Preparation of laser-induced graphene / metal nanocrystal composite electrode and its application in detection of bioactive small molecules

Abstract

The application discloses a preparation method of a graphene / metal nanocrystal composite electrode, and a product and application thereof, and belongs to the technical field of biosensing. The preparation method of the graphene / metal nanocrystal composite electrode comprises the following steps: S1, printing a reference electrode, a counter electrode, a working electrode and a wire on a polyimide substrate by means of screen printing; S2, performing first CO2 laser induction treatment on a working electrode area printed on the polyimide substrate; S3, applying a metal precursor solution on the working electrode area; and S4, performing second CO2 laser induction treatment on the working electrode area coated with the metal precursor solution. The graphene / metal nanocrystal composite electrode prepared by the preparation method can be used for electrochemical biosensing detection of bioactive small molecules.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology, and in particular to a method for preparing a graphene / metal nanocrystal composite electrode, its products, and applications. Background Technology

[0002] Electrochemical biosensors are devices that convert the information generated by the biochemical reaction of analytes into electrical signals and output them based on electrochemical principles. Their advantages include high sensitivity, low detection limits, rapid detection, and compatibility with other micro / nano fabrication technologies. They are currently widely used in biochemical analysis, disease monitoring, food safety, and other fields. Research on high-performance electrochemical biosensors is progressing rapidly, and the development of micro / nano integrated electrochemical sensors with ultra-sensitive and selective detection capabilities has become a research trend. In-situ integration and growth of nanomaterials in the sensitive element region of the sensor can simplify the sensor electrode fabrication process, eliminate undesirable interfaces caused by additional binders / additives, and provide a larger surface area and fully exposed active sites. Furthermore, reasonable integration and growth techniques are expected to improve the stability and consistency of the sensor device.

[0003] Graphene, a hexagonal planar thin-layer material composed of sp2 hybridized carbon atoms, possesses a high specific surface area, excellent electrical conductivity, and catalytic activity, making it an ideal sensitive material for constructing electrochemical biosensors and attracting widespread attention in the biomedical field. Graphene is typically synthesized via mechanical exfoliation, chemical exfoliation, and chemical vapor deposition. However, these methods can cause severe pollution or require complex synthesis steps under stringent conditions, resulting in high production costs. To meet the demands of practical applications, the development of new, simple, and efficient graphene preparation methods is essential.

[0004] Bioactive small molecules play a crucial role in various metabolic processes and the entire life cycle in the human body, directly affecting health. For example, dopamine (DA), an important neurotransmitter, plays a vital role in regulating various physiological functions of the central nervous system in mammals. DA deficiency can lead to various neurological disorders, such as schizophrenia and Parkinson's disease. Uric acid (UA) is the main end product of purine metabolism, and excessively high concentrations of UA in urine and serum are associated with various diseases, including gout and hyperuricemia. Therefore, monitoring the levels of bioactive small molecules in the body is particularly important. Highly sensitive and rapid detection of bioactive small molecules such as DA and UA in body fluids is essential for disease prevention, monitoring, and clinical diagnosis. The development of electrochemical biosensors constructed from graphene for detecting bioactive small molecules using simple methods would have a very broad application prospect. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a graphene / metal nanocrystal composite electrode, as well as its products and applications (specifically, its application in the detection of bioactive small molecules). This invention involves screen printing an electrochemical sensing electrode onto a polyimide (PI) substrate. The working electrode and counter electrode are printed using carbon paste, the reference electrode using Ag / AgCl paste, and the wires using silver paste. Then, the working electrode area is laser-induced to transform the polyimide substrate in the working electrode area into a graphene (LEG) electrode (the pre-printed carbon electrode is used for laser etching and positioning; the carbon paste is removed during the laser treatment, and graphene is etched onto the PI). Next, a metal precursor solution is coated onto the graphene electrode, and laser-induced treatment is performed again, achieving in-situ integration of the graphene / metal nanocrystal composite material on the surface of the electrochemical sensor electrode, resulting in a graphene / metal nanocrystal composite electrode.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of the present invention: a method for preparing a graphene / metal nanocrystal composite electrode, comprising the following steps:

[0008] S1. The reference electrode, counter electrode, working electrode, and wires are printed onto a polyimide substrate by screen printing.

[0009] S2. Perform a CO2 laser-induced treatment on the working electrode area printed on the polyimide substrate;

[0010] S3. Apply the metal precursor solution to the working electrode area;

[0011] S4. Perform secondary CO2 laser-induced treatment on the working electrode area coated with the metal precursor solution.

