Calmodulin electrochemical biosensor based on laser-induced graphene as well as preparation method and application of calmodulin electrochemical biosensor
By preparing an electrochemical biosensor by laser-induced deposition of specific substances on the graphene surface, the problem of complex and high cost of protein S100A12 detection in the existing technology is solved, and high-sensitivity and low-cost rapid detection is achieved, which has broad application prospects.
Patent Information
- Application Number
- CN202510880315.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-10-03
AI Technical Summary
Existing protein S100A12 detection methods such as ELISA are complex, costly and inconvenient. There is an urgent need for a simple, fast, sensitive and low-cost detection method.
Laser-induced graphene (LIG) was used as a carrier and combined with an electrochemical method to prepare a highly sensitive calmodulin electrochemical biosensor by depositing pyrrole-1-propionic acid, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride, target protein, bovine serum albumin and horseradish peroxidase-labeled immunoglobulin on its surface.
The method achieves high-sensitivity and rapid-response detection of protein S100A12 with good stability and reproducibility at low cost, making it suitable for fields such as biomedical research, clinical diagnosis, and biological monitoring.
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Figure CN120741602A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of electrochemical biosensors, and in particular relates to a calmodulin electrochemical biosensor based on laser-induced graphene, and a preparation method and application thereof. Background Art
[0002] The protein S100A12 is a crucial calmodulin. Upon binding to calcium ions, its conformation changes accordingly, leading to crucial roles in various life processes, including cell proliferation, differentiation, apoptosis, gene expression, secretion, and muscle contraction. S100A12 is generally overexpressed under inflammatory conditions. This protein has a wide range of intracellular and extracellular functions. Intracellular S100A12 functions include regulation of calcium homeostasis and cytoskeletal components, transcription factors, protein phosphorylation, and enzyme activity. Extracellular S100A12 exists in various forms, including homodimers and hexamers. Extracellularly, the S100A12 protein binds to the cell surface receptor for advanced glycation end products (RAGE) and endogenous Toll-like receptor 4 (TLR4), thereby activating the endogenous ERK1 / 2 and phosphatidylinositol 3-kinase (PI-3K) / AKT signaling pathways in lymphocytes, neutrophils, and monocytes. This activates the production of cytokines such as IL-18, IL-6, IL-1β, and TNF-α, promoting the progression of inflammation. In human tissues, the S100A12 gene is almost exclusively expressed by neutrophils.
[0003] Currently, there are two main methods for detecting the protein S100A12: enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). While RIA requires a radioactive tracer, ELISA does not. ELISA offers superior sensitivity, and commercially available kits are also mature. However, these methods are complex, inconvenient, and expensive. Given that ELISA is currently the primary detection method, there is an urgent need to develop a simple, rapid, sensitive, and low-cost method for detecting S100A12.
[0004] Compared to traditional methods, sensors are low-cost, fast-response, and sensitive analytical tools. This method uses specific biological receptors (such as nucleic acids, antibodies, proteins, etc.) to identify analytes in samples and uses transducers to evaluate readable signals (such as electricity, light, heat, mass, frequency changes, etc.). Laser-induced graphene (LIG) is a three-dimensional porous material that can be obtained by simply irradiating various carbon materials with a specific type of laser. It has made good progress in the field of sensor preparation. LIG obtained by "one-step" engraving has the characteristics of high porosity, excellent conductivity, and good mechanical flexibility. The 3D porous structure of LIG not only provides more binding sites for biomacromolecules and has good biocompatibility, but also increases the effective electrode area and electron conduction efficiency, which is conducive to improving the sensitivity of biosensors.
[0005] Based on this, the present invention proposes to utilize the excellent properties of LIG and combine it with the rapid and sensitive characteristics of electrochemical methods to prepare a protein S100A12 biosensor with fast response, high sensitivity, small size and low cost. Summary of the Invention
[0006] In view of the above shortcomings of the prior art, one of the objectives of the present invention is to provide a method for preparing a calmodulin electrochemical biosensor based on laser-induced graphene. The sensor prepared by the method of the present invention not only exhibits instant, rapid, and highly sensitive detection, but also has good stability and reproducibility.
