A method for detecting the conductivity of protein molecules and its application

Abstract

The present invention provides a method for detecting the electrical conductivity of protein molecules and its application. By asymmetrically modifying a tunneling electrode pair and a protein molecule, the protein molecule can enter the tunneling region in a fixed posture and form an asymmetric single-molecule junction with the tunneling electrode pair, which can maximize the activity of the protein molecule. This constructs a device that can stably detect the electrical conductivity of protein molecules over a long period of time. The device can be used for dynamic conformational studies of the protein itself, screening of small molecule drugs targeting the protein molecule, and analysis of the interaction between small molecule drugs and protein molecules. The present invention utilizes tunneling electrode devices and real-time electrical measurement technology to achieve protein detection at the single-molecule level, comprehensively depicting the conformational change process of proteins such as biological enzymes and their interaction characteristics with small molecules on a high time resolution scale, and is expected to improve the efficiency and success rate of existing drug design and optimization.

Description

Technical Field

[0001] The present invention relates to the field of biosensing and analysis technology, and in particular to a method for detecting the electrical conductivity of protein molecules and an application thereof. Background Art

[0002] Thanks to the rapid development of cutting-edge technologies such as cryo-electron microscopy, researchers are now able to analyze the static structure of proteins at the nanoscale. This breakthrough has made the structure-based drug design strategy possible, and it has gradually become one of the mainstream strategies for the development of small molecule drugs that target proteins.

[0003] However, in physiological environments, protein conformations are often highly dynamic. This is especially true for classic drug target proteins, such as enzymes, which often undergo dramatic conformational changes during biochemical reactions, often interacting with biomacromolecules such as substrates and cofactors. Simply analyzing a protein's static structure makes it difficult to truly capture its highly dynamic transformations, severely hindering the rational design and optimization of corresponding small molecule drugs.

[0004] The recent rise of single-molecule bioelectronics research based on the tunneling effect provides a potential solution to the above problems. Its core is to use nanogap electronic devices and real-time electrical measurement technology to achieve protein detection at the single-molecule level. Using this strategy, by detecting and analyzing the changes in the tunneling current of protein molecules, it is possible to comprehensively depict the conformational change process of proteins such as biological enzymes and their interaction characteristics with small molecules on a high time resolution scale. This not only helps to screen small molecule drugs targeting the protein and improve the efficiency and success rate of screening optimization, but also can further enhance the specificity and effect of small molecule drugs by combining the characteristics of the two "dimensions" of "protein spatial structure" and "protein dynamics". At present, there have been no reports on the construction of a device that can stably detect the conductivity of target protein molecules and use it as an analytical screening platform for screening small molecule drugs and analyzing their protein dynamics specificity.

[0005] To achieve conductivity detection of protein molecules, it is typically necessary to modify the tunneling electrode to enable it to form a single-molecule junction with the protein molecule. However, this is typically achieved through symmetrical modifications. Due to the asymmetric structure of protein molecules, with N-termini and C-termini, the process by which a protein molecule enters the tunneling region and forms a single-molecule junction varies each time it enters the tunneling region, making it difficult to ensure stable and accurate conductivity detection. Furthermore, existing modifications can affect protein activity, thereby affecting the protein's dynamic conformation and even occupying pockets within the protein molecule that are intended for small molecule binding. This not only affects the protein's structure but also the screening of small molecule drugs targeting the protein, making it difficult to identify suitable small molecule drugs and to accurately reflect the highly dynamic changes in the protein. This severely restricts the practical application of protein conductivity detection. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a method for detecting the electrical conductivity of protein molecules and its application. By asymmetrically modifying the tunneling electrode pair and the protein molecule, the protein molecule can enter the tunneling region in a fixed posture and form an asymmetric single-molecule junction with the tunneling electrode pair, which can maintain the activity of the protein molecule to the greatest extent. Thus, a device that can stably detect the electrical conductivity of protein molecules in the long term is constructed. The device can be used for studying the dynamic conformation of the protein itself, screening small molecule drugs targeting the protein molecule, and analyzing the interaction between small molecule drugs and protein molecules.

[0007] The present invention utilizes tunneling electrode devices and real-time electrical measurement technology to achieve protein detection at the single-molecule level, comprehensively depicting the conformational change process of proteins such as biological enzymes and their interaction characteristics with small molecules on a high time resolution scale, which is expected to improve the efficiency and success rate of existing drug design and optimization. Subsequently, by combining the characteristics of the two "dimensions" of "protein spatial structure" and "protein dynamics", the specificity and effect of drugs can be improved, and the application prospects are broad.

[0008] On the one hand, the present invention provides a device for detecting the conductivity of protein molecules, the device comprising a tunneling electrode pair, the tunneling electrode pair comprising a first electrode, a second electrode and a nanogap formed therebetween; the tunneling electrode pair having an asymmetric modification and being connected to the protein molecule through the asymmetric modification; the asymmetric modification comprising the first electrode being modified with a first connecting element, the second electrode being modified with a second connecting element, and the first connecting element and the second connecting element being different.

[0009] Since the structure of protein molecules is asymmetric with an N-terminus and a C-terminus, each time the protein molecule enters the tunneling region, the N-terminus may face the first electrode or the C-terminus may face the second electrode. This causes the protein molecules to have different postures in the tunneling region, and the detected tunneling current will also be different.

[0010] The tunneling electrode pair is modified so that when a protein molecule enters, it can form a single-molecule junction with the tunneling electrode. However, if the first and second electrodes are modified with the same connecting element, either the N-terminus or the C-terminus of the protein molecule may connect to the first electrode, and the formed single-molecule junction is still unstable. Therefore, the tunneling electrode pair must be asymmetrically modified. Through asymmetric modification, the N-terminus of the protein must be connected to a specific first or second electrode. This can truly fix the posture of the protein molecule in the tunneling region, thereby making the detected tunneling current more stable.

[0011] The asymmetric modification described in the present invention refers to the modification of different connecting elements on the first electrode and the second electrode, so that the first electrode and the second electrode are respectively connected to the N-terminus or C-terminus of the protein molecule through specific connecting elements. For example, the first electrode is modified with a nucleic acid aptamer, which can be connected to the N-terminus of the protein molecule, and the second electrode is modified with an antibody, which can be connected to the C-terminus of the protein molecule, so that the posture of the protein molecule in the tunneling area remains fixed, and the conductivity of the protein molecule can be stably detected.

[0012] After asymmetric modification to keep the posture of the protein molecule fixed in the tunneling region, the protein molecule needs to remain active during the tunneling current detection process. If the activity of the protein molecule decreases or the protein molecule denatures, the conductivity of the protein molecule cannot be accurately detected.

[0013] Traditional protein conductivity detection typically involves directly immobilizing the protein on a gold electrode surface via gold-sulfur bonds, without the presence of a primary and secondary linker. This direct contact between the protein and gold can affect the protein's conformation, making it difficult to maintain long-term protein activity. The asymmetric modification provided by the present invention, by separately modifying the primary and secondary linkers on a tunneling electrode pair and then attaching them to the ends of the protein, can better ensure conductivity detection of the protein in its native conformation and dynamic state, thus addressing the challenges of traditional protein conductivity detection.

[0014] In some embodiments, the nanogap of the tunneling electrode is ≤20 nm.

[0015] The tunneling effect is a unique phenomenon in quantum mechanics, whereby microscopic particles (such as electrons) can penetrate energy barriers considered insurmountable in classical physics. It is a direct manifestation of the wave-particle duality of quantum mechanics. Theoretically, the smaller the nanogap of a tunneling electrode, the better, as this allows for more accurate detection of the tunneling current. However, the smaller the nanogap, the more difficult and expensive it becomes to fabricate the tunneling electrode. In some approaches, the nanogap of the tunneling electrode is preferably controlled within a range of 0.5 to 10 nm.