[0012] Furthermore, in step S1, the reference electrode is printed using Ag / AgCl paste, the working electrode and the counter electrode are printed using carbon paste, and the wires are printed using silver paste.

[0013] Furthermore, the conditions for the CO2 laser-induced treatment in step S2 are as follows: laser wavelength 10.6μm, defocus distance 2.5-7.5mm, laser power 2.5-7.5w, scanning speed 5-15cm / s, and pixel density unit 500-1000PPI.

[0014] Furthermore, the preferred conditions for the CO2 laser-induced treatment in step S2 are: laser wavelength 10.6 μm, defocus distance 5 mm, laser power 5 W, scanning speed 10 cm / s, and pixel density unit 1000 PPI.

[0015] Furthermore, the preparation method of the metal precursor solution in step S3 is as follows: the metal salt is mixed with an ethylene glycol solution and ultrasonically dispersed evenly.

[0016] Furthermore, the metal salt is one or two of rhodium salt, platinum salt, palladium salt, gold salt, silver salt, ruthenium salt, and iridium salt.

[0017] Further, the rhodium salt is RhCl3·nH2O or Na3RhCl6, the platinum salt is H2PtCl6·nH2O or Na2PtCl6·nH2O or K2PtCl6, the gold salt is HAuCl4·nH2O or KAuCl4 or AuCl3, the silver salt is AgNO3, and n is a natural number.

[0018] Furthermore, the molar concentration of the metal element in the metal precursor solution is 2.5-10 mM. When the metal precursor solution contains two metals, the molar concentration of each metal is 2.5-10 mM, and the volume of the coated metal precursor solution is 0.25-1 μL.

[0019] Furthermore, the molar concentration of the metal element in the metal precursor solution is preferably 5 mM. When the metal precursor solution contains two metals, the molar concentration of each metal is 5 mM, and the volume of the coated metal precursor solution is preferably 0.5 μL.

[0020] Furthermore, after the metal precursor solution is coated onto the working electrode, it is first dried and then subjected to a secondary CO2 laser-induced treatment.

[0021] Furthermore, the conditions for the secondary CO2 laser-induced treatment in step S4 are as follows: laser wavelength 10.6 μm, defocus distance 2.5-7.5 mm, laser power 2.5-7.5 W, scanning speed 5-15 cm / s, and pixel density unit 500-1000 PPI.

[0022] Furthermore, the preferred conditions for the secondary CO2 laser-induced processing in step S4 are: laser wavelength 10.6 μm, defocus distance 5 mm, laser power 5 W, scanning speed 10 cm / s, and pixel density unit 1000 PPI.

[0023] The second technical solution of the present invention: a graphene / metal nanocrystal composite electrode prepared according to the above preparation method.

[0024] Furthermore, when there are two types of metals, the resulting graphene / metal nanocrystal composite electrode is a graphene / bimetal nanocrystal composite electrode.

[0025] The third technical solution of the present invention: the application of the above-mentioned graphene / metal nanocrystal composite electrode in the detection of bioactive small molecules.

[0026] Furthermore, the bioactive small molecule is dopamine, uric acid, ascorbic acid, acetaminophen, or tyrosine.

[0027] Furthermore, the detection is an electrochemical biosensing detection.

[0028] The present invention discloses the following technical effects:

[0029] (1) In this invention, electrochemical sensing electrodes are printed on a polyimide (PI) substrate by screen printing. Then, the working electrode area is laser-induced by CO2 laser-induced graphene technology to convert the polyimide in the working electrode area into a graphene (LEG) electrode (the CO2 laser converts sp3 hybrid carbon into sp2 hybrid carbon through photothermal or photochemical effects to obtain graphene. The pre-printed carbon electrode is used for laser etching positioning. During the laser treatment, the carbon paste is removed and graphene is etched on the PI). After that, a metal precursor solution is coated on the graphene electrode and laser-induced treatment is performed again to generate metal nanocrystals (the metal particles obtained by laser etching are smaller, more uniform, and have better consistency, which is more conducive to improving the electrochemical sensing performance). The graphene / metal nanocrystal composite material is integrated in situ on the surface of the electrochemical sensor electrode to obtain a graphene / metal nanocrystal composite electrode.

[0030] (2) The present invention prepares graphene by laser-induced technology. This laser-induced graphene preparation technology can selectively process microstructure regions, is easy to operate, does not require the introduction of additional chemical reagents, is inexpensive, and has the potential for large-scale industrial manufacturing.

