Surface potential sensor taking allosteric transcription factor as recognition molecule as well as preparation method and application of surface potential sensor

Abstract

The invention discloses a surface potential sensor taking an allosteric transcription factor as a recognition molecule as well as a preparation method and application of the surface potential sensor, and belongs to the technical field of surface potential detection. The surface potential sensor comprises an electrode, desoxyribonucleic acid and an allosteric transcription factor, the electrode is sequentially connected with desoxyribonucleic acid and an allosteric transcription factor; the number of base pairs in the nucleotide sequence of the deoxyribonucleic acid is more than 30. An allosteric transcription factor is used as a recognition molecule, so that the type of the recognition molecule can be changed according to the change of a target object, and the application range is wide; the construction method of the surface potential sensor is simple, and when the surface potential sensor is adopted as a working electrode for surface potential detection, rapid and high-sensitivity detection on a target object can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of surface potential detection, and in particular to a surface potential sensor using an allosteric transcription factor as a recognition molecule, and a preparation method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Potentiometric sensors have been widely used in the direct determination of various electrolyte ions, and their applications involve clinical testing, environmental monitoring, biology, food and other fields. Compared with conventional potentiometric sensors, surface potential sensors have the advantages of not requiring the preparation of polymer sensitive membranes, a wide target range, and simple electrode preparation. The currently reported surface potential direct detection methods are mainly used for the detection of metal ions, proteins, pathogenic bacteria, etc. However, since the recognition of its recognition molecules and targets cannot cause large charge changes, its potential response is small and its sensitivity is low, which seriously limits the use of surface potential methods. At the same time, the construction of existing surface potential sensors is relatively complex.

[0004] Therefore, how to provide a surface potential sensor that is simple to construct and can achieve high-sensitivity detection of the target analyte is an urgent problem to be solved. Summary of the invention

[0005] In view of this, the present invention provides a surface potential sensor using an allosteric transcription factor as a recognition molecule, and a preparation method and application thereof. The surface potential sensor provided by the present invention can achieve rapid and highly sensitive detection of the target analyte, and the construction method is simple.

[0006] In the first aspect, the present invention provides a surface potential sensor using an allosteric transcription factor as a recognition molecule, comprising an electrode, deoxyribonucleic acid and an allosteric transcription factor; the electrode is connected to the deoxyribonucleic acid and the allosteric transcription factor in sequence; the number of base pairs in the nucleotide sequence of the deoxyribonucleic acid is greater than 30.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned surface potential sensor using the allosteric transcription factor as a recognition molecule, comprising the following steps: The electrode is activated, and then the activated electrode is first immersed in a deoxyribonucleic acid solution for incubation, and then immersed in an allosteric transcription factor solution for incubation to obtain the electrode.

[0008] In a third aspect, the present invention provides an application of the surface potential sensor, wherein the surface potential sensor is used to detect the concentration of a target to be detected, and the detection of the concentration of the target to be detected comprises the following steps: Using the surface potential sensor as the working electrode, a two-electrode system is used to perform potential measurement on a series of target solutions with known concentrations, and a standard working curve of open circuit potential versus target concentration is plotted; then, using the surface potential sensor as the working electrode, a potential measurement is performed on the target solution to be measured, and the concentration of the target to be measured is obtained according to the standard working curve; the target is a substance that can specifically bind to the allosteric transcription factor.

[0009] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The present invention is the first to use allosteric transcription factors as recognition molecules for the construction of surface potential sensors, and to utilize the excellent affinity of allosteric transcription factors for targets to achieve rapid and highly sensitive detection of targets. The recognition molecules can be changed according to changes in the target, so the surface potential sensor of the present invention has a wide range of applications.

[0010] (2) In the surface potential sensor of the present invention, deoxyribonucleic acid has the advantages of being easy to chemically modify and highly editable. It is easy to connect to electrodes, and its appropriate chain length gives it a suitable charge. When the allosteric transcription factor binds to the target, it will desorb from the deoxyribonucleic acid, causing the charge distribution on the electrode surface to change, thereby achieving highly sensitive detection of the target.

