CN101196486A - Electrochemical sensor biomimetic molecular recognition nano-sensing film and preparation method thereof - Google Patents

Abstract

The invention relates to an electrochemical sensor biomimetic molecular recognition nanometer sensing film and a preparation method thereof. It is a sensing film with specific biomimetic molecular recognition ability for template molecules, characterized in that the sensing film is formed on the surface of the metal electrode of the electrochemical sensor, the thickness is controllable, and the thickness ranges from 5 to 50 nanometers. Membrane, the thickness of the sensing membrane is controlled by the reversible addition-chain transfer radical polymerization reaction time, and has a linear relationship with time. Its metal electrodes are made of gold or platinum. Preparation method: first derivatize the surface of the metal electrode with haloalkane, then perform molecular imprinting polymerization on the metal electrode whose surface is a chain transfer agent, perform molecular imprinting polymerization on the metal electrode whose surface is a chain transfer agent, and finally elute the biomimetic molecule It can be obtained by identifying the template molecules in the nanometer sensing film. The sensor prepared by the invention has high sensitivity and good stability, and will be widely used in metallurgy, chemical industry, environmental protection and other related fields.

Description

Electrochemical sensor biomimetic molecular recognition nano-sensing film and preparation method thereof

technical field

The invention belongs to the fields of bionic molecular recognition and electrochemical sensors, and more precisely relates to a bionic molecular recognition nano-sensing film of an electrochemical sensor and a preparation method thereof.

Background technique

Molecular recognition refers to the phenomenon that in a complex mixture system, one molecule or molecular fragment specifically recognizes another molecule or molecular fragment, and combines with each other to form a complex or supramolecule. The concept of molecular recognition is gradually developed from the research on biomolecules such as enzymes, receptors, and antibodies. It generally exists in various life activities, such as antigens and antibodies, protein hormones, plant lectins, exogenous There are molecular recognition phenomena between lectins or drugs and receptors, proteases and protein substrates, proteins and nucleic acids, so molecular recognition plays a vital role in life activities. At present, biomolecular recognition and related technologies have been widely used in clinical testing, drug analysis, new drug development, gene diagnosis, cancer treatment, and environmental assessment. Molecularly Imprinted Technology (MIT), which has developed in recent years with the advantages of many disciplines such as polymer synthesis, molecular design, molecular recognition, and bionic bioengineering, is based on the perspective of bionics, using artificial synthesis methods to obtain in-space and The binding site is completely matched with a specific molecule (imprinted molecule, template molecule), and has a polymer material that specifically binds to the specific molecule, thereby realizing biomimetic molecular recognition, that is, realizing biomimetic functions. The obtained molecularly imprinted polymers (Molecularly Imprinted Polymers, MIPs) not only have the same recognition performance and selectivity as natural antibodies, but also have the same high stability and resistance to harsh environments as polymers, so they are widely used in bionic sensors. , bioengineering, clinical medicine, environmental monitoring, food industry and many other fields have become one of the most promising new materials in the new century.

At present, the use of molecularly imprinted polymers as sensitive materials/recognition units of sensors—sensing membranes to prepare biomimetic sensors has become a research direction of common concern in the fields of molecularly imprinted and sensors, and has become a common research hotspot. The specific research content is to immobilize the molecularly imprinted polymer in the form of a film on the transducer surface of the sensor in an appropriate way, and then convert it into a measurable signal by various means such as electricity, heat, light, and mass for quantitative analysis of various compound. Among many sensors, electrochemical sensors are becoming more and more popular because of their unique advantages such as low power consumption, high sensitivity, stability and reliability, small size, fast detection speed, accuracy, portability, on-site direct detection and continuous detection. It has been widely used in metallurgy, chemical industry, environmental protection and other fields, and plays its own unique role in various measurements. The existing commonly used sensing film forming methods, such as pressure film method, spin coating method, etc., the thickness of the formed sensing film is mostly in the μm level, which seriously affects the mass transfer of target molecules (template molecules) inside the sensing film. Speed and equilibration time; the made sensing film is easy to fall off from the transducer of the sensor, which affects the stability of the sensor, which has limited the application and development of molecularly imprinted polymer bionic sensors. Therefore, directly synthesizing a molecularly imprinted nano-membrane (<50nm) on the surface of the metal electrode of the bionic sensor, increasing the mass transfer rate of the target molecule (template molecule) inside the molecularly imprinted polymer film, and reducing the equilibrium time have become the most promising in the field of bionic sensor research. One of the cutting edge hotspots. Although it has been reported in the literature that the graft copolymerization method is used to synthesize nano-films on the surface of the carrier (Sulitzly C., Ruckert B., Hall A.J.et al..Macromolecules, 2002, 35:79-91.), due to the graft copolymerization method Unable to get rid of the shortcomings of solution copolymerization and uneven film thickness, it has not been widely used. The key is that the bionic sensing film prepared by the traditional method has a large thickness, poor uniformity, uncontrollable thickness (generally up to micron level), and is easy to fall off from the surface of the metal electrode, which seriously affects the sensitivity, stability and application of the bionic sensor. Scope; recent surface modification methods still cannot get rid of the shortcomings of solution copolymerization and uneven film thickness, which severely limit the application of molecularly imprinted polymer biomimetic sensors.