[0007] To achieve the above object, the specific technical solutions of the present invention are as follows:
[0008] A method for preparing a calmodulin electrochemical biosensor based on laser-induced graphene comprises the following steps:
[0009] S1. Pyrrol-1-propionic acid was deposited on the surface of a laser-induced graphene electrode, and then a solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added dropwise to activate the pyrrol-1-propionic acid.
[0010] S2. Add a phosphate-buffered saline solution of the target protein to the electrode surface where pyrrolidone was deposited. After incubation and washing, add a bovine serum albumin solution after drying to block the nonspecific active sites on the electrode surface.
[0011] S3. A phosphate buffered saline solution of a protein antibody is added dropwise to the electrode surface modified with the target protein, incubated, washed, dried, and then a horseradish peroxidase-labeled immunoglobulin solution is added dropwise, incubated, washed, and dried to obtain the calmodulin electrochemical biosensor.
[0012] This method first deposits a pyrrole-1-propionic acid (PPA) polymer on the LIG electrode surface. This improves the strength and adhesion of the polymer film to the transducer surface and facilitates subsequent surface modification using DMTMM for affinity-based sensor fabrication. Compared to traditional graphene modification techniques (such as acid reflux or monolayer formation of aromatic derivatives), the electrografting of pyrrole derivatives is fast (~260 s), controllable, and scalable (via parallel electrodes). The deposited PPA is then activated using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for subsequent covalent immobilization of target proteins. After incubating the target protein on the electrode surface modified with PPA and DMTMM, unreacted sites are inactivated with bovine serum albumin (BSA). BSA, an inert globular protein without catalytic oxidative activity, effectively binds to the modified LIG electrode surface through mechanical stacking and nonspecific adsorption. The significant steric hindrance created by BSA's self-stacking effectively prevents subsequent antibody protein from directly binding to the electrode surface and causing false positives. Anti-target protein antibodies are then specifically recognized for their binding. Finally, an immunoglobulin labeled with horseradish peroxidase (IgG-HRP) recognizes and binds to the antibody. The HRP tag catalyzes the generation of a corresponding DPV signal in a hydroquinone / hydrogen peroxide detection solution. By monitoring the change in peak current, highly sensitive and selective protein detection is achieved.
[0013] The present invention adopts the laser irradiation technology "one-step method" to prepare laser-induced graphene electrodes, which does not require chemical reducing agents and complex post-processing, and the production cost of each piece is significantly reduced compared with the traditional method.
[0014] Preferably, the preparation method of the laser-induced graphene electrode includes: designing a three-electrode pattern on a polyimide film, using a semiconductor laser to laser-inducingly print a graphene electrode on the polyimide film, pasting PVC tape under the working electrode, and coating Ag / AgCl slurry on the reference electrode.
[0015] Preferably, the process of depositing pyrrole propionic acid on the surface of the laser-induced graphene electrode in step S1 includes: dissolving pyrrole-1-propionic acid in a potassium chloride solution to obtain a pyrrole-1-propionic acid solution with a concentration of 5~10mM, and then dropping the solution on the surface of the laser-induced graphene electrode, and performing electrochemical deposition using cyclic voltammetry.
[0016] Preferably, the volume of the 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride solution added dropwise in step S1 is 5-10 μL, and the concentration is 50-250 mM.
[0017] Preferably, the activation condition in step S1 is: incubating in an incubator at 35-40° C. for 0.5-2 h.
[0018] Preferably, the volume of the phosphate buffered saline solution of the target protein added dropwise in step S2 is 2.5-5 μL, and the concentration is 0.001-10 μg / mL.
[0019] Preferably, the volume of the bovine serum albumin solution added dropwise in step S2 is 2.5-5 μL, and the concentration is 1 wt%-5 wt%.