[0011] (3) The surface potential sensor of the present invention is simple to construct. When the surface potential sensor of the present invention is used as a working electrode, the concentration of the target substance to be measured can be conveniently detected quickly and with high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 Cyclic voltammograms of the glassy carbon electrode (GC), the dsNNA-modified glassy carbon electrode (dsDNA-GC), and the HucR and dsDNA-modified glassy carbon electrode (HucR-dsDNA-GC) provided in Example 1 of the present invention; Figure 2 The surface potential sensor provided in Example 1 of the present invention is used for different concentrations of uric acid (a, 10 -7 M; b, 3×10 - 7 M; c, 10 -6 M; d, 3×10 -6 M; e, 10-5 M) Response concentration gradient graph (A) and standard working curve graph (B); Figure 3 The surface potential sensor provided in Example 2 of the present invention is used for different concentrations of uric acid (a, 10 -7 M; b, 3×10 - 7 M; c, 10 -6 M; d, 3×10 -6 M; e, 10 -5 M) Response concentration gradient graph (A) and standard working curve graph (B). DETAILED DESCRIPTION

[0014] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0015] The present invention provides a surface potential sensor using an allosteric transcription factor as a recognition molecule, comprising an electrode, deoxyribonucleic acid and the allosteric transcription factor; the electrode is sequentially connected to the deoxyribonucleic acid and the allosteric transcription factor; the number of base pairs in the nucleotide sequence of the deoxyribonucleic acid is more than 30.

[0016] Allosteric transcription factors (aTFs) are regulatory proteins produced by bacteria during the evolution process in order to sense and respond to various stimuli from the external environment. They contain two domains, one is the DNA binding domain (DBD) and the other is the effector binding domain (EBD). When EBD binds to the target in the environment, its conformation changes, causing the DBD to dissociate from the DNA to achieve the function of regulating gene expression. The present invention is the first to use allosteric transcription factors as recognition molecules for the construction of surface potential sensors, and utilizes the excellent affinity of allosteric transcription factors for the target to achieve rapid and highly sensitive detection of the target. The recognition molecules used in the surface potential sensor of the present invention can be changed according to changes in the target.

[0017] In the surface potential sensor of the present invention, the electrode is connected to the deoxyribonucleic acid and the allosteric transcription factor in sequence, wherein the deoxyribonucleic acid has the advantages of easy chemical modification and strong editability. When its length is too short, the charge is low, so the prepared surface potential sensor has low sensitivity; when its length is too long, the deoxyribonucleic acid is difficult to modify to the electrode surface, the electrode incubation time is long, and it will also cause the double electric layer on the electrode surface to be unstable, thereby causing the potential signal to be unstable. Furthermore, the number of base pairs of the nucleotide sequence of the deoxyribonucleic acid is 30 to 80.

[0018] In the present invention, the electrode and the deoxyribonucleic acid are connected via a covalent bond or an intercalation effect; the covalent bond includes but is not limited to a gold-thiol bond or an ester bond, and the intercalation effect includes, for example, intercalation into mitoxantrone; the deoxyribonucleic acid and the allosteric transcription factor are connected via a specific binding domain.

[0019] In the present invention, the deoxyribonucleic acid is a double-stranded deoxyribonucleic acid, and its 3' end or 5' end is modified with a carboxyl group, an amino group or a thiol group. The double-stranded deoxyribonucleic acid has a double helix structure, and the outer side of the base carries a negative charge, but these negative charges do not repel each other, and form ionic bonds with cations in the medium, which improves the stability of the double helix of the double-stranded deoxyribonucleic acid.

[0020] In the present invention, the electrode is selected from any one of a glassy carbon electrode, a graphite electrode, a metal electrode, an ITO conductive glass electrode, a screen-printed electrode or a paper chip electrode. In one or more embodiments of the present invention, the electrode used in the present invention is a gold electrode or a glassy carbon electrode, wherein the gold electrode is connected to the deoxyribonucleic acid by a gold-thiol bond, and the glassy carbon electrode is connected to the deoxyribonucleic acid by an ester bond.

