Dopamine photothermal enzyme electrode modified with magnetic nanoparticles, preparation method and application

Abstract

The invention discloses a dopamine photothermal enzyme electrode modified with magnetic nanoparticles, a preparation method and an application thereof. First, chitosan-coated Fe3O4 nanoparticles are synthesized, and then gold nanoparticles are deposited. The obtained magnetic nanoparticles Fe3O4@CS-Au have excellent biocompatibility and conductivity; mercaptopropionic acid is used to covalently bind laccase on the surface of Fe3O4@CS-Au nanoparticles, and then the immobilized laccase is drop-coated on a glassy carbon electrode to prepare a dopamine photothermal sensor enzyme electrode modified with magnetic nanoparticles. Under light irradiation, the surface temperature of the magnetic nanoparticles increases, which can increase the catalytic activity of laccase in oxidizing dopamine, thereby increasing the response current of the laccase-modified electrode. Compared with the prior art, the enzyme electrode modified with magnetic nanoparticles of the present invention has the advantages of simple preparation method and mild conditions; the enzyme electrode has the advantages of wide detection range, high sensitivity, good stability, etc., and the enzyme electrode can function efficiently under light conditions, expanding its application in the fields of medical detection, food safety, etc.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to a dopamine photothermal enzyme electrode modified with magnetic nanoparticles, a preparation method and an application thereof. Background Art

[0002] Dopamine is a monoamine substance that is produced in the human brain and is one of the most important neurotransmitters in the human central nervous system. It has the functions of regulating cardiovascular, kidney, human metabolism, central nervous system of the body, and stimulates emotional responses in the human brain. Detecting dopamine levels in human blood is very important for diagnosing various neurological diseases and other psychological studies. However, when electrochemical methods are used to directly detect dopamine in urine, they face some difficulties, such as: during the electrochemical detection of dopamine, the substrate can undergo a secondary reaction to produce an insulating polymer product, polydopamine, which leads to inactivation of the electrode surface due to this scaling effect; slow electron transfer, proton interaction between dopamine and the bare electrode, and interference of uric acid and ascorbic acid on dopamine reduce the sensitivity of the working electrode to dopamine detection.

[0003] In order to enhance the high sensitivity and selectivity of the electrode to dopamine, the working electrode modification material should have high conductivity and excellent electrocatalytic performance. Due to the excellent electrocatalytic activity of the modification material, the chemically modified electrode improves the detection signal of dopamine detection. However, the selectivity of the chemically modified electrode for the detection substrate needs to be further improved. Summary of the invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes a dopamine photothermal enzyme electrode modified with magnetic nanoparticles, a preparation method and an application. The preparation method is simple and efficient, and the conditions are mild. The dopamine photothermal enzyme electrode has a wide detection limit, high sensitivity and good stability, and can function efficiently under light conditions.

[0005] The preparation method of the magnetic nanoparticle-modified dopamine photothermal enzyme electrode proposed in the present invention comprises the following steps:

[0006] S1: Preparation of functionalized magnetic Fe3O4@CS-Au nanoparticles modified with mercaptopropionic acid;

[0007] S2: Preparation of dopamine photothermal enzyme electrode

[0008] The functionalized magnetic Fe3O4@CS-Au nanoparticles prepared by S1 were mixed with the laccase solution and heated in a water bath. The mixed solution was then drop-coated on the electrode surface to obtain a dopamine photothermal enzyme electrode.

[0009] Preferably, the preparation method of the mercaptopropionic acid-modified magnetic Fe3O4@CS-Au nanoparticles in S1 comprises the following steps:

[0010] S11: dispersing magnetic Fe3O4 nanoparticles in distilled water, adding chitosan and a cross-linking agent to react, and washing and separating the product to obtain magnetic Fe3O4@CS nanoparticles;

[0011] S12: mixing hydrated gold trichloride and the magnetic Fe3O4@CS nanoparticles in S11 in water, heating, and then adding a reducing agent to react. After the reaction, the product is washed and separated to obtain magnetic Fe3O4@CS-Au magnetic nanoparticles;

[0012] S13: The magnetic nanoparticles Fe3O4@CS-Au in S12 are dispersed in an ethanol solution of mercaptopropionic acid to obtain Fe3O4@CS-Au-COOH nanoparticles, and then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added for activation reaction. The product is washed and separated to obtain functionalized magnetic Fe3O4@CS-Au nanoparticles.

