap tunneling electrode pair, which is manufactured on the tip of the nanopipette, and the nanogap of the nanogap tunneling electrode pair is 0.1-100 nm.

[0022] Furthermore, the tunneling electrode is obtained by heating, softening and drawing 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 nanogap tunneling electrode pair is deposited at the tip; the material of the nanogap tunneling electrode pair is a single metal or a metal mixture.

[0023] The present invention is based on a quantum tunneling probe detection device with controllable spacing. By actively adjusting the gate voltage and the bias voltage applied to both ends of the target molecule, the matching degree between the molecular energy level and the electrode is changed, and the tunneling signal of the specific molecule is enhanced or suppressed, thereby obtaining the dynamic change information of the conductivity of the target molecule relative to the gate potential and bias voltage, constructing the "potential-bias" response matrix of the conductivity, and analyzing its dependence of the conductivity on the electrode potential.

[0024] The controllable-gap quantum tunneling probe detection device comprises a two-electrode quantum tunneling probe (or tunneling electrode), a weak current detection device, and a liquid environment containing target molecules. The two-electrode quantum tunneling probe is a double-bore glass pipette with one end pulled to a sharp point (nanometer to micrometer scale). The probe is filled with a conductive material and has a pair of electrodes with a nanometer gap at its tip. The quantum tunneling probe tip is placed in the liquid environment containing the target molecules. The tail end of the quantum tunneling probe is connected to the weak current detection device, which can also control the potential of the tunneling probe electrode relative to a reference electrode.

[0025] The present invention uses a dual-electrode quantum tunneling probe combined with a three-electrode system of weak current detection equipment to actively regulate the matching degree between molecular energy levels and electrodes, thereby achieving dynamic control and high-dimensional analysis of single-molecule conductance.

[0026] The nanogap between nanogap tunneling electrodes 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 to detect, the clearer the fingerprint, and the higher the detection sensitivity. However, the smaller the gap, the more difficult it is to prepare the tunneling electrode.

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

[0028] In some embodiments, the nanogap is preferably 0.1-10 nm.

[0029] In some embodiments, the nanogap is preferably 1.5 to 3.1 nm.

[0030] Furthermore, the tunneling electrode is obtained by heating, softening and drawing a double-channel glass pipette; the double-channel glass pipette is filled with conductive material, a metal wire is inserted as the tail end, and the nanogap tunneling electrode pair is deposited on the tip.

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

[0032] The heating method for heating and softening includes microwave heating, resistance furnace heating, laser heating, etc.

[0033] The conductive material in the nanopipette channel is pyrolytic carbon, and the conductive metal wire is copper wire.

[0034] The nanogap electrode pair is a pair of metals or metal mixtures with a nanogap. Preferably, the nanogap electrode pair is made of gold, and the nanogap gold electrode pair is prepared at the tip of a glass tube using electrochemical deposition. The gap is the minimum distance between the two electrodes in the electrode pair, and the nanogap refers to a gap in the range of 0.1 nm to 100 nm. Preferably, the nanogap range is 1.5 nm to 3.1 nm.

[0035] In some embodiments, the tip-deposited nanogap tunneling electrode pair is prepared by electrochemical deposition. The electrochemical deposition of the nanogap electrode pair specifically comprises electrochemically depositing an untouched electrode pair on the tip of a nanopipette, and depositing gold using a constant current, self-termination voltage feedback method. The method is terminated when the voltage between the electrode pair approaches 0 volts, indicating that the distance between the electrode pair is on the nanometer scale, and the electrochemical deposition is terminated.

[0036] In addition, the smaller the nanogap, the higher the detection sensitivity, but it is also more susceptible to environmental disturbances. Therefore, adding modifications to the tunneling electrode will also help improve the resistance to environmental disturbances.

[0037] Furthermore, the tail end of the tunneling electrode is connected to a weak current detection device, which 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. Two working electrodes are connected to one of the conductive wires at the end of a double-bore pipette, and the other electrode is connected to a reference electrode. The two working electrodes can independently control the voltage relative to the reference electrode and simultaneously detect the weak current flowing through them. The double-bore pipette tip and reference electrode are placed in a liquid environment containing the target molecule.