[0020] Preferably, the volume of the phosphate buffered saline solution of the protein antibody added dropwise in step S3 is 2.5-5 μL, and the concentration of the protein antibody in the phosphate buffered saline solution is 1-10 μg / mL.
[0021] Preferably, the volume of the horseradish peroxidase-labeled immunoglobulin solution added dropwise in step S3 is 2.5-5 μL, and the concentration of the immunoglobulin solution is 5-15 μg / mL.
[0022] Preferably, in steps S2 and S3, the incubation conditions are: incubation at 35-40° C. for 0.5-2 h.
[0023] Another object of the present invention is to provide an application of a calmodulin electrochemical biosensor based on laser-induced graphene prepared by the method in detecting calmodulin concentration.
[0024] Preferably, the detection method is as follows: a phosphate buffer solution containing hydrogen peroxide and hydroquinone is added dropwise to the working area of the electrochemical sensor, and detection is performed using differential pulse voltammetry.
[0025] Compared with the prior art, the present invention is beneficial in that:
[0026] (1) This invention is the first to use laser-induced graphene electrodes as carriers to prepare electrochemical biosensors with high conductivity, large effective specific surface area, and flexible substrate materials with adjustable patterns for the detection of calmodulin S100A12, and exhibits excellent detection performance.
[0027] (2) The sensor prepared by the present invention uses antibodies that specifically recognize proteins and IgG-HRP to catalyze and amplify the resulting electrochemical signal, achieving high selectivity while also exhibiting good stability and reproducibility. Furthermore, compared to the commercially used enzyme-linked immunosorbent assay (ELISA) method, the biosensor has a lower detection limit and higher sensitivity. Furthermore, the sensor is a disposable device for rapid, instant detection, and has a low single-use preparation cost.
[0028] (3) The sensor prepared by this invention has good practical value. By changing the required antibodies, it can be further extended to the real-time detection of other proteins, providing a new idea and detection method for protein detection.
[0029] (4) The sensor of the present invention has broad market prospects and huge application potential, and has important development value in many fields such as biomedical research, clinical diagnosis and biological monitoring. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The figure is a flow chart of the preparation of the calmodulin electrochemical biosensor based on laser-induced graphene according to the present invention;
[0031] Figure 2 This is a graph showing the electrochemical signal response of the biosensor prepared according to the present invention to different concentrations of the target protein. In Figure A, curve a represents the current signal of the reference PBS solution, while curve b represents the current signal of the target protein S100A12 under the same conditions. Figure B shows the linear relationship between the electrochemical response signal and the target protein concentration at different concentrations.
[0032] Figure 3 Schematic diagram of the stability of the biosensor prepared in the present invention; Figure A shows the electrochemical response signal change after the sensor was stored at 4°C for 7 days, and Figure B shows the electrochemical response signal of the same electrode after 5 consecutive scans at 4°C for 7 days;
[0033] Figure 4 Schematic diagram of the electrochemical response signals of different biosensors prepared in the present invention after being stored at 4°C for 7 days;
[0034] Figure 5 Electrochemical response signal diagram of the biosensor prepared by the present invention when used to detect serum samples of normal persons and different patients;
[0035] Figure 6 This is an electrochemical response signal diagram of the biosensor prepared by the present invention used for large-scale actual clinical sample detection of three diseases. DETAILED DESCRIPTION
[0036] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0037] The present invention provides a method for preparing a calmodulin electrochemical biosensor based on laser-induced graphene, comprising the following steps:
[0038] S1. Pyrrol-1-propionic acid was deposited on the surface of a laser-induced graphene electrode, and then a solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added dropwise to activate the pyrrol-1-propionic acid.
[0039] S2. Add a phosphate-buffered saline solution of the target protein to the electrode surface where pyrrolidone was deposited. After incubation and washing, add a bovine serum albumin solution after drying to block the nonspecific active sites on the electrode surface.
[0040] S3. A phosphate buffered saline solution of a protein antibody is added dropwise to the electrode surface modified with the target protein, incubated, washed, dried, and then a horseradish peroxidase-labeled immunoglobulin solution is added dropwise, incubated, washed, and dried to obtain the calmodulin electrochemical biosensor.