[0016] Furthermore, the first connecting element and the second connecting element are independently selected from any one or more of a small molecule connecting agent, a nucleic acid aptamer, and an antibody; the small molecule connecting agent, nucleic acid aptamer, and antibody can all bind to protein molecules;

[0017] The small molecule linker contains a sulfhydryl group and can be combined with a protein molecule through a biological tag;

[0018] When a small molecule linker is used as the first linking element and / or the second linking element, the protein molecule comprises a biological tag.

[0019] There are many types of linkers that can be connected to protein molecules, such as small molecule linkers, nucleic acid aptamers, antibodies, etc. In theory, any two different linkers can be selected for asymmetric modification, which can be used for conductivity detection of protein molecules, such as: 1) the first linker is a small molecule linker, and the second linker is a nucleic acid aptamer; 2) the first linker is a small molecule linker, and the second linker is an antibody; 3) the first linker is a small molecule linker, and the second linker is a nucleic acid aptamer; 4) the first linker is a nucleic acid aptamer, and the second linker is an antibody; 5) the first linker is a small molecule linker, and the second linker is another small molecule linker; 6) the first linker is an antibody, and the second linker is another antibody; 7) the first linker is a nucleic acid aptamer, and the second linker is another nucleic acid aptamer.

[0020] Furthermore, the small molecule linker includes any one or more of glutathione (GSH), biotin containing a thiol group (SH-biotin), nitrilotriacetic acid containing a thiol group (SH-NTA), heparin containing a thiol group (SH-Heparin), and chloroalkane containing a thiol group; the biological tags that can be respectively bound to glutathione, biotin containing a thiol group, nitrilotriacetic acid containing a thiol group, heparin containing a thiol group, and chloroalkane containing a thiol group are glutathione-S-transferase tag (GST Tag), monomeric streptavidin tag (MSA Tag), histidine tag (6xHis Tag), heparin binding peptide (HBP) tag and Halo tag.

[0021] Among them, glutathione can bind to the glutathione-S-transferase tag, biotin containing a thiol group can bind to the monomeric streptavidin tag, nitrilotriacetic acid containing a thiol group can bind to the polyhistidine tag, heparin containing a thiol group can bind to the heparin-binding peptide tag, and chloroalkanes containing a thiol group can bind to the Halo tag.

[0022] Therefore, by modifying the tunneling electrode with a small molecule linker and modifying the protein molecule with a biological tag that can bind to the small molecule linker, the protein molecule can be fixedly connected.

[0023] Furthermore, the first connecting element and the second connecting element are respectively selected from different small molecule linkers; the asymmetric modification also includes modification of the N-terminus and C-terminus of the protein molecule, and the N-terminus and C-terminus of the protein molecule are respectively modified with different biological tags.

[0024] Research has shown that choosing different linkers can have different effects on maintaining the native conformation and activity of protein molecules. For example, when using antibodies as linkers, after binding to protein molecules, the antibodies will occupy part of the protein molecule's pockets, which may affect the protein's dynamics. Another example is when nucleic acid aptamers bind to protein molecules, the aptamers will also occupy part of the protein molecule's small molecule binding pockets, which may limit the subsequent screening direction of small molecule drugs. Although it is possible to compensate for the breakage of the molecular junction caused by small molecule competition later, it is more cumbersome after all.

[0025] Binding proteins to the small molecule linkers of the tunneling electrode pair via biotags allows the proteins to maintain their highest dynamics and activity, as none of their native sites are occupied. In other words, modifying the tunneling electrode pair with small molecule linkers for binding to biotags modified on proteins is more advantageous for conductivity detection of proteins.

[0026] Therefore, different small molecule linkers are modified on the first and second electrodes of the tunneling electrode pair, and different biological labels are modified on the N-terminus and C-terminus of the protein molecule. The device constructed using this asymmetric modification method can maintain the activity and natural conformation of the protein molecule to the greatest extent, and can thus be used for long-term and stable detection of the conductivity of protein molecules.

[0027] In some embodiments, the first electrode is modified with glutathione, the second electrode is modified with biotin, the N-terminus of the protein molecule is modified with a biotag GST, and the C-terminus is modified with a biotag MSA.

[0028] In some methods, protein molecules and biological tags need to be connected via a linker. The present invention has screened a large number of methods to find the most suitable linker for connecting protein molecules and biological tags, so that the protein molecules are stably fixed between the tunneling electrode pairs, and the conductivity signal is not affected by the protein's swing.

[0029] In some embodiments, the linker is a long flexible amino acid chain at one end and a short rigid alpha helix at the other end, thereby stably fixing the protein molecule and making the conductivity signal more stable.

[0030] In another aspect, the present invention provides a method for preparing a device for detecting the electrical conductivity of protein molecules, the method comprising the following steps:

[0031] (1) preparing a tunneling electrode pair with asymmetric modification, wherein the asymmetric modification includes a first electrode modified with a first connecting element, a second electrode modified with a second connecting element, the first connecting element and the second connecting element both being connected to a target protein, and the first connecting element and the second connecting element being different;

[0032] (2) Placing the asymmetrically modified tunneling electrode pair in a solution containing protein molecules, so that the protein molecules are connected to the first connecting element and the second connecting element respectively.

[0033] In another aspect, the present invention provides a method for detecting the conductivity of a protein molecule, the method comprising the following steps:

[0034] (1) A device for detecting the electrical conductivity of protein molecules is prepared using the method described above;

[0035] (2) A device for detecting the conductivity of a protein molecule is placed in a solution to perform conductivity detection, and the solution can maintain the activity of the protein molecule.

[0036] In another aspect, the present invention provides a method for screening or analyzing small molecule drugs that can bind to a target protein, the method comprising the following steps:

[0037] (1) A device for detecting the conductivity of a protein molecule is prepared by the method described above, wherein the protein molecule immobilized in the device is a target protein; the device is placed in a solution that can maintain the activity of the target protein, and a first conductivity signal is obtained;

[0038] (2) adding a candidate small molecule solution or placing the device in the candidate small molecule solution to obtain a second conductivity signal;

[0039] (3) Compare and analyze the first conductance signal and the second signal to determine which small molecule drug the candidate small molecule is, or analyze the target site information of the candidate small molecule binding to the target protein.

[0040] Furthermore, in step 3), the method for extracting the characteristic change of conductivity includes: performing statistical analysis on instantaneous conductivity changes lasting less than 1 second, performing frequency distribution statistics on the conductivity, analyzing the conductivity signals for grouping and clustering and recording the frequency of the change sequence between different clusters, and detecting the regular waveform of the conductivity change.

[0041] The method for detecting the electrical conductivity of protein molecules provided by the present invention is a single-molecule detection technology that can be used for small molecule drug screening. It can reveal the ability of the target protein to interact with the small molecule drug at the single-molecule level, and can provide rich information about the structure and function of the target protein single molecule that cannot be obtained by existing methods. For example, it can track the dynamic changes of the protein, thereby realizing the design and optimization of small molecule drugs based on protein dynamics. It can detect the dynamic changes of the target protein and the entire process before and after binding with the small molecule. It is a new and groundbreaking small molecule screening method that transforms the traditional static screening method into dynamic screening, providing a potential breakthrough for the design and discovery of small molecules based on protein dynamics.

