Composite electrode for rapidly detecting cortisol, preparation method of composite electrode and application of composite electrode in cortisol detection

Abstract

The invention discloses a composite electrode for rapidly detecting cortisol and application of the composite electrode in rapid detection of cortisol. The composite electrode comprises a laser-induced graphene substrate, a gold nano material layer modified on the laser-induced graphene substrate and a perfluorosulfonic acid polymer modified on the gold nano material layer. The composite electrode does not need an incubation binding step, is high in timeliness, improves the detection rate, can realize real-time analysis, and is stable in film-forming interface; the method is not influenced by the dispersity of a catalytic material, so that the difficulty of the product performance consistency requirement in a quality control link in industrial production is greatly reduced; the electrocatalytic analysis of cortisol can be realized in the PBS buffer solution, and the application range is wider.

Description

TECHNICAL FIELD

[0001] The application relates to a composite electrode for rapid detection of cortisol and application thereof in cortisol detection, and belongs to the technical field of biological detection. BACKGROUND

[0002] Cortisol is an important biological stress marker, which has the characteristics of low concentration and rapid change, and therefore has higher requirements for analysis sensitivity and analysis rate. The mainstream detection method of cortisol molecules, such as liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay, has long test time, high cost and relatively complex detection process, and cannot meet the requirements of rapid real-time detection. The electrochemical method has the advantages of high sensitivity, strong specificity, low cost and fast response, but the electrochemical activity of cortisol molecules is extremely weak. The existing research mainly constructs an immunosensor based on biological antibodies or a biomimetic recognition element based on molecular imprinting to construct a biomimetic immunosensor, and the preparation process is complex, the cost is high, a certain incubation time and analysis time are required, and there is a certain hysteresis.

[0003] For example, a sensor for detecting cortisol in sweat based on laser-induced graphene is disclosed in Chinese patent application CN118795002A, which uses LIG material to prepare an electrode and uses the structure of an organic electrochemical transistor to amplify the electrical signal. According to the change of the transistor electrical characteristic curve in different concentrations of cortisol solution, the relationship between the device response current and the cortisol concentration is established, and the detection of the cortisol content in sweat is realized. That is, the organic electrochemical transistor is prepared based on laser-induced graphene technology, and the recognition element is a molecularly imprinted polymer, which is based on the mechanism of specific binding for sensing and recognition. The preparation process is complex, the binding conditions are relatively strict, and the analysis and detection need to be carried out after incubation and binding with the aid of redox probes, and rapid real-time analysis and detection of cortisol cannot be realized.

[0004] Therefore, it is urgent to construct an electrochemical sensor with a simple preparation process, low cost and based on a non-immune recognition mechanism to establish a rapid real-time detection method for cortisol. SUMMARY

[0005] To solve the above technical problems, the application provides a composite electrode for rapid detection of cortisol, which is based on a composite electrocatalytic sensing interface constructed by laser-induced graphene (LIG), nano-gold and Nafion, and realizes rapid real-time detection of cortisol.

[0006] The application is a three-electrode electrochemical sensor prepared based on laser-induced graphene technology, and its recognition element is a nano-gold / Nafion composite film with catalytic activity. It is based on an electrochemical catalytic mechanism for sensing and recognition, and does not require incubation with a redox probe. Due to the film-forming and hydrophobic properties of Nafion, the interface is more stable and firm, the preparation process is simple, the response is faster, and it is helpful for rapid real-time analysis of cortisol.

[0007] The composite electrode for rapid detection of cortisol comprises: A laser-induced graphene substrate, a layer of gold nano-material modified on the laser-induced graphene substrate, and a perfluorosulfonic acid polymer modified on the layer of gold nano-material.

[0008] Preferably, the perfluorosulfonic acid polymer is Nafion.

[0009] The structure of the composite electrode LIG / Au / Nafion makes its interface performance stable and not affected by the dispersibility of the catalytic material, greatly reducing the difficulty of its requirement for product performance consistency in industrial product control.

[0010] Optionally, the composite electrode detects the concentration of cortisol in a phosphate buffered saline solution. That is, the composite electrode can rapidly analyze cortisol in a PBS buffer, making it more suitable for rapid analysis of cortisol in biological fluids and having a wider range of applications.