[0039] Furthermore, 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] Furthermore, the nanogap tunneling electrode pair is modified, and the modification can form a single molecule junction with the molecule to be detected.

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

[0042] Therefore, if the tunneling electrode carries a modified molecule, the modified molecule can capture the molecule to be tested, and the posture of the molecule to be tested can be fixed to a certain extent (thereby fixing the tilt angle of the molecule and making the molecular structure basically fixed). For example, the hydrogen bond is modified on the nanogap tunneling electrode pair to form a single molecule junction with dopamine. The hydrogen bond of one nanoelectrode is connected to the hydroxyl group of dopamine, and the hydrogen bond at one end is connected to the amino group of dopamine. In this way, the measured conductivity signal will be more stable, and it will be easier to accurately detect the molecule to be tested.

[0043] Of course, there can be many types of modifications. For example, some modifications can form strong chemical bonds to fix the molecules to be tested, thus forming an electrode-molecule-electrode pathway, which can be measured for a long time. However, this method is only suitable for detecting a single single molecule. For example, CN115541680A can only detect hydrogen peroxide, a substance that can react with peroxidase, and cannot detect multiple different single molecules at the same time.

[0044] In order to be able to detect multiple different single molecules at the same time, it is preferred to modify the tunneling electrode with relatively weak hydrogen bonds. This modification method will 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, realizing the simultaneous detection of multiple single molecules.

[0045] In another aspect, the present invention provides a method for detecting a target molecule, comprising the following steps:

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

[0047] (2) Dynamically detect the changes in the conductivity of the molecule to be tested under different potentials, obtain the conductivity change spectrum of the molecule to be tested, and thus determine the type of the molecule to be tested.

[0048] Furthermore, the method comprises the following steps:

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

[0050] (2) Dynamically adjust the gate potential and bias voltage, record and analyze the tunneling current signal flowing through the target molecule in real time, obtain the conductivity change map of the target molecule, and build a database containing the conductivity change maps of different target molecules;

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

[0052] (4) Dynamically adjust the gate potential and bias voltage, record and analyze the tunneling current signal flowing through the molecule to be tested in real time, obtain the conductivity change spectrum of the molecule to be tested, and compare it with the database to determine which target molecule the molecule to be tested is;

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

[0054] Furthermore, under the condition of dynamically adjusting the gate potential and bias voltage, the target molecule and / or the molecule to be measured undergoes any one or more of redox reaction, molecular conformational transition reaction, and molecular conjugation mode switching reaction, thereby causing its conductivity to change.

[0055] Different single molecules may undergo different reactions when the gate potential and bias of the tunneling electrode are dynamically adjusted. For example, single molecules with redox activity will undergo redox reactions, and they can be distinguished according to the different oxidation potentials of different single molecules or the different redox conductivities of different molecules; for example, single molecules with molecular conformational reaction activity will undergo conformational reactions, and they can be distinguished according to the different conformational reaction potentials of different single molecules; for example, single molecules with molecular conjugation mode switching reaction activity will undergo molecular conjugation mode switching reactions, and they can be distinguished according to the different molecular conjugation mode switching reaction potentials of different single molecules.

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

[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 dynamic regulation of gate potential and bias voltage, the spontaneous redox reaction of a single molecule typically progresses through three stages: 1. Stable reduced state; 2. Oxidation as the bias voltage increases; 3. Continued oxidation as the bias voltage continues to increase. The three stages require different potentials for different single molecules, resulting in distinct conductivity patterns.

[0059] In addition, although the existing static detection method also uses redox reactions, it must add components that promote redox reactions or other reactions to the reaction solution. The method provided by the present invention does not require the addition of any components that promote redox reactions or other reactions. It only changes the potential to change the conductivity of the single molecule under different potential conditions, thereby spontaneously undergoing redox reactions, conformational change reactions, molecular conjugation mode switching reactions, etc. Since the changes in conductivity of different single molecules at specific potentials are different, the detection of single molecules is achieved by detecting this change process. Compared with static detection, this detection method can significantly improve detection accuracy, so that single molecules that were originally undetectable in static detection can be successfully detected in dynamic detection.

