A homocysteine ​​electrochemical sensor and its preparation method and application

Abstract

The present invention provides a homocysteine ​​electrochemical sensor, and its preparation method and application. The preparation method of the homocysteine ​​electrochemical sensor of the present invention comprises the following steps: adding a dispersion containing carbon nanomaterials to the surface of a glassy carbon electrode, baking and drying, to obtain a carbon nanomaterial-modified electrode; then placing the carbon nanomaterial-modified electrode in a gold salt aqueous solution, and using an electroplating method to deposit nanogold on the surface of the carbon nanomaterial-modified electrode, to obtain a homocysteine ​​electrochemical sensor; the high conductivity of gold nanoparticles is of great significance for improving the electrocatalytic performance of the composite material for homocysteine. The homocysteine ​​electrochemical sensor prepared by the present application has low cost, low detection limit, and good stability. In addition, the spiked recovery rate of homocysteine ​​in serum samples is good, which provides potential application value for the reliable determination of homocysteine ​​in biological systems and the study of homocysteine-related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and in particular to a homocysteine ​​electrochemical sensor and a preparation method and application thereof. Background Art

[0002] In recent years, nanotechnology has advanced rapidly, and a wide variety of unique and exotic nanomaterials have emerged. Due to their unique physical and chemical properties, they are widely used in fields such as optics, catalysis, electronics, and biosensing. Consequently, countless experts and researchers are enthusiastically interested in nanoscale materials and their applications in biomedical sensing. Carbon nanomaterials, with their large surface area, high adsorption capacity, and excellent conductivity, have attracted widespread attention worldwide in numerous research and applications. Among them, acetylene black (AB) has become one of the most attractive carbon nanomaterials for modified electrode materials worldwide. Acetylene black (AB), a special type of carbon black with a regular porous structure, possesses excellent electrical conductivity, a large surface area due to its three-dimensional structure, and a strong adsorption capacity for analytes. Furthermore, compared to other carbon materials such as carbon nanotubes and graphene, AB's ultra-low cost helps maintain the cost-effectiveness of synthesizing composite materials, making it a successful candidate for biomedical sensing.

[0003] Gold nanoparticles (AuNPs) have a significant impact on the roughening of sensing interfaces, catalysis, conductivity, and enhanced mass transfer. Therefore, they hold enormous potential for application in biomedical sensing. AuNPs can be synthesized through chemical, physical, and biological methods. Electrodeposition offers a simple, rapid, and inexpensive alternative. To increase the utilization of AuNPs and enhance their catalytic activity, they are typically loaded onto highly conductive carbon nanomaterials. This increases the number of catalytically active sites in biomedical sensing processes, thereby improving the sensor's fundamental performance.

[0004] Homocysteine, also known as homocysteine, is a sulfhydryl-containing amino acid. Recent medical research has confirmed a close correlation between changes in plasma homocysteine ​​levels and certain diseases. Therefore, measuring homocysteine ​​levels is crucial for pathological studies of related diseases. Currently, a variety of analytical methods have been established for homocysteine ​​detection, including photometry, ultraviolet spectrophotometry, fluorescence, spectrometry, and enzyme immunoassay. However, many of these methods suffer from drawbacks such as complex pretreatment, high costs, high detection limits, and poor stability. Therefore, reliable and stable homocysteine ​​detection has become an urgent challenge that needs to be addressed. Summary of the Invention

[0005] In view of this, the present invention proposes a homocysteine ​​electrochemical sensor and a preparation method and application thereof to address the defects of the prior art.

[0006] In a first aspect, the present invention provides a method for preparing a homocysteine ​​electrochemical sensor, comprising the following steps:

[0007] The carbon nanomaterial and dihexadecyl phosphoric acid are added into water and ultrasonicated to prepare a dispersion;

[0008] The dispersion is added dropwise to the surface of the glassy carbon electrode and baked to obtain a carbon nanomaterial modified electrode;

[0009] The carbon nanomaterial modified electrode is placed in a gold salt aqueous solution, and nano-gold is deposited on the surface of the carbon nanomaterial modified electrode by an electrodeposition method, thereby obtaining a homocysteine ​​electrochemical sensor.

[0010] Preferably, in the method for preparing the homocysteine ​​electrochemical sensor, the carbon nanomaterial includes at least one of acetylene black, carbon nanotubes, graphene oxide, and graphene.

[0011] Preferably, in the method for preparing the homocysteine ​​electrochemical sensor, the gold salt aqueous solution includes one of a HAuCl4 aqueous solution and a NaAuCl4·2H2O aqueous solution.

[0012] Preferably, in the preparation method of the homocysteine ​​electrochemical sensor, in the step of adding the carbon nanomaterial and dihexadecyl phosphoric acid to water to obtain a dispersion, the mass volume ratio of the carbon nanomaterial, dihexadecyl phosphoric acid and water is (2-6) mg: (2-6) mg: (2-6) mL.

[0013] Preferably, in the preparation method of the homocysteine ​​electrochemical sensor, the concentration of the gold salt aqueous solution is 3 to 7 mmol / L.

