1. A preparation process of an acupuncture needle imprinting electrochemical sensor for detecting dopamine is characterized by comprising the following steps:

step (1), pretreatment of the acupuncture needle electrode:

ultrasonically cleaning the needle tip of the stainless steel acupuncture needle electrode by using ethanol and ultrapure water in sequence, and drying by using nitrogen for later use;

step (2), modification of gold nanoparticles:

immersing the treated acupuncture needle electrode into HAuCl with stirrer₄In the aqueous solution, a gold nanoparticle modification layer is electrodeposited on the surface of the needle tip of the acupuncture needle by adopting a cyclic voltammetry method to obtain an acupuncture needle electrode with the surface covered with gold nanoparticles;

step (3), modification of boric acid:

immersing the acupuncture needle electrode with the gold nanoparticles covered on the surface into a boric acid group solution containing sulfydryl, taking out and airing to obtain a boric acid modified acupuncture needle electrode;

step (4), modification of the polymer film:

in the presence of a buffer solution, mixing a dopamine solution and an acidic chrome blue K solution to obtain a polymerization solution; then immersing the boric acid modified acupuncture needle electrode into the polymerization solution, and synthesizing a molecularly imprinted polymer film on the surface of the acupuncture needle electrode by using an electropolymerization method;

and (5) eluting the template: and immersing the acupuncture needle electrode modified by the molecular imprinting membrane into a sulfuric acid solution to elute dopamine.

2. The process according to claim 1, wherein the HAuCl is present in the step (2)₄The concentration of the aqueous solution is 1-10 mmol/L.

3. The process according to claim 1, wherein the deposition voltage of the cyclic voltammetry in step (2) is from-1.5V to 0.5V; the number of deposition turns is 3-7 turns; the deposition rate is 10mV/s to 50 mV/s.

4. The process according to claim 1, wherein the solution of the boronic acid group having a mercapto group in the step (3) is a 4-mercaptophenylboronic acid solution having a concentration of 2.5mg/mL to 10mg/mL and a soaking time of 1 to 12 hours.

5. The preparation process according to claim 1, wherein the molar ratio of dopamine to acid chrome blue K in the polymerization solution in the step (4) is 1: 1-1: 5.

6. the process according to claim 1, wherein the pH of the polymerization solution in the step (4) is in the range of 4.5 to 6.0.

7. The process according to claim 1, wherein the step (4) comprises electropolymerizing the molecularly imprinted polymer film by cyclic voltammetry, wherein the voltage is in the range of-0.4V to 1.0V, the number of polymerization cycles is 15 to 25 cycles, and the polymerization rate is 25mV/s to 75 mV/s.

8. An acupuncture needle imprinting electrochemical sensor for detecting dopamine is characterized by comprising a working electrode, a reference electrode and a counter electrode, wherein a substrate electrode of the working electrode is a stainless steel acupuncture needle electrode, a layer of gold nano material is modified on the surface of the stainless steel acupuncture needle electrode, a boric acid group with a sulfhydryl group is modified on the surface of the electrode through the high affinity of an Au-S bond, then a polymer film modified electrode is obtained by electropolymerization in a buffer solution containing acid chrome blue K and dopamine by adopting a cyclic voltammetry method, and finally the dopamine is eluted in a sulfuric acid medium to obtain the acupuncture needle imprinting electrochemical sensor with dual molecular recognition performance.

9. The electrochemical sensor according to claim 8, wherein the boronic acid group having a thiol group is 4-mercaptophenylboronic acid.

Background

Dopamine is one of the important catecholamine neurotransmitter molecules widely distributed in the nervous central system of mammals, participates in the work of the brain and the circulation of body fluid of a human body, and plays a vital role in regulating the central nerve, hormone and cardiovascular system. The health of human beings is affected by too high and too low dopamine content, the nerve degeneration diseases such as Alzheimer disease, schizophrenia, epilepsy, Parkinson syndrome and the like can be caused by too low dopamine content in the body, and the brain is excited or anxious for a long time and the body is damaged by too high dopamine concentration. Therefore, the detection of the dopamine content has important significance for the health of human beings and the diagnosis and treatment of diseases.

