CN115112738A - Preparation of a laser direct writing graphene/enzyme electrode and its application in glucose sensing - Google Patents

Abstract

The invention discloses preparation of a laser direct-writing graphene/enzyme electrode and application of glucose sensing, and belongs to the technical field of biosensing. According to the invention, a miniature flexible electrode is prepared by combining a laser-induced graphene (LIG) technology, a hydrophobic end benzene ring of 1-pyrenebutyric acid and a six-membered ring structure of graphene are subjected to carboxylation on the surface of the LIG electrode through a pi-pi superposition effect, and then glucose oxidase is covalently cross-linked, and the prepared enzyme electrode can be used for glucose detection of human serum, urine sample and sweat, so that the real-time monitoring of glucose is realized, and thus, the high-sensitivity high-selection glucose sensor based on the glucose oxidase is constructed. Further promotes the development of simple, green and low-cost biosensors.

Description

Preparation of a laser direct writing graphene/enzyme electrode and its application in glucose sensing

technical field

The invention belongs to the technical field of biological sensing, and in particular relates to a preparation method of a graphene/precious metal nanoparticle composite electrode by laser direct writing and an immunosensing application.

Background technique

With the rapid development of society and the progress of civilization, people's living standards have been greatly improved. However, environmental pollution and fast-paced life have led to the rising proportion of obese and sub-healthy people, such as diabetes, hyperlipidemia, cardiovascular disease, and neurological diseases, which seriously affect people's daily life. The treatment of these diseases is usually not achieved overnight, requiring a long recuperation process and daily monitoring of the patient's condition. For example, there is no cure for diabetes, and patients generally need lifelong testing and treatment to control the blood sugar range and prevent various complications. Using traditional methods to detect blood indicators of patients is time-consuming and labor-intensive, which brings great inconvenience to hospitals and patients. Therefore, real-time detection of patients' blood glucose levels is particularly important. Due to the limitations of diagnostic methods, the current clinical blood glucose test still adopts the invasive sampling method of fingertip or arm blood collection, which increases the pain of patients and increases the probability of external infection. Studies have shown that there is a correlation between the glucose content of biological fluids such as urine, sweat, and tears in the human body and the blood glucose concentration in the body, which can be used for blood glucose monitoring in diabetes. Therefore, the development of non-invasive blood glucose detection methods is particularly necessary.

Electrochemical biosensors are sensing devices capable of combining electrochemical sensing and specific recognition of biomolecules, which play a key role in the current laboratory and clinical analysis of various chemical and biological targets. The ultimate goal of electrochemical sensors is to build a simpler, more sensitive and more reliable sensing interface, further amplify the detection signal, and improve sensitivity and accuracy. The development of new functional nanomaterials and nanotechnology provides new possibilities for improving the performance of electrochemical sensors. The search for high-performance electrode materials is the key to the preparation of excellent electrochemical sensors, and it is also an important direction of current research.

With the increasing demand of nanostructures for high-performance devices, the study of controllable micro-nanostructures has become one of the hotspots in scientific research. The preparation of controllable micro-nano structures focuses on the application of micro-nano processing technology, which makes it develop in the direction of simplified preparation process, low cost and high repeatability. Laser beams have the advantages of directional light emission, great energy density, high precision, fast processing speed, non-mechanical contact processing and flexible control, which make them widely used in laser micromachining. In particular, laser direct writing technology can get rid of the constraints of masks, greatly improve processing efficiency and repeatability, and provide new research ideas for the next generation of intelligent health detection.

Graphene is a single atomic layer of ^two -dimensional sp hybrid carbon nanosheets that exhibits excellent properties due to large specific surface area, high electron mobility, thermal conductivity, biocompatibility, ultralow density, and mechanical flexibility. Its physical and chemical properties make it widely used in sensors, lithium batteries and supercapacitors. Laser-induced graphene (LIG) technology can induce polyimide (PI) substrates to directly generate three-dimensional porous graphene, which exhibits large specific surface area and high conductivity. The preparation process does not require high temperature and solvent. It is widely used in preparation. In addition, as the most typical embedded system, the small size of the microelectrode makes the electrochemical analysis portable, fast, and more conducive to practical applications. For example, wearable sweat sensors can easily and quickly detect target analytes without pretreatment.