[0031] (3) In the graphene / metal nanocrystal composite electrode prepared by laser-induced technology in this invention, the composite material exhibits better performance due to the synergistic effect of LEG and metal nanocrystals. Specifically, the LEG of the three-dimensional porous scaffold generated by laser-induced technology has high specific surface area, high thermal stability, and excellent electronic conductivity and catalytic activity. Moreover, its three-dimensional porous scaffold structure can increase the loading of metal nanocrystals and avoid the aggregation of metal nanocrystals in applications such as catalytic reactions. At the same time, metal or bimetallic nanocrystals have excellent catalytic activity and unique electrical properties. Their addition can improve the detectable signal and the ability to selectively detect analytes. This invention uses CO2 laser-induced technology to prepare graphene / metal nanocrystal composite electrodes. By controlling the microstructure and composition of the composite material system, each structural unit can give full play to its own advantages, thereby providing unique sensitive materials for electrochemical biosensing detection, especially suitable sensitive materials for electrochemical biosensing detection of bioactive small molecules (dopamine, uric acid, ascorbic acid, acetaminophen, or tyrosine). Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a SEM image of the LEG / Rh nanocrystalline composite electrode prepared in Example 1 of the present invention;

[0034] Figure 2 The following are the elemental analysis results of the LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of the present invention, where a is the SEM image of the LEG / PtRh bimetallic nanocrystalline composite electrode, b is the energy dispersive spectral analysis result of carbon (C) in the selected region on the electrode surface, c is the energy dispersive spectral analysis result of platinum (Pt), and d is the energy dispersive spectral analysis result of rhodium (Rh).

[0035] Figure 3 The following are the elemental analysis results of the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 of the present invention, where a is the SEM image of the LEG / AuAg bimetallic nanocrystalline composite electrode, b is the energy dispersive spectroscopy (EDS) analysis result of carbon (C) in the selected region on the electrode surface, c is the EDS analysis result of gold (Au) and d is the EDS analysis result of silver (Ag).

[0036] Figure 4The DPV test results of the LEG / Rh nanocrystalline composite electrode prepared in Example 1, the LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2, and the LEG / Pt nanocrystalline composite electrode prepared in Example 3 in a mixed solution of 500 μM AA, 200 μM UA, 100 μM MDA, and 100 μM AP.

[0037] Figure 5 The DPV test results of the LEG / Au nanocrystalline composite electrode prepared in Example 4, the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5, and the LEG / Ag nanocrystalline composite electrode prepared in Example 6 of this invention in a mixed solution of 500 μM AA, 200 μM UA, 100 μM MDA, and 100 μM AP.

[0038] Figure 6 The results of DPV testing of the LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of this invention for different concentrations of AA, DA, UA and AP are shown, where: a is AA, b is DA, c is UA and d is AP.

[0039] Figure 7 The graph shows the linear relationship between the LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of this invention when detecting AA, DA, UA and AP, where: a is AA, b is DA, c is UA and d is AP;

[0040] Figure 8 The results of DPV testing of different concentrations of AA on the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 of this invention;

[0041] Figure 9 The graph shows the linear relationship of the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 of this invention when detecting AA.

[0042] Figure 10 The results of DPV testing of different concentrations of DA on the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 of this invention;

[0043] Figure 11 The graph shows the linear relationship when the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 of this invention detects DA. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe the methods and materials associated with those references. In the event of any conflict with any incorporated reference, the contents of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] In the following examples and comparative examples, a KQ-V3545H (Shanghai Chaoyang Screen Printing Material Equipment) screen printing machine was used for screen printing, and a TROTEC Speedy 100R laser engraving machine was used for one CO2 laser-induced treatment and a two-stage CO2 laser-induced treatment.

[0050] Example 1

[0051] The preparation of graphene / rhodium (LEG / Rh) nanocrystalline composite electrodes includes the following steps:

[0052] S1. The reference electrode (printed using Ag / AgCl paste), working electrode and counter electrode (printed using carbon paste), and wires (printed using silver paste) are screen printed on a high-insulation polyimide (PI) substrate (PI film).

[0053] S2. Using a 10.6μM CO2 laser, with a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI, a CO2 laser-induced treatment is performed on the working electrode area printed with carbon paste on the PI film substrate to induce the generation of a three-dimensional porous graphene microelectrode on the PI film substrate (converting the polyimide in the working electrode area into a graphene electrode).