[0041] In the present invention, the process of depositing pyrrole propionic acid on the surface of the laser-induced graphene electrode in step S1 includes: dissolving pyrrole-1-propionic acid in a potassium chloride solution to obtain a pyrrole-1-propionic acid solution with a concentration of 5-10 mM, then dripping the solution onto the surface of the laser-induced graphene electrode, and performing electrochemical deposition using cyclic voltammetry.
[0042] The volume of the 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride solution added dropwise in step S1 can be 5 to 10 μL, and the concentration can be 50 to 250 mM; the volume of the phosphate buffered saline solution of the target protein added dropwise in step S2 can be 2.5 to 5 μL, and the concentration can be 0.001 to 10 μg / mL; the volume of the bovine serum albumin solution added dropwise in step S2 can be 2.5 to 5 μL, and the concentration can be 1 wt% to 5 wt%; the volume of the phosphate buffered saline solution of the protein antibody added dropwise in step S3 can be 2.5 to 5 μL, and the concentration of the protein antibody in the phosphate buffered saline solution can be 1 to 10 μg / mL; the volume of the horseradish peroxidase-labeled immunoglobulin solution added dropwise in step S3 can be 2.5 to 5 μL, and the concentration of the immunoglobulin solution can be 5 to 15 μg / mL.
[0043] Example 1
[0044] This embodiment provides a method for preparing a calmodulin electrochemical biosensor based on laser-induced graphene, such as Figure 1 As shown, the following steps are included:
[0045] (1) Preparation of laser-induced graphene electrodes
[0046] A three-electrode pattern was designed using PowerPoint drawings, and a semiconductor laser was used to rapidly carve a one-time, fully integrated LIG electrode onto a polyimide film according to the designed pattern. The electrode consisted of a working electrode (2 mm in diameter), a reference electrode, and a counter electrode.
[0047] The specific steps are as follows: Place a clean polyethylene terephthalate (PET) sheet flat on a clean tabletop and secure it with paper tape. Use the paper tape to remove any dust or debris from the surface of the PET sheet. Use a push plate to firmly adhere the polyimide (PI) tape to the surface of the PET sheet, ensuring that there are no dust or bubbles caused by loose adhesion. Then, cut the prepared tape to the desired size, rinse the surface with deionized water, and allow it to air dry before placing it under the laser engraver for engraving. Note that the laser engraver needs to be preheated for 5 minutes. The laser parameters are as follows: 30% laser power, 20% engraving speed, and 25 mm focal length. Finally, apply PVC tape under the working electrode and apply Ag / AgCl slurry to the reference electrode. Place the tape in a 60°C oven for 5 minutes before use.
[0048] (2) Preparation of electrochemical biosensors
[0049] (2.1) Add 10 μL of 10 mM PPA (dissolved in 0.5 M KCl) solution to the working electrode surface of the LIG electrode and perform cyclic voltammetry (potential range 0-0.85 V, scan rate 0.1 v / s, and 20 cycles). Rinse the electrode surface with deionized water, carefully remove water droplets from the electrode surface with filter paper, and allow the electrode to air dry at room temperature.
[0050] (2.2) Add 10 μL of 100 mM DMTMM solution to the electrode surface obtained in step (2.1) and incubate in a 37°C incubator for 1 hour. This step activates the carboxyl groups contained in PPA to ensure that the subsequent S100A12 protein is successfully attached to the electrode surface. Then rinse the electrode surface with deionized water, absorb the water droplets with filter paper, and allow it to air dry naturally.
[0051] (2.3) Add 3 μL of the target protein S100A12 (prepared in PBS) to the surface of the electrode obtained in step (2.2). Incubate at 37°C for 1 hour. Rinse the electrode surface with deionized water, remove water droplets with filter paper, and allow to air dry. At this time, the electrode S100A12 / PPA / LIG is obtained.