[0060] Furthermore, the molecule to be detected enters the nanogap of the nanogap tunneling electrode pair by any one or more of the following methods: free diffusion, electrostatic adsorption, hydrogen bond adsorption, covalent bond adsorption, specific binding, optical tweezers, and π-π stacking.

[0061] Different ways in which the molecules to be tested enter the nanogap can also be determined by different modification methods of the tunneling electrode.

[0062] Of course, there can be many types of modifications. For example, some modifications can form strong chemical bonds to fix the molecules to be tested, thus forming an electrode-molecule-electrode pathway, which can be measured for a long time. However, this method is only suitable for detecting a single single molecule. For example, CN115541680A can only detect hydrogen peroxide, a substance that can react with peroxidase, and cannot detect multiple different single molecules at the same time.

[0063] In order to be able to detect multiple different single molecules at the same time, it is preferred to modify the tunneling electrode with relatively weak hydrogen bonds. This modification method will 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, realizing the simultaneous detection of multiple single molecules.

[0064] In some methods, there are many connection methods that are compatible with most of the molecules to be tested, 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 the modification method using hydrogen bond connection has a binding strength that is neither too strong nor too weak, and this modification method will not fix a molecule for a long time, but 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, and multiple single molecules can be detected simultaneously, which has very high universality. Modification using other methods, such as nucleic acid aptamers, peroxidases, etc., can only detect one target substance and cannot detect multiple different single molecules at the same time.

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

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

[0067] Since the conductivity of a single molecule changes immediately with changes in gate potential and bias voltage, and the reaction occurs instantaneously, the time intervals for adjusting different gate potentials and bias voltages have no effect. Adjustments can be made every 1 second, every 1 minute, or even every 1 hour, and the conductivity change graphs obtained will show almost no difference.

[0068] Furthermore, the molecule to be detected or the target molecule is a substance whose conductivity changes under different potentials.

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

[0070] Furthermore, 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] Furthermore, the molecule to be detected or the target molecule is a substance that undergoes redox reaction under different potential conditions.

[0072] Conductance changes during gate potential and bias modulation can occur for a variety of reasons, including redox reactions, molecular conformational transitions, and molecular conjugation mode switching, with redox reactions being the most common. The methods provided by this invention can accurately detect single molecules with varying redox activity at different potentials.

[0073] Furthermore, the molecules to be detected or target molecules include molecules that are difficult to accurately detect by static detection using a tunneling electrode, wherein the static detection means that the gate potential and bias voltage remain unchanged during the detection process; the molecules that are difficult to accurately detect by static detection using a tunneling electrode include molecules in which, in static detection, the conductivity peak distributions of the molecules to be detected are very close, making it difficult to distinguish which single molecule it is.

[0074] Static detection can only detect the current changes before and after the redox reaction of a single molecule. Some static detection patterns of single molecules are very similar, making it impossible to distinguish the specific single molecule. For example, when statically detecting dopamine and norepinephrine, their conductivity distributions do not differ significantly, making it impossible to distinguish whether they are dopamine or norepinephrine. However, the dynamic detection method of the tunneling electrode provided by the present invention can accurately distinguish dopamine and norepinephrine in solution through their conductivity changes.

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

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

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

[0078] In some methods, in order to simulate human or animal body fluids to a certain extent, a buffer solution such as PBS can be used that imitates body fluids as much as possible.

[0079] In another aspect, the present invention provides a method for detecting target molecules that are difficult to detect or distinguish by static detection, the method comprising the following steps:

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

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

[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:

[0084] 1. This paper provides a new single-molecule dynamic detection method. By dynamically adjusting the gate potential and bias voltage, the tunneling current signal flowing through the single molecule is recorded and analyzed in real time. According to the conductivity change spectrum of the single molecule under different potential conditions, the dynamic detection of the single molecule is realized, which significantly improves the detection accuracy of single molecules based on tunneling electrodes.