[0054] S3. Weigh 26.3 mg of RhCl3·3H2O (0.1 mmol) and mix it with 20 mL of ethylene glycol solution. Disperse the mixture evenly by ultrasonication to obtain a metal precursor solution (RhCl3 ethylene glycol solution with a molar concentration of 5 mM).

[0055] S4. Apply 0.5 μL of the metal precursor solution to the working electrode area (laser-induced graphene area) using a pipette, and dry it in an infrared drying oven.

[0056] S5. Using a 10.6μM CO2 laser, a secondary CO2 laser-induced treatment was performed on the working electrode area coated with the metal precursor solution under the laser induction conditions of a defocus distance of 5mm, laser power of 5w, scanning speed of 10cm / s, and pixel density of 1000PPI to obtain a LEG / Rh nanocrystalline composite electrode.

[0057] Example 2

[0058] The preparation of a graphene / platinum-rhodium (LEG / PtRh) bimetallic nanocrystalline composite electrode includes the following steps:

[0059] S1. The reference electrode (printed using Ag / AgCl paste), working electrode and counter electrode (printed using carbon paste), and wires (printed using silver paste) are screen printed on a high-insulation polyimide (PI) substrate (PI film).

[0060] S2. Using a 10.6μM CO2 laser, with a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI, a CO2 laser-induced treatment is performed on the working electrode area printed with carbon paste on the PI film substrate to induce the generation of a three-dimensional porous graphene microelectrode on the PI film substrate (converting the polyimide in the working electrode area into a graphene electrode).

[0061] S3. Weigh 26.3 mg RhCl3·3H2O (0.1 mmol) and 51.8 mg H2PtCl6·6H2O (0.1 mmol) and mix them with 20 mL of ethylene glycol solution. Disperse the mixture evenly by sonication to obtain a metal precursor solution (a mixed solution of RhCl3 / H2PtCl6 in ethylene glycol with a molar concentration of 5 mM for both RhCl3 and H2PtCl6).

[0062] S4. Apply 0.5 μL of the metal precursor solution to the working electrode area (laser-induced graphene area) using a pipette, and dry it in an infrared drying oven.

[0063] S5. Using a 10.6μM CO2 laser, a secondary CO2 laser-induced treatment was performed on the working electrode area coated with the metal precursor solution under the laser induction conditions of a defocus distance of 5mm, laser power of 5w, scanning speed of 10cm / s, and pixel density of 1000PPI to obtain a LEG / PtRh nanocrystalline composite electrode.

[0064] Example 3

[0065] The preparation of graphene / platinum (LEG / Pt) nanocrystalline composite electrodes includes the following steps:

[0066] S1. The reference electrode (printed using Ag / AgCl paste), working electrode and counter electrode (printed using carbon paste), and wires (printed using silver paste) are screen printed on a high-insulation polyimide (PI) substrate (PI film).

[0067] S2. Using a 10.6μM CO2 laser, with a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI, a CO2 laser-induced treatment is performed on the working electrode area printed with carbon paste on the PI film substrate to induce the generation of a three-dimensional porous graphene microelectrode on the PI film substrate (converting the polyimide in the working electrode area into a graphene electrode).

[0068] S3. Weigh 51.8 mg of H2PtCl6·6H2O (0.1 mmol) and mix it with 20 mL of ethylene glycol solution. Disperse the mixture evenly by ultrasonication to obtain a metal precursor solution (H2PtCl6 ethylene glycol solution with a molar concentration of 5 mM).

[0069] S4. Apply 0.5 μL of the metal precursor solution to the working area electrode (laser-induced graphene area) using a pipette, and dry it in an infrared drying oven.

[0070] S5. Using a 10.6μM CO2 laser, a secondary CO2 laser-induced treatment was performed on the working electrode area coated with the metal precursor solution under the laser induction conditions of a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI to obtain a LEG / Pt nanocrystalline composite electrode.

[0071] Example 4

[0072] The preparation of graphene / gold (LEG / Au) nanocrystalline composite electrodes includes the following steps:

[0073] S1. The reference electrode (printed using Ag / AgCl paste), working electrode and counter electrode (printed using carbon paste), and wires (printed using silver paste) are screen printed on a high-insulation polyimide (PI) substrate (PI film).

[0074] S2. Using a 10.6μM CO2 laser, with a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI, a CO2 laser-induced treatment is performed on the working electrode area printed with carbon paste on the PI film substrate to induce the generation of a three-dimensional porous graphene microelectrode on the PI film substrate (converting the polyimide in the working electrode area into a graphene electrode).