[0021] In the present invention, the allosteric transcription factor is selected from any one of HucR, OtrR, HosA or TetR. It is understood that the allosteric transcription factor of the present invention is not limited to the above, and other allosteric transcription factors can also be used to construct the surface potential sensor of the present invention. In one or more embodiments of the present invention, the allosteric transcription factor is selected from HucR or OtrR, which is used to measure the concentration of uric acid and oxytetracycline, respectively.

[0022] In one or more embodiments of the present invention, the nucleotide sequence of the deoxyribonucleic acid of the present invention is shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5, and the specific sequence is as follows: SEQ ID NO. 1: 5'-TTT TAA TAA TAA CAA GCT TAG ATG TCT ACC TAA ATT AAGAGA AT-3'; which is preferably linked to HucR; SEQ ID NO. 2: 5′-CAG AGG CGT ATT TTA ATA ATA ACA AGC TTA GAT GTC TACCTA AAT TAA GAG AAT AAA CAA TGA G-3′; which is preferably linked to HucR; SEQ ID NO. 3: 5′-GAG AAC GAC AAG ACC TTG TCA AAG CTG ATG GTG AAC AACGTG TGT G-3′; which is preferably linked to OtrR; SEQ ID NO. 4: 5′-CCT CCG ATT TTC AGA TTG ACA CTC TAT CAT TGA TAG GGATAT ATT CCA ACT CTA TCA AT-3′; which is preferably linked to TetR.

[0023] SEQ ID NO. 5: 5′-CTC ATT GTT TAT TCT CTT AAT TGT TCG TAT ACG AAC G-3′; which is preferably linked to HosA.

[0024] The present invention also provides a method for preparing the surface potential sensor using the allosteric transcription factor as a recognition molecule, comprising the following steps: The electrode is activated, and then the activated electrode is first immersed in a deoxyribonucleic acid solution for incubation, and then immersed in an allosteric transcription factor solution for incubation to obtain the electrode.

[0025] In the present invention, the process of activating the electrode comprises the following steps: using a three-electrode system, performing cyclic voltammetry scanning in a sulfuric acid solution until a steady state is reached. The concentration of the sulfuric acid solution is preferably 0.05 to 2 M. The present invention does not impose any particular restrictions on the specific conditions of the above process, and those skilled in the art can select commonly used conditions to activate the electrode. Those skilled in the art can also choose other methods to activate the electrode, as long as an electrode that is easily connected to deoxyribonucleic acid can be obtained.

[0026] The present invention does not impose any special restrictions on the incubation conditions in the DNA solution, and those skilled in the art can select the incubation conditions according to the type of linking group. In one embodiment of the present invention, the carboxylated glassy carbon electrode is first activated by N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and then immersed in an amino-modified DNA solution, and incubated at 3-5°C and 40-80rpm for 6-10h to obtain a glassy carbon electrode connected to DNA. In another embodiment of the present invention, the activated gold electrode is self-assembled in a thiol-modified DNA solution at 20-40°C to obtain a gold electrode connected to DNA.

[0027] The present invention does not impose any special restrictions on the incubation process in the allosteric transcription factor solution. The present invention preferably comprises: immersing the electrode connected to the deoxyribonucleic acid in the allosteric transcription factor solution and incubating at 20-30° C. for 50-90 minutes.

[0028] In the present invention, the concentration of the deoxyribonucleic acid solution is 8-20 μM, more preferably 10-15 μM; the concentration of the allosteric transcription factor solution is 0.4-1 mg / mL, more preferably 0.6-0.8 mg / mL.

[0029] The present invention also provides an application of the surface potential sensor, wherein the surface potential sensor is used to detect the concentration of a target to be detected, and the detection of the concentration of the target to be detected comprises the following steps: Using the surface potential sensor as the working electrode, a two-electrode system is used to perform potential measurement on a series of target solutions with known concentrations, and a standard working curve of open circuit potential versus target concentration is plotted; then, using the surface potential sensor as the working electrode, a potential measurement is performed on the target solution to be measured, and the concentration of the target to be measured is obtained according to the standard working curve; the target is a substance that can specifically bind to the allosteric transcription factor.