[0085] 2. By using nanogap electrode pairs and single-molecule structures to build conductive channels, and combining an electrochemical three-electrode system to apply a gate voltage (Vg), the energy level arrangement, redox state, or protonation degree of the molecule can be directly regulated, achieving precise control of the structure, charge state, and energy state of a single molecule. The molecular conductivity fingerprint (statistical distribution of high and low conductivity states) can be obtained by regulating the gate potential and bias voltage.

[0086] 3. Compared with traditional methods, the breakthrough advantage of this technology lies in the innovation of dynamic control capabilities.

[0087] 4. Through dynamic detection, single molecules that are difficult to distinguish through static detection can be accurately distinguished and detected based on the dynamic conductivity changes.

[0088] 5. By selecting appropriate modification methods for the tunneling electrode, it can be compatible with most of the molecules to be tested, thus achieving simultaneous detection of multiple single molecules.

[0089] 6. It not only makes up for the technical shortcomings of traditional single-molecule detection, but also shows unique potential in the fields of molecular electronic devices, precise catalytic design and dynamic analysis of biomolecules.

[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, guiding the design of efficient catalysts; in the biomedical field, this technology can analyze the conformational changes of proteins and the regulation mechanisms of enzyme activity under the action of electric fields, providing new perspectives 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 spatiotemporal 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 of Example 3 in which the thiol group of the molecule can form a gold-sulfur bond with the gold electrode, and the carbonyl group and amino group on the other side of the molecule can form hydrogen bonds with the hydroxyl group and amino group on the dopamine molecule to form a unimolecular junction;

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

[0096] Figure 5 3 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 3 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 is shown in FIG1 when the gate voltage WE1 is -200, -100, 0, and 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, and 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 conductivity fingerprints integrated into one graph (where G represents the gate voltage, e.g. G:-200mV means the gate voltage is -200mV);

[0101] Figure 10 4 is the conductivity distribution diagram of DA and NE during static detection in Example 4;

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

[0103] Figure 18 Schematic diagram of the circuit in Example 4;

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

[0105] Detailed description

[0106] Tunneling electrode

[0107] 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.

[0108] A tunneling electrode is an electrode structure that utilizes the quantum tunneling effect to achieve electron transport. Quantum tunneling refers to the phenomenon in which electrons penetrate regions prohibited by classical mechanics (such as insulating layers or potential barriers) with a certain probability. It is a fundamental property of quantum mechanics. When the potential barrier between an electrode and the target material (or another electrode) is very thin (typically nanometers), electrons can pass through the barrier, forming a tunneling current. This is the quantum tunneling effect. The tunneling current is extremely sensitive to the thickness and height of the barrier and typically follows an exponential decay relationship. Unlike traditional electrodes, tunneling electrodes do not rely on ohmic contact (i.e., electrons do not need to cross the barrier through thermal excitation). Instead, they achieve electron transport directly through quantum tunneling.

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

[0110] The tunneling electrode pair includes independent nanoelectrodes separated by a nanogap, wherein the nanogap length is 0.1 nm to 100 nm. The tunneling electrodes can have any convenient shape or size and can be composed of any conductive material. Each tunneling electrode disclosed in the present invention can be made of different materials or a mixture of materials, such as an alloy.

[0111] Tunneling electrodes are used to measure an electrical current that can travel through and / or across a molecule, which can be a tunneling current. Measuring the current can be used to determine the sequence of a biopolymer, such as a nucleic acid molecule (e.g., DNA or RNA) or a protein. For mass measurement, the gap spacing between the electrodes of one or more tunneling electrode pairs can be stable and controllable.

[0112] Quantum tunneling probe detection device

[0113] Quantum tunneling probes are instruments that utilize the quantum tunneling effect to achieve highly sensitive detection. Their core principle is to monitor changes in the tunneling current across the nanoscale gap between tunneling electrodes to reveal the physical or chemical properties of the sample being measured. These devices have important applications in nanotechnology, surface science, biosensing, and other fields. The most prominent example is the scanning tunneling microscope (STM), but other specialized sensors are also available.