[0075] S3. Weigh 41.2 mg HAuCl4·4H2O (0.1 mmol) and mix it with 20 mL of ethylene glycol solution. Disperse the mixture evenly by ultrasonication to obtain a metal precursor solution (HAuCl4 ethylene glycol solution with a molar concentration of 5 mM).

[0076] S4. Apply 0.5 μL of the metal precursor solution to the working electrode area (laser-induced graphene area) using a pipette, and dry it in an infrared drying oven.

[0077] S5. Using a 10.6μM CO2 laser, a secondary CO2 laser-induced treatment was performed on the working electrode area coated with the metal precursor solution under the laser induction conditions of a defocus distance of 5mm, laser power of 5w, scanning speed of 10cm / s, and pixel density of 1000PPI to obtain a LEG / Au nanocrystalline composite electrode.

[0078] Example 5

[0079] The preparation of a graphene / platinum (LEG / AuAg) bimetallic nanocrystalline composite electrode includes the following steps:

[0080] S1. The reference electrode (printed using Ag / AgCl paste), working electrode and counter electrode (printed using carbon paste), and wires (printed using silver paste) are screen printed on a high-insulation polyimide (PI) substrate (PI film).

[0081] S2. Using a 10.6μM CO2 laser, with a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI, a CO2 laser-induced treatment is performed on the working electrode area printed with carbon paste on the PI film substrate to induce the generation of a three-dimensional porous graphene microelectrode on the PI film substrate (converting the polyimide in the working electrode area into a graphene electrode).

[0082] S3. Weigh 17.0 mg AgNO3 (0.1 mmol) and 41.2 mg HAuCl4·4H2O (0.1 mmol) and mix them with 20 mL of ethylene glycol solution. Disperse the mixture evenly by ultrasonication to obtain a metal precursor solution (a 5 mM AgNO3 / HAuCl4 ethylene glycol mixed solution).

[0083] S4. Apply 0.5 μL of the metal precursor solution to the working electrode area (laser-induced graphene area) using a pipette, and dry it in an infrared drying oven.

[0084] S5. Using a 10.6μM CO2 laser, with a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI, a secondary CO2 laser-induced treatment was performed on the working electrode area coated with the metal precursor solution to obtain a LEG / AuAg nanocrystalline composite electrode.

[0085] Example 6

[0086] The preparation of a graphene / rhodium / gold (LEG / Ag) bimetallic nanocrystalline composite electrode includes the following steps:

[0087] S1. The reference electrode (printed using Ag / AgCl paste), working electrode, counter electrode (printed using carbon paste), and silver wire (printed using silver paste) are screen printed on a high-insulation polyimide (PI) substrate (PI film).

[0088] S2. Using a 10.6μM CO2 laser, with a defocus distance of 5mm, laser power of 5W, scanning speed of 10cm / s, and pixel density of 1000PPI, a CO2 laser-induced treatment is performed on the working electrode area printed with carbon paste on the PI film substrate to induce the generation of a three-dimensional porous graphene microelectrode on the PI film substrate (converting the polyimide in the working electrode area into a graphene electrode).

[0089] S3. Weigh 17.0 mg AgNO3 and mix it with 20 mL of ethylene glycol solution. Disperse the mixture evenly by ultrasonication to obtain a metal precursor solution (a 5 mM AgNO3 mixed solution in ethylene glycol).

[0090] S4. Apply 0.5 μL of the metal precursor solution to the working electrode area (laser-induced graphene area) using a pipette, and dry it in an infrared drying oven.

[0091] S5. Using a 10.6μM CO2 laser, a secondary CO2 laser-induced treatment was performed on the working electrode area coated with the metal precursor solution under the laser induction conditions of a defocus distance of 5mm, laser power of 5w, scanning speed of 10cm / s, and pixel density of 1000PPI to obtain a LEG / Ag nanocrystalline composite electrode.