[0013] Preferably, the molar ratio of magnetic Fe3O4 nanoparticles, chitosan and cross-linking agent in S11 is 5:(1-3):(4-6).

[0014] Preferably, the concentration of the magnetic Fe3O4 nanoparticles is 1-5 g / L.

[0015] Preferably, the cross-linking agent in S11 is an aqueous solution of glutaraldehyde.

[0016] Preferably, the reaction temperature in S11 is 10-80° C., and the reaction time is 0.5-5 h.

[0017] Preferably, the mass ratio of magnetic Fe3O4@CS nanoparticles, hydrated gold trichloride and reducing agent in S12 is 20:(1-2):(2-6).

[0018] Preferably, the reducing agent in S12 is one of sodium citrate, ascorbic acid and sodium borohydride.

[0019] Preferably, the reaction temperature in S12 is 80-100° C., and the reaction time is 15-60 min.

[0020] Preferably, the mass ratio of magnetic nanoparticles Fe3O4@CS-Au and mercaptopropionic acid in S13 is 500-5000:8.

[0021] Preferably, the mass ratio of Fe3O4@CS-Au-COOH nanoparticles, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in S13 is 1:4-16:6-24.

[0022] Preferably, the activation reaction is carried out under stirring at 20-40° C. for 20-60 min.

[0023] Preferably, the water bath heating temperature in S2 is 20-40° C. and the heating time is 1-4 h.

[0024] Preferably, the activity of the laccase solution is 2.2-18.4 U / mL.

[0025] Preferably, the concentration of the functionalized magnetic Fe3O4@CS-Au nanoparticles in the laccase solution is 1-8 mg / mL.

[0026] The invention provides a dopamine photothermal enzyme electrode modified with magnetic nanoparticles prepared by the above method.

[0027] The present invention provides an application of the above-mentioned magnetic nanoparticle-modified dopamine photothermal enzyme electrode in dopamine detection.

[0028] Mechanism of action

[0029] The surface temperature of the Fe3O4@CS-Au-lac electrode of the present invention gradually increases under light conditions, thereby improving the catalytic activity of laccase, thereby improving the detection signal of the dopamine photothermal sensor enzyme electrode modified with magnetic nanoparticles. The substrate dopamine undergoes a redox reaction with laccase and is oxidized to dopamine quinone in the presence of O2. Laccase is converted from an oxidized state (Cu 2+ ) becomes reduced state (Cu + ), and with the conversion of O2 into water and back to the oxidized state, subsequently, the electrons (e - ) is quickly transferred to the oxidation product through a highly conductive carrier, reducing dopamine quinone to dopamine. Therefore, in the electrochemical workstation, the concentration of dopamine is obtained by detecting the amount of electron transfer (response current size); thus, the detection of dopamine can be achieved, especially the detection of dopamine under acidic conditions, with a wide detection limit, high sensitivity and good stability.

[0030] In addition, Fe3O4 in the electrode is easy to separate and recover and has good electrocatalytic performance because Fe 2+ with Fe 3+ The mutual conversion of Fe3O4 accelerates the transfer of electrons and promotes electrochemical detection. The surface of Fe3O4 coated with chitosan has abundant amine and hydroxyl groups, which can be used as the deposition sites of nano-gold. The combination with nano-gold particles can improve the conductivity and biocompatibility of the electrode, further improving the detection effect. At the same time, Fe3O4 and gold nanoparticles have a photothermal effect. Under light, the surface temperature rises and gradually reaches the optimal temperature of laccase, which increases the activity of laccase, thereby increasing the reaction rate of laccase oxidation of dopamine and increasing the response current of the laccase-modified electrode.