Contents of the invention

The invention aims to provide a biomimetic molecular recognition nano-sensing film of an electrochemical sensor and a preparation method thereof, improve the sensitivity, stability and application range of the electrochemical sensor, and make up for the deficiencies of the prior art.

The technical idea of the present invention is to combine the molecular imprinting technology with the reversible addition-fragmentation chain transfer radical polymerization reaction, so that the "growth" on the surface of the metal electrode has bionic molecular recognition ability for imprinted molecules, and the thickness can be reversible. Addition-fragmentation chain transfer radical polymerization reaction time-controlled nano-sensing film, as an identification device of electrochemical sensor, is widely used in metallurgy, chemical industry, environmental protection and other fields.

Therefore, the biomimetic molecular recognition nano-sensing film of the present invention is a sensing film with a biomimetic molecular recognition ability specific to template molecules, and is characterized in that the sensing film is formed on the surface of a metal electrode of an electrochemical sensor, The film with a controllable thickness and a thickness ranging from 5 to 50 nm is only at the nanometer level.

The thickness of the sensing film of the present invention is controlled by the reversible addition-chain transfer radical polymerization reaction time, and has a linear relationship with time. The metal electrode is a metal electrode made of gold or platinum.

The preparation method of the present invention is as follows: first, active groups such as amino groups and alcohol groups are connected on the surface of the metal electrode of the electrochemical sensor, and the active groups are converted into haloalkanes as chain transfer agents for reversible addition-fragmentation chain transfer free radical polymerization. Under the action of the Grignard reagent, the alkyl halide is converted into a chain transfer agent, and then under the action of the initiator, a reversible addition-fragmentation chain transfer radical polymerization reaction (simultaneous molecular imprinting polymerization reaction occurs), on the surface of the metal electrode A biomimetic molecular recognition nano-sensing membrane containing template molecules is formed, and a biomimetic molecular recognition nano-sensing membrane with controllable thickness is obtained by elution of the template molecules.

The concrete method that the present invention prepares is as follows:

a. Haloalkane derivatization on the surface of the metal electrode:

(1) Put the metal electrode in an airtight glass vessel, soak it with Piranha solution (98% H ₂ SO ₄ : 30% H ₂ O ₂ =7: 3, volume ratio) at 90°C for 5 minutes, and then use Rinse with ultrapure water and absolute ethanol, blow dry with nitrogen; then place the metal electrode in another airtight glass vessel, soak it in the absolute ethanol solution of mercapto compound with a concentration of 0.01-10mmol/L for 2-48 hours, Take it out and wash it with absolute ethanol, and dry it with nitrogen gas to obtain a mercaptolated metal electrode;

(2) Put the above-mentioned thiolated metal electrode in a closed glass container, add 10-100 mL of dry dichloromethane, 10-500 μL of triethylamine, and 5-50 mg of dimethylaminopyridine in sequence, and cool the reaction vessel in an ice-water bath to 0-5°C to obtain System A; 10-500 μL of dibromodimethylpropyl bromide was dissolved in another 10-100 mL of dry dichloromethane, and added dropwise to System A; while maintaining ice bath conditions, continue stirring for 1 ~ 24 hours, then turn to room temperature and stir for 1 ~ 48 hours; take out the metal electrode, rinse it with absolute ethanol, dry it with nitrogen, and obtain a metal electrode derivatized with haloalkane;

b. Convert the haloalkane on the surface of the metal electrode into a chain transfer agent