[0014] Preferably, the preparation method of the homocysteine ​​electrochemical sensor adopts the electrochemical deposition method to deposit gold nanoparticles on the surface of the carbon nanomaterial modified electrode, and the electrochemical parameters controlled are: a scan rate of 80 to 120 mV / s, a potential range of -1.5 to 0 V, and a cycle scan of 5 to 35 times.

[0015] Preferably, the preparation method of the homocysteine ​​electrochemical sensor further includes treating the glassy carbon electrode before adding the dispersion dropwise to the surface of the glassy carbon electrode. The treatment is specifically as follows: polishing the glassy carbon electrode with a polishing cloth; and then ultrasonically cleaning the polished glassy carbon electrode with an aqueous nitric acid solution, an aqueous anhydrous ethanol solution, and water in sequence, thereby completing the treatment of the glassy carbon electrode.

[0016] Preferably, in the preparation method of the homocysteine ​​electrochemical sensor, 2 to 10 μL of the dispersion is added dropwise to the surface of the glassy carbon electrode.

[0017] In a second aspect, the present invention further provides a homocysteine ​​electrochemical sensor, which is prepared using the preparation method.

[0018] In a third aspect, the present invention further provides a homocysteine ​​electrochemical sensor prepared by the preparation method or use of the homocysteine ​​electrochemical sensor in detecting homocysteine ​​concentration.

[0019] The preparation method of the homocysteine ​​electrochemical sensor of the present invention has the following beneficial effects compared with the prior art:

[0020] The present invention discloses a homocysteine ​​electrochemical sensor preparation method, comprising the steps of dropwise adding a dispersion containing carbon nanomaterials to the surface of a glassy carbon electrode and baking to dry, thereby obtaining a carbon nanomaterial-modified electrode. The carbon nanomaterial-modified electrode is then placed in an aqueous gold salt solution, and nanogold is deposited on the surface of the carbon nanomaterial-modified electrode by electrodeposition, thereby obtaining a homocysteine ​​electrochemical sensor. The present invention combines a simple electrochemical deposition method with a drop coating method to prepare a homocysteine ​​electrochemical sensor. The high conductivity of gold nanoparticles is of great significance for improving the electrocatalytic performance of the composite material for homocysteine. The homocysteine ​​electrochemical sensor prepared in the present invention is low-cost, has a low detection limit, and exhibits excellent stability. Furthermore, the spiked recovery rate of homocysteine ​​in serum samples is good, providing potential applications for the reliable determination of homocysteine ​​in biological systems and the study of homocysteine-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0022] Figure 1 The effect of the homocysteine ​​electrochemical sensor prepared in Examples 1 to 4 of the present invention on the oxidation peak potential and oxidation peak current of 200 μmol / L HcySH;

[0023] Figure 2 The effect of homocysteine ​​electrochemical sensor on the oxidation peak current of HcySH under different detection conditions;

[0024] Figure 3 Surface morphologies of bare GCE, AuNPs / GCE, AB-DHP / GCE, and AuNPs / AB-DHP / GCE;

[0025] Figure 4Energy spectrum of AuNPs / AB-DHP / GCE;

[0026] Figure 5 Differential pulse voltammetry curves of bare GCE, AB-DHP / GCE, AuNPs / GCE, and AuNPs / AB-DHP / GCE in 0.15 mol / L pH = 6.0 phosphate buffer solution containing 200 μmol / L HcySH;

[0027] Figure 6 Cyclic voltammetry scan of AuNPs / AB-DHP / GCE in 5 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 1 mol / L KCl;

[0028] Figure 7 For bare GCE and AuNPs / AB-DHP / GCE, the charge Q is related to the square root of time t (t 1 / 2 ) linear relationship diagram between;

[0029] Figure 8 Electrochemical impedance spectroscopy (EIS) graphs of GCE, AB-DHP / GCE, and AuNPs / AB-DHP / GCE in 5 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 1 mol / L KCl;

[0030] Figure 9 Linear sweep voltammograms of 200 μmol / L HcySH on AuNPs / AB-DHP / GCE at different scan rates;

[0031] Figure 10 The differential pulse voltammetry curves of AuNPs / AB-DHP / GCE monitoring 200 μmol / L HcySH in 0.15 mol / L PBS buffer solutions at different pH values;

[0032] Figure 11 The amperometric response diagram of different concentrations of homocysteine ​​on the AuNPs / AB-DHP / GCE electrode. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] The present invention provides a method for preparing a homocysteine ​​electrochemical sensor, comprising the following steps:

[0035] S1, adding carbon nanomaterials and dihexadecyl phosphoric acid into water and preparing a dispersion by ultrasonication;

[0036] S2, adding the dispersion dropwise to the surface of the glassy carbon electrode and baking to dry, thereby obtaining a carbon nanomaterial modified electrode;

[0037] S3. Placing the carbon nanomaterial modified electrode in a gold salt aqueous solution, and using an electrodeposition method to deposit nano-gold on the surface of the carbon nanomaterial modified electrode, thereby obtaining a homocysteine ​​electrochemical sensor.