[0114] Gate potential

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

[0116] bias

[0117] Bias refers to the DC voltage (or voltage difference) applied between two electrodes in an electronic device or circuit to control the device's operating state (e.g., conduction, cutoff, amplification) or achieve specific functions (e.g., carrier injection, bandgap control, etc.). It is a fundamental concept in electronics, semiconductor devices, and quantum technology. It refers to the voltage applied between two tunneling electrodes (i.e., the voltage applied across a single molecule) to drive electron tunneling. Bias is the "foundation voltage" for the operation of electronic devices. Active regulation of DC voltage enables device functions such as switching, amplification, and sensing.

[0118] Conductivity

[0119] Conductivity is a physical quantity that measures the ability of a material or component to conduct electricity, indicating how easily current flows through it. It is the reciprocal of resistance, meaning G = R1. The unit is Siemens (symbol: S), with 1 S = 1 A / V (ampere / volt). Physical meaning: The greater the conductance, the less resistance the material or component offers to current flow, and the greater its conductivity. DETAILED DESCRIPTION

[0120] The present invention will be described in further detail below in conjunction with the accompanying drawings and Examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not serve to limit the present invention in any way. The reagents used in this example are all known products and were obtained by purchasing commercially available products.

[0121] Example 1. Preparation of tunneling electrode

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

[0123] Step 1: Pull a double-bore glass pipette into a probe with a single-sided tip

[0124] A double-bore glass pipette treated with a plasma cleaner was placed in a P-2000 laser drawing instrument and drawn using a two-step process. The double-bore 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 involves applying a laser beam to heat the center of a thin glass tube while simultaneously drawing it. The drawing parameters are set to: Heat: 850°, Filament: 4°, Velocity: 30°, Delay: 160°, and Pull: 100°. In the second step, the parameters are set to: Heat: 860°, Filament: 3°, Velocity: 20°, Delay: 140°, and Pull: 160°. At the end of the drawing process, 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 m3 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 emits a bright yellow light and remains for 10 seconds. At this time, the butane in the double-hole tube is pyrolyzed into carbon element and deposited on the tip of the double-hole tube. Then slowly move the butane flame toward 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: Use electrochemical deposition to deposit a nano-gap gold electrode pair on the tip of the double-hole glass tube

[0129] A metal wire was inserted from the end of a double-bore glass tube, ensuring full contact with the pyrolytic carbon. Hot melt adhesive was used to secure the wire to ensure device stability. The tip of the double-bore glass tube was then placed in a gold plating solution. The end wire was connected to an electrochemical workstation using a three-electrode setup. The working electrode was connected to one wire, and the counter and reference electrodes were connected to the other wires. These electrodes were periodically swapped throughout the deposition process to achieve simultaneous gold plating at both ends. Gold electrochemical deposition was performed using a constant current mode. The voltage between the two electrodes was monitored. When the voltage between the electrode pair approached 0 volts, indicating a nanometer-scale separation between the electrodes, electrochemical deposition was terminated. The tunneling probe was then removed from the gold plating solution and stored in ultrapure water. The metal wire was a copper wire with an outer diameter of 0.2 mm. The gold plating solution was a custom-made gold plating solution containing 4.4 mM NH4AuSO3 and 52 mM (NH4)2SO3. The ultrapure water was 18.2 MΩ·cm. The entire electrochemical deposition process was performed in a Faraday cage.

[0130] Example 2: Single molecule dynamic detection method provided by the present invention

[0131] A quantum tunneling probe detection device was constructed using the nanogap quantum tunneling probe (tunneling electrode) prepared in Example 1. The device comprises 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, with two working electrodes connected to one of the conductive wires at the end of a two-hole pipette, and the other electrode connected to a reference electrode. The two working electrodes can independently control the voltage relative to the reference electrode, while simultaneously detecting the weak current flowing through them.