Dissolve 5-50 mL of Grignard reagent in 10-100 mL of ultra-dry tetrahydrofuran, preheat the oil bath to 30-60 °C, add 100-1000 μL of dry carbon disulfide dropwise, and keep the oil bath at 50 °C for 1-24 hours; then add the above The metal electrode derivatized by haloalkane was passed through nitrogen for 10 minutes, and then reacted in an oil bath at 50°C under the protection of nitrogen for 1 to 48 hours; after the reaction, the metal electrode was washed three times with tetrahydrofuran, methanol, and acetone, and dried in vacuum at 60°C to obtain a chain Transfer agent derivatized metal electrodes;

c. Molecularly imprinted polymerization reaction is carried out on the metal electrode whose surface is a chain transfer agent

(1) Add a porogen into a sealed glass vessel, add functional monomers, template molecules, and a crosslinking agent in sequence, and stir to completely dissolve it to obtain a transparent solution B; add the above-mentioned metal electrode that has been derivatized by a chain transfer agent;

(2) In another airtight glass vessel, add the initiator into the porogen and stir to make it completely dissolved, then add the above-mentioned solution B and mix evenly to obtain solution C;

(3) The above solution C is blown with nitrogen for 1 to 60 minutes to remove oxygen and moisture; then place the airtight glass vessel in an oil bath at 50 to 120°C and stir for 1 to 72 hours; by controlling the length of time, the metal electrode can A biomimetic molecular recognition nano-sensing film containing template molecules with a thickness ranging from 5 to 50 nm is obtained on the surface;

d. Final elution of template molecules in the biomimetic molecular recognition nanosensing membrane

Rinse the metal electrode of the above-mentioned biomimetic molecular recognition nano-sensing film containing template molecules with absolute ethanol; then soak it in a mixed solution with a volume ratio of acetic acid/methanol of 10/1 to 1/10, and shake for 1 to 10 minutes. 72 hours; filter out the soaking solution, wash repeatedly with methanol until neutral, then dry in a vacuum oven at 50-120°C until constant weight, and finally obtain a metal electrode derivatized with a bionic molecular recognition nano-sensing film on the surface, that is, in A biomimetic molecular recognition nano-sensing film is obtained on the surface of the metal electrode. of which the above

Metal electrodes are: electrodes made of gold, platinum, etc.;

Mercapto compounds: 2-mercaptoethanol, 2-mercaptoethylamine, 2-mercaptopropanol, 2-mercaptopropylamine, mercapto-n-undecanol, mercapto-n-undecylamine;

Grignard reagents are: phenylmagnesium bromide (PMB), phenylmagnesium chloride (PMC);

The chain transfer agent is: dithioester compounds such as dithiobenzoate;

The porogens are: benzene and its derivatives, methyl halide, tetrahydrofuran, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile;

Template molecules are: amino acids and their derivatives, chiral compounds, drugs, pesticides and other compounds;

Functional monomers are: acrylic acid and its esters, vinylpyridines;

The crosslinking agent is: ethylene glycol dimethacrylate (EGDMA), divinylbenzene (DVB);

The initiator is: an azo initiator such as azobisisobutyronitrile (AIBN).

The electrochemical sensor biomimetic molecular recognition nano-sensing film of the present invention combines the biomimetic specific recognition of molecularly imprinted polymers and the controllability of reversible addition-fragmentation chain transfer radical polymerization technology, and is directly synthesized on the surface of metal electrodes. The molecularly imprinted nano-sensing film makes the thickness of the sensing film reach the nm level. The sensor prepared by the invention has high sensitivity and good stability, and will be widely used in metallurgy, chemical industry, environmental protection and other related fields.

Detailed ways

The biomimetic molecular recognition nano-sensing film of the present invention is a sensing film with a biomimetic molecular recognition ability specific to template molecules, and is characterized in that the sensing film is formed on the surface of a metal electrode of an electrochemical sensor and has a thickness of Controlled film with a thickness ranging from 5 to 50 nm, and the thickness is only at the nanometer level.