[0038] It should be noted that the preparation method of the homocysteine ​​electrochemical sensor of the present application is to add a dispersion containing carbon nanomaterials dropwise to the surface of a glassy carbon electrode and bake it to dry, thereby obtaining a carbon nanomaterial-modified electrode; then, the carbon nanomaterial-modified electrode is placed in an aqueous gold salt solution, and nanogold is deposited on the surface of the carbon nanomaterial-modified electrode by electrodeposition, thereby obtaining a homocysteine ​​electrochemical sensor. The present application combines a simple electrochemical deposition method with a drop coating method to prepare a homocysteine ​​electrochemical sensor. The high conductivity of gold nanoparticles is of great significance for improving the electrocatalytic performance of the composite material for homocysteine. The homocysteine ​​electrochemical sensor prepared by the present application is low-cost, has a low detection limit, and has good stability. In addition, the spiked recovery rate of homocysteine ​​in serum samples is good, providing potential application value for the reliable determination of homocysteine ​​in biological systems and the study of homocysteine-related diseases.

[0039] In some embodiments, the carbon nanomaterial includes at least one of acetylene black, carbon nanotubes, graphene oxide, and graphene.

[0040] In some embodiments, the gold salt aqueous solution includes one of a HAuCl 4 aqueous solution and a NaAuCl 4 ·2H 2 O aqueous solution.

[0041] In some embodiments, in the step of adding carbon nanomaterials and dicetyl phosphoric acid to water to obtain a dispersion, the mass volume ratio of carbon nanomaterials, dicetyl phosphoric acid and water is (2-6) mg: (2-6) mg: (2-6) mL.

[0042] In some embodiments, the concentration of the gold salt aqueous solution is 3-7 mmol / L.

[0043] In some embodiments, a three-electrode system is used (a carbon nanomaterial-modified glassy carbon electrode is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode). With a gold salt aqueous solution as the base liquid, the gold salt is electrochemically reduced to generate gold nanoparticles deposited on the surface of the carbon nanomaterial-modified electrode by means of an electrodeposition method. The controlled electrochemical parameters are: a scan rate of 80 to 120 mV / s, a potential range of -1.5 to 0 V, and a cyclic scan of 5 to 35 times.

[0044] In some embodiments, the glassy carbon electrode is treated before the dispersion is added to the surface of the glassy carbon electrode. Specifically, the treatment includes: polishing the glassy carbon electrode with a polishing cloth; and then ultrasonically cleaning the polished glassy carbon electrode with nitric acid aqueous solution, anhydrous ethanol and ultrapure water in sequence, thereby completing the treatment of the glassy carbon electrode.

[0045] Specifically, the polishing cloth contains a slurry containing Al2O3 polishing powder, and the particle diameter of the Al2O3 polishing powder is 50nm; the volume ratio of nitric acid to water in the nitric acid aqueous solution is 1:1; the polished glassy carbon electrode is ultrasonically cleaned using nitric acid aqueous solution, anhydrous ethanol and ultrapure water in sequence, and each stage of ultrasonic cleaning is carried out for 2 to 5 minutes.

[0046] In some embodiments, 2-10 μL of the dispersion is dropped onto the surface of the glassy carbon electrode.

[0047] Based on the same inventive concept, an embodiment of the present application further provides a homocysteine ​​electrochemical sensor, which is prepared using the above-mentioned preparation method.

[0048] Based on the same inventive concept, an embodiment of the present application further provides an application of the above-mentioned homocysteine ​​electrochemical sensor in detecting homocysteine ​​concentration.

[0049] The following further illustrates the preparation method of the homocysteine ​​electrochemical sensor of the present application with specific examples. This section further illustrates the content of the present invention in conjunction with specific examples, but should not be understood as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The acetylene black (particle size 30-45 nm) used in the following examples was purchased from Tianjin Youmeng Chemical Technology Co., Ltd., and dihexadecyl phosphoric acid was purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0050] Example 1

[0051] The present invention provides a method for preparing a homocysteine ​​electrochemical sensor, comprising the following steps:

[0052] S1. Place 4 mg of acetylene black (AB) and 4 mg of dihexadecyl phosphoric acid (DHP) in 4 mL of ultrapure water and perform ultrasonic dispersion treatment for 10 h to obtain an AB-DHP dispersion.

[0053] S2. A bare GCE (i.e., a glassy carbon electrode) with a diameter of 3.0 mm was polished on a polishing cloth containing a slurry of Al2O3 polishing powder with a particle diameter of 50 nm. The surface was then ultrasonically cleaned with aqueous nitric acid, anhydrous ethanol, and ultrapure water for 3 min each to obtain a clean and smooth electrode surface. Subsequently, 6 μL of AB-DHP dispersion was drop-coated on the treated GCE surface using a single-point adjustable pipette with a range of 0.5 to 10 μL, and then dried under an infrared lamp to obtain an AB-DHP / GCE modified electrode.

[0054] S3. The AB-DHP / GCE modified electrode was placed in a 5 mmol / L HAuCl4 aqueous solution for electrochemical deposition. The electrochemical parameters controlled by the electrochemical deposition were: a scan rate of 100 mV / s, a potential range of -1.5 to 0 V, and 25 cycles of cyclic scanning to allow AuNPs (gold nanoparticles) to successfully combine with AB-DHP, thereby obtaining AuNPs / AB-DHP / GCE, i.e., a homocysteine ​​electrochemical sensor.