[0011] Optionally, within a range of 10-2000 nM of the concentration of cortisol, the oxidation peak current of the composite electrode between +0.1 V and +0.4 V has a linear relationship with the concentration of cortisol, R 2 ≥0.99.

[0012] Preferably, the oxidation peak of the composite electrode current and the concentration of cortisol has a linear relationship, which is located at +0.1 V, +0.15 V, +0.2 V, +0.25 V, +0.3 V, +0.31 V, +0.32 V, +0.33 V, +0.34 V, +0.35 V, +0.36 V, +0.37 V, +0.38 V, +0.39 V, +0.4 V, or any range between any two of them.

[0013] According to another aspect of the application, a preparation method of the composite electrode is provided. The method has a simple preparation process, low cost, and high product performance consistency, and is suitable for large-scale batch production.

[0014] The preparation method of the composite electrode comprises the following steps: a) preparing a laser-induced graphene electrode LIG; b) depositing a layer of gold nano-material on the working electrode area of the laser-induced graphene electrode (LIG) by potentiostatic electrodeposition to form a LIG / Au electrode; c) dropping a dispersion liquid containing a perfluorosulfonic acid polymer on the working electrode area of the LIG / Au electrode to obtain the composite working electrode after drying.

[0015] Preferably, step a) is: preparing a laser-induced graphene electrode on the surface of an aromatic carbon-based substrate material, removing impurities, drying, coating a reference electrode position with Ag / AgCl slurry, drying, forming a reference electrode, performing insulation packaging, and obtaining the laser-induced graphene electrode (LIG).

[0016] Preferably, step b) is: dropping a solution containing an Au source onto the surface of the laser-induced graphene electrode (LIG) electrode, and performing electrodeposition by potentiostatic method; after electrodeposition is completed, dropping a sulfuric acid solution onto the electrode interface, and performing activation again by cyclic voltammetry until the cyclic voltammetry curve is stable, thereby obtaining a LIG / Au electrode.

[0017] Preferably, step c) is: dropping a Nafion dispersion liquid on the working electrode area of the LIG / Au electrode to obtain the composite electrode after drying.

[0018] Preferably, the solution containing an Au source in step b) is a chloroauric acid solution with a mass concentration of 0.5-2%.

[0019] Optionally, the mass concentration of the chloroauric acid solution is selected from any value or a range value between any two values selected from 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, and 2.0%.

[0020] Preferably, the mass concentration of Nafion in the Nafion dispersion liquid in step c) is 0.1-1%.

[0021] Optionally, the mass concentration of Nafion in the Nafion dispersion liquid is selected from any value or a range value between any two values selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%.

[0022] As a specific embodiment, the preparation method of the composite electrode comprises the following steps: (1) preparing a laser-induced graphene electrode (LIG): introducing an electrode pattern into a computer connected to a laser engraving instrument, setting laser parameters, and starting the program to prepare a laser-induced graphene electrode (LIG) on the surface of a polyimide film; (2) LIG electrode pretreatment and activation: the laser-induced graphene electrode LIG obtained in step (1) is surface cleaned and dried, then Ag / AgCl paste is coated on the reference electrode position, dried to form a reference electrode, and then insulated and packaged with polyimide tape; then phosphate buffered saline solution is added to the electrode interface, and the electrode is activated by cyclic voltammetry; (3) Preparation of LIG / Au electrode: a solution containing Au source is added to the surface of the LIG electrode activated in step (2), and electrodeposition is performed by constant potential method; after electrodeposition is completed, sulfuric acid solution is added to the electrode interface, and activation is performed again by cyclic voltammetry until the cyclic voltammetry curve is stable, and the LIG / Au electrode is obtained; (4) Preparation of the composite electrode: Nafion dispersion liquid is added to the working electrode area of the LIG / Au electrode obtained in step (3), and the composite electrode is prepared after drying.

[0023] According to another aspect of the present application, a cortisol sensor is provided, comprising at least one of the composite electrode and the composite electrode prepared according to the above method.