The thickness of the sensing film is controlled by the reversible addition-chain transfer radical polymerization reaction time, and has a linear relationship with time. The metal electrode is a metal electrode made of gold or platinum.

The specific method prepared by the present invention is further described in detail with the following four examples:

Example 1.

a. First derivatize the surface of the metal electrode with haloalkane

(1) Put the metal electrode in a closed glass vessel, soak it with Piranha solution (98%H ₂ SO ₄ : 30% H ₂ O ₂ =7:3, volume ratio) at 90°C for 5 minutes, and then use super Rinse with pure water and absolute ethanol, blow dry with nitrogen; then place the metal electrode in another airtight glass container, soak it in the absolute ethanol solution of 2-mercaptoethanol with a concentration of 0.1mmol/L for 48 hours, take it out with Rinse with absolute ethanol and dry with nitrogen to obtain a mercaptolated metal electrode;

(2) Put the above-mentioned thiolated metal electrode in a sealed glass vessel, add 10 mL of dry dichloromethane, 10 μL of triethylamine, and 5 mg of dimethylaminopyridine in sequence, and cool the reaction vessel to 0-5 °C in an ice-water bath. System A was obtained; 10 μL of dibromodimethylpropyl bromide was dissolved in another 10 mL of dry dichloromethane, and added dropwise to System A; the stirring was continued for 2 hours under ice-bath conditions, and then turned to room temperature for 6 hours; Take out the metal electrode, rinse it with absolute ethanol, and dry it with nitrogen to obtain a metal electrode derivatized with haloalkane;

b. Convert the haloalkane on the surface of the metal electrode into a chain transfer agent

Dissolve 5 mL of Grignard reagent in 10 mL of ultra-dry tetrahydrofuran, preheat the oil bath to 40 °C, add 100 μL of dry carbon disulfide dropwise, and maintain the oil bath at 50 °C for 2 hours; then add the metal electrode derivatized by the chain transfer agent precursor, Nitrogen was passed for 10 minutes, and then reacted in an oil bath at 50°C for 6 hours under the protection of nitrogen; after the reaction, the metal electrode was washed with tetrahydrofuran, methanol, and acetone three times in sequence, and dried in vacuum at 60°C to obtain a metal electrode derivatized with a chain transfer agent;

c. Molecularly imprinted polymerization reaction on the metal electrode whose surface is a chain transfer agent

(1) Add 20mL porogen tetrahydrofuran into a sealed glass vessel, add 1mmol of functional monomer methacrylic acid, 20mg of template molecule theophylline, and 5mmol of cross-linking agent EGDMA in sequence, stir to dissolve completely, and obtain transparent solution A; Metal electrodes derivatized with chain transfer agents;

(2) In another airtight glass vessel, the initiator AIBN was added into the porogen tetrahydrofuran and stirred to completely dissolve it, then solution A was added and mixed uniformly to obtain solution B;

(3) Nitrogen was passed through solution B for 10 minutes to remove oxygen and moisture; then the airtight glass vessel was placed in an oil bath at 60° C., and stirred for 6 hours; a biomimetic molecular recognition nano-sensing film containing template molecules was obtained on the surface of the metal electrode;

d. Elution of template molecules in biomimetic molecular recognition nanosensing membranes

The metal electrode derivatized by the biomimetic molecular recognition nano-sensing membrane containing template molecules prepared above was rinsed with absolute ethanol; then soaked in a mixed solution with a volume ratio of acetic acid/methanol of 10/1, and oscillated for 1 hour; filtered After removing the soaking solution, wash it repeatedly with methanol until it is neutral, and then dry it in a vacuum oven at 50°C to constant weight to obtain a metal electrode derivatized with a bionic molecular recognition nano-sensing film, which is obtained on the surface of the metal electrode of the electrochemical sensor. Biomimetic molecular recognition nano-sensing film.

The prepared electrochemical sensor is an electrochemical sensor derivatized with a biomimetic molecular recognition nano-sensing film on the surface of a metal electrode. The identification lower line (detection line) of Caffeine is 3nmol/L, and the linear range is 10-500nmol/L; compared with caffeine, its separation factor for theophylline is 6.92.