Currently, a plurality of methods for detecting dopamine exist, and the methods mainly include a gas chromatography method, a fluorescence method, a liquid chromatography method, a capillary electrophoresis method and the like. Although these methods have the advantages of low detection limit, the disadvantages of expensive instrument, expensive operation cost, uneasy carrying and incapability of on-line detection are common, so that they have limitations in practical application. In comparison, the electrochemical method is receiving more and more attention due to the advantages of simple instrument, fast detection speed, low price, easy carrying, and on-line monitoring.

Electrochemical detection of dopamine is very susceptible to interference from other electroactive molecules, such as uric acid and ascorbic acid. In order to eliminate the interference of co-existing species, molecular imprinting techniques have been used to design and construct sensors capable of specifically recognizing target molecules. The molecular imprinting technology takes target molecules as templates, takes proper substances as functional monomers, the templates and the functional monomers form polymers through covalent bonds or non-covalent bonds, and cavities matched with the spatial structures of the template molecules are left in the polymers after the template molecules are eluted from the polymers. Therefore, the molecular imprinting technology can improve the selectivity of dopamine detection.

Disclosure of Invention

The invention aims to provide a preparation process of an acupuncture needle imprinting electrochemical sensor for detecting dopamine, wherein a working electrode substrate of the sensor is a stainless steel acupuncture needle electrode, and the sensor has the advantages of low price and simple and convenient operation and is widely applied to the medical field. The electrochemical sensor has high specific recognition capability and excellent anti-interference capability on DA, and the preparation method is simple.

In order to achieve the purpose, the invention adopts the technical scheme that:

step (1), pretreatment of the acupuncture needle electrode:

ultrasonically cleaning the needle tip of the stainless steel acupuncture needle electrode by using ethanol and ultrapure water in sequence, and drying by using nitrogen for later use;

step (2), modification of gold nanoparticles:

immersing the treated acupuncture needle electrode into HAuCl with stirrer₄In the aqueous solution, a gold nanoparticle modification layer is electrodeposited on the surface of the needle tip of the acupuncture needle by adopting a cyclic voltammetry method to obtain an acupuncture needle electrode with the surface covered with gold nanoparticles;

step (3), modification of boric acid:

immersing the acupuncture needle electrode with the gold nanoparticles covered on the surface into a boric acid group solution containing sulfydryl, taking out and airing to obtain a boric acid modified acupuncture needle electrode;

step (4), modification of the polymer film:

in the presence of a buffer solution, mixing a dopamine solution and an acidic chrome blue K solution to obtain a polymerization solution; then immersing the boric acid modified acupuncture needle electrode into the polymerization solution, and synthesizing a molecularly imprinted polymer film on the surface of the acupuncture needle electrode by using an electropolymerization method;

and (5) eluting the template: immersing the acupuncture needle electrode modified by the molecular imprinting membrane into a sulfuric acid solution to elute dopamine;

the HAuCl of the preparation step (2)₄The concentration of the aqueous solution is 1-10 mmol/L;

the deposition voltage of the cyclic voltammetry in the preparation step (2) is-1.5V-0.5V; the number of deposition turns is 3-7 turns; the deposition rate is 10mV/s to 50 mV/s.

The boric acid group solution with sulfydryl in the preparation step (3) is a 4-mercaptophenylboronic acid solution, the concentration is 2.5mg/mL-10mg/mL, and the soaking time is 1h-12 h.

In the polymerization solution obtained in the preparation step (4), the molar ratio of dopamine to acidic chrome blue K is 1: 1-1: 5.

in the polymerization liquid in the preparation step (4), the pH range of the polymerization liquid is 4.5-6.0.

The preparation step (4) adopts a cyclic voltammetry electropolymerization molecular imprinting polymer film, the voltage range is-0.4V-1.0V, the polymerization cycle number is 15-25 cycles, and the polymerization rate is 25mV/s-75 mV/s.

The invention also aims to provide an acupuncture needle imprinting electrochemical sensor for detecting dopamine, which comprises a working electrode, a reference electrode and a counter electrode, wherein a substrate electrode of the working electrode is a stainless steel acupuncture needle electrode, a layer of gold nano material is modified on the surface of the stainless steel acupuncture needle electrode, a boric acid group with a sulfhydryl group is modified on the surface of the electrode through the high affinity of an Au-S bond, then a polymer film modified electrode is obtained by electropolymerization in a buffer solution containing acid chrome blue K and dopamine by adopting a cyclic voltammetry method, and finally the dopamine is eluted in a sulfuric acid medium to obtain the acupuncture needle imprinting electrochemical sensor with double molecular recognition performance.