Diabetes is a disease that affects millions of people worldwide, so research on glucose biosensors remains in high demand, with efforts to find cheaper, more precise, minimal or non-invasive methods to quantify glucose levels in real time. Glucose oxidase (GOx) has been widely studied due to its important catalytic activity. It is inexpensive, stable and practical, and is an ideal model molecule for the preparation of enzyme sensors. In view of the high selectivity and high catalytic activity of the enzyme, the glucose oxidase method is currently the most used blood glucose detection method on the market. However, the difficulty of the work is to immobilize the enzyme on the desired platform, the enzyme immobilization process should be cheap, fast and biocompatible, and the immobilization process should improve the stability, reusability and activity of the enzyme. There are many different techniques for anchoring enzymes to desired substrates, such as adsorption, entrapment or covalent cross-linking. However, there is a dynamic equilibrium in the adsorption process, which is easy to fall off, which leads to the instability of the detection method. The embedding method affects the activity of the enzyme, and the covalent crosslinking has better stability and reusability.

Typically, GOx is immobilized on rigid substrates, such as surfaces covered with thin fluorine tin oxide or indium tin oxide, gold, or glassy carbon electrode surfaces, which hinders its application on the human body. The present invention adopts the LIG electrode, which has good biocompatibility, as well as a good environment as an enzyme and the effect of promoting electron transfer. The combined electrochemical and laser approach can provide a favorable environment for the covalent bonding of glucose oxidase in the graphene matrix and the electron transfer of GOx without adding additional mediators to enhance the activity of the enzyme. The proposed glucose sensing method, owing to its excellent electrochemical performance, biocompatibility, and relatively simple preparation route, shows potential in glucose monitoring, especially in terms of non-invasiveness and painlessness.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a preparation method of laser direct writing graphene/enzyme electrode and its application in glucose sensing.

The present invention designs a three-electrode pattern, combines laser induced graphene (LIG) technology to prepare a miniature flexible graphene electrode, and utilizes the π-π superposition effect of the hydrophobic end benzene ring of 1-pyrene butyric acid and the six-membered ring structure of graphene, and has the advantages of The surface working area of LIG electrode is carboxylated, and then GOx is covalently cross-linked. By using a portable electrochemical workstation and chronoamperometry, the current signal generated by GOx catalyzing glucose at a constant voltage is directly related to the glucose concentration, and a portable glucose sensor is prepared. , the prepared enzyme electrode can be used for glucose detection in human serum, urine and sweat, to realize real-time monitoring of glucose, thereby constructing a highly sensitive and high-selective glucose sensing based on glucose oxidase. Further promote the development of portable, green, and low-cost biosensors.

To achieve the above object, the present invention adopts the following technical solutions:

A portable preparation method of a laser-induced graphene/enzyme electrode, comprising the following steps:

1) Design a three-electrode system micro-electrode pattern, and use a laser to print graphene micro-electrodes on a high-insulation PI film;

2) Ag/AgCl paste is coated on the reference electrode, conductive silver paste is coated on the end of the three electrodes as a signal output connector, and polydimethylsiloxane PDMS fixes the working area area, thereby forming a laser direct writing graphene electrode;

3) Add 1-pyrene butyric acid solution dropwise to the working area of the working electrode, let it stand, rinse the electrode with acetic acid, ethanol and water in turn, and dry it at room temperature;

4) First infiltrate the surface of the working electrode with 2 μL of ethanol, air to semi-dry to reduce the surface tension, then dropwise add 10 μL of the mixed solution of glucose oxidase GOx and glutaraldehyde, activate overnight at 4°C, rinse with pure water, and air dry. The graphene/enzyme electrode is obtained.

Further, the laser printing conditions in step 1) are: laser wavelength 450 nm, output power 5.5 W, relative laser intensity 20-50%, relative printing depth 5-30%, and circular diameter of electrode working area 4 mm.

Further, in the step 2) the curing temperature of the Ag/AgCl paste and the conductive silver paste is 80° C., and the curing time is 2 hours.

Further, in the step 2), the specific operation of the fixed working area of the polydimethylsiloxane PDMS is as follows: the PDMS silicone elastomer (SYLGARD™ 184 Silicone Elastomer base) and the curing agent are in a mass ratio of 10:1. The ratio is fully stirred and mixed evenly, and it is allowed to stand at 85°C for 3-4 minutes to make it semi-cured. Use a syringe needle to take a small amount and apply it to the circular tangent next to the electrode working area to fix the circular area of the working area.