Example 2.

a. Haloalkane derivatization on the electrode surface

(1) Put the metal electrode in a closed glass vessel, soak it with Piranha solution (98%H ₂ SO ₄ : 30% H ₂ O ₂ =7:3, volume ratio) at 90°C for 5 minutes, and then use super Rinse with pure water and absolute ethanol, blow dry with nitrogen; then place the metal electrode in another airtight glass vessel, soak it in the absolute ethanol solution of 2-mercaptoethylamine with a concentration of 1mmol/L for 24 hours, take it out with Rinse with absolute ethanol, blow dry with nitrogen, and obtain the mercaptolated metal electrode; (2) Place the above-mentioned mercaptolated metal electrode in a closed glass vessel, add 20 mL of dry dichloromethane, 20 μL of triethylamine, and two Methylaminopyridine 10 mg, cooled the reaction vessel to 0-5°C in an ice-water bath to obtain System A; 20 μL of dibromodimethylpropyl bromide was dissolved in another 20 mL of dry dichloromethane, and added dropwise to System A; Stirring was continued for 6 hours while maintaining the ice bath, and then turned to room temperature and stirred for 12 hours; the metal electrode was taken out, rinsed with absolute ethanol, and dried with nitrogen to obtain a metal electrode derivatized with haloalkane;

b. Convert the haloalkane on the surface of the metal electrode into a chain transfer agent

Dissolve 10 mL of Grignard reagent in 20 mL of ultra-dry tetrahydrofuran, preheat the oil bath to 50 °C, add 200 μL of dry carbon disulfide dropwise, and maintain the oil bath at 50 °C for 6 hours; then add the metal electrode derivatized by the chain transfer agent precursor, Nitrogen was passed for 10 minutes, and then reacted in an oil bath at 50°C for 12 hours under the protection of nitrogen; after the reaction, the metal electrode was washed three times with tetrahydrofuran, methanol, and acetone in sequence, and dried in vacuum at 60°C to obtain a metal electrode derivatized with a chain transfer agent;

c. Molecularly imprinted polymerization reaction on the metal electrode whose surface is a chain transfer agent

(1) Add 20 mL of porogen dichloromethane into a sealed glass vessel, sequentially add 1 mmol of functional monomer 4-vinylpyridine, 20 mg of template molecule caffeine, and 5 mmol of cross-linking agent DVB, stir to dissolve completely, and obtain Transparent solution A; add metal electrodes derivatized with chain transfer agents;

(2) In another airtight glass vessel, the initiator AIBN was added into the porogen dichloromethane and stirred to make it completely dissolved, then added to solution A and mixed uniformly to obtain solution B;

(3) Nitrogen was passed through solution B for 20 minutes to remove oxygen and moisture; then the airtight glass vessel was placed in an oil bath at 60° C., stirred and reacted for 12 hours, and a biomimetic molecular recognition nano-sensing film containing template molecules was obtained on the surface of the metal electrode;

d. Elution of template molecules in biomimetic molecular recognition nanosensing membranes

Rinse the prepared metal electrode of the biomimetic molecular recognition nano-sensing film containing template molecules with absolute ethanol; then soak in a mixed solution with a volume ratio of acetic acid/methanol of 1/1, and shake for 5 hours; filter the soaking solution Afterwards, it was washed repeatedly with methanol until it was neutral, and then dried in a vacuum oven at 60°C to constant weight to obtain a metal electrode derivatized with a biomimetic molecular recognition nano-sensing film, that is, biomimetic molecular recognition was obtained on the surface of the metal electrode of the electrochemical sensor. Nano-sensing film.

The prepared electrochemical sensor is an electrochemical sensor derivatized with a biomimetic molecular recognition nano-sensing film on the surface of a metal electrode. The sensing film is a nano-sensing film with biomimetic molecular recognition ability for caffeine. The lower line of identification (detection line) was 1.5nmol/L, and the linear range was 5-500nmol/L; taking theophylline as the reference molecule, its separation factor for caffeine was 7.38.