[0031] Beneficial technical effects

[0032] The highly sensitive photothermal laccase electrode prepared by the present invention has the advantages of simple method, mild conditions, wide detection limit, high sensitivity, good stability, etc., and can function efficiently under light conditions, so that it has broad application prospects in the fields of biomedicine, food safety, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a transmission electron microscope photo of the Fe3O4@CS-Au nanoparticles prepared in Example 1 of the present invention;

[0034] Figure 2 This is a high-power transmission electron microscope partial magnified image of the Fe3O4@CS-Au nanoparticles prepared in Example 1 of the present invention;

[0035] Figure 3 X-ray powder diffraction patterns of Fe3O4@CS and Fe3O4@CS-Au nanoparticles prepared in Example 1 of the present invention;

[0036] Figure 4 It current response diagram of the laccase electrode prepared in Example 1 of the present invention to different concentrations of dopamine under light and without light;

[0037] Figure 5 This is a graph showing the current response of the laccase electrode prepared in Example 1 of the present invention to different concentrations of dopamine under light conditions. DETAILED DESCRIPTION

[0038] The present invention will be further explained below in conjunction with specific embodiments.

[0039] Example 1

[0040] A method for preparing a dopamine sensor enzyme electrode modified with magnetic nanoparticles comprises the following steps:

[0041] 1) The preparation process of magnetic Fe3O4 nanoparticles and magnetic Fe3O4@CS nanoparticles is as follows:

[0042] 1-1) Weigh 0.54g of ferric chloride hexahydrate into a three-necked flask, add 35mL of distilled water, disperse by ultrasonication, pass argon, add 0.2g of ferrous chloride tetrahydrate, add 1mL of ammonia water at 65°C every 20min, add three times in total, react for 1.5h, raise the temperature to 93°C, react for 1h, separate by magnetic separation, wash with ethanol 3 times, wash with water 3 times, and separate to obtain magnetic Fe3O4 nanoparticles;

[0043] 1-2) 0.1 g Fe3O4 prepared in 1-1) was placed in a three-necked flask, 20 mL of distilled water and 10 mL of CS solution (4 mg / mL) were added, argon was passed through, 2 mL of 5 wt% glutaraldehyde was added dropwise at 50°C, reacted for 60 min, washed once with ethanol and once with water to obtain magnetic nanoparticles Fe3O4@CS;

[0044] 2) The preparation process of magnetic Fe3O4@CS-Au nanoparticles and functionalized magnetic Fe3O4@CS nanoparticles is as follows:

[0045] 2-1) Add 30 mL of distilled water and 20 uL of gold chloride solution to Fe3O4@CS, boil, add 2 mL of 1 wt% trisodium citrate, continue the reaction for 30 min, separate by magnetic separation, wash with ethanol 3 times, and wash with water 3 times to obtain Fe3O4@CS-Au;

[0046] 2-2) Take 5 mg of Fe3O4@CS-Au, add 4 mL of 20 uM mercaptopropionic acid solution, shake at 25°C for 2 h, magnetically separate, and wash with water twice to obtain mercaptopropionic acid-modified magnetic Fe3O4@CS-Au nanoparticles.

[0047] 2-3) adding 40 mg EDC, 60 mg NHS, and 4 mL pH 4.5 acetic acid buffer solution to the Fe3O4@CS-Au nanoparticles modified with mercaptopropionic acid in 2-2, reacting at 37° C. for 30 min, and washing with water twice to obtain the functionalized magnetic Fe3O4@CS-Au nanoparticles;

[0048] Depend on Figure 1 , 2 It can be seen that the Fe3O4@CS-Au nanoparticles obtained in Example 1 are uniformly dispersed, and the average diameter of the Fe3O4@CS-Au nanoparticles is about 10-20nm. The surface modification of Fe3O4 nanoparticles by CS can be judged from the appearance. Chitosan is uniformly coated on the outside of Fe3O4 to form a core-shell structure. The HRTEM image shows that the interplanar spacing is 0.25nm, corresponding to the (311) crystal plane of the Fe3O4 cubic system; the interplanar spacing is 0.23nm, corresponding to the Au (111) crystal plane.