[0042] In some embodiments, the present invention constructs a platform for analyzing and screening small molecule drugs using an electrical chip based on quantum tunneling electrodes, and the construction method is as follows:

[0043] Nanoelectrode pairs with a spacing of less than 20 nm are fabricated through physical, chemical, electrochemical, or optical means. The shapes and materials of the two electrodes are such that quantum tunneling occurs between the pairs. Single molecules of target proteins (such as enzymes) with active and spatial structure are immobilized between the tunneling electrodes, creating a tunneling analysis and screening platform capable of measuring changes in the electrical conductivity of target proteins in real time on a microsecond timescale.

[0044] In some embodiments, the tunneling electrode pair can be prepared by electrochemical deposition, mechanically controlled cleavage, mechanical processing, etching, or field emission.

[0045] In some embodiments, the present invention is based on a tunneling electrode pair prepared by a nanopipette, and the method is as follows:

[0046] A multi-channel capillary is drawn into a nanopipette with a tip on one side by external force. Carbon deposition is used to precipitate and fill the drawn tip to form a carbon electrode pair. A metal wire is then inserted from the tail of the nanopipette until it is in full contact with the filled carbon. Hot melt glue is melted and condensed at the tail of the nanopipette using a glue gun to fix the relative position of the metal wire and the nanopipette. The top of the carbon electrode pair is then etched by electroetching to form a concave surface that is conducive to subsequent deposition. Finally, gold atoms are deposited on the concave surface by electrochemical deposition to form a tunneling electrode pair with a gap of less than 20 nm.

[0047] In some embodiments, the method of bridging and fixing a target protein with activity and spatial structure between a tunneling electrode pair includes: binding to a target protein with a biotag by modifying different small molecules on each electrode pair, or binding to a target protein by modifying different nucleic acid aptamers on each electrode pair, or binding to a target protein by modifying different antibodies on each electrode pair.

[0048] In some approaches, the target protein with a biotag is bound by a small molecule modified with different small molecules on each electrode pair. The modified small molecules on the electrode pair include glutathione, biotin with a thiol group, nitrilotriacetic acid with a thiol group, heparin with a thiol group, and other small molecules with thiol groups that can bind to biotags. The thiol groups on the small molecules form gold-sulfur bonds with the gold surface of the tunneling electrode pair, forming a self-assembled monolayer.

[0049] In some approaches, different small molecules are modified on the electrode pairs to bind to the biotagged target protein. Biotags, such as glutathione-S-transferase tags, monomeric streptavidin tags, polyhistidine tags, heparin-binding peptide tags, and Halo tags, are added to the N- or C-terminus of the target protein via protein fusion. These tags, which have small molecule affinity, include glutathione-S-transferase tags, monomeric streptavidin tags, polyhistidine tags, heparin-binding peptide tags, and Halo tags. The tags at the N- and C-termini bind with high affinity to the small molecule layers modified on the electrode pairs, respectively, to immobilize the target protein.

[0050] It is necessary to select a suitable linker between the target protein and the biotag, and to ensure that the target protein is stably fixed between the tunneling electrode pair and that the conductivity signal is not affected by protein fluctuations. Through extensive screening, the present invention has found the most suitable linker for connecting the target protein and the biotag, thereby improving the stability of the target protein in the tunneling region and enhancing the accuracy of the target protein d conductivity detection.

[0051] In some methods, a fusion protein with a biological tag is prepared, and the synthesis scheme is as follows:

[0052] Through gene editing, the gene fragments for the biotag and linker are inserted into the plasmid expressing the target protein. The gene fragment for the glutathione-S-transferase tag is inserted within the multiple cloning region and at the N-terminus of the target protein sequence through enzymatic digestion and homologous recombination. A gene fragment of 5-20 amino acids that cannot form a secondary structure is inserted between the GST tag gene fragment and the target protein gene fragment to serve as a flexible linker for the N-terminal biotag. A monomeric streptavidin tag (MSA tag) is inserted within the multiple cloning region and at the C-terminus of the target protein sequence through enzymatic digestion and homologous recombination. A five-amino acid fragment (EAAAK) that can specifically form an alpha-helical secondary structure is inserted between the monomeric streptavidin tag gene fragment and the target protein gene fragment to serve as a rigid linker for the C-terminal protein tag. The plasmid obtained after genetic modification is amplified and transcribed in large quantities in the engineered bacteria. The engineered bacteria expressing the fusion protein are then broken and the solution containing the target protein is extracted. The fusion protein is purified by affinity chromatography columns, ion exchange columns, size exclusion columns, and gel filtration columns.

[0053] When the target protein is labeled with GST and MSA, the tunneling electrode pair is first immersed in a solution containing biotin with a thiol group and incubated for 4-12 hours. After the biotin is completely bound to the gold surface of the tunneling electrode pair, the modification on the single-sided tunneling electrode is removed by high voltage through electrochemical means. The tunneling electrode pair is then inserted into a solution containing glutathione. At this time, the simple tunneling electrode that has shed the modification is modified with glutathione, while the other one remains modified with biotin, thereby achieving asymmetric modification.

[0054] In some embodiments, a method for analyzing and screening small molecule drugs for a target protein based on the platform includes the following steps:

[0055] S1. Place the platform in a solution that maintains the activity of the target protein. Use an ammeter to monitor the changes in the target protein's conductivity signal in real time to obtain the target protein's kinetic conductivity signal changes. Compare the conductivity signal changes with the static spatial structure obtained by the target protein's spatial structure analysis technology to obtain the target protein's structural dynamics.

[0056] S2. Add candidate small molecules to the liquid phase environment of the substance. By real-time monitoring and analyzing the regular change characteristics of the measured conductivity signal caused by the added small molecules, the molecular structure and target information of the small molecules are obtained, and further high-throughput small molecule screening acting on the target protein is achieved through the liquid mobile phase.

[0057] In some embodiments, the electronic chip based on quantum tunneling electrodes includes one or more pairs of tunneling electrodes.

[0058] In some embodiments, in step S1, there is only one target protein modified between each pair of tunneling electrodes, and the changes in the conductivity signal obtained are all caused by structural changes, surface hydrophilicity changes, protonation, deprotonation, polymerization, binding to partner proteins, binding to cofactors, and binding to small drug molecules of the target protein.

[0059] In some embodiments, in step S1, the protein spatial structure analysis technology includes X-ray diffraction technology, nuclear magnetic resonance technology, cryo-electron microscopy technology, circular dichroism spectroscopy, infrared spectroscopy, small-angle X-ray scattering, dynamic light scattering, and surface plasmon resonance.

[0060] In some embodiments, in step S2, the characteristic change in conductivity after the addition of the small molecule is analyzed, which is the real-time conductivity calculated based on the bias voltage and the real-time current. The characteristic change extraction method includes: statistically analyzing the instantaneous conductivity changes with a duration of less than 1 s, performing frequency distribution statistics on the conductivity, analyzing the conductivity signal for grouping and clustering and recording the frequency of the change order between different clusters, and detecting the regular waveform of the conductivity change.

[0061] In some embodiments, in step S2, the step of obtaining small molecule target information includes:

[0062] S2-1. Site-Targeted Identification: The tunneling electrode pair described above is placed in a solution containing a small molecule that binds to the target protein bridged to the tunneling electrode. An ammeter is used to monitor the changes in the target protein's conductivity in real time, generating characteristic signals indicating the effect of small molecules targeting different sites on the target protein's conductivity. Subsequently, small molecules targeting unknown sites are added to the solution, and the protein site targeted by the small molecule is analyzed by analyzing the characteristic signals.