Example 3.

a. Haloalkane derivatization on the surface of the metal electrode

(1) Put the metal electrode in a closed glass vessel, soak it with Piranha solution (98%H ₂ SO ₄ : 30% H ₂ O ₂ =7:3, volume ratio) at 90°C for 5 minutes, and then use super Rinse with pure water and absolute ethanol, blow dry with nitrogen; then place the metal electrode in another airtight glass vessel, soak it in the absolute ethanol solution of 5mmol/L mercapto n-undecanol for 12 hours, take it out with Rinse with absolute ethanol, and blow dry with nitrogen to obtain a mercaptolated metal electrode; (2) Place the above-mentioned mercaptolated metal electrode in a closed glass vessel, add 50 mL of dry dichloromethane, 200 μL of triethylamine, and two Methylaminopyridine 20 mg, cooled the reaction vessel to 0-5°C in an ice-water bath to obtain System A; 200 μL of dibromodimethylpropyl bromide was dissolved in another 50 mL of dry dichloromethane, and added dropwise to System A; Stirring was continued for 12 hours while maintaining the ice bath, and then turned to room temperature and stirred for 24 hours; the metal electrode was taken out, rinsed with absolute ethanol, and dried with nitrogen to obtain a metal electrode derivatized with haloalkane;

b. Convert the haloalkane on the surface of the metal electrode into a chain transfer agent

Dissolve 20 mL of Grignard reagent in 50 mL of ultra-dry tetrahydrofuran, preheat the oil bath to 50 °C, add 500 μL of dry carbon disulfide dropwise, and maintain the oil bath at 50 °C for 12 hours; then add the metal electrode derivatized by the chain transfer agent precursor, Nitrogen was passed for 10 minutes, and then reacted in an oil bath at 50°C under nitrogen protection for 24 hours; after the reaction, the metal electrode was washed with tetrahydrofuran, methanol, and acetone three times in sequence, and vacuum-dried at 60°C to obtain a metal electrode derivatized with a chain transfer agent;

c. Molecularly imprinted polymerization reaction on the metal electrode whose surface is a chain transfer agent

(1) Add 40 mL of porogen acetonitrile into a closed glass vessel, add 1 mmol of functional monomer 4-vinylpyridine, 50 mg of template molecule 4,4′-DDT, and 5 mmol of cross-linking agent EGDMA in sequence, and stir to dissolve it completely , to obtain a transparent solution A; add a metal electrode derivatized with a chain transfer agent;

(2) In another airtight glass vessel, the initiator AIBN was added into the porogen acetonitrile and stirred to make it completely dissolved, then solution A was added and mixed uniformly to obtain solution B;

(3) Nitrogen was passed through solution B for 30 minutes to remove oxygen and moisture; then the airtight glass vessel was placed in an oil bath at 50° C., stirred and reacted for 48 hours, and a biomimetic molecular recognition nano-sensing film containing template molecules was obtained on the surface of the metal electrode;

d. Elution of template molecules in biomimetic molecular recognition nanosensing membranes

The prepared metal electrode derivatized with biomimetic molecular recognition nano-sensing membrane containing template molecules was rinsed with absolute ethanol; then soaked in a mixed solution with a volume ratio of acetic acid/methanol of 1/5, and oscillated for 10 hours; filtered off After soaking in the solution, wash it repeatedly with methanol until it is neutral, and then dry it in a vacuum oven at 60°C until it reaches a constant weight to obtain a metal electrode derivatized with a biomimetic molecular recognition nano-sensing film, that is, a biomimetic sensor is obtained on the surface of the metal electrode of the electrochemical sensor. Molecular recognition nanosensing membrane.

The prepared electrochemical sensor is an electrochemical sensor derivatized with a biomimetic molecular recognition nano-sensing film on the surface of a metal electrode. The sensing film is a nano-sensing film that has the biomimetic molecular recognition ability for 4,4'-DDT, and its size and thickness are controlled at 15nm; The lower line of recognition (detection line) for 4,4'-DDT is 1nmol/L, and the linear range is 5-500nmol/L; with 2,4'-DDT as the reference molecule, its separation of 4,4'-DDT The factor is 4.16.