[0030] In the present invention, when the allosteric transcription factor is HucR, the target is uric acid; when the allosteric transcription factor is OtrR, the target is oxytetracycline; when the allosteric transcription factor is HosA, the target is p-hydroxybenzoic acid (preservative); when the allosteric transcription factor is TetR, the target is tetracycline.

[0031] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0032] In the following examples, UA: uric acid; dsDNA: double-stranded DNA; H2N-dsDNA: amino-modified double-stranded DNA; GC: glassy carbon electrode; Tris-HCl: tris(hydroxymethylaminomethane) hydrochloride; HS-dsDNA: thiol-modified double-stranded DNA; NHS: N-hydroxysuccinimide; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0033] Example 1 This embodiment provides a surface potential sensor for detecting uric acid (UA) and a method for detecting uric acid.

[0034] (1) Construction of surface potential sensor: ① Cleaning of glassy carbon electrode: Use 0.05 μm aluminum oxide to repeatedly polish the purchased glassy carbon electrode (Gaoshi Ruilian Optoelectronics Technology Co., Ltd.) on suede in circular motions, and then rinse with deionized water. Use a three-electrode system to perform cyclic voltammetry on the polished electrode to ensure that the peak potential difference is within 90 mV.

[0035] ② Carboxylation of glassy carbon electrode: Place the electrode in 0.1 M H2SO4 solution, use a three-electrode system, activate the electrode at +2.0 V for 30 s and -1.0 V for 10 s. Then scan the potential at 100 mV / s in the potential range of 0 to +1.0 V until a stable cyclic voltammogram is obtained. In this process, the glassy carbon electrode is activated and a large number of carboxyl groups are formed on the surface.

[0036] ③Glass carbon electrode modified with dsDNA: After rinsing the carboxylated glassy carbon electrode with deionized water, immerse it in a solution containing 11 mg / mL NHS and 15 mg / mL EDC (prepared with Tris-HCl buffer at pH=8.5), and incubate it in a shaker at room temperature and 60 rpm for 6 h. After washing with deionized water, immerse the obtained glassy carbon electrode in a H2N-dsDNA solution with a concentration of 11.21 μM (double-stranded DNA: 5'-TTT TAA TAA TAA CAA GCT TAG ATG TCT ACC TAA ATT AAGAGA AT-3', as shown in SEQ ID NO. 1), and incubate it in a shaker at 4°C and 60 rpm for 8 h.

[0037] ④ Modification of HucR: Rinse the obtained glassy carbon electrode with deionized water, and then immerse it in HucR protein with a concentration of 0.73 mg / mL and pH = 8.5 (prepared with Tris-HCl buffer with pH = 8.5), select room temperature and 200 rpm on a magnetic stirrer, incubate for 1 hour, and obtain the constructed surface potential sensor.

[0038] (2) Electrode characterization: Cyclic voltammetry characterization was performed using a three-electrode system. The cleaned glassy carbon electrode (GC), dsNNA-modified glassy carbon electrode (dsDNA-GC) electrode, and HucR and dsDNA-modified glassy carbon electrode (HucR-dsDNA-GC) were subjected to cyclic voltammetry scanning in potassium ferricyanide solution (5 mM K3[Fe(CN)6]), as shown in Figure 1 As shown in the figure, compared with the oxidation peak potential of the cleaned glassy carbon electrode (GC), the oxidation peak potential of the dsNNA-modified glassy carbon electrode (dsDNA-GC) becomes lower; compared with the oxidation peak potential of the dsNNA-modified glassy carbon electrode (dsDNA-GC), the oxidation peak potential of the HucR and dsDNA-modified glassy carbon electrode (HucR-dsDNA-GC) is lower; this is mainly because the conductivity of double-stranded DNA and allosteric transcription factors is poor. The test results prove the successful modification of dsDNA and HucR.