[0063] S2-2. Targeted Identification of Protein Catalytic Intermediates: Analyze regular characteristic signals and identify corresponding enzyme catalytic intermediates. Add small molecules to the liquid phase of the substance. By real-time monitoring and analysis of the enzyme conductivity state selectively affected by the added small molecules, the enzyme catalytic intermediates specifically targeted by the small molecules are analyzed.

[0064] In some embodiments, in step S2, when performing high-throughput drug screening using a small molecule drug analysis and screening platform, 100 small molecules are first selected from a small molecule compound library as a group, and each small molecule is mixed into a small molecule mixed solution at a final concentration of 2.5 uM to obtain multiple groups of small molecule mixed solutions. The small molecule mixed solutions are injected into a liquid flow cell by liquid mobile phase grouping, and each is incubated with a tunneling analysis and screening platform for 10 minutes. By observing and analyzing the changes in the conductivity signal of the target protein, a small molecule mixed solution that acts on a specific site or a specific structural intermediate state of the target protein is screened. The mixed solution is further divided into groups of 10, and the above operation is repeated. Finally, it is subdivided into groups of 1, and the above operation is repeated to obtain a specific small molecule that acts on a specific site or a specific structural intermediate state of the target protein.

[0065] In some embodiments, in step S2, the small molecule drugs subjected to high-throughput screening include small molecules that bind to known or unknown characteristic sites of the target protein, and small molecules that bind to known or unknown specific structural intermediates of the target protein.

[0066] In some embodiments, in step S2, when performing high-throughput screening, the tunneling analysis screening platform is placed in a liquid mobile phase, including a quartz capillary, a quartz microfluidic, and a quartz liquid flow cell.

[0067] In another aspect, the present invention provides a method for detecting the affinity between a small molecule and a target protein, the method comprising the following steps:

[0068] (1) A device for detecting the conductivity of a protein molecule is prepared by the method described above, wherein the protein molecule immobilized in the device is a target protein; the device is placed in a solution that can maintain the activity of the target protein, and a first conductivity signal is obtained;

[0069] (2) adding a candidate small molecule solution or placing the device in the candidate small molecule solution to obtain a second conductivity signal;

[0070] (3) Compare and analyze the first conductivity signal and the second signal.

[0071] On the other hand, the present invention provides a device for screening and sorting small molecule drugs based on tunneling electrodes, the device comprising a tunneling electrode, a characteristic signal acquisition module, a conductance characteristic acquisition module and a data analysis module; the characteristic signal acquisition module is used to obtain a first conductance signal of the tunneling electrode pair in a solution that can maintain the activity of the target protein; the conductance characteristic acquisition module is used to obtain a second conductance signal of the tunneling electrode pair in a solution containing a candidate compound; and the data analysis module is used to compare and analyze the first signal and the second signal.

[0072] The input for analysis in this device is the change in the target protein's conductivity before and after the addition of a candidate small molecule. Features that can be categorized as characteristics include: uniform and frequent conductivity peaks, sustained current increases or decreases, sustained and regular changes in conductivity states, and interference with the target protein's original conductivity without changes. Based on these input features, the outputs include the small molecule's target, characteristic intermediate states of action, function, or binding affinity.

[0073] In some embodiments, an output module is further included.

[0074] In another aspect, the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein the program implements the method described above when executed by a processor.

[0075] In another aspect, the present invention provides a terminal, comprising: a processor and a memory, wherein the memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory, so that the device executes the method described above.

[0076] In another aspect, the present invention provides an application of the above-described device in constructing a small molecule drug screening platform, wherein the application includes any one or more of the following:

[0077] (1) Dynamic molecular conformation study of target proteins;

[0078] (2) Screening small molecules that can bind to target proteins;

[0079] (3) Identify the binding site between small molecules and target proteins;

[0080] (4) Detecting the effect of small molecule binding to target protein on the kinetic characteristics of target protein;

[0081] (5) Detecting the effect of small molecule binding to target protein on target protein activity;

[0082] (6) Detect the binding or dissociation constant of small molecules with target proteins.

[0083] The present invention has the following beneficial effects:

[0084] 1. Utilize tunneling electrode devices and real-time electrical measurement technology to achieve protein detection at the single-molecule level, and stably detect the tunneling current of protein molecules for a long time (up to 6 hours or more).

[0085] 2. Asymmetric modification is used to keep the posture of the protein molecule fixed in the tunneling region, thereby more stably detecting the conductivity of the protein molecule.

[0086] 3. Select a modification method that combines a small molecule linker with a biological tag to keep the protein molecule at its most dynamic and active state. No natural sites on the protein molecule are occupied, thus maintaining the activity and natural conformation of the protein molecule to the greatest extent. This can be used for long-term and stable detection of the conductivity of the protein molecule and is more conducive to the screening of small molecule drugs.

[0087] 4. The constructed device has a wide range of applications and can comprehensively depict the conformational change process of proteins such as biological enzymes on a high time resolution scale. It can also be used to construct a small molecule drug screening platform that binds to target proteins, with higher efficiency and success rate in small molecule drug design and optimization. By combining the characteristics of the two "dimensions" of "protein spatial structure" and "protein dynamics", the specificity and effect of screening small molecule drugs can be further improved. It can also be used to analyze the interaction characteristics between target proteins and small molecules, including identifying the binding sites of small molecules and target proteins, detecting the effect of the binding of small molecules to target proteins on the activity of target proteins, detecting the affinity of small molecules to target proteins, obtaining binding or dissociation constants, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is a scanning electron microscope image of the first electrode and the second electrode of the tunneling electrode in Example 1;

[0089] Figure 2 is the current signal mutation (It) occurring during the binding process between the protein molecule and the target protein on the tunneling electrode in Example 2;

[0090] Figure 3 Schematic diagram of the small molecule drug analysis and screening platform based on the quantum tunneling electrode electrical chip and its application in Example 3;

[0091] Figure 4 The characteristic signal is obtained when the WIN and WBM inhibitors in Example 3 bind to the target protein on the tunneling electrode;

[0092] Figure 5 is the cluster diagram of enzyme catalytic conductance in Example 3;

[0093] Figure 6 This is the detection result of asymmetric modification in Example 4;

[0094] Figure 7 This is the test result of the symmetrical modification in Example 4;

[0095] Figure 8 This is the effect of different connecting elements on the conductivity detection of protein molecules in Example 5. DETAILED DESCRIPTION

[0096] To describe the present invention in more detail, the technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are merely intended to illustrate how the present invention is implemented and are not intended to limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0097] Example 1. Preparation of tunneling electrode

[0098] The method for preparing the tunneling electrode provided in this embodiment is as follows:

[0099] 1) Drawing nanopore double-pore tubes

[0100] Specifically: the raw material capillary is a double-bore tube made of quartz material with an outer diameter of 1.2 mm, an inner diameter of 0.9 mm, and a length of 100 mm.

[0101] First, the quartz double-bore capillary was cleaned by rinsing the inner and outer surfaces with 18.2MΩ•cm ultrapure water, then placed in a plasma cleaner for 30 minutes to remove surface debris. After cleaning, the quartz double-bore capillary was drawn using a P-2000 laser drawing instrument. A CO2 laser beam was applied to the middle of the quartz double-bore capillary, causing the middle of the capillary to melt and form an hourglass-shaped structure. SEM characterization of the double holes at the tip of the hourglass showed a pore size of less than 100nm. The parameters used were:

[0102] Line1: HEAT=820, FIL=4, VEL=30, DEL=160, PUL=100;

[0103] Line2: HEAT=830, FIL=3, VEL=20, DEL=140, PUL=170

[0104] 2) High-temperature carbon deposition at the tip of the double-hole tube

[0105] Specifically, the hourglass-shaped part of the nanopore double-hole tube is placed in a protective quartz tube, and 0.2m 3 / min argon, 0.2m 3 / min butane. Butane is added to a double-bore tube at high temperature, depositing it at the tip of the hourglass portion of the tube, forming a nanopore carbon nanoelectrode at the very tip. A metal wire is inserted at the end of the double-bore tube to connect the carbon nanoelectrode and secure it.

