阅读说明：本技术 一种用于弧菌检测的比率型电化学传感平台及其制备方法和应用 (Ratio type electrochemical sensing platform for vibrio detection and preparation method and application thereof ) 是由 王新星 罗细亮 齐有啸 申优 张丽 徐立强 崔久英 于 2020-01-02 设计创作，主要内容包括：本发明提供了一种用于弧菌检测的比率型电化学传感平台,包括工作电极和设置在所述工作电极表面的修饰层,所述修饰层包括设置在所述工作电极表面的树脂-金属螯合物层、沉积在所述树脂-金属螯合物层表面的金纳米颗粒层和固定在所述金纳米颗粒层表面的探针DNA,所述探针DNA为弧菌的特异性靶DNA序列的互补序列,所述探针DNA的3’端和5’端分别修饰有二茂铁和巯基。本发明提供的比率型电化学传感平台操作简单,响应迅速,不受样品颜色和浑浊度的影响,并具有高的检测灵敏度、特异性和重现性。这使得本发明制备的比率型电化学传感平台显示出有别于传统微生物学检测方法、现代实时荧光定量PCR检测方法和一般电化学传感平台的独特优势,实际应用前景广阔。(The invention provides a ratio-type electrochemical sensing platform for vibrio detection, which comprises a working electrode and a modification layer arranged on the surface of the working electrode, wherein the modification layer comprises a resin-metal chelate layer arranged on the surface of the working electrode, a gold nanoparticle layer deposited on the surface of the resin-metal chelate layer and a probe DNA fixed on the surface of the gold nanoparticle layer, the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, and the 3 'end and the 5' end of the probe DNA are respectively modified with ferrocene and sulfydryl. The ratio type electrochemical sensing platform provided by the invention is simple to operate, is quick in response, is not influenced by the color and turbidity of a sample, and has high detection sensitivity, specificity and reproducibility. Therefore, the ratio-type electrochemical sensing platform prepared by the invention has unique advantages different from the traditional microbiological detection method, the modern real-time fluorescence quantitative PCR detection method and the common electrochemical sensing platform, and has wide practical application prospect.)

1. A ratio-type electrochemical sensing platform for vibrio detection comprises a working electrode and a modification layer arranged on the surface of the working electrode, wherein the modification layer comprises a resin-metal chelate layer arranged on the surface of the working electrode, a gold nanoparticle layer deposited on the surface of the resin-metal chelate layer and probe DNA fixed on the surface of the gold nanoparticle layer, the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, and ferrocene and sulfydryl are modified at the 3 'end and the 5' end of the probe DNA respectively.

2. The ratiometric electrochemical sensing platform of claim 1, wherein the resin-metal chelate is a multi-coordinated chelate obtained by a chelating reaction between a resin and a metal ion, and the resin has a pore structure and a chelating functional group.

3. A ratiometric electrochemical sensing platform according to claim 2, wherein said chelating functional groups are one or more of amino, imino, carboxyl, hydroxyl, thiol and Schiff base bonds;

the pore diameter of the pore channel structure is less than 3 nm.

4. A ratiometric electrochemical sensing platform according to any one of claims 1 to 3, wherein the metal ions corresponding to the metal in the resin-metal chelate are metal ions that undergo redox reaction at an electrode potential of from-1.5V to 1.0V.

5. The ratiometric electrochemical sensing platform of claim 4, wherein the metal ion is Cu²⁺、Pb²⁺、Cd²⁺Or Zn²⁺。

6. A method for preparing a ratiometric electrochemical sensing platform of any one of claims 1 to 5, comprising the steps of:

providing a resin dispersion;

mixing the resin dispersion liquid with a metal ion solution for chelation reaction to obtain a resin-metal chelate;

dispersing the resin-metal chelate in water to obtain a resin-metal chelate dispersion;

dripping the resin-metal chelate dispersion liquid on the surface of a working electrode to obtain a chelate modified working electrode;