[0049] 3) The preparation process of Fe3O4@CS-Au nanoparticles immobilized laccase and magnetic nanoparticles modified laccase electrode is as follows:

[0050] 3-1) Add the obtained functionalized Fe3O4@CS-Au nanoparticles to 1 mL of distilled water and 1 mL of laccase, the laccase activity is 4.4 U / mL, and the Fe3O4@CS-Au nanoparticle concentration is 5 mg / mL; shake at 30°C for 60 min to obtain the Fe3O4@CS-Au nanoparticle-immobilized laccase;

[0051] 3-2) A glassy carbon electrode with a diameter of 3 mm was polished using 1.0, 0.3 and 0.05 μm alumina powders, and the glassy carbon electrode was cleaned with ethanol and ultrapure water and dried in an argon flow; 5 μL of 5 mg / mL Fe3O4@CS-Au nanoparticle-immobilized laccase solution was drop-coated on the surface of the glassy carbon electrode, dried at 4°C, and then the surface of the laccase electrode was rinsed with ultrapure water to obtain a laccase electrode.

[0052] Figure 3 The X-ray powder diffraction patterns of Fe3O4@CS and Fe3O4@CS-Au nanoparticles prepared in Example 1; Fe3O4@CS nanoparticles can be easily indexed as cubic phases of Fe3O4 (PDF#88-0866). The XRD pattern of Fe3O4@CS-Au nanocomposite materials consists of cubic phases of Fe3O4 and Au (PDF#99-0056). The XRD patterns show that Fe3O4@CS and Fe3O4@CS-Au have high crystallinity, and the crystal form does not change significantly during the coating process.

[0053] The prepared magnetic nanoparticle-modified dopamine sensor enzyme electrode is used to detect the magnetic nanoparticle-modified dopamine photothermal sensor enzyme electrode.

[0054] The specific detection steps are as follows:

[0055] 1) Weigh 0.1 mol sodium acetate and 0.1 mol glacial acetic acid respectively, dissolve them in ultrapure water, transfer them to a 500 mL volumetric flask, dilute to obtain 0.2 M sodium acetate solution and 0.2 M acetic acid solution respectively, and then prepare them into 0.2 M acetic acid / sodium acetate buffer solution according to the proportion, pH = 4.5;

[0056] 2) Add 20 mL of the above buffer solution to the electrolytic cell, use the above prepared laccase electrode as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode, add different concentrations of dopamine to the buffer solution under light and lightless conditions, and the final concentrations of dopamine are 1, 5, 25, 50, 100, 200, 400, 600, 800, 1000, 1200, 1400 μM respectively; test the It curve of the electrode response to dopamine on the electrochemical workstation. Figure 4 As shown, the data obtained is an It curve graph, with time as the horizontal axis and current as the vertical axis. Figure 5 As shown, according to the It curve, the working curve is obtained with the dopamine concentration as the horizontal axis and the response current value as the vertical axis; under light conditions, when the concentration is c=1-1400μM: ΔI=0.008c(μmol / L)(R 2 =0.997).

[0057] The Fe3O4@CS-Au nanoparticles prepared by the present invention are uniform in size and have a particle size of 10-20nm. They are then connected to the surface of gold nanoparticles using mercaptopropionic acid and the carboxyl groups are activated to form carboxyl functionalized Fe3O4@CS-Au nanoparticles. The obtained enzyme electrode modified with magnetic nanoparticles can effectively detect dopamine in water, with a detection range of 1-1400μM.