[0055] Example 2

[0056] The preparation method of the homocysteine ​​electrochemical sensor provided in the embodiment of the present application is the same as that in Example 1, except that 4 mg of carbon nanotubes (MWCNTs) are used instead of 4 mg of acetylene black in step S1. The rest of the process is the same as that in Example 1.

[0057] Example 3

[0058] The preparation method of the homocysteine ​​electrochemical sensor provided in the embodiment of the present application is the same as that in Example 1, except that 4 mg of graphene oxide (GO) is used instead of 4 mg of acetylene black in step S1, and the rest of the process is the same as that in Example 1.

[0059] Example 4

[0060] The preparation method of the homocysteine ​​electrochemical sensor provided in the embodiment of the present application is the same as that in Example 1, except that 4 mg of graphene (GR) is used instead of 4 mg of acetylene black in step S1, and the rest of the process is the same as that in Example 1.

[0061] Comparative Example 1

[0062] This comparative example provides a method for preparing a homocysteine ​​electrochemical sensor, comprising the following steps:

[0063] S1. Polish a bare GCE (i.e., glassy carbon electrode) with a diameter of 3.0 mm on a polishing cloth containing a slurry of Al2O3 polishing powder with a particle diameter of 50 nm. Then, ultrasonically clean the surface with aqueous nitric acid solution, aqueous anhydrous ethanol solution, and ultrapure water for 3 minutes each to obtain a clean and smooth electrode surface.

[0064] S2. The treated GCE was placed in a 5 mmol / L HAuCl4 aqueous solution for electrochemical deposition. The electrochemical parameters controlled by the electrochemical deposition were: a scan rate of 100 mV / s, a potential range of -1.5 to 0 V, and a cyclic scan of 25 cycles to deposit AuNPs (gold nanoparticles) on the GCE surface to obtain AuNPs / GCE.

[0065] Performance Testing

[0066] Electrochemical Detection of Homocysteine

[0067] The electrochemical detection system for homocysteine ​​typically utilizes a three-electrode system. The working electrode is a bare GCE, AB-DHP / GCE, AuNPs / GCE, or AuNPs / AB-DHP / GCE, which is the site of the electrochemical reaction of homocysteine. A saturated calomel electrode serves as a reference electrode, acting as a reference for comparison during the detection process. A platinum electrode serves as an auxiliary electrode, forming a polarization circuit with the working electrode. The detection substrate is a 0.15 mol / L phosphate buffer solution (pH = 6.0) containing a specific concentration of HcySH (DL-homocysteine, purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.). The phosphate buffer solution is prepared by dissolving 30 g NaCl, 0.75 g KCl, 13.425 g Na₂HPO₄·12H₂O, and 0.9 g KH₂PO₄ in 200 mL of ultrapure water, diluting the volume to 250 mL and adjusting the pH to 6.0. During the electrochemical characterization process, the scanning potential range was set to 0 V to 0.7 V. In addition, before detection, it was necessary to enrich at a potential of -0.2 V for 20 s to facilitate the accumulation of homocysteine ​​on the electrode surface and achieve good detection results.

[0068] 1. Optimization of carbon nanomaterials

[0069] Biomedical sensors modified with carbon nanomaterials can effectively increase the specific surface area of ​​electrodes and improve electrocatalytic and electron transfer activities. Among them, acetylene black (AB), carbon nanotubes (MWCNT), graphene oxide (GO), and graphene (GR) have made great progress in the preparation and research of biomedical sensors. In order to determine the carbon nanomaterial with the best detection effect for HcySH, the performance of the homocysteine ​​electrochemical sensors prepared in Examples 1 to 4 was investigated. The results are as follows: Figure 1Specifically, according to the above detection method, a three-electrode system was used, with the homocysteine ​​electrochemical sensor prepared in Examples 1 to 4 as the working electrode, and the detection base solution was a 0.15 mol / L phosphate buffer solution containing 200 μmol / L HcySH.

[0070] Figure 1 a: AuNPs / AB-DHP / GCE is the homocysteine ​​electrochemical sensor prepared in Example 1, b: AuNPs / MWCNT-DHP / GCE is the homocysteine ​​electrochemical sensor prepared in Example 2, c: AuNPs / GO-DHP / GCE is the homocysteine ​​electrochemical sensor prepared in Example 3, d: AuNPs / GR-DHP / GCE is the homocysteine ​​electrochemical sensor prepared in Example 4; Figure 1 A represents the effect of the homocysteine ​​electrochemical sensor prepared in Examples 1 to 4 on the oxidation peak potential of 200 μmol / L HcySH, and B represents the effect of the homocysteine ​​electrochemical sensor prepared in Examples 1 to 4 on the oxidation peak current of 200 μmol / L HcySH.

[0071] from Figure 1 It can be seen that the same concentration of HcySH has the lowest oxidation peak potential and the largest oxidation peak current on AuNPs / AB-DHP / GCE, indicating that the AB-DHP and AuNPs nanocomposite film has a significant catalytic effect on the electrochemical oxidation of homocysteine, so AuNPs / AB-DHP / GCE is the best electrode for detecting HcySH.