placing the chelate modified working electrode in a chloroauric acid solution, forming a three-electrode system with a reference electrode and a counter electrode, performing electrodeposition, and forming a gold nanoparticle layer on the surface of the resin-metal chelate to obtain a nanogold/chelate modified working electrode;

fixing probe DNA on the surface of the nanogold/chelate modified working electrode, wherein the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, modifying ferrocene and sulfydryl at the 3 'end and the 5' end of the probe DNA respectively, and fixing the probe DNA on the surface of the nanogold/chelate modified working electrode through Au-S bonds to obtain the ratiometric electrochemical sensing platform for detecting the vibrio.

7. The method according to claim 6, wherein the mass concentration of the resin-metal chelate dispersion is 0.1 to 5.0mg/m L.

8. The preparation method according to claim 7, wherein the volume of the resin-metal chelate dispersion liquid dropped onto the surface of the working electrode is 3 to 20 μ L.

9. The method of claim 6, wherein the electrodeposition is potentiostatic electrochemical deposition or cyclic voltammetry electrochemical deposition;

the time of the constant potential electrochemical deposition is 100-800 s, and the potential of the constant potential electrochemical deposition is-0.5V;

the cyclic potential interval of the electrochemical deposition of the cyclic voltammetry is-0.4-0.5V, the sweep rate of the electrochemical deposition of the cyclic voltammetry is 0.01-0.1V/s, and the number of the sweep sections of the electrochemical deposition of the cyclic voltammetry is 10-100.

10. Use of the ratiometric electrochemical sensing platform of any one of claims 1 to 5 or the ratiometric electrochemical sensing platform obtained by the preparation method of any one of claims 6 to 9 in vibrio detection.

Technical Field

The application relates to the technical field of vibrio detection, in particular to a ratio-type electrochemical sensing platform for vibrio detection and a preparation method and application thereof.

Background

Vibrio is an important pathogenic bacterium causing marine culture animal diseases and human food-borne diseases, and the development of advanced analysis and detection technology of pathogenic vibrio in seawater and marine products has great significance. The traditional microbiological detection method needs a series of steps such as pathogen separation, culture, morphology and biochemical identification, and has the disadvantages of complex operation, long time consumption and low specificity (GB 4789.7-2013); the modern real-time fluorescent quantitative PCR technology needs to carry out complex primer design, has high requirements on the technical level of operators, and also has high requirements on the transparency of samples by fluorescent detection signals, so the technology is difficult to popularize and use in basic laboratories. These methods are not conducive to rapid diagnosis of aquatic diseases and rapid inspection of seafood safety.

The electrochemical DNA biosensor has unique advantages in the development of an instant detection technology with miniaturized instruments, simplified operation and rapid result response. The core component of modern electrochemical DNA biosensors is the chemically modified electrode (i.e., the electrochemical sensing platform). Its properties directly determine the performance of the electrochemical DNA biosensor. However, differences such as self characteristics (such as material morphology and electrode effective surface area), probe fixing density, reagent signal change caused by non-target objects and the like inevitably exist between parallel chemical modified electrodes, so that the constructed electrochemical DNA biosensor has large signal fluctuation and poor reproducibility and reliability, and the practical application is greatly limited.

Disclosure of Invention

The invention aims to provide a ratio type electrochemical sensing platform for vibrio detection, which improves the repeatability and reliability of vibrio detection.

The invention provides a ratio-type electrochemical sensing platform for vibrio detection, which comprises a working electrode and a modification layer arranged on the surface of the working electrode, wherein the modification layer comprises a resin-metal chelate layer arranged on the surface of the working electrode, a gold nanoparticle layer deposited on the surface of the resin-metal chelate layer and a probe DNA fixed on the surface of the gold nanoparticle layer, the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, and the 3 'end and the 5' end of the probe DNA are respectively modified with ferrocene and sulfydryl.