[0058] Example 2

[0059] A method for preparing a dopamine sensor enzyme electrode modified with magnetic nanoparticles comprises the following steps:

[0060] 1) The preparation process of magnetic Fe3O4 nanoparticles and magnetic Fe3O4@CS nanoparticles is as follows:

[0061] 1-1) Weigh 0.54g of ferric chloride hexahydrate into a three-necked flask, add 35mL of distilled water, disperse by ultrasonication, pass argon, add 0.2g of ferrous chloride tetrahydrate, add 1mL of ammonia water at 65°C every 20min, add three times in total, react for 1.5h, raise the temperature to 93°C, react for 1h, separate by magnetic separation, wash with ethanol 3 times, wash with water 3 times, and separate to obtain magnetic Fe3O4 nanoparticles;

[0062] 1-2) 0.1 g of Fe3O4 prepared in 1-1) was placed in a three-necked flask, 20 mL of distilled water and 5 mL of CS solution (4 mg / mL) were added, argon was passed through, 2 mL of 5 wt% glutaraldehyde was added dropwise at 50°C, the reaction was carried out for 60 min, and the mixture was washed once with ethanol and once with water to obtain magnetic nanoparticles Fe3O4@CS;

[0063] 2) The preparation process of magnetic Fe3O4@CS-Au nanoparticles and functionalized magnetic Fe3O4@CS nanoparticles is as follows:

[0064] 2-1) Add 30 mL of distilled water and 20 uL of gold chloride solution to Fe3O4@CS, boil, add 2 mL of 1 wt% trisodium citrate, continue the reaction for 30 min, separate by magnetic separation, wash with ethanol 3 times, and wash with water 3 times to obtain Fe3O4@CS-Au;

[0065] 2-2) Take 5 mg of Fe3O4@CS-Au, add 4 mL of 20 uM mercaptopropionic acid solution, shake at 25°C for 2 h, magnetically separate, and wash with water twice to obtain mercaptopropionic acid-modified magnetic Fe3O4@CS-Au nanoparticles.

[0066] 2-3) adding 40 mg EDC, 60 mg NHS, and 4 mL pH 4.5 acetic acid buffer solution to the Fe3O4@CS-Au nanoparticles modified with mercaptopropionic acid in 2-2, reacting at 37° C. for 30 min, and washing with water twice to obtain the functionalized magnetic Fe3O4@CS-Au nanoparticles;

[0067] 3) The preparation process of Fe3O4@CS-Au nanoparticles immobilized laccase and magnetic nanoparticles modified laccase electrode is as follows:

[0068] 3-1) Add the obtained functionalized Fe3O4@CS-Au nanoparticles to 1 mL of distilled water and 1 mL of laccase, the laccase activity is 4.4 U / mL, and the Fe3O4@CS-Au nanoparticle concentration is 5 mg / mL; shake at 30°C for 60 min to obtain the Fe3O4@CS-Au nanoparticle-immobilized laccase;

[0069] 3-2) A glassy carbon electrode with a diameter of 3 mm was polished using 1.0, 0.3 and 0.05 μm alumina powders, cleaned with ethanol and ultrapure water, and dried with an argon flow; 5 μL of 5 mg / mL Fe3O4@CS-Au nanoparticle-immobilized laccase solution was drop-coated on the surface of the glassy carbon electrode, dried at 4°C, and then the surface of the enzyme electrode was rinsed with ultrapure water.

[0070] The operation steps of using the laccase electrode for photothermal detection of dopamine are the same as those in Example 1;

[0071] The Fe3O4@CS-Au nanoparticles obtained are uniform in size, with a particle size of 8-18nm. They are then connected to the surface of gold nanoparticles using mercaptopropionic acid and the carboxyl groups are activated to form carboxyl-functionalized Fe3O4@CS-Au nanoparticles. The enzyme electrode modified with the obtained magnetic nanoparticles can effectively detect dopamine in water, with a detection range of 10-1000μM.