[0106] 3) Formation of gold tunneling electrode pairs by electrochemistry

[0107] Specifically, the carbon nanoelectrodes were inserted into a 0.1M KOH, 0.1M KCl etching solution, with two carbon electrodes as working electrodes, and Ag / AgCl and platinum wires as reference electrodes and counter electrodes, respectively. A CHI760C potentiostat was used for cyclic scanning, with an initial potential of 0V, a final potential of 2V, a scanning speed of 0.1V / s, and 4-6 scans.

[0108] Using a constant potential instrument, the carbon nanoelectrode pair was immersed in undiluted ECF64D electroplating solution (4.4mMNH4AuSO3, 52mM (NH4)2SO3), with silver wire as the reference and counter electrode, and the carbon electrode pair as the two working electrodes. The working potentials were set to -730mV and -750mV respectively. Pre-electroplating was performed for 60s, and gold was deposited on the carbon nanoelectrode to form a gold electrode pair.

[0109] Using a potentiostat, set a gold electrode pair as the working electrode and reference / counter electrode in the same plating solution, preset a constant current of 2nA for feedback until a tunneling current is observed between the electrode pair, and stop the feedback.

[0110] The prepared gold tunneling electrode pair was stored in 18.2MΩ·cm ultrapure water. The scanning electron microscope image of the obtained tunneling electrode pair is shown in the figure below. Figure 1 shown.

[0111] Example 2: Construction of a device for detecting the electrical conductivity of protein molecules

[0112] In this example, WDR5 protein (WD repeat domain protein 5) was used as a protein molecular element to construct a device for detecting the electrical conductivity of WDR5 protein molecules. The specific method is as follows:

[0113] 1. Preparation of GST-WDR5-MSA plasmid by gene editing

[0114] Using the Pgex-4T-1 plasmid (SEQ ID NO. 1) as a blank vector, the target fragment WDR5-Linker-MSA-taa was inserted into the MCS region following the GST-thrombin site (the Pgex-4T-1 plasmid comes with GST and a long flexible linker (SEQ ID NO. 3)) to generate a recombinant plasmid. The first 22 unnecessary amino acids of WDR5 were truncated, and only the 23-334 fragment (SEQ ID NO. 2) was inserted. The C-terminal linker fragment was selected to form a rigid alpha-helical linker upon expression. The linker sequence is shown below: EAAAK (SEQ ID NO. 4). The MSA fragment (SEQ ID NO. 5) represents the engineered streptavidin gene sequence. Finally, the taa fragment was added at the end to terminate expression.

[0115] The nucleotide sequence of the constructed GST-WDR5-MSA recombinant plasmid is shown in SEQ ID No.6.

[0116] 2. Fusion protein expression

[0117] The recombinant plasmid in 1) was transformed into competent E. coli Transetta (DE3) cells, and IPEG was added for low-temperature induced expression.

[0118] After obtaining the protein expression lysate, it was purified by GSH affinity chromatography, ion exchange column and molecular sieve coacervation chromatography to obtain the purified GST-WDR5-MSA fusion protein, the amino acid sequence of which is shown in SEQ ID No. 7. The N-terminus of the fusion protein contains the biological tag GST, and the C-terminus contains the biological tag MSA.

[0119] 3. Asymmetric modification of tunneling electrode pairs

[0120] 1) Immerse the tunneling electrode pair in a 0.2 mM solution of biotin with a thiol group (Biotin-PEG-SH purchased from Aladdin, model B163355) and incubate for 12 hours. The thiol groups will self-assemble with the gold surfaces of the two electrodes to form gold-sulfur bonds, thereby modifying the biotin to immobilize small molecules.

[0121] 2) Using a potentiostat, immerse the tunneling electrode pair in a 0.1 M KOH solution. Use the first electrode as the working electrode and insert an AgCl / Ag wire as the reference and counter electrodes. Set the constant voltage to -1.1 V and apply a constant bias for 60 seconds to break the gold-sulfur bonds on the gold surface of the first electrode.

[0122] 3) The gold tunneling electrode that was electrochemically interrupted in step 2) and only has one end modified with biotin, while the other end is blank, is immersed in a 0.2 mM reduced glutathione solution and incubated for 4 hours, so that the first electrode is modified with reduced glutathione and the second electrode is still modified with biotin.

[0123] 4. Assembling protein molecules

[0124] 1) Dilute the GST-WDR5-MSA fusion protein in 1× PBS buffer to obtain a final concentration of 1 μM GST-WDR5-MSA protein dilution solution.

[0125] 2) Insert the asymmetrically modified tunneling electrode pair and the reference electrode (AgCl / Ag) into a dilution of the GST-WDR5-MSA protein. Connect the glutathione-modified first electrode (GSH-modified gold electrode) to the AgCl / Ag. Apply a bias voltage cycling between -300mV and +300mV, and observe the current changes in real time. When a current jump is observed, rinse the tunneling electrode pair with 1× PBS buffer. If the current remains at the same level after the jump, the protein molecule has been attached to the second electrode of the tunneling electrode, and protein assembly is complete.

[0126] The steps of assembling protein molecules and tunneling electrodes and the corresponding current changes are as follows: Figure 2 As shown in the figure, 1 is the first electrode, 2 is the second electrode, 3 is the first connector, 4 is the second connector, 5 is the protein molecule, 6 is the biotag bound to the first connector, and 7 is the biotag bound to the second connector. When the protein molecule is connected to a single electrode at one end, it switches back and forth between two conductance states at high frequency. When connected to two electrodes at both ends, it exhibits a stable high conductance state.

[0127] Example 3: Construction of a small molecule drug screening platform binding to target proteins and its application

[0128] The device for detecting the electrical conductivity of protein molecules constructed in accordance with the method provided in Example 2 in this embodiment can be used as a screening probe for candidate small molecule drugs, thereby constructing a small molecule drug screening platform that can bind to target proteins. It can be directly used for dynamic molecular conformation research of target proteins, screening of small molecule drugs, analysis of binding sites of small molecule drugs and target proteins, the effect of binding of small molecule drugs to target proteins on target protein activity, and detection of affinity, binding or dissociation constants of small molecules and target proteins. The small molecule drug analysis and screening platform based on quantum tunneling electrode electrical chips provided in this embodiment and its applications are as follows: Figure 3 shown.

[0129] 1. Small molecule site analysis

[0130] 1.1. Recording site characteristic signals

[0131] The tunneling electrode pair assembled with the target protein WDR5 protein was placed in a liquid mobile phase containing 1×PBS buffer solution, and the protein conductance under a bias voltage of 100 mV was recorded.

[0132] A 1× PBS solution containing 2.5 μM of the WBM site inhibitor SADS-1 was injected into the mobile phase. The characteristic current changes after the WBM site inhibitor bound to the target protein were observed and recorded. The structural formula of the WBM site inhibitor SADS-1 is as follows:

[0133]

[0134] After recording, 1× PBS buffer solution was injected into the liquid mobile phase for flushing, and cyclic voltammetry was used to switch the bias between -300 mV and +300 mV to allow SADS-1 to fall off the target protein.