Preferably, the resin-metal chelate is a multi-coordination chelate obtained by carrying out a chelation reaction on resin and metal ions, and the resin has a pore structure and a chelating functional group.

Preferably, the chelating functional group is one or more of amino, imino, carboxyl, hydroxyl, sulfydryl and Schiff alkali bond;

the pore diameter of the pore channel structure is less than 3 nm.

Preferably, the metal ion corresponding to the metal in the resin-metal chelate is a metal ion that undergoes an oxidation-reduction reaction at an electrode potential of-1.5V to 1.0V.

Preferably, the metal ion is Cu²⁺、Pb²⁺、Cd²⁺Or Zn²⁺。

The invention provides a preparation method of the ratio-type electrochemical sensing platform in the technical scheme, which comprises the following steps:

providing a resin dispersion;

mixing the resin dispersion liquid with a metal ion solution for chelation reaction to obtain a resin-metal chelate;

dispersing the resin-metal chelate in water to obtain a resin-metal chelate dispersion;

dripping the resin-metal chelate dispersion liquid on the surface of a working electrode to obtain a chelate modified working electrode;

placing the chelate modified working electrode in a chloroauric acid solution, forming a three-electrode system with a reference electrode and a counter electrode, performing electrodeposition, and forming a gold nanoparticle layer on the surface of the resin-metal chelate to obtain a nanogold/chelate modified working electrode;

fixing probe DNA on the surface of the nanogold/chelate modified working electrode, wherein the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, modifying ferrocene and sulfydryl at the 3 'end and the 5' end of the probe DNA respectively, and fixing the probe DNA on the surface of the nanogold/chelate modified working electrode through Au-S bonds to obtain the ratiometric electrochemical sensing platform for detecting the vibrio.

Preferably, the mass concentration of the resin-metal chelate dispersion is 0.1-5.0 mg/m L.

Preferably, the volume of the resin-metal chelate dispersion liquid dripped onto the surface of the working electrode is 3-20 mu L.

Preferably, the electrodeposition is potentiostatic electrochemical deposition or cyclic voltammetry electrochemical deposition;

the time of the constant potential electrochemical deposition is 100-800 s, and the potential of the constant potential electrochemical deposition is-0.5V;

the cyclic potential interval of the electrochemical deposition of the cyclic voltammetry is-0.4-0.5V, the sweep rate of the electrochemical deposition of the cyclic voltammetry is 0.01-0.1V/s, and the number of the sweep sections of the electrochemical deposition of the cyclic voltammetry is 10-100.

The invention provides an application of the ratio-type electrochemical sensing platform in the technical scheme or the ratio-type electrochemical sensing platform prepared by the preparation method in the technical scheme in vibrio detection.

The invention provides a ratio-type electrochemical sensing platform for vibrio detection, which comprises a working electrode and a modification layer arranged on the surface of the working electrode, wherein the modification layer comprises a resin-metal chelate layer arranged on the surface of the working electrode, a gold nanoparticle layer deposited on the surface of the resin-metal chelate layer and a probe DNA fixed on the surface of the gold nanoparticle layer, the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, and the 3 'end and the 5' end of the probe DNA are respectively modified with ferrocene and sulfydryl. In the present invention, the probe DNA hybridizes with a specific target DNA fragment of a specific Vibrio and undergoes a change in configuration; the ferrocene modified by the 3' end of the probe DNA is used as a signal molecule, and the change of the electrochemical signal can indicate whether a specific target DNA fragment exists and the quantity of the specific target DNA fragment, so that the qualitative and quantitative determination of vibrio is realizedDetecting; the resin-metal chelate in the electrochemical sensing platform is used as an internal reference probe molecule to provide an internal reference signal required by ratio type detection; the final detection signal for qualitative and quantitative vibrio detection is the current signal of ferrocene (I)_Fc) Current Signal (I) with Metal ions in resin-Metal chelate_M) Ratio of (A) to (B), I_Fc/I_M.I_Fc/I_MThe specific target DNA sequence of the specific vibrio can be quantitatively detected in a certain concentration range in relation to the concentration of the specific target DNA sequence of the specific vibrio; i is_Fc/I_MErrors caused by different electrode characteristic differences and different experimental conditions can be reduced or eliminated, and the reproducibility and reliability of the detection result are improved; the gold nanoparticles in the electrochemical sensing platform can improve the conductivity and the effective surface area of the electrode of the electrochemical sensing platform, improve the detection sensitivity and be used for fixing probe DNA through Au-S bonds in the follow-up process. Compared with the traditional microbiological detection method, the ratio-type electrochemical sensing platform provided by the invention has the advantages of lower operation complexity, quick response, higher detection sensitivity and specificity. Compared with the modern real-time fluorescent quantitative PCR detection method, the detection of the ratio-type electrochemical sensing platform provided by the invention is not influenced by the color and turbidity of the sample. In addition, compared with a common electrochemical sensing platform, the ratio type electrochemical sensing platform prepared by the invention has higher reproducibility and reliability of detection results.