[0072] Example 3

[0073] A method for preparing a dopamine sensor enzyme electrode modified with magnetic nanoparticles comprises the following steps:

[0074] 1) The preparation process of magnetic Fe3O4 nanoparticles and magnetic Fe3O4@CS nanoparticles is as follows:

[0075] 1-1) Weigh 0.54g of ferric chloride hexahydrate into a three-necked flask, add 35mL of distilled water, disperse by ultrasonication, pass argon, add 0.2g of ferrous chloride tetrahydrate, add 1mL of ammonia water at 65°C every 20min, add three times in total, react for 1.5h, raise the temperature to 93°C, react for 1h, separate by magnetic separation, wash with ethanol 3 times, wash with water 3 times, and separate to obtain magnetic Fe3O4 nanoparticles;

[0076] 1-2) 0.1 g Fe3O4 prepared in 1-1) was placed in a three-necked flask, 20 mL of distilled water and 10 mL of CS solution (4 mg / mL) were added, argon was passed through, 2 mL of 5 wt% glutaraldehyde was added dropwise at 50°C, reacted for 60 min, washed once with ethanol and once with water to obtain magnetic nanoparticles Fe3O4@CS;

[0077] 2) The preparation process of magnetic Fe3O4@CS-Au nanoparticles and functionalized magnetic Fe3O4@CS nanoparticles is as follows:

[0078] 2-1) Add 30 mL of distilled water and 20 uL of gold chloride solution to Fe3O4@CS, boil, add 2 mL of 1 wt% trisodium citrate, continue the reaction for 30 min, separate by magnetic separation, wash with ethanol 3 times, and wash with water 3 times to obtain Fe3O4@CS-Au;

[0079] 2-2) Take 5 mg of Fe3O4@CS-Au, add 4 mL of 10 uM mercaptopropionic acid solution, shake at 25°C for 2 h, magnetically separate, and wash with water twice to obtain mercaptopropionic acid-modified magnetic Fe3O4@CS-Au nanoparticles.

[0080] 2-3) adding 40 mg EDC, 60 mg NHS, and 4 mL pH 4.5 acetic acid buffer solution to the Fe3O4@CS-Au nanoparticles modified with mercaptopropionic acid in 2-2, reacting at 37° C. for 30 min, and washing with water twice to obtain the functionalized magnetic Fe3O4@CS-Au nanoparticles;

[0081] 3) The preparation process of Fe3O4@CS-Au nanoparticles immobilized laccase and magnetic nanoparticles modified laccase electrode is as follows:

[0082] 3-1) Add the obtained functionalized Fe3O4@CS-Au nanoparticles to 1 mL of distilled water and 1 mL of laccase, the laccase activity is 4.4 U / mL, and the Fe3O4@CS-Au nanoparticle concentration is 5 mg / mL; shake at 30°C for 60 min to obtain the Fe3O4@CS-Au nanoparticle-immobilized laccase;

[0083] 3-2) A glassy carbon electrode with a diameter of 3 mm was polished using 1.0, 0.3 and 0.05 μm alumina powders, cleaned with ethanol and ultrapure water, and dried with an argon flow; 5 μL of 5 mg / mL Fe3O4@CS-Au nanoparticle-immobilized laccase solution was drop-coated on the surface of the glassy carbon electrode, dried at 4°C, and then the surface of the enzyme electrode was rinsed with ultrapure water.

[0084] The operation steps of using the laccase electrode for photothermal detection of dopamine are the same as those in Example 1;

[0085] The Fe3O4@CS-Au nanoparticles obtained are uniform in size, with a particle size of 10-20nm. They are then connected to the surface of gold nanoparticles using mercaptopropionic acid and the carboxyl groups are activated to form carboxyl-functionalized Fe3O4@CS-Au nanoparticles. The enzyme electrode modified with the obtained magnetic nanoparticles can effectively detect dopamine in water, with a detection range of 50-800μM.

[0086] Example 4

[0087] A method for preparing a dopamine sensor enzyme electrode modified with magnetic nanoparticles comprises the following steps:

[0088] 1) The preparation process of magnetic Fe3O4 nanoparticles and magnetic Fe3O4@CS nanoparticles is as follows:

[0089] 1-1) Weigh 0.54g of ferric chloride hexahydrate into a three-necked flask, add 35mL of distilled water, disperse by ultrasonication, pass argon, add 0.2g of ferrous chloride tetrahydrate, add 1mL of ammonia water at 65°C every 20min, add three times in total, react for 1.5h, raise the temperature to 93°C, react for 1h, separate by magnetic separation, wash with ethanol 3 times, wash with water 3 times, and separate to obtain magnetic Fe3O4 nanoparticles;