[0072] 2. Optimization of detection conditions

[0073] In the field of biomedical sensing, different experimental detection prerequisites lead to very different final monitoring results. In order to achieve the best detection effect, the following conditions were optimized in this application, and the results are as follows: Figure 2 Specifically, Figure 2 According to the above detection method, a three-electrode system was used, with the homocysteine ​​electrochemical sensor prepared in Example 1 as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The detection base solution was a phosphate buffer solution containing 200 μmol / L HcySH. Specifically, Figure 2A shows the effect of the detection base solution concentration on the HcySH oxidation peak current; B shows the effect of the volume of the AB-DHP dispersion added in step S2 of Example 1 on the HcySH oxidation peak current; C shows the effect of the number of cycle scans in step S3 of Example 1 on the HcySH oxidation peak current; D shows the relationship between the HcySH oxidation peak current and the enrichment potential; and E shows the relationship between the HcySH oxidation peak current and the enrichment time.

[0074] from Figure 2 It can be seen that the optimal base solution (i.e. phosphate buffer solution) concentration is 0.15 mol / L; the optimal droplet coating amount of AB-DHP dispersion is 6 μL; the optimal cycle scanning number of gold nanoparticles is 25 circles; the optimal enrichment potential and enrichment time are -0.2 V and 20 s, respectively.

[0075] 3. Analysis of the mechanism of homocysteine ​​electrochemical sensor

[0076] 3.1 SEM characterization and energy spectrum analysis of different electrode surfaces

[0077] The surface morphology and properties of the bare GCE (A) in Example 1, the AuNPs / GCE (B) in Comparative Example 1, the AB-DHP / GCE (C) in Example 1, and the AuNPs / AB-DHP / GCE (D) were analyzed by scanning electron microscopy (SEM). Figure 3 shown.

[0078] from Figure 3 It can be seen that the bare GCE surface is relatively smooth ( Figure 3 A); After electrodeposition of AuNPs, a dense, uniform and shiny golden film is produced on the surface of GCE. It can be clearly observed that the film is covered with gold nanoparticles ( Figure 3 Middle B); After AB-DHP was drop-coated on the GCE surface, the electrode surface was uneven and had a loose pore structure ( Figure 3 Middle C); After electrodeposition of AuNPs on AB-DHP / GCE ( Figure 3 The entire electrode surface is relatively loose, and the AuNPs are clearly visible attached to the acetylene black (AB) lattice in the magnified image of 3D. The nanostructured effect of the AuNPs, combined with the loose and porous structure of acetylene black (AB), increases the active area of ​​the electrode, promoting the enrichment of homocysteine ​​and thus improving the detection sensitivity of homocysteine ​​on the sensor.

[0079] In order to further analyze and confirm that AuNPs were successfully integrated into the matrix of AB-DHP, the energy spectrum analysis of AuNPs / AB-DHP / GCE in Example 1 was performed. The results are as follows: Figure 4 shown.

[0080] from Figure 4 It can be seen that the elements present on the surface of AuNPs / AB-DHP / GCE are C and Au. The C element may come from AB-DHP and the supporting carrier glassy carbon sheet used in the test, and the Au element mainly comes from AuNPs. Figure 4 On the right, from top to bottom, are the distribution maps of Au and C elements on the surface of the AuNPs / AB-DHP / GCE electrode. In the figure, white represents the Au element and blue represents the C element. It can be seen that Au and C elements are evenly distributed on the electrode surface, that is, the AuNPs / AB-DHP composite film is successfully modified to the GCE surface.

[0081] 3.2 Electrochemical response of homocysteine ​​at different electrodes

[0082] The differences in electrocatalytic performance of homocysteine ​​were studied using DPV on four different electrodes. Figure 5 Specifically, in Example 1, the bare GCE ( Figure 5 Curve a), AB-DHP / GCE in Example 1 ( Figure 5 Curve b), AuNPs / GCE in Comparative Example 1 ( Figure 5 Curve c), AuNPs / AB-DHP / GCE in Example 1 ( Figure 5 Middle curve d) DPV graph in 0.15 mol / L pH=6.0 phosphate buffer solution containing 200 μmol / L HcySH.

[0083] from Figure 5 As can be seen in the graph, the homocysteine ​​response signal on bare GCE is poor, almost nonexistent (curve a). On AB-DHP / GCE, only a weak homocysteine ​​oxidation peak is observed at 0.51 V, with a peak current of 0.17 μA (curve b). On AuNPs / GCE (curve c), a sharp homocysteine ​​oxidation peak appears at 0.46 V, with an electrochemically measured peak current of 4.7 μA. On AuNPs / AB-DHP / GCE, the oxidation peak at 0.404 V is sharper, with an electrochemically measured peak current of 8.3 μA. Furthermore, on AuNPs / AB-DHP / GCE, the oxidation peak potential of HcySH undergoes a relatively large negative shift to 0.106 V. This is likely due to the large surface area of ​​gold nanoparticles and the strong adsorption capacity of acetylene black, which accelerate the electron transfer rate of homocysteine ​​at the modified electrode. In addition, the AuNPs / AB-DHP film presents a three-dimensional loose and porous network structure on the electrode surface with more electroactive sites, which is more conducive to improving the electrochemical response of homocysteine ​​compared with AuNPs or AB-DHP alone.