[0092] Effect verification

[0093] 1. Morphological and structural characterization

[0094] The surface morphology of the LEG / Rh nanocrystalline composite electrode prepared in Example 1 and the LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of this invention were characterized. The SEM image of the LEG / Rh nanocrystalline composite electrode prepared in Example 1 is shown below. Figure 1 As shown, by Figure 1 It can be seen that porous foam-structured graphene can be successfully integrated in situ on the electrode surface using laser etching. The graphene structure remains intact after secondary laser treatment, and secondary laser irradiation can deposit Rh nanocrystals in situ on the graphene surface. The elemental analysis results of the LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 are as follows: Figure 2 As shown, a) is the SEM image of the LEG / PtRh bimetallic nanocrystalline composite electrode, b) is the energy dispersive spectral analysis (EDS) result of carbon (C) in the selected region of the electrode surface, c) is the EDS result of platinum (Pt), and d is the EDS result of rhodium (Rh). From a), it can be seen that the nanocrystals obtained by laser etching are uniformly and consistently dispersed on the graphene surface. The EDS characterization results of b and d show that C, Pt, and Rh are uniformly distributed in the characterization region, proving that the nanocrystals prepared by laser etching are PtRh nanocrystals. Introducing a bimetallic precursor salt onto the graphene electrode surface and then treating it with laser irradiation can successfully prepare the LEG / PtRh bimetallic nanocrystalline composite electrode. The elemental analysis results of the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 are as follows: Figure 3As shown, a is the SEM image of the LEG / AuAg bimetallic nanocrystalline composite electrode, b is the energy dispersive spectral analysis result of carbon (C) in the selected area on the electrode surface, c is the energy dispersive spectral analysis result of gold (Au), and d is the energy dispersive spectral analysis result of silver (Ag). It can be seen from a that the nanocrystals obtained by laser etching are uniformly and consistently dispersed on the graphene surface. The energy dispersive spectral characterization results of bd show that C, Au and Ag are uniformly distributed in the characterization area, proving that the nanocrystals prepared by laser etching are AuAg nanocrystals.

[0095] 2. Electrochemical analysis (analysis of the detection effect on bioactive small molecules)

[0096] The electrochemical analysis was performed on a VSP-300 electrochemical workstation (BioLogic, France).

[0097] (1) Analysis of the detection effect of each bioactive small molecule in the mixed solution

[0098] 1) The LEG / Rh nanocrystalline composite electrode prepared in Example 1, the LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2, and the LEG / Pt nanocrystalline composite electrode prepared in Example 3 were connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry (DPV). A 10 mM PBS solution was prepared, and different concentrations of bioactive small molecules were added to obtain a mixed solution of 500 μM ascorbic acid (AA), 200 μM uric acid (UA), 100 μM dopamine (DA), and 100 μM acetaminophen (AP). 10 μL of the mixed solution was dropped onto the working area surface of the LEG / Rh nanocrystalline composite electrode, the LEG / Pt nanocrystalline composite electrode, and the LEG / PtRh bimetallic nanocrystalline composite electrode, respectively, and differential pulse voltammetry was performed. The scanning range was -0.6 to 0.9 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms. The DPV test results of LEG / PtRh bimetallic nanocrystalline composite electrodes, LEG / Rh nanocrystalline composite electrodes, and LEG / Pt nanocrystalline composite electrodes in a mixed solution of 500 μM AA, 200 μM UA, 100 μM DA, and 100 μM AP are as follows: Figure 4 As shown. By Figure 4It can be seen that the LEG / PtRh bimetallic nanocrystalline composite electrode exhibited four distinct oxidation peaks during DPV testing (the graphene / metal nanocrystalline composite electrode detects different small molecules through direct catalytic oxidation; different small molecules have different catalytic oxidation potentials, which manifest as DPV oxidation peaks at different positions in the electrochemical test). This indicates that the electrode can effectively distinguish four electrochemically active small molecules, and the DPV peak current value of the LEG / PtRh electrode is significantly higher than that of the LEG / Pt and LEG / Rh electrodes. Furthermore, the LEG / Rh electrode struggled to distinguish the responses of UA and AP small molecules during DPV testing, while the current response value of the LEG / Pt electrode was significantly lower than that of the LEG / PtRh electrode.

[0099] 2) The LEG / Au nanocrystalline composite electrode prepared in Example 4, the LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5, and the LEG / Ag nanocrystalline composite electrode prepared in Example 6 were connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry. A 10 mM PBS solution was prepared, and different concentrations of bioactive small molecules were added to obtain a mixed solution of 500 μM ascorbic acid (AA), 200 μM uric acid (UA), 100 μM dopamine (DA), and 100 μM acetaminophen (AP). 10 μL of the mixed solution was dropped onto the working area surface of the LEG / Au nanocrystalline composite electrode, the LEG / Ag nanocrystalline composite electrode, and the LEG / AuAg bimetallic nanocrystalline composite electrode, respectively, and differential pulse voltammetry was performed. The scanning range was -0.3 to 0.8 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms.