Drawings

FIG. 1 is a graph showing a ratio-type standard curve obtained in example 2 of the present invention;

FIG. 2 is a non-ratiometric standard curve obtained in comparative example 1 of the present invention;

FIG. 3 is a graph showing the reproducibility of electrochemical sensing platforms according to embodiments of the present invention;

fig. 4 shows the selectivity results of the electrochemical sensing platform provided by the embodiment of the present invention.

Detailed Description

The invention provides a ratio-type electrochemical sensing platform for vibrio detection, which comprises a working electrode and a modification layer arranged on the surface of the working electrode, wherein the modification layer comprises a resin-metal chelate layer arranged on the surface of the working electrode, a gold nanoparticle layer deposited on the surface of the resin-metal chelate layer and a probe DNA fixed on the surface of the gold nanoparticle layer, the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, and the 3 'end and the 5' end of the probe DNA are respectively modified with ferrocene and sulfydryl.

The ratiometric electrochemical sensing platform provided by the invention comprises a working electrode, and in the embodiment of the invention, the working electrode is a glassy carbon electrode.

The present invention provides a ratiometric electrochemical sensing platform comprising a resin-metal chelate layer disposed on a surface of the working electrode. Unit area (cm)²) The mass of the resin-metal chelate compound on the surface of the working electrode is preferably 0.004-1.4 mg. in the embodiment of the invention, the resin-metal chelate compound layer is preferably obtained from a resin-metal chelate compound dispersion liquid with a concentration of 10 mu L of 1.0mg/m L.

In the invention, the resin-metal chelate is a multi-coordination chelate obtained by chelating resin and metal ions, and the resin preferably has a pore structure and a chelating functional group. In the invention, the chelating functional group is preferably one or more of amino, imino, carboxyl, hydroxyl, sulfydryl and Schiff alkali bond; the pore size of the channel structure is preferably less than 3 nm. In an embodiment of the present invention, the resin may be specifically a phenolic resin. The present invention is not particularly limited in the kind and source of the phenolic resin, and a phenolic resin known to those skilled in the art may be used. In the present invention, the method for preparing the phenolic resin preferably comprises the steps of:

providing a 3-aminophenol solution;

adding an ammonia water solution into the 3-aminophenol solution, mixing and stirring to obtain an alkaline aminophenol solution;

and adding a formaldehyde solution into the alkaline aminophenol solution, mixing and stirring, and carrying out condensation reaction to obtain the phenolic resin.

In the invention, the solvent in the 3-aminophenol solution is preferably a mixed solvent of ethanol and water, the concentration of the 3-aminophenol solution is preferably 0.1 mol/L, and the volume ratio of the ethanol to the water is preferably 1: 2.5.