[0090] 1-2) 0.1 g Fe3O4 prepared in 1-1) was placed in a three-necked flask, 20 mL of distilled water and 10 mL of CS solution (4 mg / mL) were added, argon was passed through, 2 mL of 5 wt% glutaraldehyde was added dropwise at 50°C, reacted for 60 min, washed once with ethanol and once with water to obtain magnetic nanoparticles Fe3O4@CS;

[0091] 2) The preparation process of magnetic Fe3O4@CS-Au nanoparticles and functionalized magnetic Fe3O4@CS nanoparticles is as follows:

[0092] 2-1) Add 30 mL of distilled water and 20 uL of gold chloride solution to Fe3O4@CS, boil, add 2 mL of 1 wt% trisodium citrate, continue the reaction for 30 min, separate by magnetic separation, wash with ethanol 3 times, and wash with water 3 times to obtain Fe3O4@CS-Au;

[0093] 2-2) Take 5 mg of Fe3O4@CS-Au, add 4 mL of 20 uM mercaptopropionic acid solution, shake at 25°C for 2 h, magnetically separate, and wash with water twice to obtain mercaptopropionic acid-modified magnetic Fe3O4@CS-Au nanoparticles.

[0094] 2-3) adding 40 mg EDC, 60 mg NHS, and 4 mL pH 4.5 acetic acid buffer solution to the Fe3O4@CS-Au nanoparticles modified with mercaptopropionic acid in 2-2, reacting at 37° C. for 30 min, and washing with water twice to obtain the functionalized magnetic Fe3O4@CS-Au nanoparticles;

[0095] 3) The preparation process of Fe3O4@CS-Au nanoparticles immobilized laccase and magnetic nanoparticles modified laccase electrode is as follows:

[0096] 3-1) Add the obtained functionalized Fe3O4@CS-Au nanoparticles to 1 mL of distilled water and 1 mL of laccase, the laccase activity is 4.4 U / mL, and the Fe3O4@CS-Au nanoparticle concentration is 5 mg / mL; shake at 30°C for 60 min to obtain the Fe3O4@CS-Au nanoparticle-immobilized laccase;

[0097] 3-2) A glassy carbon electrode with a diameter of 3 mm was polished using 1.0, 0.3 and 0.05 μm alumina powders, cleaned with ethanol and ultrapure water, and dried with an argon flow; 2 μL of 5 mg / mL Fe3O4@CS-Au nanoparticle-immobilized laccase solution was drop-coated on the surface of the glassy carbon electrode, dried at 4°C, and then the surface of the enzyme electrode was rinsed with ultrapure water.

[0098] The operation steps of using the laccase electrode for photothermal detection of dopamine are the same as those in Example 1;

[0099] The Fe3O4@CS-Au nanoparticles obtained are uniform in size, with a particle size of 10-20nm. They are then connected to the surface of gold nanoparticles using mercaptopropionic acid and the carboxyl groups are activated to form carboxyl-functionalized Fe3O4@CS-Au nanoparticles. The enzyme electrode modified with the obtained magnetic nanoparticles can effectively detect dopamine in water, with a detection range of 30-1100μM.

[0100] Only the laccase modified electrode cannot be detected: 5 μL of laccase solution (4.4 U / mL) was dropped on the electrode surface polished in the same way as above, dried at 4°C, and the enzyme electrode surface was rinsed with ultrapure water. 20 mL of acetic acid buffer solution was added to the electrolytic cell, and the laccase electrode prepared above was used as the working electrode, the saturated Ag / AgCl electrode was used as the reference electrode, and the platinum wire electrode was used as the auxiliary electrode. Different concentrations of dopamine were added to the buffer solution, and the cyclic voltammetry curve of the electrode was tested on the electrochemical workstation. Since the laccase surface is a non-conductive protein shell, the transfer of electrons on the electrode is hindered, and the redox peak cannot be detected in the cyclic voltammetry curve.