Further, the 1-pyrene butyric acid solution in the step 3) is specifically 5 mmol/L 1-pyrene butyric acid solution in acetic acid, the dosage is 2-5 μL, and the standing time is 1 hour.

Further, in step 4), the mixed solution of glucose oxidase GOx and glutaraldehyde is obtained by mixing GOx solution and glutaraldehyde solution in a volume ratio of 7:3 (V:V). The GOx solution was prepared by dissolving GOx with a final concentration of 4 mg/mL and BSA with a final concentration of 2 mg/mL in 0.1 mol/L PBS buffer (pH 7). The concentration of glutaraldehyde aqueous solution is 0.5% (W/W), which is currently used and prepared.

A preparation method of a graphene/enzyme electrode glucose sensor, comprising the following steps:

(1) The sample solution was added dropwise to the surface of the graphene/enzyme electrode, covering the three electrodes, and the chronoamperometry was used to monitor the curve of the current over time, thereby constructing a glucose sensor.

The volume of the sample solution in the above step 1) is 50-100 μL. The sample solution can be serum, sweat, urine, and the detection limit of glucose is 0.05-5.0 mmol/L; the instrument used is Radiometer Sensit BT Mini Electrochemical The analyzer is connected to a smart phone via Bluetooth to realize smart phone detection. The APP software used is PStouch 2.7; the constant voltage of the timing current is 0.3-0.8V, and the timing interval is 80 s.

A laser direct writing graphene/enzyme electrode prepared by the above method.

Application of the above graphene/enzyme electrode in a glucose sensor.

The significant advantages of the present invention are:

1) The laser direct writing technology prepares flat microelectrodes, which is easy to operate, does not require organic solvents, is environmentally friendly, and has extremely low cost. At the same time, the electrodes can be patterned at the microscale and can be prepared in batches, which is an electrode system in the micrometer size range. Special needs have been created to facilitate the design of miniaturized electrochemical sensors. LIG technology can induce polyimide (PI) substrate to directly generate three-dimensional porous graphene, which exhibits large specific surface area and high electrical conductivity, which is conducive to the assembly of more GOx to the electrode surface and maintains the good electrochemical performance of the modified electrode. nature.

2) The enzyme immobilization process should be cheap, fast and biocompatible, and the immobilization process should improve the stability, reusability, and activity of the enzyme. There are many different techniques for anchoring enzymes to desired substrates, such as adsorption, entrapment or covalent cross-linking. However, there is a dynamic equilibrium in the adsorption process, which is easy to fall off, which leads to the instability of the detection method. The embedding method affects the activity of the enzyme, and the covalent crosslinking has better stability and reusability. In the present invention, 1-pyrene butyric acid mediator is selected, the hydrophobic end pyrene ring and the benzene ring structure of graphene (that is, the hexagonal honeycomb structure) are firmly combined with the π-π superposition effect, and the hydrophilic end carboxyl group can effectively bond GOx. The assembly process is simple and straightforward, and the GOx can be assembled in two steps.

3) The prepared electrodes have good stability, selectivity and reusability for glucose sensing.

4) The sensor developed in the present invention can be used not only for serum samples, but also for glucose sensing of biological fluids such as human sweat and urine, and can be used for non-invasive monitoring of diabetes. The microelectrode requires a very small amount of sample, only about 50-100 μL, which is conducive to the detection of biological fluid samples, and the sample does not require pretreatment. At the same time, the flexibility and biocompatibility of the electrodes have promising applications in wearable sweat sensing.

Description of drawings

Fig. 1 Schematic diagram of preparation process and glucose sensing analysis of LIG/enzyme electrode;

Fig. 2 SEM images of LIG (A, B) and LIG/GOx (C, D) electrodes;

Fig. 3 CV diagrams of different modified electrodes in potassium ferricyanide solution, potassium ferricyanide solution: 5 mmol/L containing 0.1 mol/L KCl, scan rate: 0.1 V/s;

Fig.4 CV curves of LIG and LIG/GOx electrodes in PBS and glucose solution, glucose solution: 0.1 mol/L, PBS: 0.1 mol/L, pH=7;

Fig. 5 The i-t curves of glucose detection by LIG/GOx electrode at different potentials;

Fig. 6 The i-t curves of different concentrations of glucose solution at LIG and LIG/GOx electrodes, constant potential: 0.7 V;

Fig. 7 Working curve of chronoamperometry and glucose concentration, glucose concentration unit: mol/L, constant potential: 0.7 V.