[0100] The DPV test results of LEG / AuAg bimetallic nanocrystalline composite electrodes, LEG / Au nanocrystalline composite electrodes, and LEG / Ag nanocrystalline composite electrodes in a mixed solution of 500 μM AA, 200 μM UA, 100 μM DA, and 100 μM AP are as follows: Figure 5 As shown. By Figure 5 It is known that the LEG / AuAg electrode can be used to detect three small molecules: AA, DA, and UA, while the LEG / Au and LEG / Ag electrodes can only detect DA and UA. Furthermore, the LEG / AuAg electrode provides a higher detection signal for the same concentration of small molecules compared to the LEG / Au and LEG / Ag electrodes.

[0101] (2) Sensing performance test of LEG / PtRh electrode

[0102] 1) DPV test of different concentrations of AA using LEG / PtRh electrode

[0103] The LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of this invention was connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry. 10 mM PBS solution was prepared, and different amounts of AA were added to obtain AA solutions with concentrations of 100, 200, 300, 400, and 500 μM, respectively. 10 μL of AA solution was dropped onto the working area surface of the LEG / PtRh electrode, and differential pulse voltammetry was performed. The scan range was -0.6 to 0.0 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms.

[0104] 2) DPV test of LEG / PtRh electrode with different concentrations of DA

[0105] The LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of this invention was connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry. A 10 mM PBS solution was prepared, and different amounts of DA were added to obtain DA solutions with concentrations of 10, 20, 30, 40, and 50 μM, respectively. 10 μL of DA solution was dropped onto the working area surface of the LEG / PtRh electrode, and differential pulse voltammetry was performed. The scan range was -0.3 to 0.4 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms.

[0106] 3) DPV test of different concentrations of UA using LEG / PtRh electrode

[0107] The LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of this invention was connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry. 10 mM PBS solution was prepared, and different amounts of UA were added to obtain UA solutions with concentrations of 10, 20, 30, 40, and 50 μM, respectively. 10 μL of UA solution was dropped onto the working area surface of the LEG / PtRh electrode, and differential pulse voltammetry was performed. The scan range was 0.0–0.7 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms.

[0108] 4) DPV testing of different AP concentrations using LEG / PtRh electrode

[0109] The LEG / PtRh bimetallic nanocrystalline composite electrode prepared in Example 2 of this invention was connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry. A 10 mM PBS solution was prepared, and different amounts of AP were added to obtain AP solutions with concentrations of 5, 10, 20, 30, and 50 μM, respectively. 10 μL of AP solution was dropped onto the working area surface of the LEG / PtRh electrode, and differential pulse voltammetry was performed. The scan range was 0.0–0.6 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms.

[0110] The DPV test results of the LEG / PtRh electrode for different concentrations of AA, DA, UA and AP are as follows: Figure 6 As shown, a represents the test result for AA, b represents the test result for DA, c represents the test result for UA, and d represents the test result for AP. Figure 6 It is known that the DPV response current of four small molecules, ascorbic acid (AA), dopamine (DA), uric acid (UA), and acetaminophen (AP), increases with increasing concentration. The LEG / PtRh electrode can detect AA in the concentration range of 100-500 μM, DA in the concentration range of 10-50 μM, AA in the concentration range of 10-50 μM, and AP in the concentration range of 5-50 μM, respectively.

[0111] Based on the DPV test results of the LEG / PtRh electrode for different concentrations of AA, DA, UA, and AP, a linear fit was performed on the peak current values ​​and concentrations of ascorbic acid (AA), dopamine (DA), uric acid (UA), and acetaminophen (AP) to obtain the linear relationship graphs for the detection of AA, DA, UA, and AP by the LEG / PtRh electrode, as shown below. Figure 7 As shown, where a represents the linear relationship between AA, b represents the linear relationship between DA, c represents the linear relationship between UA, and d represents the linear relationship between AP. From... Figure 7 It can be seen that the linear fitting equation for AA is y = 0.00353x + 0.86717, R0 2 The value is 0.94583; the linear fitting equation for DA is y = 0.01166x + 0.67911, R0 = 0.94583. 2 The value is 0.98066; the linear fitting equation for UA is y = 0.04349x + 0.77447, R0 2 The value is 0.94633; the linear fitting equation for AP is y = 0.05671x + 3.25054, R0. 2 It is 0.96531.