Example 4.

a. Haloalkane derivatization on the surface of the metal electrode

(1) Put the metal electrode in a closed glass vessel, soak it with Piranha solution (98%H ₂ SO ₄ : 30% H ₂ O ₂ =7:3, volume ratio) at 90°C for 5 minutes, and then use super Rinse with pure water and absolute ethanol, blow dry with nitrogen; then place the metal electrode in another airtight glass vessel, soak it in the absolute ethanol solution of 10mmol/L mercapto n-undecylamine for 6 hours, take it out with Rinse with absolute ethanol, blow dry with nitrogen, and obtain a mercaptolated metal electrode; (2) Place the above-mentioned mercaptolated metal electrode in a closed glass vessel, add 100 mL of dry dichloromethane, 500 μL of triethylamine, and two Methylaminopyridine 50 mg, cooled the reaction vessel to 0-5°C in an ice-water bath to obtain System A; 500 μL of dibromodimethylpropyl bromide was dissolved in another 100 mL of dry dichloromethane, and added dropwise to System A; Stirring was continued for 24 hours while maintaining the ice bath, and then turned to room temperature and stirred for 48 hours; the metal electrode was taken out, rinsed with absolute ethanol, and dried with nitrogen to obtain a metal electrode derivatized with haloalkane;

b. Convert the haloalkane on the surface of the metal electrode into a chain transfer agent

Dissolve 50 mL of Grignard reagent in 100 mL of ultra-dry tetrahydrofuran, preheat the oil bath to 60 °C, add 1000 μL of dry carbon disulfide dropwise, and maintain the oil bath at 50 °C for 24 hours; then add the metal electrode derivatized by the chain transfer agent precursor, Nitrogen was passed for 10 minutes, and then reacted in an oil bath at 50°C under nitrogen protection for 48 hours; after the reaction, the metal electrode was washed with tetrahydrofuran, methanol, and acetone three times in sequence, and dried in vacuum at 60°C to obtain a metal electrode derivatized with a chain transfer agent;

c. Molecularly imprinted polymerization reaction on the metal electrode whose surface is a chain transfer agent

(1) Add 40 mL of porogen toluene into a closed glass vessel, add 1 mmol of functional monomer 4-vinylpyridine, 20 mg of template molecule 3-chlorophenol, and 5 mmol of cross-linking agent cross-linking agent EGDMA in sequence, and stir to completely dissolve it to obtain Transparent solution A; add metal electrodes derivatized with chain transfer agents;

(2) In another airtight glass vessel, the initiator AIBN was added into the porogen toluene and stirred to make it completely dissolved, then solution A was added and mixed uniformly to obtain solution B;

(3) Nitrogen was passed through solution B for 60 minutes to remove oxygen and moisture; then the airtight glass vessel was placed in an oil bath at 100° C., stirred and reacted for 72 hours, and a biomimetic molecular recognition nano-sensing film containing template molecules was obtained on the surface of the metal electrode;

d. Elution of template molecules in biomimetic molecular recognition nanosensing membranes

The prepared metal electrode derivatized with the biomimetic molecular recognition nano-sensing membrane containing template molecules was rinsed with absolute ethanol; then soaked in a mixed solution with a volume ratio of acetic acid/methanol of 1/10, and oscillated for 12 hours; filtered off After soaking in the solution, wash it repeatedly with methanol until it is neutral, and then dry it in a vacuum oven at 60°C until it reaches a constant weight to obtain a metal electrode derivatized with a biomimetic molecular recognition nano-sensing film, that is, a biomimetic sensor is obtained on the surface of the metal electrode of the electrochemical sensor. Molecular recognition nanosensing membrane.

The prepared electrochemical sensor is an electrochemical sensor derivatized with a biomimetic molecular recognition nano-sensing film on the surface of a metal electrode. The identification lower line (detection line) of -chlorophenol is 0.6nmol/L, and the linear range is 3-500nmol/L; taking 5-chlorophenol as a comparison molecule, its separation factor for 3-chlorophenol is 8.23.

Claims (7)

1. bionic molecular recognition nano sensing film of electrochemical transducer, be the sensing membrane that has the single-minded bionical molecule distinguishability of template molecule, it is characterized in that this sensing membrane is that to be created on the surface of metal electrode of electrochemical sensor, controllable thickness, thickness range be the film of 5～50nm.

2. bionic molecular recognition nano sensing film of electrochemical transducer as claimed in claim 1 is characterized in that described metal electrode is is the metal electrode of material with gold or platinum.

3. bionic molecular recognition nano sensing film of electrochemical transducer as claimed in claim 1, the thickness that it is characterized in that sensing membrane are by reversible addition-chain transfer Raolical polymerizable time control, and linear with the time.