Fig. 8 The i-t curve of the selectivity test of LIG/GOx electrode for different small molecule interfering substances, the concentration of each sample: 1 mmol/L, the potential: 0.7 V.

Fig. 9 The i-t curve of the repeated use of LIG/GOx electrodes;

Fig. 10 The i-t curve of LIG/GOx electrode precision effect;

Fig. 11 The actual picture of the curling state of the flexible electrode;

Fig. 12 i-t curves of LIG/GOx electrodes before and after crimping;

Fig. 13 The i-t curve of the actual sample addition and recovery test of LIG/GOx electrode.

Detailed ways

In order to better understand the present invention, the present invention will be further described below with reference to the accompanying drawings and embodiments, but it is not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified.

Instrument: Nano Pro-Ⅲ laser printer (Tianjin Jiayin Nanotechnology Co., Ltd.).

The preparation of LIG/GOx electrode and the glucose sensing analysis process are shown in Figure 1: the specific process includes, a) laser direct writing three-electrode patterned graphene; b) preparation of LIG electrode; c) self-assembly of 1-pyrene butyric acid; d) Glutaraldehyde covalently bonded to GOx; e) Chronoamperometry.

Example 1

The micro-electrode pattern of the three-electrode system was designed, using a 450 nm laser with an output power of 5.5 W, a relative printing depth of 15%, and a relative laser intensity of 30%. The LIG electrode graphene pattern was printed on a high-insulation PI film. Rinse with ethanol and air dry. Ag/AgCl paste was coated on the reference electrode, and conductive silver paste was coated on the end of the three electrodes as a signal output connector, at 80°C, and the curing time was 2 hours. Mix the polymer and curing agent in SYLGARD 184 PDMS in a mass ratio of 10:1, stir and mix well, and make it semi-cured for 3 minutes at 85 °C. Take a small amount of syringe needle to fix the circular area of the working area to prevent dripping. The sample solution was tested for diffusion, thereby preparing a LIG electrode.

Example 2

In the working area of the LIG electrode prepared in Example 1, 3 μL of 5 mmol/L 1-pyrenebutyric acid solution in acetic acid was added dropwise, dispersed evenly, and allowed to stand for 1 hour. The electrode was rinsed with acetic acid, ethanol and water in turn. After drying, a LIG/pyrene butyric acid electrode was obtained, and the surface of the electrode was carboxylated.

Example 3

Prepare GOx solution: 4 mg/mL GOx and 2 mg/mL BSA in PBS (0.1 mol/L, pH 7).

In the working area of the LIG/pyrene butyric acid electrode prepared in Example 2, the surface was first wetted with 2 μL of ethanol, aired to semi-dry to reduce the surface tension, and then 10 μL of GOx solution and 0.5wt% glutaraldehyde solution were added dropwise by volume ratio The mixed solution prepared at 7:3 was activated at 4°C overnight, rinsed with pure water, and dried for use to obtain LIG/GOx electrodes.

Example 4

The surface morphologies of LIG and LIG/GOx composite electrodes were characterized by scanning electron microscopy (SEM) (Figure 2). In Figure 2 (A, B) are the scanning electron microscope (SEM) images of the graphene electrode. It can be seen from the figure that the surface of the graphene electrode presents a spatial network structure, showing a uniform layered structure, uniform dispersion and tight texture, indicating that Graphene electrodes have a large specific surface area. The LIG/GOx composite electrode (Figures C, D) still retains a complete mesoporous structure, indicating that the electrode surface morphology was not destroyed during the GOx modification process, and the graphene film layer was thickened, indicating that the graphene surface is rich in carboxyl groups Glucose oxidase was successfully immobilized on the electrode through covalent cross-linking with glutaraldehyde.