[0024] According to another aspect of the present application, a method for rapidly detecting cortisol is provided. Not only can the rapid real-time detection of cortisol be realized, but also the rapid analysis of cortisol in PBS buffer can be realized, which is more suitable for the rapid analysis of cortisol in biological fluids and has a wider application range.

[0025] The method for rapidly detecting cortisol comprises the following steps: S1. Adding a cortisol-containing test liquid to the interface of the composite electrode for pre-enrichment; S2. Scanning the electrode interface after enrichment by cyclic voltammetry, and recording and obtaining the characteristic oxidation peak current value of cortisol; S3. Comparing the characteristic oxidation peak current value with the standard curve of cortisol concentration to determine the concentration of cortisol in the test liquid; The composite electrode is selected from at least one of the composite electrode and the composite electrode prepared according to the above method.

[0026] Preferably, the cortisol-containing test liquid in step S1 is a phosphate buffered saline solution containing cortisol.

[0027] Preferably, the pre-enrichment potential of the pre-enrichment in step S1 is -0.4~ -0.1 V, and the pre-enrichment time is 30~90 s.

[0028] Preferably, the scanning potential range in step S2 is -0.1 V to +0.5 V, and the scanning rate is 0.02 V / s.

[0029] Preferably, the characteristic oxidation peak of cortisol in S3 is located between +0.1 V and +0.4 V.

[0030] Optionally, the characteristic oxidation peak of cortisol in S3 is located within any value of +0.1 V, +0.15 V, +0.2 V, +0.25 V, +0.3 V, +0.31 V, +0.32 V, +0.33 V, +0.34 V, +0.35 V, +0.36 V, +0.37 V, +0.38 V, +0.39 V, or +0.4 V, or any range between both.

[0031] Preferably, the characteristic oxidation peak of cortisol in S3 is located between +0.33 V and +0.35 V.

[0032] More preferably, the characteristic oxidation peak of the cortisol is located at +0.34 V.

[0033] The electrocatalytic detection method of the present invention can rapidly analyze cortisol in PBS buffer, making it more suitable for rapid analysis of cortisol in biological fluids and with a wider range of applications.

[0034] In this application, Nafion is a synonym for perfluorosulfonic acid ionomer (tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer).

[0035] "PBS buffer" is short for Phosphate-Buffered Saline, and its pH value is 5-8.

[0036] The beneficial effects of this application include, but are not limited to: (1) The composite electrode for rapid detection of cortisol provided in this application does not require an incubation and binding step, has high timeliness, improves the detection rate, and enables real-time analysis.

[0037] (2) The method for preparing the composite electrode provided in this application is simple, low-cost, and has high product performance consistency, making it suitable for mass production.

[0038] (3) The composite electrode provided in this application has a stable film-forming interface.

[0039] (4) The composite electrode provided in this application can realize the electrocatalytic analysis of cortisol in PBS buffer, and has a wider range of applications. Attached Figure Description

[0040] Figure 1The figure shows a comparison of the electrochemical behavior of electrode sample 1# with comparative electrode samples D1# and D2# for cortisol; LIG / Au / Nafion corresponds to electrode sample 1#, LIG ​​corresponds to electrode sample D1#, and LIG / Au corresponds to electrode sample D2#.

[0041] Figure 2 The oxidation peak current corresponding to +0.34 V for cortisol standard solution was detected by electrode sample 1# in the range of 10~2000 nM.

[0042] Figure 3 This is a standard curve for cortisol measurement using electrode sample #1 in the range of 10–2000 nM. R 2 = 0.9979. Detailed Implementation

[0043] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0044] Unless otherwise specified, all raw materials and reagents used in this application are commercially purchased and used directly without processing. The instruments and equipment used adopt the manufacturer's recommended scheme and parameters.

[0045] In the examples, the Nafion solution used was purchased from DuPont.

[0046] In this embodiment, the laser-induced graphene preparation was carried out using a laser engraving machine from Tianjin Jiayin Nanotechnology Co., Ltd.

[0047] In this embodiment, electrochemical analysis was performed using a PalmSens4 electrochemical workstation from Radeont Technology.