4. the preparation method of a bionic molecular recognition nano sensing film of electrochemical transducer comprises following a-d step:

A. surface of metal electrode being carried out alkyl halide derives:

(1) metal electrode is placed airtight glassware, with Piranha solution (98%H ₂SO ₄: 30%H ₂O ₂=7: 3, volume ratio) under 90 ℃ of conditions, soaked 5 minutes, use ultrapure water, absolute ethyl alcohol drip washing then successively, nitrogen dries up; Metal electrode is placed another airtight glassware again, soaked 2～48 hours in concentration is the ethanol solution of sulfhydryl compound of 0.01～10mmol/L, take out with absolute ethyl alcohol drip washing totally, nitrogen dries up, and obtains the metal electrode of sulfhydrylation;

(2) metal electrode with above-mentioned sulfhydrylation places airtight glassware, adds dry methylene chloride 10～100mL successively, triethylamine 10～500 μ L, and dimethyl aminopyridine 5～50mg, ice-water bath is cooled to 0～5 ℃ with reaction vessel, obtains system A; Dibromo dimethyl propyl acylbromide 10～500 μ L are dissolved in the methylene chloride of other 10～100mL drying, dropwise add among the system A; Keep and continue under the condition of ice bath to stir 1～24 hour, transferred stirring at room afterwards to 1～48 hour; It is clean with absolute ethyl alcohol drip washing to take out metal electrode, and nitrogen dries up, and obtains the metal electrode of alkyl halide derivatization;

B. the alkyl halide with above-mentioned surface of metal electrode is converted into chain-transferring agent

5～50mL Grignard reagent is dissolved in the super dry tetrahydrofuran of 10～100mL, and oil bath is preheated to 30～60 ℃, dropwise adds dry carbon disulphide 100～1000 μ L, keeps 50 ℃ of reactions of oil bath 1～24 hour; The metal electrode that adds above-mentioned alkyl halide derivatization afterwards, logical nitrogen 10 minutes, the following 50 ℃ of oil baths reaction of nitrogen protection afterwards 1～48 hour; Reaction finishes the back metal electrode and uses tetrahydrofuran successively, and methyl alcohol, acetone respectively wash 3 times, and 60 ℃ of vacuum drying obtain the metal electrode of chain-transferring agent derivatization;

C. be that the metal electrode of chain-transferring agent carries out the molecular engram polyreaction to above-mentioned surface:

(1) add pore-foaming agent in the airtight glassware, add function monomer, template molecule, crosslinking chemical successively, stirring makes it to dissolve fully, obtains the transparence solution B; The metal electrode that adds the above-mentioned derivatization of chain-transferring agent;

(2) in the airtight glassware of another one, initiating agent added to stir in the pore-foaming agent and make it to dissolve fully, add above-mentioned solution B then and mix, obtain solution C;

(3) above-mentioned solution C is led to nitrogen 1～60 minute, remove deoxidation and moisture; Then airtight glassware is placed 50～120 ℃ oil bath, stirring reaction 1～72 hour; Can on surface of metal electrode, obtain the bionic molecular recognition nano sensing film that contains template molecule that thickness range is 5～50nm by control time length;

D. the template molecule in the last wash-out bionic molecular recognition nano sensing film:

With the metal electrode of the bionic molecular recognition nano sensing film that contains template molecule of above-mentioned preparation, clean with absolute ethyl alcohol drip washing; Be that 10/1～1/10 mixed solution soaks with acetate/methyl alcohol volume ratio then, vibrated 1～72 hour; Use the methyl alcohol cyclic washing until neutrality after the elimination soak solution, then in 50～120 ℃, dry to constant weight with vacuum drying chamber, obtain the metal electrode of surface biomimetic molecular recognition nano sensing film derivatization at last, promptly obtain bionic molecular recognition nano sensing film at surface of metal electrode.

5. as claim 1 or 4 described bionic molecular recognition nano sensing film of electrochemical transducer, it is characterized in that described template molecule is one of them of theophylline, caffeine, chlorophenol or DDT molecule.

6. bionic molecular recognition nano sensing film of electrochemical transducer as claimed in claim 4 is characterized in that described chain-transferring agent is the dithiobenzoic acid ester.

7. bionic molecular recognition nano sensing film of electrochemical transducer is applied on the surface of metal electrode of electrochemical sensor.