[0132] The method for single-molecule dynamic detection using a quantum tunneling probe detection device is as follows:

[0133] Step 1: Connect a two-electrode quantum tunneling probe (tunneling electrode) to a reference electrode (e.g., Ag / AgCl electrode) and a weak current detection device, then place it in a liquid containing the target molecule. Set a set of gate potentials, Vg = [Vg0, Vg1, Vg2…Vgn], and a set of bias voltages, Vb = [Vb0, Vb1, Vb3…Vbm], to control the potentials of the two working electrodes to (Vgi, Vgi+Vbj), where i∈[0,n] and j∈[0,m]. Tunneling current signals are collected for different (Vgi, Vbj) combinations, and real-time conductance values ​​are calculated, with the distribution of high and low conductance states statistically analyzed. The target molecule's conductivity response matrix is ​​analyzed to reveal the dynamic correlation between conformational changes, charge states, and energy states. Conductance fingerprints are generated for various target molecules, and a database of these fingerprints is constructed.

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

[0135] Example 3: Specific single molecule dynamic detection example

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

[0137] First, the quantum tunneling detection device prepared in Example 1 was functionalized and the quantum tunneling probe (nanogap of 1.5-3.1 nm, preferably 1.5 nm) was placed in a 0.5 mM 4(5)-(2-mercaptoethyl)1H-imidaz ole-2-carboxamide (ICA) solution dissolved in ethanol for 24 h. Figure 3 As shown, the thiol group of the molecule can form a gold-sulfur bond with the gold electrode to stabilize the connection, and the carbonyl group and amino group on the other side of the molecule can form hydrogen bonds with the hydroxyl group and amino group on the dopamine molecule to form a single molecule junction.

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

[0139] Different voltages were applied to WE1 and WE2, and the current flowing through the single-molecule junction was monitored. The voltages applied to (WE1 and WE2) were (-200mV, -100mV), (-200mV, 0mV), (-100mV, 0mV), (-100mV, 100mV), (0mV, 100mV), (0mV, 200mV), (100mV, 200mV), (100mV, 300mV), (200mV, 300mV), (200mV, 400mV), (300mV, 400mV), and (300mV, 500mV). Some characteristic currents after applying different voltages are shown in the figure below. 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 ~ 300 mV, the voltage difference Bias is 200 mv, 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 a frequent transition 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 and the difference in duration under different potentials and biases are analyzed to form a molecular conductivity fingerprint, such as Figures 7-9 As shown, Figure 7 The conductance diagrams are as follows: WE1 is -200, -100, 0, 100mV, the voltage difference Bias is 100mv, and WE2 is WE1+Bias; Figure 8 The conductance 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, respectively detecting two single molecules of dopamine (DA) and norepinephrine (NE). When no gate potential control is applied (static detection), the conductivity fingerprints of these two single molecules are very similar (e.g. 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 40,000 pA and 55,000 pA (the highest peak is considered to be the baseline, i.e., the average signal of the solution, and the lower peak is considered to be the tunneling signal); at a gate potential of -100 mV, the conductance peaks are 40,000 pA and 47,500 pA; at a gate potential of 0 mV, the conductance peaks are 50,000 pA and 58,000 pA; at a gate potential of 100 mV, the conductance peaks are 50,000 pA and 55,000 pA; at a gate potential of 200 mV, the conductance peaks are 57,000 pA and 65,000 pA; at a gate potential of 300 mV, the conductance peaks are 70,000 pA and 75,000 pA; at a gate potential of 400 mV, the conductance peaks are 75,000 pA and 82,000 pA; while for molecule NE, at a gate potential of -200 When the gate potential is 0 mV, the conductance peaks are at 60,000 pA and 74,000 pA; when the gate potential is -100 mV, the conductance peaks are at 55,000 pA and 70,000 pA; when the gate potential is 0 mV, the conductance peaks are at 45,000 pA and 60,000 pA; when the gate potential is 100 mV, the conductance peaks are at 47,000 pA and 57,000 pA; when the gate potential is 200 mV, the conductance peaks are at 47,000 pA and 67,000 pA; when the gate potential is 300 mV, the conductance peaks are at 55,000 pA and 90,000 pA; when the gate potential is 400 mV, the conductance peaks are at 55,000 pA and 65,000 pA.