[0039] (3) UA determination: The surface potential sensor constructed above was rinsed with deionized water and used as the working electrode to form a two-electrode system with the reference electrode (silver / silver chloride electrode). The two-electrode system was immersed in 2 mL of 10 -7 mol / L、3×10 -7 mol / L、10 -6 mol / L、3×10 -6 mol / L and 10 -5 mol / L uric acid in Tris-HCl (pH=8.5) buffer, stirred at 400 rpm under magnetic stirring until the potential was stable, and the potential was measured using an electrochemical workstation. The open circuit potential was plotted against the UA concentration to obtain a standard working curve, as shown in Figure 2 As shown, Figure 2 A is the surface potential sensor for different concentrations of uric acid (a, 10 -7 mol / L; b, 3×10 -7 mol / L; c, 10 -6 mol / L; d, 3×10 -6 mol / L; e, 10 -5 mol / L) concentration gradient diagram, Figure 2 B is the surface potential sensor for different concentrations of uric acid (a, 10 -7 mol / L; b, 3×10 -7 mol / L; c, 10 -6 mol / L; d, 3×10 - 6 mol / L; e, 10 -5mol / L) standard working curve. It can be seen that the surface potential sensor of this embodiment can be used in 10 -6.5 ~10 -5 It showed good linear response in the UA concentration range of mol / L, with a detection limit of 10 -7 mol / L.

[0040] Example 2 This embodiment provides a surface potential sensor for detecting uric acid (UA) and a method for detecting uric acid.

[0041] (1) Construction of surface potential sensor: ① Cleaning of glassy carbon electrode: Use 0.05 μm aluminum oxide to repeatedly polish the purchased glassy carbon electrode (Gaoshi Ruilian Optoelectronics Technology Co., Ltd.) on suede in circular motions, and then rinse with deionized water. Use a three-electrode system to perform cyclic voltammetry on the polished electrode to ensure that the peak potential difference is within 90 mV.

[0042] ② Carboxylation of glassy carbon electrode: Place the electrode in 0.1 M H2SO4 solution, use a three-electrode system, activate the electrode at +2.0 V for 30 s and -1.0 V for 10 s. Then scan the potential at 100 mV / s in the potential range of 0 to +1.0 V until a stable cyclic voltammogram is obtained. In this process, the glassy carbon electrode is activated and a large number of carboxyl groups are formed on the surface.

[0043] ③ Glassy carbon electrode modified with dsDNA: After rinsing the carboxylated glassy carbon electrode with deionized water, immerse it in a solution containing 11 mg / mL NHS and 15 mg / mL EDC (prepared with Tris-HCl buffer at pH=8.5), and incubate it in a shaker at room temperature and 60 rpm for 6 h. After washing with deionized water, the obtained glassy carbon electrode was immersed in 11.21 μM H2N-dsDNA (double-stranded DNA: 5'-CAG AGG CGT ATT TTA ATA ATA ACA AGC TTA GAT GTC TAC CTAAAT TAA GAG AAT AAA CAA TGA G-3', as shown in SEQ ID NO. 2), and incubate it in a shaker at 4°C and 60 rpm for 8 h.

[0044] ④ Modification of HucR: Rinse the obtained glassy carbon electrode with deionized water, and then immerse it in HucR protein with a concentration of 0.73 mg / mL and pH = 8.5 (prepared with Tris-HCl buffer with pH = 8.5), select room temperature and 200 rpm on a magnetic stirrer, incubate for 1 hour, and obtain the constructed surface potential sensor.

[0045] (2) UA determination: The surface potential sensor constructed above was rinsed with deionized water and used as the working electrode to form a two-electrode system with the reference electrode (silver / silver chloride electrode). The two-electrode system was immersed in 2 mL of 10 -7 mol / L、3×10 -7 mol / L、10 -6 mol / L、3×10 -6 mol / L and 10 -5 mol / L uric acid in Tris-HCl (pH=8.5) buffer, stirred at 400 rpm under magnetic stirring until the potential was stable, and the potential was measured using an electrochemical workstation. The open circuit potential was plotted against the UA concentration to obtain a standard working curve, as shown in Figure 3 As shown, Figure 3 A in the figure is the concentration gradient diagram of the surface potential sensor's response to uric acid. Figure 3 B in FIG. 1 is a standard working curve of the surface potential sensor in response to uric acid. -7 ~10 -5 mol / L UA concentration range showed good linear response, and the detection limit was 3×10 -8 mol / L.