[0084] 3.3 Determination of the effective area of ​​the electrode by cyclic voltammetry

[0085] Fe(CN)6 3- / Fe(CN)6 4- The effective surface area of ​​AuNPs / AB-DHP / GCE prepared in Example 1 was measured using a probe. Specifically, AuNPs / AB-DHP / GCE was placed in a 5 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 1 mol / L KCl, and cyclic voltammetry was performed at different scan rates within the potential range of -0.2V-0.8V. The experimental graphs and I pc With v 1 / 2 The linear relationship is as follows Figure 6 As shown, Figure 6 A is the cyclic voltammetry scan, B is the I pc With v 1 / 2 linear relationship.

[0086] According to the Randles–Sevcik formula: p =(2.69×10 5 )×n 3 / 2 v 1 / 2 D 1 / 2 AC, where n is the number of electron transfers, for Fe(CN)6 3- / Fe(CN)6 4- probe, its value is 1; D is Fe(CN)6 3- / Fe(CN)6 4- The diffusion coefficient in solution is 7.63×10 -6 cm 2 / s; c is Fe(CN)6 3- / Fe(CN)6 4- The concentration is 5.0 mol / cm 3 , combined with the i in this system p With v 1 / 2 The linear equation i p =539.99v 1 / 2 -3.43, R = 0.999, the effective surface area A of AuNPs / AB-DHP / GCE can be calculated to be 0.145 cm 2 , compared with the surface area of ​​bare GCE (disk electrode with a diameter of 3 mm) of 0.07 cm 2 , increased by about 1.1 times, which fully proved that the modification of AuNPs / AB-DHP nanocomposite film on the GCE surface can effectively increase the electrode surface area, thereby enhancing the enrichment of homocysteine ​​at the sensing interface and providing a guarantee for the high sensitivity of the sensor.

[0087] 3.4 Electrocatalytic mechanism of AuNPs / AB-DHP membrane for homocysteine

[0088] To investigate the electrocatalytic mechanism of the AuNPs / AB-DHP composite film on homocysteine, the electrochemical behavior of homocysteine ​​at the same concentration on the bare GCE and AuNPs / AB-DHP / GCE described in Example 1 was studied using chronocoulometry. Specifically, a three-electrode system was employed, using the bare GCE and AuNPs / AB-DHP / GCE as working electrodes, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The detection solution consisted of 0.15 mol / L phosphate buffer (pH 6.0) containing 200 μmol / L HcySH.

[0089] Specifically, for bare GCE and AuNPs / AB-DHP / GCE, the linear relationship between charge Q and time t is as follows: Figure 7 As shown in A, the charge Q and the square root of time (t 1 / 2 ) is a linear relationship between Figure 7 As shown in B.

[0090] For bare GCE, the linear relationship between Q and t1 / 2 is: Q(μC)=5.72×10-3t1 / 2(s1 / 2)-4.16×10-3, R=0.99. According to the Cottrell equation, the diffusion coefficient of homocysteine ​​on bare GCE can be calculated as: D=1.37×10-17cm2 / s, and the adsorption amount is: 6.07×10-13mol / cm2; for AuNPs The linear relationship between Q and t1 / 2 is: Q(μC) = 39.53t1 / 2(s1 / 2) - 1.99, R = 0.99. According to the Cottrell equation, the diffusion coefficient of homocysteine ​​on AuNPs / AB-DHP / GCE is calculated to be D = 6.53 × 10-10 cm2 / s, and the adsorption capacity is 2.90 × 10-10 mol / cm2. Clearly, the synergistic effect of AuNPs and AB-DHP increases the diffusion coefficient and adsorption capacity of homocysteine, promoting the enrichment of homocysteine.

[0091] 3.5 Electrochemical impedance spectroscopy

[0092] Electrochemical impedance spectroscopy (EIS) was used to characterize the interface characteristics before and after electrode treatment. Figure 8 shown. Figure 8 The bare GCE ( Figure 8Curve a), AB-DHP / GCE in Example 1 ( Figure 8 Curve b), AuNPs / AB-DHP / GCE in Example 1 ( Figure 8 Middle curve c) EIS diagram in 5 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 1 mol / L KCl.

[0093] from Figure 8 It can be seen that the semicircle radius of the bare GCE is larger (curve a), indicating that [Fe(CN)6] 3- / 4- The electron transfer resistance is large during the electrode reaction. After the bare GCE is modified with AB-DHP, the semicircle radius is reduced, indicating that [Fe(CN)6] 3- / 4- During the electrode reaction, the electron transfer resistance decreases (curve b), which indicates that the pore structure of AB-DHP is [Fe(CN)6] 3- / 4- The reaction on the electrode surface provides numerous active sites. Based on this, the modified AuNPs yielded a lower resistance (curve c), demonstrating that AuNPs are excellent conductive materials that accelerate electron transfer. This fully demonstrates the success of the proposed biomedical sensor design.