Example 5

The LIG, LIG/pyrenebutyric acid and LIG/GOx electrodes prepared in Examples 1, 2 and 3 were tested for electrochemical activity by cyclic voltammetry. A 5 mmol/L potassium ferricyanide solution containing 0.1 mol/L KCl was prepared. Take out the above-mentioned LIG, LIG/pyrenebutyric acid, and LIG/GOx electrodes, pipette 100 µL potassium ferricyanide solution dropwise onto the electrode surface, and perform electrochemical scanning by cyclic voltammetry. Speed: 0.1 V/s. It can be seen from Figure 3 that the LIG electrode has obvious redox peaks. When the bare electrode was modified by pyrene butyric acid, the response current decreased significantly. This is because pyrene butyric acid is a small molecular group with poor electrical conductivity, which obviously hinders the electron transfer on the surface of the electrode, but still shows good oxidation. peaks and reduction peaks. The peak current of the LIG/GOx electrode is further reduced, indicating that GOx has been successfully modified on the electrode surface. The LIG/pyrene butyric acid electrode fully immobilizes GOx, which promotes the electron transfer between the GOx electroactive center and the electrode surface, and retains the The biological activity of GOx gives GOx a suitable microenvironment for direct electrochemical behavior. The CV curve of the GOx-modified LIG electrode still has obvious redox peaks, indicating that the surface of the LIG/GOx electrode still has a good electrochemical response.

Example 6

Take the LIG electrode prepared in Example 1 and the LIG/GOx electrode prepared in Example 3, and use a pipette to pipette 100 μL of PBS solution or 0.1 mol/L glucose solution and drop them on the electrodes, respectively. Electrochemical scanning was performed by Anfa, scanning range: 0.0~0.8 V, scanning speed: 0.1 V/s. Figure 4 shows that compared with the LIG electrode, the LIG/GOx electrode has a more sensitive current signal to glucose, but there is no current peak, so the chronoamperometry is used in the later analysis.

Example 7

Using PBS as the solvent, the series of concentrations are 0, 5.0×10 ^-5 , 1.0×10 ^-4 , 5.0×10 ^-4 , 1.0×10 ^-3 , 2.5×10 ^-3 , 5.0×10 ^-3 , 1.0×10 ^-2 mol/L glucose solution.

The LIG/GOx electrode prepared in the series of Example 3 was taken out, and 100 μL of glucose solution of series concentration was pipetted from low to high, and the electrode was scanned by i-t. Each time the solution is changed, the electrode needs to be rinsed three times with a higher concentration solution, and the i-t scan is performed at a potential of 0.5 V, and the i-t curve is obtained by timing each concentration for 80 s. Taking different LIG/GOx electrodes, changing the potential to 0.6 V, and performing a series of glucose concentration scans at 0.7 V, the results are shown in Figure 5. When the potential is 0.5 V, 0.6 V, and 0.7 V, the sensitivity of LIG/GOx electrode to glucose detection has obvious differences. With the increase of voltage, the sensitivity of LIG/GOx electrode to glucose increases. The i-t current of LIG/GOx electrode has the most significant response to glucose at 0.7 V, and the highest sensitivity response to glucose; its excellent performance may be related to the catalytic activity and stability of glucose oxidase. Combining the influence of different potentials, 0.7 V with a larger current signal was selected as the best working potential for the LIG/GOx electrode to detect the chronocurrent of glucose. This potential can effectively stimulate the catalytic activity of GOx and has a better effect on glucose. current response.

Example 8

Take the LIG electrode prepared in Example 1 and the LIG/GOx electrode prepared in Example 3, and use a pipette to pipette 100 μL of a series of concentrations of 0, 5.0×10 ^-5 , 1.0×10 ^-4 , 5.0×10 from low to high ^-4 , 1.0×10 ^-3 , 2.5×10 ^-3 , 5.0×10 ^-3 , 1.0×10 ^-2 mol/L glucose solution was added dropwise to the electrode, each time the solution was changed, it was necessary to use a higher concentration of glucose The electrode was rinsed with the solution three times, and it was scanned at a potential of 0.7 V for 80 s for each concentration to obtain the it curve. The results are shown in Fig. 6. Compared with the LIG electrode, the LIG/GOx electrode responds rapidly to glucose. With the increase of the glucose concentration in the system, the response current increases step by step, indicating that the LIG/GOx electrode is sensitive to glucose. Has a very high affinity and enzymatic catalytic activity.

The change of the current results with the concentration is shown in Figure 7. When the potential is 0.7 V, the current response of the LIG/GOx electrode has an exponential relationship with the logarithm of the glucose concentration, indicating that the LIG/GOx electrode has a more sensitive current response to glucose detection. , while the LIG electrode is basically unresponsive to glucose, which proves that GOx plays a directional catalytic role for glucose in the electrochemical reaction. This shows that the LIG/GOx electrode prepared by the present invention can measure the glucose concentration through the chronoamperometry generated by the GOx catalytic reaction, and the minimum detection concentration is 5.0×10 ^-5 mol/L, and its sensitivity is much higher than that of the conventional blood glucose meter (Roche's excellent blood glucose meter, the detection range is 0.6-33.3mM; the detection range of Sannuo GA-3 blood glucose meter is 1.1-33.3mM).