[0135] The mobile phase was injected with 2.5 μM WIN site inhibitor OICR-9429 (molecular formula: C 29 H 32 F3N5O3, CAS number: 1801787-56-3) in 1× PBS solution, and observe and record the characteristic current changes after the WIN site inhibitor binds to the protein element.

[0136] 1.2 Analysis of small molecule inhibitors of unknown sites

[0137] The tunneling electrode pair assembled with the target protein was placed in a liquid mobile phase of 1× PBS buffer solution containing 2.5 μM of an unknown small molecule. The current change at 100 mV was recorded for 10 minutes and compared with the characteristic current signals of the measured WBM inhibitor and WIN inhibitor to analyze the target site of the unknown small molecule.

[0138] 1.3. Screening for WBM or WIN site-specific inhibitors

[0139] The small molecule compounds to be screened were mixed in 1×PBS buffer solution, with 50 small molecule compounds as a group. The concentration of a single small molecule in the group was 250nM. The small molecule mixtures were injected into the liquid mobile phase in groups. A 100mV bias was applied to the electrode pair and the current was recorded for 10 minutes. If the current did not change, the next group of small molecule mixtures was injected. If the current changed, it was compared with the characteristic signal of WIN or WBM.

[0140] The group of 50 small molecule compounds was further subdivided into 10 small molecule compounds as a group, with a single small molecule concentration of 1 μM, and the above steps were repeated.

[0141] Finally, the 10 small molecules in the modified group were tested individually, and the above steps were repeated to screen for WBM or WIN site-specific inhibitors.

[0142] The obtained WIN inhibitors and WBM inhibitors produced different characteristic signals after binding to the protein element WDR5, such as Figure 4 As shown, it can be used for site differentiation.

[0143] 2. Protein dynamics specificity analysis

[0144] The device for detecting the conductivity of protein molecules was prepared using Example 2. The WDR5 (23-334) gene fragment to be expressed was replaced with the phi29 DNA polymerase gene fragment (1-575) (SEQ ID No. 8). A small molecule drug screening platform was constructed, and protein kinetic specificity analysis was performed.

[0145] 2.1. Recording the current pattern during phi29 DNA polymerase catalysis

[0146] The tunneling electrode pair assembled with protein elements was placed in a liquid mobile phase containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM (NH4)2S04, 4 mM DTT, and 100 μM dNTPs (the solution required for enzyme translation). The temperature was maintained in the range of 30°C to 42°C. Single-stranded template DNA and primers were additionally added. The current changes under bias voltages of +300mV, +200mV, +100mV, -100mV, -200mV, and -300mV were recorded for 10 minutes. The changes in current characteristics when the DNA polymerase continued to catalyze under different bias voltages were analyzed. The current characteristics were classified by clustering to reflect the catalytic stages of DNA polymerase, including primer binding, initiation synthesis, strand displacement, continuous synthesis, proofreading, and termination.

[0147] 2.2 Analysis of protein kinetic specificity of unknown small molecules

[0148] Apply a 100mV bias and add 2.5μM of a small molecule with unknown protein kinetics through the liquid mobile phase. Observe the current change to identify the DNA polymerase catalytic phase where the characteristic signal of small molecule binding occurs. Rinse the tunneling electrode pair with a solution containing 50mM Tris-HCl pH 7.5, 10mM MgCl2, 10mM (NH4)2SO4, 4mM DTT, and 100μM dNTPs, which are required for enzyme translation. Apply a cyclic bias from -300mV to +300mV to allow the small molecule to dissociate from the protein element. Repeat the experiment by adding 2.5μM of the small molecule again to eliminate chance.

[0149] 2.3. Screening for protein dynamics-specific inhibitors

[0150] The small molecule compounds to be screened were mixed in a solution required for enzyme translation (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM (NH4)2S04, 4 mM DTT, and 100 μM dNTPs). The small molecule mixtures were grouped into 50 groups, with the concentration of each small molecule within the group being 250 nM. The small molecule mixtures were injected into the liquid mobile phase in groups. A 100 mV bias was applied to the electrode pair and the current was recorded for 10 minutes. If no characteristic signal change in small molecule binding was observed, the next group of small molecule mixtures was injected. If a change in current was observed, the group of 50 small molecules was further subdivided into groups of 10 compounds, with the concentration of each small molecule being 1 μM, and the above steps were repeated. Finally, the 10 small molecules in the group that showed changes were tested individually, and the above steps were repeated.

[0151] The DNA enzyme catalytic stage acted upon by a small molecule that results in a characteristic signal is recorded and repeated to ensure specificity of the kinetics within the protein.

[0152] The conductivity state changes of the obtained phi29 protein measured as a protein element related to its enzymatic activity biological process correspond to the primer binding, synthesis, proofreading, and termination in its biological process, such as Figure 5 shown.

[0153] 3. Analyze the inhibitory characteristics of small molecules on enzyme catalysis

[0154] 3.1 Preparation of protein components

[0155] The device for detecting the conductivity of protein molecules was prepared using Example 2. The WDR5 (23-334) gene fragment to be expressed was replaced with the Sir2 enzyme gene fragment (1-389) (SEQ ID No. 9). A small molecule drug screening platform was constructed, and the inhibitory characteristics of small molecules on the enzyme catalysis process were analyzed.

[0156] 3.1. Recording the current pattern during Sir2 enzyme catalysis

[0157] The tunneling electrode pair, assembled with the protein component, was placed in a mobile phase containing 50 mM Tris-HCl (pH 8.0) and 5 mM NAD+ buffer, maintained at a temperature between 30°C and 37°C. The current was recorded for 10 minutes at bias voltages of +300mV, +200mV, +100mV, -100mV, -200mV, and -300mV. This also records the conductance of the Sir2 enzyme in the non-catalytic state. The substrate peptide KKGQSTSRHKK(Ac)LMFKTEG was then added, and the current was recorded for 10 minutes at bias voltages of +300mV, +200mV, +100mV, -100mV, -200mV, and -300mV. This analysis analyzed the current characteristics during substrate deacetylation catalysis under different bias voltages. The chronological order corresponds to the three sequential steps of deacetylation: substrate binding, covalent intermediate formation, and deacetylation product release.

[0158] 3.3.2 Analysis of the enzyme catalytic inhibitory effect of unknown small molecules and the stage of inhibition

[0159] A 100mV bias voltage was applied, and 2.5μM Sir2 enzyme inhibitory small molecules were added through the liquid mobile phase. The current changes were observed to identify the conductance stage where the Sir2 enzyme conductance mutated and stagnated. Whether the conductance stagnation could be restored was observed to analyze the enzyme catalytic stage at which the inhibitor small molecule bound and inhibited, as well as its category (non-covalent inhibitor or covalent inhibitor).

[0160] 4. Analyze the binding Kd of small molecules

[0161] A device for detecting the conductivity of protein molecules constructed according to the method provided in Example 2 was used to construct a small molecule drug screening platform, and the binding Kd analysis of small molecules and target proteins was performed.

[0162] 4.1. Recording the Conductance of the Dynamic Equilibrium State of Inhibitor Binding and Debinding from Protein Elements

[0163] The tunneling electrode pair assembled with protein elements was placed in a liquid mobile phase containing 1×PBS buffer solution, and the protein conductance under a bias of 100 mV was recorded.

[0164] A 1×PBS solution containing a very low concentration of 10 nM WIN site inhibitor OICR-9429 was injected into the liquid mobile phase, and the characteristic current changes of OICR-9429 in the dynamic equilibrium state of binding to and detaching from the protein element were observed and recorded.