After the 3-aminophenol solution is obtained, the ammonia water solution is added into the 3-aminophenol solution to be mixed and stirred. In the invention, the mass percentage of the ammonia water in the ammonia water solution is preferably 28%; the volume ratio of the ammonia water solution to the 3-aminophenol solution is preferably 1: 150; the stirring time is preferably 10 minutes; the stirring temperature is preferably 30 ℃.

After obtaining the alkaline aminophenol solution, adding the formaldehyde solution into the alkaline aminophenol solution, mixing and stirring the mixture, and carrying out condensation reaction to obtain the phenolic resin. In the present invention, the mass percentage of formaldehyde in the formaldehyde solution is preferably 37%; the volume of the formaldehyde solution and the basic aminophenol solution is preferably 1: 135. In the present invention, the time of the condensation reaction is preferably 4 hours; the temperature of the condensation reaction is preferably 30 ℃. In the present invention, the condensation reaction is carried out under stirring conditions; the stirring method is not particularly limited, and the technical scheme of stirring well known to those skilled in the art can be adopted, for example, magnetic stirring can be adopted in laboratory lab; in the present invention, the stirring rate is preferably 400 rpm.

After the condensation reaction, the present invention preferably centrifuges the obtained reaction feed liquid, and washes the precipitate obtained by the centrifugation to obtain the phenolic resin. In the present invention, the rotation speed of the centrifugation is preferably 4500 rmp; the time for the centrifugation is preferably 40 minutes. In the present invention, the washing is preferably water washing; the cleaning method is preferably ultrasonic cleaning; the ultrasonic cleaning time is 10 minutes; the number of times of the centrifugation and ultrasonic cleaning is preferably 10.

In the present invention, the metal ion is preferably a metal ion that undergoes an oxidation-reduction reaction at an electrode potential of-1.5V to 1.0V, and specifically Cu²⁺、Pb²⁺、Cd²⁺Or Zn²⁺。

In the present invention, the chelating reaction of the resin with the metal ion preferably includes the steps of:

providing an aqueous resin dispersion;

and mixing the resin aqueous dispersion with a metal ion aqueous solution, and carrying out a chelation reaction to obtain a resin-metal chelate.

In the present invention, the mass concentration of the aqueous resin dispersion is preferably 0.5mg/m L, and in the embodiment of the present invention, the water used for preparing the aqueous resin dispersion may be specifically ultrapure water.

After obtaining the resin aqueous dispersion, the invention mixes the resin aqueous dispersion with the metal ion aqueous solution. In the present invention, the metal ion is preferably provided by a soluble metal salt of the metal according to the above technical aspect; specifically, when the metal ion is preferably Cu²⁺When the soluble metal salt is copper chloride; when the metal ion is preferably Pb²⁺When the corresponding soluble metal salt is lead nitrate; when the metal ion is preferably Cd²⁺When the metal salt is cadmium nitrate tetrahydrate, the corresponding soluble metal salt is preferably cadmium nitrate tetrahydrate; when the metal ion is preferably Zn²⁺The molar concentration of the aqueous solution of metal ions is preferably 200 mmol/L. in the present invention, the volume ratio of the aqueous resin dispersion to the aqueous solution of metal ions is preferably 1: 1.

In the present invention, the chelation reaction is performed at room temperature; the chelation reaction is preferably carried out under stirring; the stirring method is not particularly limited, and the technical scheme of stirring known to those skilled in the art can be adopted; in the embodiment of the present invention, the stirring rate is preferably 500 rpm; the time of the chelation reaction is preferably 4 hours.

After the chelation reaction, the present invention preferably centrifuges the obtained chelation reaction solution, washes the precipitate obtained by the centrifugation, and centrifuges the washed precipitate again to obtain the resin-metal chelate.

The centrifugation method is not particularly limited in the invention, and the technical scheme of centrifugation well known to those skilled in the art can be adopted; in the present example, the centrifugation conditions are preferably 10000 rmp. In the present invention, the rinsing is preferably performed with ultrapure water; the number of washing and centrifugation cycles is preferably 3.