[0048] Example 1: Preparation of Electrode Sample 1# Step 1: Import the electrode pattern into the computer connected to the laser engraving machine, set the laser power to 1300 mW and the scanning rate to 1.0 cm / s, start the program, and prepare a laser-induced graphene electrode on the surface of the polyimide film.

[0049] Step 2: Clean the electrode surface with pure water to remove impurities and dry it with nitrogen. Coat the reference electrode with Ag / AgCl slurry and dry it in a 70 ℃ oven for 30 min to form the reference electrode. Insulate and encapsulate it with polyimide tape to complete the preparation of the LIG electrode.

[0050] Step 3: Add 100 μL of 0.01 M PBS (pH=7.4) to the electrode interface and activate the electrode using cyclic voltammetry. The pretreatment potential is -0.3 V, the time is 60 s, the scan potential is -0.1 V to +0.5 V, and the scan rate is 0.02 V / s.

[0051] Step 4: Prepare a 1% chloroauric acid aqueous solution. Add 100 μL of the chloroauric acid solution to the LIG electrode surface and perform electrodeposition using a potentiostatic method. Set the potential to -0.7 to -0.1 V and the time to 30 to 90 s. This completes the preparation of the LIG / Au electrode. Add 100 μL of 0.5 M sulfuric acid to the electrode interface and activate the gold using cyclic voltammetry. Set the potential to -0.2 to -0.9 V and the time to 100 mV / s until the curve stabilizes.

[0052] Step 5: Dissolve 5% Nafion solution in deionized water to prepare a 0.5% Nafion solution. Add 6 μL of 0.1% Nafion dispersion to the working electrode region of the LIG / Au electrode and dry in an oven at 35 °C for 1 hour to obtain the LIG / Au / Nafion composite electrocatalytic sensing interface.

[0053] Example 2 Preparation of Electrode Sample 2# The preparation conditions and steps are the same as in Example 1, except that the concentration of the chloroauric acid aqueous solution is 0.5%.

[0054] Example 3 Preparation of Electrode Sample 3# The preparation conditions and steps were the same as in Example 1, except that the concentration of the chloroauric acid aqueous solution was 1.5% and the concentration of the Nafion solution was 0.8%. Example 4 Preparation of electrode sample 4# The preparation conditions and steps are the same as in Example 1, except that the concentration of the chloroauric acid aqueous solution is 2% and the concentration of the Nafion solution is 1%.

[0055] Example 5 Preparation of Electrode Sample 5# The preparation conditions and steps were the same as in Example 1, except that the concentration of the Nafion solution was 0.1%.

[0056] Comparative Example 1: Comparative electrode samples D1# and D2# The LIG prepared in Example 1 was used as the control electrode sample D1#, and the LIG / Au was used as the control electrode sample D2#.

[0057] Example 6 Electrochemical analysis of electrode samples 1# to 5# and control samples D1# and D2# Electrode samples 1# to 5# and control samples D1# and D2# were connected to the electrochemical workstation, and cortisol solution was analyzed and detected by cyclic voltammetry (pre-enrichment potential -0.1~-0.4 V, enrichment time 30~90 s, scan potential -0.1V~+0.5 V, scan rate 0.02 V / s).

[0058] The results showed that electrode samples 1 to 5 all had significant electrochemical catalytic responses to cortisol.

[0059] Taking electrode sample 1# as a typical example, its electrochemical behavior towards cortisol is compared with that of control samples D1# and D2#. Figure 1 As shown.

[0060] As can be seen from the figure, compared with the comparison samples D1# and D2#, sample 1#, which has a composite electrocatalytic sensing interface, has a more obvious electrochemical catalytic response to cortisol.

[0061] Example 6: Relationship curves between electrode samples 1# to 5# and cortisol concentration detection With the phosphate electrolyte at pH 7.4, the CV method was used to detect cortisol standard solutions of different concentrations (10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM, 2000 nM) using electrode samples 1# to 5#. The oxidation peak current corresponding to the +0.34 V position was recorded, and a linear relationship curve was plotted.

[0062] The results showed that, within the range of 10–2000 nM, the oxidation peak currents corresponding to positions 1# to 5# +0.34 V of electrode samples all exhibited a linear relationship with cortisol concentration, and the coefficient of determination of the standard curve was [missing information]. R 2 All are greater than 0.99.