Example 9

The LIG/GOx electrode prepared in Example 3 was taken out, and 100 μL of 1 mmol/L solutions of tartaric acid, sucrose, galactose, lactobionic acid and glucose were taken out in turn, and the electrode was scanned by i-t. Each time the solution is changed, it needs to be washed with water, and then the electrode is rinsed three times with the next test solution, and the i-t scan is performed at a potential of 0.7 V for 80 s to obtain the i-t curve. It can be seen from Figure 8 that the LIG/GOx electrode exhibits a significant current response to glucose, while sucrose, galactose, and lactobionic acid have little effect on the detection of glucose, and tartaric acid has a slight effect. This shows that small sugar molecules such as galactose hardly interfere with the detection of glucose, indicating that the prepared LIG/GOx electrode can well eliminate the interference of these active substances, indicating that the LIG/GOx electrode has good selectivity for glucose , has a strong affinity with glucose, and has strong anti-interference performance.

Example 10

Take the LIG/GOx electrode prepared in Example 3, and pipette 100 μL series of glucose with concentrations of 0, 1.0×10 ^-4 , 1.0×10 ^-3 , 5.0×10 ^-3 mol/L from low to high in turn with a pipette. The solution is added dropwise to the electrode. Each time the solution is replaced, the electrode needs to be rinsed three times with a solution with a higher concentration. , washed with water, air-dried, and reused 5 times, as shown in Figure 9. It can be seen from Fig. 9 that after continuous repeated use for 5 times, the current curve decreases slightly with the increase of the number of scans, but still has a good current performance, indicating that the LIG/GOx electrode has good reusability.

Example 11

Take a batch of LIG/GOx electrodes prepared in Example 3, and use a pipette to pipette 100 μL of serial concentrations of 0, 1.0×10 ^-4 , 1.0×10 ^-3 , 5.0×10 ^-3 mol/L from low to high. The glucose solution was dripped onto the electrode. Each time the solution was changed, the electrode was rinsed three times with a solution with a higher concentration. It can be seen from Figure 10 that the current curves of 5 electrodes in the same batch are almost overlapped after continuous measurement of a series of concentrations of glucose, the current performance is good, and the response performance is basically stable, and the concentrations are 1.0×10 ^-4 and 1.0×10 ^-3 The relative standard deviations (RSD) of 5.0×10 ^-3 mol/L glucose solution were 7.69%, 6.78% and 5.05%, respectively, indicating that the LIG/GOx electrode has good precision.

Example 12

Take the LIG/GOx electrode prepared in Example 3, and pipette 100 μL series of glucose with concentrations of 0, 1.0×10 ^-4 , 1.0×10 ^-3 , 5.0×10 ^-3 mol/L from low to high in turn with a pipette. The solution was added dropwise to the electrode. Each time the solution was replaced, the electrode was rinsed three times with a higher concentration solution, and it was scanned at a potential of 0.7 V, and the it curve was obtained by timing each concentration for 80 s. Another LIG/GOx electrode prepared in Example 3 was taken, and it was in a curled state according to Figure 11, and it was scanned by the same method as above. The obtained it curve was shown in Figure 12. The curves almost overlap, and the current performance is good, indicating that the LIG/GOx electrode can still effectively detect the glucose concentration under a certain curling state.

Example 13

Select fetal bovine serum (FBS) diluted 20 times with PBS buffer, urine diluted 1 times, and sweat diluted 1 times as biological samples, and glucose solutions were added respectively to make the final concentrations 0, 0.1, 1.0, 5.0 mmol. /L, according to the steps of Example 11, different biological samples use different electrodes in the same batch, and use chronoamperometry to detect serum, sweat, and urine with different concentrations of glucose solutions. The i-t curve is shown in Figure 13, and it can be seen from the figure Different concentrations of glucose solution showed obvious step change in different samples. With the increase of glucose solution concentration, the current value of the electrode response also gradually increased, which has a good current response. The recorded current signal values, the results are shown in Table 1. It can be drawn from Table 1 that the recovery rates of adding different concentrations of glucose to urine, serum and sweat samples were 69.29%~97.77%, 105.28%~161.72%, 79.09%~103.78%, and the urine and sweat were 69.29%~97.77%, 79.09%~103.78%. The detection interference is small, the serum sample is slightly interfered, and the recovery rate is high, but considering the low concentration detected, it is still suitable for serum sample detection. It shows that the prepared LIG/GOx electrode can be well applied to the detection of glucose in biological samples such as urine, serum and sweat.