The ratio-type electrochemical sensing platform provided by the invention further comprises a gold nanoparticle layer deposited on the surface of the resin-metal chelate layer, wherein the gold nanoparticle layer improves the conductivity and the effective surface area of an electrode of the electrochemical sensing platform and is used for fixing probe DNA through Au-S bonds subsequently, in the invention, the preparation method of the gold nanoparticle layer is preferably electrodeposition in a chloroauric acid solution, the molar concentration of the chloroauric acid solution is preferably 5.0 mmol/L, in the invention, the electrodeposition is preferably constant potential electrochemical deposition or cyclic voltammetry electrochemical deposition, the time of the constant potential electrochemical deposition is preferably 100-800S, more preferably 150-500S, most preferably 180-300S, the potential of the constant potential electrochemical deposition is preferably-0.5V, more preferably-0.2V, the cyclic potential interval of the cyclic voltammetry electrochemical deposition is preferably-0.4-0.5V, the sweeping speed of the cyclic voltammetry electrochemical deposition is preferably 0.01-0.1V/S, more preferably 0.05V/S, the cyclic voltammetry electrochemical deposition is preferably 10-40 sections, and the most preferably 20-40 sections.

The ratiometric electrochemical sensing platform provided by the invention further comprises probe DNA fixed on the surface of the gold nanoparticle layer, wherein the probe DNA is a complementary sequence of a specific target DNA sequence (tlh) of vibrio parahaemolyticus, and the 3 'end and the 5' end of the probe DNA are respectively modified with ferrocene and sulfydryl. In the present invention, the probe DNA (pDNA) is a complementary sequence of a specific target DNA sequence (tDNA) of Vibrio parahaemolyticus, and it can hybridize with the tDNA of Vibrio parahaemolyticus to undergo a change in configuration. In the invention, the 3 'end of the pDNA is modified with ferrocene, the 5' end of the pDNA is modified with sulfydryl, and Au-S bond is formed between the sulfydryl and the gold nanoparticles, so that the pDNA is fixed on the gold nanoparticle layer, the ferrocene is used as a signal molecule, and the change of electrochemical signals can indicate whether tDNA molecules exist and how much the tDNA molecules are in quantity, thereby realizing the qualitative and quantitative determination of vibrios. The source of the probe DNA modified with ferrocene and thiol is not particularly limited in the present invention, and the probe DNA can be purchased from commercially available sources well known to those skilled in the art.

In the present invention, the tlh gene fragment of Vibrio parahaemolyticus has the following base sequence:

pDNA：TTTTTGATGACACTGCCAGATGCGACGA

tDNA：TCGTCGCATCTGGCAGTGTCATC

the detection principle of the electrochemical sensing platform provided by the invention is as follows: when the pDNA is fixed on the surface of the nano gold/chelate modified electrode, the pDNA is in a lodging state on the surface of the modified electrode because single-stranded DNA (ssDNA) presents flexibility. The pDNA covers the surface of Au-RS-M (Au represents electrodeposited gold nanoparticle layer; RS represents resin sphere; and M represents chelated metal ion), and the electron transfer of M in RS on the surface of the electrode is blocked, so that I_MIs smaller. At the same time, because of the pDNA fixation, another signal molecule ferrocene is introduced, and the current signal (I) of ferrocene is generated_Fc). And, since pDNA is in the lodging state at this time, the ferrocene molecule at the 3' end of pDNA is closer to the electrode surface, so that I_FcIs relatively large. In the presence of tDNA, pDNA hybridizes to tDNA, changes configuration from the previously "flexible" ssDNA to "rigid" double-stranded DNA (dsDNA), and stands up on the Au-RS-M surface. At this time, electron transfer at the electrode surface is enhanced in the interior M of RS, I_MIncreasing; and the ferrocene molecule at the 3' end of pDNA is far away from the electrode surface, I_FcAnd decreases. I is_Fc/I_MThe size of tDNA can be changed along with the introduction of tDNA, and the change and the concentration of tDNA are in a linear relation in a certain range, thereby realizing the quantitative detection of the tDNA. According to the detection result of tDNA, the specific vibrio can be qualitatively and quantitatively detected. And I_Fc/I_MErrors caused by different electrode characteristic differences and different experimental conditions can be reduced or eliminated, and the reproducibility and reliability of the detection result are improved.