[0046] Example 3 This embodiment provides a surface potential sensor for detecting uric acid (UA) and a method for detecting uric acid.

[0047] (1) Construction of surface potential sensor: ① Activation of gold electrode: polish the purchased gold electrode to a mirror surface, use a three-electrode system (gold electrode as working electrode, silver / silver chloride electrode as reference electrode, platinum wire electrode as auxiliary electrode), and perform cyclic voltammetry test in 1 M sulfuric acid until it is stable.

[0048] ② Gold electrode modified dsDNA: Immerse the activated gold electrode in -5 M HS-dsDNA (double-stranded DNA: 5'-CAG AGGCGT ATT TTA ATA ATA ACA AGC TTA GAT GTC TAC CTA AAT TAA GAG AAT AAA CAA TGAG-3', as shown in SEQ ID NO. 2) was incubated (self-assembly) in a shaker at 25°C and 60 rpm for 4 h.

[0049] ④ Modification of HucR: Rinse the modified gold electrode obtained in step ② with deionized water, and then immerse it in HucR protein with a concentration of 0.73 mg / mL and pH = 8.5 (prepared with Tris-HCl buffer with pH = 8.5), select room temperature and 200 rpm on a magnetic stirrer, incubate for 1 hour, and obtain the constructed surface potential sensor.

[0050] (2) UA determination: The surface potential sensor constructed above was rinsed with deionized water and used as the working electrode to form a two-electrode system with the reference electrode (silver / silver chloride electrode). The two-electrode system was immersed in 2 mL of 10 -7 mol / L、3×10 -7 mol / L、10 -6 mol / L、3×10 -6 mol / L and 10 -5 mol / L uric acid in Tris-HCl (pH=8.5) buffer, stirred at 400 rpm under magnetic stirring until the potential was stable, and the potential was measured using an electrochemical workstation. -7 ~10 -5 mol / L UA concentration range showed good linear response, and the detection limit was 3×10 -8 mol / L.

[0051] Example 4 This embodiment provides a surface potential sensor for detecting oxytetracycline and a method for detecting oxytetracycline.

[0052] (1) Construction of surface potential sensor: ① Cleaning of glassy carbon electrode: Use 0.05 μm aluminum oxide to repeatedly polish the purchased glassy carbon electrode (Gaoshi Ruilian Optoelectronics Technology Co., Ltd.) on suede in circular motions, and then rinse with deionized water. Use a three-electrode system to perform cyclic voltammetry on the polished electrode to ensure that the peak potential difference is within 90 mV.

[0053] ② Carboxylation of glassy carbon electrode: Place the electrode in 0.1 M H2SO4 solution, use a three-electrode system, activate the electrode at +2.0 V for 30 s and -1.0 V for 10 s. Then scan the potential at 100 mV / s in the potential range of 0 to +1.0 V until a stable cyclic voltammogram is obtained. In this process, the glassy carbon electrode is activated and a large number of carboxyl groups are formed on the surface.

[0054] ③Glass carbon electrode modified with dsDNA: After rinsing the carboxylated glassy carbon electrode with deionized water, immerse it in a solution containing 11 mg / mL NHS and 15 mg / mL EDC (prepared with Tris-HCl buffer at pH=8.5), and incubate it for 6 h at room temperature and 60 rpm in a shaker. After washing with deionized water, immerse the obtained glassy carbon electrode in 11.21 μM H2N-dsDNA (double-stranded DNA: 5'-GAG AAC GAC AAG ACC TTG TCA AAG CTG ATG GTG AAC AAC GTGTGT G-3', as shown in SEQ ID NO. 3), and incubate it for 8 h at 4°C and 60 rpm in a shaker.