[0112] (3) Sensing performance test of LEG / AuAg electrode

[0113] 1) DPV testing of different concentrations of AA using LEG / AuAg electrode

[0114] The LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 of this invention was connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry. 10 mM PBS solution was prepared, and different amounts of AA were added to obtain AA solutions with concentrations of 1, 5, 10, 20, 50, and 100 μM, respectively. 10 μL of AA solution was dropped onto the working area surface of the LEG / AuAg electrode, and differential pulse voltammetry was performed. The scan range was -0.6 to 0.8 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms.

[0115] 2) DPV testing of different concentrations of DA using LEG / AuAg electrode

[0116] The LEG / AuAg bimetallic nanocrystalline composite electrode prepared in Example 5 of this invention was connected to an electrochemical workstation, and electrochemical scanning was performed using differential pulse voltammetry. 10 mM PBS solution was prepared, and different amounts of DA were added to obtain DA solutions with concentrations of 0.2, 2, 5, 10, 50, and 100 μM, respectively. 10 μL of DA solution was dropped onto the working area surface of the LEG / AuAg electrode, and differential pulse voltammetry was performed. The scan range was -0.6 to 0.8 V, the amplitude was 50 mV, the pulse width was 50 ms, and the pulse period was 500 ms.

[0117] The DPV test results of LEG / AuAg electrode for different concentrations of AA are as follows: Figure 8 As shown, by Figure 8 It is known that the LEG / AuAg electrode can detect AA molecules in the concentration range of 1-100 μM and has a good linear response; the detection limit for AA molecules using the DPV detection method is 1 μM.

[0118] Based on the DPV test results of different concentrations of amino acids (AA) using the LEG / AuAg electrode, a linear fit was performed on the peak current value and concentration of AA to obtain a linear relationship graph for AA detection using the LEG / AuAg electrode, as shown in the figure. Figure 9 As shown, by Figure 9 It can be seen that the linear fitting equation for AA is y = 0.1035x + 10.242, R0 2 It is 0.9451.

[0119] The DPV test results of LEG / AuAg electrode for different concentrations of DA are as follows: Figure 10 As shown, based on the DPV test results of the LEG / AuAg electrode for different concentrations of DA, a linear fit was performed on the peak current value and concentration of DA to obtain the linear relationship graph of DA detection by the LEG / AuAg electrode, as shown in the figure. Figure 11 As shown. By Figure 10 and Figure 11It is known that the LEG / AuAg electrode can detect DA molecules in the concentration range of 0.2-100 μM. The linear relationship in the 0.2-10 μM range conforms to y = 1.2903x + 11.724, and the linear relationship in the 10-100 μM range conforms to y = 0.234x + 22.15. Using the DPV detection method, the detection limit for DA molecules is 0.2 μM.

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An application of a graphene / metal nanocrystal composite electrode in the detection of bioactive small molecules, characterized in that, The preparation steps of the graphene / metal nanocrystal composite electrode include: S1. The reference electrode, counter electrode, working electrode, and wires are printed onto a polyimide substrate by screen printing. S2. Perform a CO2 laser-induced treatment on the working electrode area printed on the polyimide substrate; S3. Apply the metal precursor solution to the working electrode area; S4. Perform secondary CO2 laser-induced treatment on the working electrode area coated with the metal precursor solution. The method for preparing the metal precursor solution in step S3 is as follows: the metal salt is mixed with an ethylene glycol solution and ultrasonically dispersed evenly; The metal salts are rhodium salts and platinum salts; The bioactive small molecules are dopamine, uric acid, ascorbic acid, and acetaminophen.

2. The application as described in claim 1, characterized in that, In step S1, the working electrode and counter electrode are printed using carbon paste, the reference electrode is printed using Ag / AgCl paste, and the wires are printed using silver paste.

3. The application as described in claim 1, characterized in that, The conditions for the CO2 laser-induced treatment in step S2 are as follows: laser wavelength 10.6μm, defocus distance 2.5-7.5mm, laser power 2.5-7.5w, scanning speed 5-15cm / s, and pixel density unit 500-1000PPI.

4. The application as described in claim 1, characterized in that, The molar concentration of each metal element in the metal precursor solution is 2.5-10 mM, and the volume of the coated metal precursor solution is 0.25-1 μL.

5. The application as described in claim 1, characterized in that, The conditions for the secondary CO2 laser-induced treatment in step S4 are: laser wavelength 10.6μm, defocus distance 2.5-7.5mm, laser power 2.5-7.5w, scanning speed 5-15cm / s, and pixel density unit 500-1000PPI.

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