[0101] Reproducibility test of the present invention: Take 10 electrodes polished according to the same method as above, drip 5μL 5mg / mL Fe3O4@CS-Au nanoparticle immobilized laccase solution, dry at 4°C, and rinse the surface of the enzyme electrode with ultrapure water. Add 20mL of acetic acid buffer solution to the electrolytic cell, use the above-prepared laccase electrode as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode. Add 200μM dopamine to the buffer solution, and test the cyclic voltammetry curve of the electrode on the electrochemical workstation. The relative standard deviation of the peak current of the oxidation peak of the 10 electrodes is 3.31%, indicating that the present invention has a small error and good reproducibility.

Claims

1. A method for preparing a dopamine photothermal enzyme electrode modified with magnetic nanoparticles, characterized in that: The steps are as follows: S1: Preparation of functionalized magnetic Fe3O4@CS-Au nanoparticles modified with mercaptopropionic acid S11: dispersing magnetic Fe3O4 nanoparticles in distilled water, adding chitosan and a cross-linking agent to react, and washing and separating the product to obtain magnetic Fe3O4@CS nanoparticles; S12: mixing hydrated gold trichloride and the magnetic Fe3O4@CS nanoparticles in S11 in water, heating, and then adding a reducing agent to react. After the reaction, the product is washed and separated to obtain magnetic Fe3O4@CS-Au magnetic nanoparticles; S13: The magnetic nanoparticles Fe3O4@CS-Au in S12 are dispersed in an ethanol solution of mercaptopropionic acid to obtain Fe3O4@CS-Au-COOH nanoparticles, and then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added for activation reaction. The product is washed and separated to obtain functionalized magnetic Fe3O4@CS-Au nanoparticles; S2: Preparation of dopamine photothermal enzyme electrode The functionalized magnetic Fe3O4@CS-Au nanoparticles prepared in S1 were mixed with the laccase solution and heated in a water bath, and then the mixed solution was drop-coated on the electrode surface to form a dopamine photothermal enzyme electrode; In S11, the cross-linking agent is an aqueous solution of glutaraldehyde; The reducing agent in S12 is one of sodium citrate, ascorbic acid and sodium borohydride.

2. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 1, characterized in that: The molar ratio of magnetic Fe3O4 nanoparticles, chitosan and cross-linking agent in S11 is 5:1-3:4-6.

3. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 2, characterized in that: The concentration of the magnetic Fe3O4 nanoparticles is 1-5 g / L.

4. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 2, characterized in that: The reaction temperature in S11 is 10-80°C, and the reaction time is 0.5-5h.

5. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 1, characterized in that: The mass ratio of magnetic Fe3O4@CS nanoparticles, hydrated gold trichloride and reducing agent in S12 is 20:1-2:2-6.

6. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 1, characterized in that: The reaction temperature in S12 is 80-100°C, and the reaction time is 15-60min.

7. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 1, characterized in that: The mass ratio of magnetic nanoparticles Fe3O4@CS-Au and mercaptopropionic acid in S13 is 500-5000:

8.

8. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 1, characterized in that: The mass ratio of Fe3O4@CS-Au-COOH nanoparticles, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in S13 is 1:4-16:6-24.

9. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 8, characterized in that: The conditions for the activation reaction in S13 are stirring and culturing at 20-40°C for 20-60 min.

10. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 1, characterized in that: The water bath heating temperature in S2 is 20-40°C and the time is 1-4h.

11. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 10, characterized in that: The activity of the laccase solution in S2 was 2.2-18.4 U / mL.

12. The method for preparing the dopamine photothermal enzyme electrode modified with magnetic nanoparticles according to claim 10, characterized in that: The concentration of functionalized magnetic Fe3O4@CS-Au nanoparticles in laccase solution in S2 was 1-8 mg / mL.

13. A dopamine photothermal enzyme electrode modified with magnetic nanoparticles prepared by the method according to any one of claims 1 to 12.

14. Use of the dopamine photothermal enzyme electrode modified with magnetic nanoparticles as claimed in claim 13 in dopamine detection.

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