[0055] ④ Modification of OtrR: Rinse the obtained glassy carbon electrode with deionized water, and then immerse it in 0.7 mg / mL, pH=8.5 OtrR protein (prepared with Tris-HCl buffer, pH=8.5), select room temperature and 200 rpm on a magnetic stirrer, incubate for 1 hour, and obtain the constructed surface potential sensor.

[0056] (2) Oxytetracycline determination: The surface potential sensor constructed above was rinsed with deionized water and used as the working electrode to form a two-electrode system with the reference electrode (silver / silver chloride electrode). The two-electrode system was immersed in 2 mL of 10 -7 mol / L、10 -6.5 mol / L、10 -6 mol / L、10 -5.5 mol / L and 10 -5 mol / L oxytetracycline in Tris-HCl (pH=8.5) buffer, stirred at 400 rpm under magnetic stirring until the potential was stable, and the potential was measured using an electrochemical workstation. The surface potential sensor of this embodiment can show a good linear response in the concentration range of 1.778-17.78nmol / L oxytetracycline, and the detection limit is 10 -9 mol / L.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A surface potential sensor using an allosteric transcription factor as a recognition molecule, characterized in that: It comprises an electrode, deoxyribonucleic acid and an allosteric transcription factor; the electrode is sequentially connected to the deoxyribonucleic acid and the allosteric transcription factor; the number of base pairs of the nucleotide sequence of the deoxyribonucleic acid is more than 30.

2. The surface potential sensor using an allosteric transcription factor as a recognition molecule according to claim 1, characterized in that: The electrode is connected to the deoxyribonucleic acid via a covalent bond or an intercalation reaction; the deoxyribonucleic acid is connected to the allosteric transcription factor via a specific binding domain.

3. The surface potential sensor using an allosteric transcription factor as a recognition molecule according to claim 2, characterized in that: The deoxyribonucleic acid is a double-stranded deoxyribonucleic acid, and the 3' end or the 5' end thereof is modified with a carboxyl group, an amino group or a thiol group.

4. The surface potential sensor using an allosteric transcription factor as a recognition molecule according to claim 1, characterized in that: The electrode is selected from any one of a glassy carbon electrode, a graphite electrode, a metal electrode, an ITO conductive glass electrode, a screen-printed electrode or a paper chip electrode.

5. The surface potential sensor using an allosteric transcription factor as a recognition molecule according to claim 1, characterized in that: The allosteric transcription factor is selected from any one of HucR, OtrR, HosA or TetR.

6. The surface potential sensor using an allosteric transcription factor as a recognition molecule according to claim 1, characterized in that: The nucleotide sequence of the deoxyribonucleic acid is shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO.

5.

7. The method for preparing a surface potential sensor using an allosteric transcription factor as a recognition molecule according to any one of claims 1 to 6, characterized in that: The steps include: The electrode is activated, and then the activated electrode is first immersed in a deoxyribonucleic acid solution for incubation, and then immersed in an allosteric transcription factor solution for incubation to obtain the electrode.

8. The preparation method according to claim 7, characterized in that: The concentration of the deoxyribonucleic acid solution is 8-20 μM; the concentration of the allosteric transcription factor solution is 0.4-1 mg / mL.

9. Use of a surface potential sensor using an allosteric transcription factor as a recognition molecule according to any one of claims 1 to 6, characterized in that: The surface potential sensor is used to detect the concentration of the target to be detected, and the detection of the concentration of the target to be detected includes the following steps: Using the surface potential sensor as the working electrode, a two-electrode system is used to perform potential measurement on a series of target solutions with known concentrations, and a standard working curve of open circuit potential versus target concentration is plotted; then, using the surface potential sensor as the working electrode, a potential measurement is performed on the target solution to be measured, and the concentration of the target to be measured is obtained according to the standard working curve; the target is a substance that can specifically bind to the allosteric transcription factor.

10. The use according to claim 9, characterized in that When the allosteric transcription factor is HucR, the target is uric acid; when the allosteric transcription factor is OtrR, the target is oxytetracycline; when the allosteric transcription factor is HosA, the target is p-hydroxybenzoic acid; when the allosteric transcription factor is TetR, the target is tetracycline.

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