Table 1 Detection results of LIG/GOx electrodes in samples

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (10)

1. A portable preparation method of a laser-induced graphene/enzyme electrode is characterized by comprising the following steps: the method comprises the following steps:

1) designing a microelectrode pattern of a three-electrode system, and printing a graphene microelectrode on a high-insulation PI film by adopting a laser;

2) coating Ag/AgCl slurry on a reference electrode, coating conductive silver slurry on the tail end of a three-electrode to serve as a signal output connector, and fixing the area of a working area by polydimethylsiloxane PDMS (PDMS) so as to form a laser direct writing graphene electrode;

3) dropwise adding a 1-pyrenebutyric acid solution to a working area of a working electrode, standing, sequentially leaching the electrode with acetic acid, ethanol and water, and airing at room temperature;

4) soaking the surface of the working electrode with 2 mu L of ethanol, airing to be half-dry, reducing the surface tension, then dropwise adding 10 mu L of mixed solution of glucose oxidase GOx and glutaraldehyde, activating at 4 ℃ overnight, leaching with pure water, and airing to obtain the graphene/enzyme electrode.

2. The portable preparation method of laser-induced graphene/enzyme electrode according to claim 1, characterized in that: the laser printing conditions in the step 1) are as follows: the wavelength of the laser is 450 nm, the output power is 5.5W, the relative intensity of the laser is 20-50%, the relative printing depth is 5-30%, and the diameter of the circle of the working area of the electrode is 4 mm.

3. The portable preparation method of laser-induced graphene/enzyme electrode according to claim 1, characterized in that: the curing temperature of the Ag/AgCl paste and the conductive silver paste in the step 2) is 80 ℃, and the curing time is 2 hours.

4. The portable preparation method of laser-induced graphene/enzyme electrode according to claim 1, characterized in that: the specific operation of fixing the area of the working area by polydimethylsiloxane PDMS in the step 2) is as follows: mixing a PDMS organic silicon elastomer and a curing agent according to a mass ratio of 10: 1, standing for 3-4 min at 85 ℃ for semi-curing, taking a small amount of the solution by using a syringe needle, and coating the solution on a circular tangent line beside an electrode working area for fixing the circular area of the working area.

5. The portable preparation method of laser-induced graphene/enzyme electrode according to claim 1, characterized in that: the 1-pyrenebutyric acid solution in the step 3) is specifically an acetic acid solution of 5 mmol/L1-pyrenebutyric acid, the dosage of the solution is 2-5 mu L, and the standing time is 1 hour.

6. The portable preparation method of the laser-induced graphene/enzyme electrode according to claim 1, characterized in that: the mixed solution of the glucose oxidase GOx and the glutaraldehyde in the step 4) is obtained by mixing a GOx solution and a glutaraldehyde solution according to the volume ratio of 7: 3; wherein the GOx solution is prepared by dissolving GOx with a final concentration of 4mg/mL and BSA with a final concentration of 2 mg/mL in 0.1mol/L PBS buffer with pH = 7; the concentration of the glutaraldehyde solution is 0.5wt%, and the glutaraldehyde solution is prepared as it is.

7. The application of the graphene/enzyme electrode prepared by the preparation method according to claim 1 in a glucose sensor is characterized by comprising the following steps:

(1) and dropwise adding the sample solution on the surface of the graphene/enzyme electrode, covering the graphene/enzyme electrode with three electrodes, and monitoring the change curve of current along with time by adopting a chronoamperometry so as to construct the glucose sensor.

8. Use according to claim 7, characterized in that: the volume of the sample solution in the step (1) is 50-100 mu L, the sample solution is serum, sweat or urine, and the detection concentration of glucose is 0.05-5.0 mmol/L.

9. Use according to claim 7, characterized in that: the instrument adopted in the step (1) is a Mini electrochemical analyzer of a Redtest Sensit BT, the mini electrochemical analyzer is connected with a smart phone through Bluetooth, detection of the smart phone is achieved, and APP software is PStouch 2.7.

10. Use according to claim 7, characterized in that: the constant voltage of the timing current adopted in the step (1) is 0.3-0.8V, and the timing interval time is 80 s.

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