The invention also provides a preparation method of the ratio-type electrochemical sensing platform in the technical scheme, which comprises the following steps:

providing a resin dispersion;

mixing the resin dispersion liquid with a metal ion solution for chelation reaction to obtain a resin-metal chelate;

dispersing the resin-metal chelate in water to obtain a resin-metal chelate dispersion;

dripping the resin-metal chelate dispersion liquid on the surface of a working electrode to obtain a chelate modified working electrode;

placing the chelate modified working electrode in a chloroauric acid solution, forming a three-electrode system with a reference electrode and a counter electrode, performing electrodeposition, and forming a gold nanoparticle layer on the surface of the resin-metal chelate to obtain a nanogold/chelate modified working electrode;

fixing probe DNA on the surface of the nanogold/chelate modified working electrode, wherein the probe DNA is a complementary sequence of a specific target DNA sequence of vibrio, modifying ferrocene and sulfydryl at the 3 'end and the 5' end of the probe DNA respectively, and fixing the probe DNA on the surface of the nanogold/chelate modified working electrode through Au-S bonds to obtain the ratiometric electrochemical sensing platform for detecting the vibrio.

In the present invention, the preparation method of the resin-metal chelate is the same as the technical scheme of the resin-metal chelate preparation described in the above technical scheme, and details are not repeated here.

After obtaining the resin-metal chelate, the present invention disperses the resin-metal chelate in water to obtain a resin-metal chelate dispersion, in the present invention, the water is preferably ultrapure water, and the mass concentration of the resin-metal chelate dispersion is preferably 1.0mg/m L.

After the resin-metal chelate dispersion liquid is obtained, the resin-metal chelate dispersion liquid is dripped on the surface of a working electrode, and the chelate modified electrode is obtained after drying. In the present invention, the working electrode is preferably a glassy carbon electrode, and the working electrode is preferably subjected to a cleaning treatment before use, and the method of the cleaning treatment in the present invention is not particularly limited, and a pretreatment method of the working electrode known to those skilled in the art may be used.

In the present invention, the volume of the resin-metal chelate dispersion liquid dripped onto the surface of the working electrode is preferably 10 μ L. in the present invention, the obtained working electrode dripped with the dispersion liquid is preferably dried at room temperature to obtain a chelate-modified working electrode.

According to the method, a chelate modified working electrode is immersed into a chloroauric acid solution, electrochemical deposition is carried out by adopting a three-electrode system, a gold nanoparticle layer is formed on the surface of a resin-metal chelate, and a nanogold/chelate modified working electrode is obtained.

After obtaining the nanogold/chelate modified working electrode, the invention fixes pDNA on the surface of the nanogold/chelate modified working electrode to obtain the ratio type electrochemical sensing platform for vibrio detection. In the invention, the pDNA is a complementary sequence of a specific target DNA sequence of vibrio, the 3 'end and the 5' end of the pDNA are respectively modified with ferrocene and sulfydryl, and the pDNA is fixed on the surface of the nanogold/chelate modified working electrode through Au-S bonds.

In the present invention, the pDNA is activated before modifying the electrode, and in the present invention, the activation method comprises the steps of:

mixing a Tris-HCl solution of pDNA with Tris-HCl, Tris (2-carboxyethyl) phosphine (TCEP) and sodium chloride, and then carrying out ultrasonic treatment;

and (3) carrying out reaction activation on the mixed system subjected to the ultrasonic treatment in a shaking table.