[0063] Taking electrode sample 1# as a typical example, the oxidation peak current at 0.34 V is as follows: Figure 2 As shown in the figure, the relationship curve between cortisol concentration and concentration is as follows: Figure 3 As shown, within the range of 10–2000 nM, the sensing interface exhibits a linear relationship with cortisol levels. R 2 = 0.9979.

[0064] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A composite electrode for rapid detection of cortisol, characterized in that, include: Laser-induced graphene substrate, gold nanomaterial layer modified on the laser-induced graphene substrate, and perfluorosulfonic acid polymer modified on the gold nanomaterial layer.

2. The composite electrode according to claim 1, characterized in that, The composite electrode detects the concentration of cortisol in a phosphate buffer solution.

3. The composite electrode according to claim 1 or 2, characterized in that, Within the cortisol concentration range of 10–2000 nM, the oxidation peak current of the composite electrode, located between +0.1 V and +0.4 V, exhibits a linear relationship with the cortisol concentration. R 2 ≥0.

99.

4. The method for preparing the composite electrode according to any one of claims 1 to 3, characterized in that, Includes the following steps: a) Fabrication of laser-induced graphene electrodes (LIG); b) In the working electrode region of the laser-induced graphene electrode (LIG), a gold nanomaterial layer is electrodeposited using a constant potential method to form a LIG / Au electrode. c) A dispersion containing a perfluorosulfonic acid polymer is drop-coated onto the working electrode region of the LIG / Au electrode, and the composite working electrode is obtained after drying.

5. The method according to claim 4, characterized in that, Step a) is as follows: a laser-induced graphene electrode is prepared on the surface of an aromatic carbon-based material, impurities are removed, the electrode is dried, an Ag / AgCl slurry is applied to the reference electrode position, the electrode is dried to form a reference electrode, and then it is encapsulated in an insulating manner to obtain the laser-induced graphene electrode LIG. Step b) is as follows: A solution containing an Au source is dropped onto the surface of the laser-induced graphene electrode (LIG electrode), and electrodeposition is performed using a constant potential method. After electrodeposition, a sulfuric acid solution is dropped onto the electrode interface, and activation is performed again using cyclic voltammetry until the cyclic voltammetry curve is stable, thus obtaining the LIG / Au electrode. Step c) involves drop-coating a Nafion dispersion onto the working electrode region of the LIG / Au electrode and then drying it to obtain the composite electrode.

6. The method according to claim 5, characterized in that, The solution containing the Au source in step b) is a chloroauric acid solution with a mass concentration of 0.5-2%; The mass concentration of Nafion in the Nafion dispersion described in step c) is 0.1-1%.

7. A cortisol sensor, characterized in that, It includes at least one of the composite electrode according to any one of claims 1 to 3 and the composite electrode prepared by the method according to any one of claims 4 to 6.

8. A method for rapid detection of cortisol, characterized in that, Includes the following steps: S1. The test solution containing cortisol is added dropwise to the interface of the composite electrode for pre-enrichment; S2. Cyclic voltammetry was used to scan the enriched electrode interface, and the characteristic oxidation peak current values ​​of cortisol were recorded and obtained. S3. Compare the characteristic oxidation peak current value with the standard curve of cortisol concentration to determine the concentration of cortisol in the test solution; The composite electrode is selected from at least one of the composite electrodes according to any one of claims 1 to 3 and the composite electrodes prepared by the method according to any one of claims 4 to 6.

9. The method according to claim 8, wherein the test solution containing cortisol in step S1 is a phosphate buffer solution containing cortisol; The pre-enrichment potential in step S1 is -0.4 to -0.1 V, and the pre-enrichment time is 30 to 90 s; In step S2, the scanning potential range is -0.1 V to +0.5 V, and the scanning rate is 0.02 V / s.

10. The method according to claim 8, characterized in that, The characteristic oxidation peak of cortisol described in S3 is located between +0.1 V and +0.4 V; Preferably, the characteristic oxidation peak of cortisol is located at +0.34 V.

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