The invention has the following beneficial effects:

the invention adopts the stainless steel acupuncture needle as the matrix, and has the advantages of simple manufacture, low cost, small volume and high sensitivity.

The invention utilizes the high affinity of Au-S bond to soak the acupuncture needle covered with gold nano-particles in the 4-mercaptophenylboronic acid solution, and provides a method which is simple and convenient to operate and modifies the boric acid groups on the surfaces of the gold nano-particles. The boric acid group can be covalently combined with dopamine, and the dopamine can be rapidly recognized.

According to the invention, the acid chrome blue K is used as a template molecule, and the good adsorption effect of the acid chrome blue K on dopamine is utilized, so that an accurate imprinting site can be formed. In addition, the acid chrome blue K has an oxidation electrocatalysis effect on dopamine, oxidation peak electricity of dopamine and interferents (such as ascorbic acid) can be separated, the acid chrome blue K has good anti-interference capability, and dopamine detection in the presence of the interferents is realized.

The invention provides a preparation method of a sensor which has quick response to dopamine molecules, high sensitivity and good molecular recognition capability.

Drawings

FIG. 1 is a graph of electrochemical performance of various materials prepared in example 1. Wherein a is a bare acupuncture needle electrode, b is an acupuncture needle electrode modified by gold nanoparticles, c is an acupuncture needle electrode modified by gold nanoparticles and boric acid, d is an acupuncture needle electrode modified after electropolymerization, and e is an acupuncture needle electrode after elution.

Fig. 2 is a cyclic voltammogram of the electrodes prepared in example 1 and comparative example 1. Wherein A is before the elution of the blotting electrode, B is after the elution of the blotting electrode, C is before the elution of the non-blotting electrode, and D is after the elution of the non-blotting electrode.

Fig. 3 is a graph showing differential impulse responses of the needle-blot electrochemical sensor of example 1 to different concentrations of dopamine in pH 7.4 phosphate buffer. The concentration of the curves a-r corresponding to dopamine is as follows in sequence: 5X 10^-7，1×10^-6，2.5×10^-6，5×10^-6，1×10^-5，2×10^-5，4×10^-5，6×10^-5，8×10^-5，1×10^-4，2×10^-4，4×10^-4，6×10^-4，8×10^-4，1×10^-3mol/L。

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Example 1

(1) Pretreatment of the acupuncture needle electrode: ultrasonically cleaning the needle tip of the acupuncture needle electrode for 300s by using ethanol and ultrapure water in sequence, and drying the needle tip by using nitrogen for later use.

(2) Modification of gold nanoparticles: taking the treated acupuncture needle electrode as a working electrode, a platinum wire electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and 8mmol/L HAuCl₄In the aqueous solution as electrolyte, cyclic voltammetry is adopted, and the electrodeposition range is-1.5-0.5V; the electrodeposition rate is 25 mv/s; the number of the electro-deposition circles is 5, and the acupuncture needle electrode with the needle point covered with the gold nanoparticles is obtained.

(3) Modification of boronic acid groups: and (3) immersing the needle tip of the acupuncture needle electrode with the surface covered with the gold nanoparticles into 5mg/mL 4-mercaptophenylboronic acid solution for 6h, taking out and drying to obtain the acupuncture needle electrode modified by the gold nanoparticles and the boric acid group film.

(4) Modification of polymer film: and (2) taking 10mL of acetic acid-sodium acetate (pH 5.2) containing 3mmol/L of acid chrome blue K and 1mmol/L of dopamine as a polymerization base solution, immersing the acupuncture needle modified with the gold nano-particles and the boric acid groups into the polymerization solution, electropolymerizing by adopting a cyclic voltammetry, and circularly scanning for 20 circles at a scanning speed of 50mv/s within a potential range of-0.4-1.0V to obtain the polymer film modified electrode.

(5) And (3) template elution: and (3) immersing the polymer membrane modified electrode in a 0.5mol/L sulfuric acid solution for 5h, and eluting the template molecule dopamine to obtain the working electrode of the electrochemical sensor based on the molecular imprinting.

Example 2

(1) Pretreatment of the acupuncture needle electrode: ultrasonically cleaning the needle tip of the acupuncture needle electrode for 300s by using ethanol and ultrapure water in sequence, and drying the needle tip by using nitrogen for later use.