[0165] 4.2. Extracting the binding time (τ_on) and non-binding time (τ_off) of inhibitor molecules

[0166] Recording began with the first OICR-9429-induced current dip. A threshold was set to distinguish the current signal. The binding time (τ_on) was recorded from the start of each current dip to the return to baseline. The non-binding time (τ_off) was recorded from the return to baseline to the start of each current dip. Current was recorded continuously for 10 minutes at a bias of 100 mV to allow for sufficient τ_on and τ_off measurements for subsequent statistical analysis.

[0167] 4.3. Calculation of Kd of Small Molecule Inhibitors

[0168] The Kon (association rate constant) and Koff (dissociation rate constant) of small molecule-protein binding were calculated using the following formulas.

[0169] Koff = 1 / τ_on

[0170] Kon = 1 / (τ_off × [L])

[0171] where [L] is the concentration of free small molecules (unit: M).

[0172] After obtaining Kon and Koff, calculate the Kd of the inhibitor small molecule relative to the target protein, where Kd=Kon / Koff.

[0173] Example 4: Effect of asymmetric modification

[0174] This example uses the method provided in Example 2 to construct a device for detecting the electrical conductivity of protein molecules. The tunneling electrode is modified using the following methods: 1. Asymmetric modification (Example 2); 2. Symmetric modification. Biotin is modified at both ends of the tunneling electrode, and GST is modified at both ends of the WDR5 molecule. The nucleotide sequence of the constructed GST-WDR5-GST recombinant plasmid is shown in SEQ ID No. 10, and the amino acid sequence of the GST-WDR5-GST fusion protein is shown in SEQ ID No. 11. Two devices were constructed, each of which was used to detect the electrical conductivity of protein molecules. A WBM inhibitor (WDR5-IN-6, molecular formula: C 13 H8Cl2N2O2S, CAS No.: 326901-92-2, the concentration used was 20 μM saturation concentration). The test results of the two devices are as follows. Figure 6 and Figure 7 As shown, Figure 6 is the detection result of asymmetric modification, Figure 7 This is the detection result for symmetric modification.

[0175] The ▲|molecular junction refers to the increased current when the target protein is connected to the tunneling electrode, that is, the tunneling current flowing through the target protein. This current = the molecular junction current flowing through the protein |molecular junction + the basic tunneling current |0. The detected protein molecular signal is correlated with |molecular junction and has little correlation with |0.

[0176] according to Figure 7 As can be seen, the second symmetrical modification allows for the detection of two different conductance signals, one at the ▲| molecular junction and the other at the 3 / 5▲| molecular junction. However, the first asymmetric modification yields only one stable conductance signal, corresponding to the ▲| molecular junction. This suggests that asymmetric modification results in more stable signals for the detected protein molecules.

[0177] Example 5: Effects of different linker elements on protein molecule activity

[0178] 1. Differences between different connection elements

[0179] This example first employed symmetrical modification using different linking elements to investigate the effects of different linking methods on protein tunneling signal detection. The first linking element was a small molecule ligand-protein tag, with biotin modified at both ends of the tunneling electrode (Type 2 in Example 4); the second linking element was a DNA aptamer, with a nucleic acid aptamer (SEQ ID No. 12) modified at both ends of the tunneling electrode; and the third linking element was a polyclonal antibody, with WDR5 antibody (purchased from HUABIO, R1512-17) modified at both ends of the tunneling electrode. The three devices were examined for WDR5 attachment ease, immobilization uniformity (detected by determining the reproducibility of the characteristic current signal generated after the addition of the small molecule based on the detection signal), and protein flexibility (detected by observing the electrical signal to determine whether the formed protein molecular junction still functioned properly). For example, during catalysis, enzymes undergo conformational changes. A more flexible linking method allows the protein to undergo acceptable conformational changes while forming the molecular junction, resulting in different electrical signals without hindrance. If hindrance is present, the electrical signals at different stages will be inconsistent. The difference between them is not obvious enough and confusion occurs), the original conductance refers to the unprocessed electrical signal with time as the horizontal axis and current as the vertical axis, which is used to obtain the frequency distribution data. Frequency distribution means: the given original conductance is composed of countless points, and one point is collected every 2 microseconds, corresponding to the current intensity at that time. Here, all points within 100ms are statistically analyzed and grouped according to current intensity to show the concentration and discrete trends of the data. The clearer the frequency distribution is, the more it can distinguish the current signals of different peaks, and the better the flexibility of the protein. The more confused the different peaks are, the worse the flexibility of the protein. The test results are as follows Figure 8 shown.

[0180] according to Figure 8 It can be seen that compared with DNA aptamers and polyclonal antibodies, the small molecule ligand-protein tag connection method has obvious advantages. It is easier to connect, has better fixation uniformity, and has higher protein flexibility. Therefore, the small molecule ligand-protein tag connection method is preferred for conductivity detection of protein molecules.

[0181] 2. Effects of different small molecule linkers

[0182] This example uses the method provided in Example 2 to construct a device for detecting the conductivity of protein molecules, wherein asymmetric modification is used for the modification of the tunneling electrode, but different types of linking elements are used, specifically: 1. The first linking element is glutathione, the N-terminus of WDR5 contains the biotag GST, and the second linking element is a nucleic acid aptamer (sequence No. 12); 2. The first linking element is glutathione, the N-terminus of WDR5 contains the biotag GST, and the second linking element is a WDR5 polyclonal antibody (purchased from HUABIO, R1512-17); 3. The first linking element is glutathione, the second linking element is biotin, the N-terminus of WDR5 contains the biotag GST, and the C-terminus contains the biotag MSA; 4. The first linking element is nitrilotriacetic acid, the second linking element is biotin, the N-terminus of WDR5 contains a histidine tag, and the C-terminus contains the biotag MSA; 5. The first linking element is heparin with a thiol group, the second linking element is biotin, the N-terminus of WDR5 contains a heparin-binding peptide tag, and the C-terminus contains the biotag MSA. The activity of WDR5 in the three devices was examined, as well as the maximum duration and stability that it could maintain for conductivity detection. The protein conformation detection method is as follows: when the target protein is connected between the tunneling electrodes, a jump in the molecular junction conductance is formed. This conductance is a fixed value in a single experiment. If the protein conformation is destroyed, the molecular junction conductance will decrease or even disappear, thereby judging whether the protein conformation is stable. The biological activity detection method is to add a small molecule that can bind to the WBM site (WDR5-0103, molecular formula: C 21 H 25 N₃O₄ (CAS number: 890190-22-4, saturation concentration of 20 μM) was used to observe the appearance of characteristic peaks and the uniformity of the characteristic peak signals. The maximum detection time was calculated based on the time it took for the characteristic peak to stably appear. The stability test method was to verify whether multiple different conductance signals appeared during the test. The WDR5 protein has a rigid molecular structure, and the conductance signal should remain stable after the molecular junction is formed. The presence of different conductance signals indicates unstable binding and Brownian motion. The uniformity of the conductance signal was used for analysis. The test results are shown in Table 1.

[0183] Table 1. Effects of different linker elements on protein activity

[0184]

[0185] Table 1 shows that asymmetric modification using different linkers has different effects on maintaining the native conformation and activity of protein molecules. A comparison of methods 1 to 3 shows that when antibodies or aptamers are used as linkers, protein activity decreases, the maximum detection time shortens, and stability decreases. This may be due to the direct binding of antibodies or aptamers to the protein molecules, which may have a certain impact on the protein itself and make it difficult to maintain the native conformation of the protein molecules. The antibodies or aptamers also occupy some small molecule binding pockets on the protein molecules. When small molecule linkers are used, since the protein molecules are connected to the small molecule linker via a biotag, the protein molecules do not change before and after the connection, thus maintaining the highest dynamics and activity. Moreover, no native sites on the protein molecules are occupied. This connection method is more favorable for conductivity detection of protein molecules, with the longest detection time reaching over 6 hours and the highest stability.