[0094] 3.6 Effect of scan rate on the electrochemical response of homocysteine

[0095] Figure 9 The linear sweep voltammograms of 200 μmol / L HcySH on the AuNPs / AB-DHP / GCE electrode in Example 1 (the base solution is 0.15 mol / L phosphate buffer solution at pH = 6.0) at different scan rates (scan rates from inside to outside are 20, 40, 60, 80, 100, and 120 mV / s, respectively); specifically, Figure 9 A is the linear sweep voltammogram, B is the I of HcySH p The linear relationship between v and C is the E of HcySH p Linear relationship diagram between ρ and lnv.

[0096] To investigate the kinetics of the chemical reaction of homocysteine ​​on the surface of AuNPs / AB-DHP / GCE electrode, the linear sweep voltammetry (LSV) response of AuNPs / AB-DHP / GCE in 0.15 mol / L PBS (pH 6.0) solution containing 200 μmol / L HcySH was investigated. p Closely related to v: I p=0.53v-0.99, R = 0.97, clearly demonstrating a linear positive correlation. Therefore, the HcySH electrochemical reaction on the AuNPs / AB-DHP / GCE electrode is controlled by adsorption. Furthermore, the low-positive-charge oxidation peak shifts toward the high-positive-charge direction with increasing scan rate, demonstrating irreversible electrochemical behavior. Based on the Laviron theory, the electron transfer number is 1.4≈1, indicating that the HcySH oxidation reaction on the AuNPs / AB-DHP / GCE electrode is accompanied by one electron transfer.

[0097] 3.7 Effect of pH value of substrate on electrochemical response of homocysteine

[0098] The differential pulse voltammetry curves of 200 μmol / L HcySH were monitored using the AuNPs / AB-DHP / GCE in Example 1 in 0.15 mol / L PBS buffer solutions of different pH values. Figure 10 shown.

[0099] The pH value of the solution directly affects the dissociation state of the analyte in the solution, and on the other hand, it affects the charge state of the composite membrane modified electrode surface. These factors significantly affect the electrochemical response of the analyte, so it is very necessary to optimize the pH value of the detection solution. Figure 10 It can be seen from the figure that the electrochemical response signal of HcySH on AuNPs / AB-DHP / GCE is the strongest when pH is 6.0. Therefore, the optimal pH of PBS is 6.0. p It is linearly negatively correlated with pH change: E p =0.79-0.063pH, R=0.94, indicating that the electrochemical reaction of HcySH on AuNPs / AB-DHP / GCE transferred an electron and at the same time, a proton was involved. The reaction mechanism is speculated as follows:

[0100] Step 1: HcySH combines with AuNPs in the electrode material: AuNPs+HcySH→HcyS-AuNPs+H + +e - ;

[0101] Step 2: In acidic solution: HcyS-AuNPs+H + →(HcyS-AuNPs)H + ;

[0102] Step 3: Scanning towards a more negative potential: (HcyS-AuNPs)H + +e - →HcySH+AuNPs.

[0103] 3.8 Performance evaluation of homocysteine ​​electrochemical sensor

[0104] 3.8.1 Investigation of linear range

[0105] Amperometric alternating current (IT) detection was performed in a continuously stirred 0.1 mol / L serum-containing 0.15 mol / L PBS (pH 6.0) solution to investigate the biomedical sensing effect of AuNPs / AB-DHP / GCE on HcySH in Example 1. The results are shown in FIG. Figure 11 shown.

[0106] Specifically, Figure 11 Figure 2 shows the amperometric response of different homocysteine ​​concentrations on AuNPs / AB-DHP / GCE, as well as the linear relationship between the Ip of HcySH and its c (inset). The working potential was 0.6 V, and the substrate was a 0.15 mol / L PBS buffer solution (pH 6.0) containing 0.1 mol / L serum.

[0107] from Figure 11 As can be seen from the figure, the linear range is 3 to 1000 μmol / L. The electrode can generate HcySH concentration signals in a relatively short time (3 s), with a detection limit of 0.01 μmol / L and a sensitivity of 18 nA / (μmol / L).

[0108] 3.8.2 Anti-interference ability, stability and reproducibility

[0109] The anti-interference ability of the homocysteine ​​biomedical sensor AuNPs / AB-DHP / GCE prepared in Example 1 was investigated in a 0.15 mol / L PBS (pH 6.0) solution containing 200 μmol / L HcySH by time-current response method. The results are shown in Table 1. + , 200 times K + , Mg 2+ , 40 times the Ca 2+ 0.4 times of dopamine and uric acid, and 0.3 times of ascorbic acid had almost no effect on the detection of 50 μmol / L HcySH, indicating that the sensor has significant differences in its anti-interference ability against inorganic ions and small biological molecules.