(2) Modification of gold nanoparticles: taking the treated acupuncture needle electrode as a working electrode, a platinum wire electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and 1mmol/L HAuCl₄In the aqueous solution as electrolyte, cyclic voltammetry is adopted, and the electrodeposition range is-1.5-0.5V; the electrodeposition rate is 10 mv/s; the number of the electro-deposition circles is 3, and the acupuncture needle electrode with the needle point covered with the gold nanoparticles is obtained.

(3) Modification of boronic acid groups: and (3) immersing the needle tip of the acupuncture needle electrode with the surface covered with the gold nanoparticles into a 2.5mg/mL 4-mercaptophenylboronic acid solution for 12h, taking out and drying in the air to obtain the acupuncture needle electrode modified by the gold nanoparticles and the boric acid group film.

(4) Modification of polymer film: and (2) taking 10mL of acetic acid-sodium acetate (pH 4.5) containing 1mmol/L of acid chrome blue K and 1mmol/L of dopamine as a polymerization base solution, immersing the acupuncture needle modified with the gold nano-particles and the boric acid groups into the polymerization solution, electropolymerizing by adopting a cyclic voltammetry, and circularly scanning for 15 circles at a scanning speed of 25mv/s within a potential range of-0.4-1.0V to obtain the polymer film modified electrode.

(5) And (3) template elution: and (3) immersing the polymer membrane modified electrode in a 0.5mol/L sulfuric acid solution for 5h, and eluting the template molecule dopamine to obtain the working electrode of the electrochemical sensor based on the molecular imprinting.

Example 3

(1) Pretreatment of the acupuncture needle electrode: ultrasonically cleaning the needle tip of the acupuncture needle electrode for 300s by using ethanol and ultrapure water in sequence, and drying the needle tip by using nitrogen for later use.

(2) Modification of gold nanoparticles: taking the treated acupuncture needle electrode as a working electrode, a platinum wire electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and 4mmol/L HAuCl₄In the aqueous solution as electrolyte, cyclic voltammetry is adopted, and the electrodeposition range is-1.5-0.5V; the electrodeposition rate was 50 mv/s; the number of the electro-deposition circles is 7, and the acupuncture needle electrode with the needle point covered with the gold nanoparticles is obtained.

(3) Modification of boronic acid groups: and (3) immersing the needle tip of the acupuncture needle electrode with the surface covered with the gold nanoparticles into a 10mg/mL 4-mercaptophenylboronic acid solution for 1h, taking out and drying to obtain the acupuncture needle electrode modified by the gold nanoparticles and the boric acid group film.

(4) Modification of polymer film: and (2) taking 10mL of acetic acid-sodium acetate (pH is 6.0) containing 5mmol/L of acid chrome blue K and 1mmol/L of dopamine as a polymerization base solution, immersing the acupuncture needle modified with the gold nano-particles and the boric acid groups into the polymerization base solution, electropolymerizing by adopting a cyclic voltammetry, and circularly scanning for 25 circles at a sweeping speed of 75mv/s within a potential range of-0.4-1.0V to obtain the polymer film modified electrode.

(5) And (3) template elution: and (3) immersing the polymer membrane modified electrode in a 0.5mol/L sulfuric acid solution for 5h, and eluting the template molecule dopamine to obtain the working electrode of the electrochemical sensor based on the molecular imprinting.

Comparative example 1

(1) Pretreatment of the acupuncture needle electrode: ultrasonically cleaning the needle tip of the acupuncture needle electrode for 300s by using ethanol and ultrapure water in sequence, and drying the needle tip by using nitrogen for later use.

(2) Modification of gold nanoparticles: taking the treated acupuncture needle electrode as a working electrode, a platinum wire electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and 8mmol/L HAuCl₄In the aqueous solution as electrolyte, cyclic voltammetry is adopted, and the electrodeposition range is-1.5-0.5V; the electrodeposition rate is 25 mv/s; the number of the electro-deposition circles is 5, and the acupuncture needle electrode with the needle point covered with the gold nanoparticles is obtained.

(3) Modification of boronic acid groups: and (3) immersing the needle tip of the acupuncture needle electrode with the surface covered with the gold nanoparticles into 5mg/mL 4-mercaptophenylboronic acid solution for 6h, taking out and drying to obtain the acupuncture needle electrode modified by the gold nanoparticles and the boric acid group film.