[0186] At the same time, it can be seen from Examples 3 to 5 of the comparative documents that different small molecule linkers have different effects on protein activity. This may be because some small molecule linkers have nonspecific binding and a high probability of binding to impurities in the solution, affecting the proper immobilization of the target protein. Furthermore, small molecules with complex structures and high molecular weights, such as heparin, are not conducive to electron transfer from the electrode to the protein interior. Therefore, the most preferred modification method is Example 3 (Example 2), which can maximize the maintenance of protein activity, allowing for long-term and stable detection of protein conductivity and more conducive to the construction of a small molecule drug screening platform.

[0187] Example 6: Linker Screening

[0188] In this example, the GST-WDR5-MSA plasmid was prepared according to the method provided in Example 1. Three linker sequences were used, as shown in Table 2. SEQ ID No. 3 represents a single-ended, long, flexible amino acid chain, and SEQ ID No. 4 represents a short, rigid alpha helix. The effects of different linker types on the fabrication of devices for detecting protein molecular conductance were investigated, as was the stability of the conductance signals in the three devices.

[0189] The results are shown in Table 2.

[0190] Table 2. Effects of different linkers on protein activity

[0191]

[0192] As can be seen in Table 2, the detection systems prepared using the first or second linker have very stable conductivity signals when detecting the conductivity of protein small molecules. However, in actual operation, the first linker connection method is easier to implement because under the test conditions, the surface potential of the single-end electrode with GSH modified on the surface is controlled by AgCl. When a +100mV bias is applied to the single-end electrode modified with Biotin on the other end, the target protein using the first linker method is more likely to form a molecular junction; while the second linker method does not show obvious surface potential preference. Therefore, the first linker is the most preferred, and when the third or fourth linker is used, the conductivity signal is prone to obvious oscillation.

[0193] The first or second linker types have a long, flexible amino acid chain at one end and a short, rigid alpha helix at the other. This type of linker allows the target protein to be stably captured between the electrode pair. The third type of linker, however, has long, flexible amino acid chains at both ends. This may result in the protein being unable to be stably fixed between the electrode pair, resulting in significant oscillations in the conductivity signal. The fourth type of linker has a short, rigid structure at both ends. This may prevent both ends from binding to the electrodes due to the lack of flexibility of the connecting portion, making it difficult to achieve a stable molecular junction. Therefore, the most preferred linker is one with a long, flexible amino acid chain at one end and a short, rigid alpha helix at the other end, such as the first type. This structure provides a certain degree of flexibility, facilitates molecular junction formation, and ensures signal stability after the molecular junction is formed. However, it is understandable that there are many different sequences of linkers with short, rigid alpha helices or long, flexible amino acid chains. As long as one end is a long, flexible amino acid chain and the other end is a short, rigid alpha helix, the stability of the protein molecular conductivity signal can basically be achieved.

[0194] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A device for detecting the electrical conductivity of protein molecules, characterized in that: The invention comprises a tunneling electrode pair, wherein the tunneling electrode pair comprises a first electrode, a second electrode and a nanogap formed therebetween; the tunneling electrode pair has an asymmetric modification and is connected to the protein molecule through the asymmetric modification; the asymmetric modification comprises that the first electrode is modified with a first connecting element, the second electrode is modified with a second connecting element, and the first connecting element and the second connecting element are different.

2. The device according to claim 1, wherein The first connecting element and the second connecting element are independently selected from any one or more of a small molecule connecting agent, a nucleic acid aptamer, and an antibody; the small molecule connecting agent, nucleic acid aptamer, and antibody can all bind to protein molecules; The small molecule linker contains a sulfhydryl group and can be combined with a protein molecule through a biological tag; When a small molecule linker is used as the first linking element and / or the second linking element, the protein molecule comprises a biological tag.

3. The device according to claim 2, wherein The small molecule linker includes any one or more of glutathione, biotin containing a thiol group, nitrilotriacetic acid containing a thiol group, heparin containing a thiol group, and chloroalkane containing a thiol group; the biological tags that can be respectively combined with glutathione, biotin containing a thiol group, nitrilotriacetic acid containing a thiol group, heparin containing a thiol group, and chloroalkane containing a thiol group are glutathione-S-transferase tags, monomeric streptavidin tags, histidine tags, heparin-binding peptide tags, and Halo tags.

4. The device according to claim 3, characterized in that The first connecting element and the second connecting element are respectively selected from different small molecule linkers; the asymmetric modification also includes modification of the N-terminus and C-terminus of the protein molecule, and the N-terminus and C-terminus of the protein molecule are respectively modified with different biological tags.

5. A method for preparing a device for detecting the conductivity of protein molecules, characterized in that: The following steps are involved: (1) preparing a tunneling electrode pair with asymmetric modification, wherein the asymmetric modification includes a first electrode modified with a first connecting element, a second electrode modified with a second connecting element, the first connecting element and the second connecting element both being connected to a target protein, and the first connecting element and the second connecting element being different; (2) Placing the asymmetrically modified tunneling electrode pair in a solution containing protein molecules, so that the protein molecules are connected to the first connecting element and the second connecting element respectively.

6. A method for detecting the conductivity of protein molecules, characterized in that: The following steps are involved: (1) A device for detecting the conductivity of protein molecules is prepared by the method according to claim 5; (2) A device for detecting the conductivity of a protein molecule is placed in a solution to perform conductivity detection based on the tunneling effect, and the solution can maintain the activity of the protein molecule.

7. A method for screening or analyzing small molecule drugs that can bind to a target protein, characterized in that: The steps include: (1) A device for detecting the conductivity of a protein molecule is prepared by the method according to claim 5, wherein the protein molecule immobilized in the device is a target protein; the device is placed in a solution that can maintain the activity of the target protein, and a first conductivity signal is obtained; (2) adding a candidate small molecule solution or placing the device in the candidate small molecule solution to obtain a second conductivity signal; (3) Compare and analyze the first conductance signal and the second signal to determine which small molecule drug the candidate small molecule is, or analyze the target site information of the candidate small molecule binding to the target protein.

8. The screening or analysis method according to claim 7, wherein In step 3), the method for extracting the characteristic change of conductivity includes: performing statistical analysis on instantaneous conductivity changes lasting less than 1 second, performing frequency distribution statistics on the conductivity, analyzing the conductivity signals for grouping and clustering, recording the frequency of the change sequence between different clusters, and detecting the regular waveform of the conductivity change.

9. A method for detecting the affinity between a small molecule and a target protein, characterized in that: The steps include: (1) A device for detecting the conductivity of a protein molecule is prepared by the method according to claim 5, wherein the protein molecule immobilized in the device is a target protein; the device is placed in a solution that can maintain the activity of the target protein, and a first conductivity signal is obtained; (2) adding a candidate small molecule solution or placing the device in the candidate small molecule solution to obtain a second conductivity signal; (3) Compare and analyze the first conductivity signal and the second signal.

10. Use of the device according to any one of claims 1 to 4 in constructing a small molecule drug screening platform, characterized in that: The application includes any one or more of the following: (1) Dynamic molecular conformation study of target proteins; (2) Screening small molecules that can bind to target proteins; (3) Identify the binding site between small molecules and target proteins; (4) Detecting the effect of small molecule binding to target protein on the kinetic characteristics of target protein; (5) Detecting the effect of small molecule binding to target protein on target protein activity; (6) Detect the binding or dissociation constant of small molecules with target proteins.

Images