[0110] Table 1 - Effects of inorganic ions and small biological molecules on HcySH peak current

[0111] Interference Concentration (μmol / L) Error caused (%) <![CDATA[Na + ]]> 500 -4.72 <![CDATA[K + ]]> 10000 -4.99 <![CDATA[Ca 2+ ]]> 2000 -4.28 <![CDATA[Cl - ]]> 20000 -4.13 <![CDATA[Mg 2+ ]]> 10000 -4.13 ascorbic acid 15 3.5 uric acid 20 4.7 Dopamine 20 2.81

[0112] The performance of the homocysteine ​​biomedical sensor was investigated using differential pulse voltammetry. The homocysteine ​​biomedical sensor AuNPs / AB-DHP / GCE prepared in Example 1 was allowed to stand for 7 days before being tested in parallel. The detected signal was 93.6% of the initial signal, indicating the sensor's stability. Under the optimal conditions, eight parallel measurements were performed using eight different AuNPs / AB-DHP / GCEs in a 0.15 mol / L PBS (pH 6.0) solution containing 200 μmol / L HcySH, yielding a relative standard deviation of 3.5%. Eight parallel measurements using a single AuNPs / AB-DHP / GCE on 200 μmol / L HcySH yielded a relative standard deviation of 6.4%, demonstrating the reproducibility of the homocysteine ​​biomedical sensor.

[0113] 3.8.3 Detection of Homocysteine ​​in Human Serum Samples

[0114] The practical application of the AuNPs / AB-DHP / GCE was tested by measuring homocysteine ​​concentrations in human serum samples (purchased from Nanjing Xinfan Biotechnology Co., Ltd.) using the standard addition technique. The results, shown in Table 2, show that in a 0.1 mol / L serum solution in 0.15 mol / L PBS (pH 6.0), the recoveries for six measurements ranged from 92.9% to 107.6%, with an average recovery of 98.3%. This demonstrates that the homocysteine ​​biosensor exhibits good recovery of spiked homocysteine ​​in serum samples.

[0115] Table 2 - Recovery rate experimental results

[0116]

[0117] In summary, this application combines a simple electrochemical deposition method with a drop coating method to prepare AuNPs / AB-DHP / GCE and construct a homocysteine ​​biomedical sensing interface. Electrochemical characterization shows that the high conductivity of gold nanoparticles and the high porosity of acetylene black are of great significance for improving the electrocatalytic performance of the composite material for homocysteine. The AuNPs / AB-DHP nanocomposite film was characterized by SEM and EDS, and the reaction mechanism of homocysteine ​​at the sensing interface was explored. The results show that the sensor has low cost, low detection limit, and good stability. In addition, the spiked recovery rate of homocysteine ​​in serum samples is good, which provides potential application value for the reliable determination of homocysteine ​​in biological systems and the study of homocysteine-related diseases.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a homocysteine ​​electrochemical sensor, characterized in that: The following steps are involved: The carbon nanomaterial and dihexadecyl phosphoric acid are added into water and ultrasonicated to prepare a dispersion; The dispersion is added dropwise to the surface of the glassy carbon electrode and baked to obtain a carbon nanomaterial modified electrode; The carbon nanomaterial modified electrode is placed in a gold salt aqueous solution, and nano-gold is deposited on the surface of the carbon nanomaterial modified electrode by an electrodeposition method, thereby obtaining a homocysteine ​​electrochemical sensor. The carbon nanomaterial includes at least one of acetylene black, carbon nanotubes, graphene oxide, and graphene; The gold salt aqueous solution includes one of a HAuCl4 aqueous solution and a NaAuCl4·2H2O aqueous solution.

2. The method for preparing a homocysteine ​​electrochemical sensor according to claim 1, wherein: In the step of adding carbon nanomaterials and dicetyl phosphoric acid into water to obtain a dispersion, the mass volume ratio of carbon nanomaterials, dicetyl phosphoric acid and water is (2-6) mg: (2-6) mg: (2-6) mL.

3. The method for preparing a homocysteine ​​electrochemical sensor according to claim 1, wherein: The concentration of the gold salt aqueous solution is 3-7 mmoL / L.

4. The method for preparing a homocysteine ​​electrochemical sensor according to claim 1, wherein: The electrochemical parameters controlled by the electrodeposition method for depositing nano-gold on the surface of the carbon nanomaterial modified electrode are: a scan rate of 80 to 120 mV / s, a potential range of -1.5 to 0 V, and a cycle scan of 5 to 35 circles.

5. The method for preparing a homocysteine ​​electrochemical sensor according to claim 1, wherein: Before adding the dispersion dropwise to the surface of the glassy carbon electrode, the glassy carbon electrode is pretreated. Specifically, the pretreatment comprises: polishing the glassy carbon electrode with a polishing cloth; and then ultrasonically cleaning the polished glassy carbon electrode in sequence with a 1:1 volume ratio of nitric acid aqueous solution, anhydrous ethanol, and ultrapure water, thereby completing the pretreatment of the glassy carbon electrode.

6. The method for preparing a homocysteine ​​electrochemical sensor according to claim 1, wherein: 2-10 μL of the dispersion was added dropwise to the surface of the glassy carbon electrode.

7. A homocysteine ​​electrochemical sensor, characterized in that: The preparation method is as described in any one of claims 1 to 6.

8. Use of the homocysteine ​​electrochemical sensor prepared by the preparation method according to any one of claims 1 to 6 or the homocysteine ​​electrochemical sensor according to claim 7 in detecting homocysteine ​​concentration.

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