(4) Modification of polymer film: and (2) taking 10mL of acetic acid-sodium acetate (pH is 5.2) containing 3mmol/L of acid chrome blue K as a polymerization base solution, immersing the acupuncture needle modified with the gold nano and the boric acid group into the polymerization base solution, electropolymerizing by adopting a cyclic voltammetry, and performing cyclic scanning for 20 circles at a scanning speed of 50mv/s within a potential range of-0.4-1.0V to obtain the polymer film modified electrode.

(5) And (3) template elution: and (3) immersing the polymer membrane modified electrode in a 0.5mol/L sulfuric acid solution for 5h, and eluting the template molecule dopamine to obtain the working electrode of the electrochemical sensor based on the molecular imprinting.

Test example 1

In a medium containing 1mmol/L K₃[Fe(CN)₆]^3-/4-The bare needle electrode (a), the gold nanoparticle-modified needle electrode (b) (the modified electrode prepared in the step (2) of example 1), the gold nanoparticle-and boric acid-modified needle electrode (c) (the modified electrode prepared in the step (3) of example 1), the electropolymerized modified electrode (d) (the modified electrode prepared in the step (4) of example 1), and the eluted modified electrode (e) (the modified electrode prepared in the step (5) of example 1) were used as the industrial needle electrode (a), the gold nanoparticle-modified needle electrode (b) (the modified electrode prepared in the step (2) of example 1), the gold nanoparticle-and boric acid-modified needle electrode (c) (the modified electrode prepared in the step (3) of example 1), the electropolymerized modified electrode (d), and the eluted modified electrode (e) (the modified electrode prepared in the step (5) of example 1), respectivelyThe electrode is used as a reference electrode, the saturated calomel electrode is used as a counter electrode, and the electrochemical performance of the prepared electrode is detected by using cyclic voltammetry. As shown in FIG. 1, the current value of the acupuncture needle electrode (c) modified with gold nanoparticles and boric acid was relatively large and larger than that of the acupuncture needle electrode (b) modified with gold nanoparticles, indicating that boric acid groups had been modified. In the subsequent electropolymerization process, the current value of the modified electrode (d) after electropolymerization is smaller, which indicates that the molecularly imprinted membrane is completely electropolymerized on the surface of the acupuncture needle electrode (c) modified by gold nano-particles and boric acid. During the elution process, as the template molecule dopamine is eluted, the molecularly imprinted membrane develops holes, and a current value is detected, which indicates that the molecularly imprinted electrochemical sensor (e) is obtained.

Test example 2

In 0.05mol/L Phosphate (PBS) solution with pH 7.4, before eluting with imprinted electrode (A) (modified electrode prepared in step (4) of example 1), after eluting with imprinted electrode (B) (modified electrode prepared in step (5) of example 1), before eluting with non-imprinted electrode (C) (modified electrode prepared in step (4) of comparative example 1), after eluting with non-imprinted electrode (D) (modified electrode prepared in step (5) of comparative example 1) were used as working electrodes, saturated calomel electrode was used as reference electrode, platinum wire electrode was used as counter electrode, and 1 × 10 detection was performed by circulation method^-4The scanning potential of a current signal of mol/L dopamine is-0.2-0.8V. As shown in fig. 2, there was no significant redox peak before elution of the imprinted electrode, since the electrode surface was covered by the imprinted polymer film, preventing electron transfer to the electrode surface. After elution, an obvious redox peak appears, which provides a plurality of electroactive surface sites and effectively promotes the electron transfer between the electrode and the dopamine after the dopamine is eluted, so that the dopamine generates redox on the surface of the electrode to generate an obvious electric signal; and the change before and after the non-imprinted electrode is eluted is not obvious, because the non-imprinted electrode is not added with dopamine in the polymerization process, and no electroactive site is generated after the non-imprinted electrode is eluted.

Test example 3

The molecularly imprinted electrochemical sensor prepared in example 1 was used as a working electrodeThe saturated calomel electrode is a reference electrode, the platinum wire electrode is a counter electrode, and the 5 multiplied by 10 is performed in 0.05mol/L PBS solution with pH 7.4 by adopting differential pulse voltammetry^-7～1.0×10^-3And detecting electrochemical signals of dopamine in a mol/L range. As shown in FIG. 3, the oxidation peak current was significantly increased with increasing dopamine concentration.

The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.