[0052] (2.4) After the protein is attached to the electrode, 5 μL of 1 wt% BSA (prepared in PBS) is added to the surface of the electrode obtained in step (2.3) and incubated at 37°C for 45 minutes to block the electrode. Since BSA itself has a large volume and is prone to deposition and aggregation, it can prevent the subsequent antibodies from directly attaching to the electrode surface. The electrode surface is then rinsed with deionized water, the water droplets are absorbed by filter paper, and the mixture is allowed to air dry. At this time, the electrode BSA / S100A12 / PPA / LIG is obtained.
[0053] (2.5) Add 3 μL of 5 μg / mL anti-S100A12 antibody protein (prepared in PBS, referred to as S100A12-ab) to the electrode surface obtained in step (2.4) and react at 37°C for 1 hour. Then rinse the electrode surface with deionized water, remove water droplets with filter paper, and allow to air dry.
[0054] (2.6) Add 3 μL of 10 μg / mL IgG-HRP (prepared in PBS) to the surface of the electrode obtained in step (2.5) and react at 37°C for 1 hour. Then rinse the electrode surface with deionized water, absorb the water droplets with filter paper, and allow it to air dry naturally to obtain an electrochemical biosensor for detection.
[0055] (3) Electrochemical detection
[0056] A 0.1 M phosphate buffer solution containing 5 mM hydrogen peroxide (H2O2) and hydroquinone (HQ) was added dropwise to the working electrode of the electrochemical biosensor, and detection was performed using differential pulse voltammetry (DPV). The relevant parameters were set as follows: the potential scan range was set to +0.4 to -0.6, the pulse amplitude was 0.05 V, and the pulse width was 0.05 s.
[0057] The current signals of the control PBS solution and the target protein S100A12 under the same conditions are shown in Figure 2 As shown in (A), it can be seen from the figure that the current response signal detected by S100A12 in the experimental group is significantly higher than that in the control group PBS, indicating that the sensor can be used to detect protein S100A12.
[0058] The prepared sensor was used to detect different concentrations of protein S100A12 standards, and the current response of the electrochemical biosensor to different concentrations of protein S100A12 standards was investigated; the results are shown in Figure 2. Figure 2As shown in (B), as the concentration of target protein S100A12 increases, the corresponding DPV response signal also increases. In the concentration range of 0.001μg / mL to 10μg / mL, the measured current value has a good linear relationship with the logarithm of the concentration. The linear regression equation obtained by fitting is: I (μA) = 5.834 Log c (μg / mL) + 55.307 (R 2 =0.998); where c (μg / mL) represents the concentration of protein S100A12, and I (μA) represents the magnitude of the current response signal).
[0059] Example 2
[0060] This example analyzes the stability of electrochemical biosensors.
[0061] The sensor prepared in Example 1 was stored at 4°C for 7 days to verify the stability of the sensor during storage. Figure 3 As shown in (A), curve a is the detection result on the same day, and curve b is the detection result 7 days later. It is calculated that the peak current value detected 7 days later decreased by about 7.23%; at the same time, the same electrode was scanned continuously. The results are shown in Figure 3 (B). The peak current value of the electrode increased slightly during the 5 consecutive scans, and the RSD was 0.95%, indicating that the established sensor has relatively good stability during short-term storage.
[0062] Example 3
[0063] This example conducts reproducibility analysis of electrochemical biosensors
[0064] Six sensors were made according to the preparation method of Example 1. The reproducibility of the sensors was verified by monitoring their current responses on the LIG electrode. The results are shown in Figure 1. Figure 4 The RSD of the six sensors was 7.09%, which proved that the fabrication of the sensors and the measurement using differential pulse voltammetry showed good reproducibility.