[0143] When testing an unknown solution, the following are seen in the conductivity distribution diagram of the gate potential -200~400: Figures 11 to 17 From the conductivity distribution peaks at different positions shown, we can know that there are two molecules, DA and NE, in the solution.

[0144] It can be seen from this that when statically detecting unknown single molecules, it is impossible to distinguish what kind of single molecule it is (for example, it is impossible to distinguish whether it is DA or NE) because the static detection spectra of some single molecules are very similar. However, when changing to dynamic detection, because there is conductivity information under different potentials, the type of molecule measured can be known by comparing the information library. Moreover, during dynamic detection, the smaller the step size of the gate potential Vg adjustment, the finer the molecular fingerprint information, the better the resolution effect, and theoretically, more substances can be distinguished.

[0145] Example 5: Effect of Tunneling Electrode Modification on Detection Results

[0146] 1. Comparison of different modification methods

[0147] This example employed the method provided in Example 3 to detect single-molecule sample solutions containing two single molecules, dopamine and norepinephrine, at 100 nM concentrations. The solvent was a 0.1x PBS solution at pH 7.4. The quantum tunneling probes were modified using the following different methods: 1. ICA modification (same as in Example 3), forming a single-molecule junction with dopamine via hydrogen bonding; 2. Mercaptophenylboronic acid modification, forming a single-molecule junction with dopamine via covalent bonding; 3. Dopamine aptamer modification, forming a single-molecule junction with dopamine via specific binding; and 4. Direct detection using an unmodified tunneling electrode. The effects of the four different modification methods on dopamine single-molecule detection were investigated and analyzed, with the results shown in Table 1.

[0148] Table 1. Effects of different tunneling electrode modification methods on detection results

[0149]

[0150] As can be seen from Table 1, the first modification method uses hydrogen bonds to form single-molecule junctions with the molecule to be tested, and the detected conductivity change signal is stable and clear, and multiple single molecules can be detected simultaneously. In contrast, the second and third methods use covalent bonds or strong chemical bonds to connect single molecules, and the connection is very strong. After connection, it cannot be detached and replaced with other single molecules, so only one single molecule can be detected. The fourth method, due to the lack of modification, has unstable detected conductivity signals. This is because each single molecule has a different structure (not a completely symmetrical structure). When it enters the tunneling region, its posture will lead to different tunneling currents, which will directly affect the accuracy of the detection results and make it difficult to achieve one-to-one matching of the conductivity change fingerprint.

[0151] In addition, comparing the first and second methods, covalent bond capture is more robust than hydrogen bond capture, but the flexibility of switching between different single molecules during detection is not as good as hydrogen bond capture, which can result in some single molecules not being captured. Sometimes, the signals of most single molecules in the solution are stable, while the signals of some single molecules are unstable, resulting in low universality. However, hydrogen bond capture is more universal and is therefore preferred for capturing the molecules to be detected for dynamic detection.

[0152] 2. Comparison of hydrogen bond modification

[0153] The method provided in Example 3 was used to detect single-molecule sample solutions containing dopamine and norepinephrine single molecules. The concentrations of both dopamine and norepinephrine were 100 nM, and the solvent was a 0.1x PBS solution at pH 7.4. The quantum tunneling probe formed a single-molecule junction with the single molecules using hydrogen bonds. The following hydrogen bond modification methods were investigated (see Table 2): 1. ICA modification (same as in Example 3); 2. Mercaptobenzoic acid modification. The effects of each modification method on dopamine single-molecule detection were analyzed, 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 hydrogen bonds are used to modify tunneling electrodes, the hydrogen bonds obtained by modification with different substances 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. It is most preferred to use ICA for hydrogen bond modification. When used for dynamic detection, it has the best universality, the most stable signal, and more accurate detection results.

[0157] All patents and publications mentioned in this specification are intended to indicate that they are publicly available in the art and that the present invention may be used. All patents and publications cited herein are also listed in the references, just as if each publication were specifically cited individually. It is understood that the embodiments described in the present invention are only preferred embodiments and features, and anyone skilled in the art may make some modifications and variations based on the essence of the description of the present invention, and such modifications and variations are considered to be within the scope of the present invention and the scope of the independent claims and appended claims.