In the present invention, the Tris-HCl solution of pDNA preferably has a molar concentration of 5.0 × 10^-6mol/L, the Tris-HCl solution of pDNA is obtained by dilution of mother solution, and the molarity of the mother solution is preferably 1.0 × 10^-4mol/L, the mixing is carried out in a centrifugal tube, the ratio of the volume of Tris-HCl solution of pDNA, the volume of Tris-HCl and the mass of TCEP to the mass of sodium chloride in the mixed system is preferably 250 mu L: 250 mu L: 2.0mg:8.0mg, the frequency of the ultrasonic is preferably 40KHz, the time of the ultrasonic is preferably 1-2 minutes, the temperature of the shaking table is set to 37 ℃, and the time of the reaction activation is preferably 1 hour.

After the activation, the activated pDNA solution is dripped on the surface of the nano gold/chelate modified electrode, and probe immobilization reaction is carried out under the condition of normal temperature and moisture retention to obtain a ratio type electrochemical sensing platform, wherein the concentration of the activated pDNA solution is 2.5 × 10^-6mol/L, the dropping volume is preferably 10 mu L, in the invention, the humidity of the moisture is preferably 10%, and the time of the probe immobilization reaction is preferably 8 hours.

The invention also provides application of the ratio type electrochemical sensing platform in the technical scheme in vibrio detection. In the present invention, the vibrio is preferably vibrio parahaemolyticus, and the method of detection is preferably differential pulse voltammetry scanning. In the present invention, before performing quantitative detection, a standard curve is preferably obtained, and the method for obtaining the standard curve preferably includes the following steps:

preparing tDNA standard working solution with series concentration;

respectively dripping the tDNA standard working solution on the surface of the electrochemical sensing platform in the technical scheme, and incubating for DNA hybridization to obtain a dsDNA modified electrode;

placing the dsDNA modified electrode in PBS buffer solution for differential pulse voltammetry scanning to obtain a differential pulse voltammetry curve, and recording the peak current signal ratio of ferrocene to metal ions in the differential pulse voltammetry curve;

and drawing to obtain a standard curve by taking the logarithm of the current signal ratio of the ferrocene to the metal ions as a vertical coordinate and the negative logarithm of the concentration of the tDNA standard working solution as a horizontal coordinate.

In the present invention, when preparing the standard working solution, it is preferable to prepare a tDNA stock solution and dilute the tDNA stock solution into tDNA standard working solutions of a series of concentrations, and in the present invention, the concentration of the tDNA stock solution is preferably 1.0 × 10^{- 4}mol/L, and the solvent in the tDNA stock solution is Tris-HCl.

In the present invention, the dropping volume of the standard working solution is preferably 10. mu. L. the incubation temperature is preferably 37 ℃ and the incubation time is preferably 2.5 hours.

In the present invention, the concentration of the PBS buffer solution is preferably 0.1 mol/L, and the pH value of the PBS buffer solution is preferably 7.4.

In the present invention, the conditions of the differential pulse voltammetry scan are preferably: initial potential is 0.5V, final potential is-0.2V, potential increment is 0.004V, amplification is 0.05V, pulse width is 0.05s, and standing time is 2 s.

In the present invention, after the differential pulse voltammetry scan, the electrodes are preferably washed with Tris-HCl.

In the present invention, the linear range of the standard curve is 1.0 × 10^-15～1.0×10^-9mol/L, detection limit of about 3.2 × 10^-16mol/L(3σ/m)。

In the invention, when vibrio detection is carried out, the extracted DNA sample is preferably detected according to the technical scheme to obtain the ferrocene and metal ion current signal ratio of the sample, and a quantitative detection result is obtained according to the standard curve obtained by the technical scheme.

The following examples are provided to illustrate the ratio-type electrochemical sensing platform for vibrio detection and the preparation method and application thereof, but they should not be construed as limiting the scope of the present invention.