[0065] Example 4
[0066] This example uses an electrochemical biosensor to detect protein S100A12 in serum
[0067] In the actual test, serum samples from patients with diabetes (T2D), coronary heart disease (CHD), and patients with both diseases (T2D+CHD) were collected and compared with those of normal people (Normal) and the control PBS to verify that the prepared biosensor still showed a good detection level in the complex actual sample test. Figure 5As shown, the protein S100A12 was significantly elevated in the serum of patients with both diabetes and coronary artery disease compared to the PBS solution in the control group and the serum samples of healthy individuals. Furthermore, the concentration of S100A12 in the serum of patients with both diseases was higher than that of patients with diabetes or coronary artery disease. This demonstrates that the sensor can effectively distinguish between healthy individuals and patients, as well as between patients with a single disease and those with diabetes and coronary artery disease.
[0068] Example 5
[0069] Based on Example 4, this example collected more actual clinical samples, including 13 serum samples from diabetic patients, 11 serum samples from patients with coronary heart disease, and 11 serum samples from patients with diabetes and coronary heart disease. Figure 6 The serum current of patients with T2D-CHD was significantly higher than that of patients with isolated T2D (p<0.0001), indicating that the serum S100A12 protein level in patients with T2D-CHD was significantly higher than that in patients with isolated T2D. This suggests that this invention has great potential for the initial screening and diagnosis of patients with diabetes and coronary heart disease.
[0070] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a calmodulin electrochemical biosensor based on laser-induced graphene, characterized in that: The following steps are involved: S1. Pyrrol-1-propionic acid was deposited on the surface of a laser-induced graphene electrode, and then a solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added dropwise to activate the pyrrol-1-propionic acid. S2. Add a phosphate-buffered saline solution of the target protein to the electrode surface where pyrrolidone was deposited. After incubation and washing, add a bovine serum albumin solution after drying to block the nonspecific active sites on the electrode surface. S3. A phosphate buffered saline solution of a protein antibody is added dropwise to the electrode surface modified with the target protein, incubated, washed, dried, and then a horseradish peroxidase-labeled immunoglobulin solution is added dropwise, incubated, washed, and dried to obtain the calmodulin electrochemical biosensor.
2. The preparation method according to claim 1, characterized in that The preparation method of laser-induced graphene electrode includes: designing a three-electrode pattern on a polyimide film, using a semiconductor laser to laser-inducingly print graphene electrodes on the polyimide film, pasting PVC tape under the working electrode, and coating Ag / AgCl slurry on the reference electrode.
3. The preparation method according to claim 1, characterized in that Step S1 is a process for depositing pyrrole propionic acid on the surface of the laser-induced graphene electrode, comprising: dissolving pyrrole-1-propionic acid in a potassium chloride solution to obtain a pyrrole-1-propionic acid solution with a concentration of 5 to 10 mM, and then dropping the solution onto the surface of the laser-induced graphene electrode, and performing electrochemical deposition using cyclic voltammetry.
4. The preparation method according to claim 1, characterized in that The volume of the 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride solution added dropwise in step S1 is 5 to 10 μL, and the concentration is 50 to 250 mM; And / or, the activation condition is: incubating in an incubator at 35-40° C. for 0.5-2 h.
5. The preparation method according to claim 1, characterized in that The volume of the phosphate buffered saline solution of the target protein added dropwise in step S2 is 2.5-5 μL, and the concentration is 0.001-10 μg / mL.
6. The preparation method according to claim 1, characterized in that The volume of the bovine serum albumin solution added dropwise in step S2 is 2.5-5 μL, and the concentration is 1 wt %-5 wt %.
7. The preparation method according to claim 1, characterized in that The volume of the phosphate buffered saline solution of the protein antibody added dropwise in step S3 is 2.5-5 μL, and the concentration of the protein antibody in the phosphate buffered saline solution is 1-10 μg / mL.
8. The preparation method according to claim 1, characterized in that The volume of the horseradish peroxidase-labeled immunoglobulin solution added dropwise in step S3 is 2.5-5 μL, and the concentration of the immunoglobulin solution is 5-15 μg / mL.
9. A calmodulin electrochemical biosensor based on laser-induced graphene prepared by the method according to any one of claims 1 to 8.
10. Use of a calmodulin electrochemical biosensor based on laser-induced graphene prepared by the method according to any one of claims 1 to 8 in detecting calmodulin concentration.