Claims

1. A method for detecting a molecule to be detected, characterized in that: The following steps are involved: (1) Place the tunneling electrode in a solution containing the molecule to be detected; (2) Dynamically detecting the change in the conductivity of the molecule to be tested under different potentials, obtaining a conductivity change spectrum of the molecule to be tested, and thereby determining the type of the molecule to be tested; the different potentials, including different gate potentials and bias voltages, record and analyze the tunneling current signal flowing through the molecule to be tested in real time to obtain a conductivity change spectrum of the molecule to be tested; The tail end of the tunneling electrode is connected to a weak current detection device, which 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 the electrodes; The gate potential refers to the overall offset of the voltage of the two working electrodes relative to the reference electrode voltage; the bias voltage refers to the voltage applied between the two tunneling electrodes; The molecule to be detected is a substance whose conductivity changes under different potentials.

2. The detection method according to claim 1, wherein The molecules to be detected include molecules that are difficult to accurately detect through static detection of tunneling electrodes.

3. The detection method according to claim 2, wherein The molecules to be detected include any one or more of dopamine, serotonin, and norepinephrine.

4. The detection method according to claim 1, wherein The molecule to be detected enters the nanogap of the nanogap tunneling electrode pair in any one or more of the following ways: free diffusion, electrostatic adsorption, hydrogen bond adsorption, covalent bond adsorption, specific binding, optical tweezers, and π-π stacking.

5. The detection method according to claim 4, wherein The molecule to be detected enters the nanogap of the nanogap tunneling electrode pair by hydrogen bond adsorption.

6. The detection method according to claim 1, wherein The following steps are involved: (1) Place the tunneling electrode in a solution containing the target molecule; (2) Dynamically adjust the gate potential and bias voltage, record and analyze the tunneling current signal flowing through the target molecule in real time, obtain the conductivity change map of the target molecule, and build a database containing the conductivity change maps of different target molecules; (3) Place the tunneling electrode in a solution containing the molecule to be detected; (4) Dynamically adjust the gate potential and bias voltage, record and analyze the tunneling current signal flowing through the molecule to be tested in real time, obtain the conductivity change spectrum of the molecule to be tested, and compare it with the database to determine which target molecule the molecule to be tested is; Both the target molecule and the molecule to be detected are single molecules.

7. The detection method according to claim 6, wherein Under the condition of dynamically adjusting the gate potential and bias voltage, the target molecule and / or the molecule to be measured undergoes any one or more of redox reaction, molecular conformational transition reaction, and molecular conjugation mode switching reaction, thereby causing its conductivity to change.

8. A device for detecting single molecules, characterized in that The system comprises a tunneling electrode, wherein the molecule to be detected is located between the nano-gap of the tunneling electrode and is placed under different potentials to detect changes in the conductivity of the molecule to determine the type of the molecule to be detected. The different potentials include different gate potentials and bias voltages, and the tunneling current signal flowing through the molecule to be detected is recorded and analyzed in real time to obtain a conductivity change spectrum of the molecule to be detected. The molecule to be detected is a substance whose conductivity changes under different potentials. The tunneling electrode comprises a nanogap tunneling electrode pair, wherein the nanogap of the nanogap tunneling electrode pair is 0.1 to 100 nm; the tail end of the tunneling electrode is connected to a weak current detection device, wherein the weak current detection device comprises 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 the electrodes; The gate potential refers to the overall offset of the voltage of the two working electrodes relative to the voltage of the reference electrode; the bias voltage refers to the voltage applied between the two tunneling electrodes.

9. The device according to claim 8, wherein The tunneling electrode is obtained by heating, softening and drawing a double-channel glass pipette; the double-channel glass pipette is filled with conductive material, a metal wire is inserted as the tail end, and a nanogap tunneling electrode pair is deposited at the tip; the material of the nanogap tunneling electrode pair is a single metal or a metal mixture.

10. The device according to claim 8, wherein The nanogap tunneling electrode pair is modified, and the modification can form a single molecule junction with the molecule to be detected.

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