CN115112738A - Preparation of laser direct-writing graphene/enzyme electrode and glucose sensing application - Google Patents
Preparation of laser direct-writing graphene/enzyme electrode and glucose sensing application Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of biosensing, and particularly relates to a preparation method of a laser direct-writing graphene/precious metal nanoparticle composite electrode and application of immunosensing.
Background
With the rapid development of society and the advancement of civilization, the living standard of people is greatly improved. However, the pollution to the environment and the fast-paced life lead to the increasing proportion of obese and sub-healthy people, and the daily life of people is seriously affected by diabetes, hyperlipidemia, cardiovascular diseases, neurological diseases and the like. Treatment of these ailments is often not accomplished at one glance, requiring a long nursing procedure and routine monitoring of the patient's condition. For example, diabetes mellitus is not yet treated completely, and patients generally need lifelong detection and treatment to control the blood sugar range and prevent various complications. The traditional method for detecting the blood index of the patient wastes time and labor, brings great inconvenience to hospitals and patients, and is particularly important for detecting the blood sugar level of the patient in real time. Due to the limitation of the diagnosis method, the current clinical blood sugar detection still adopts an invasive sampling mode of sampling blood by fingertips or arms, so that the pain of a patient is increased, and the probability of external infection is increased. Research shows that the glucose content of biological fluids such as urine, sweat, tears and the like of a human body is related to the blood glucose concentration in the human body, and the glucose monitoring instrument can be used for blood glucose monitoring of diabetes. Therefore, it is particularly necessary to develop a non-invasive blood glucose detection method.
An electrochemical biosensor is a sensing device that can combine electrochemical sensing with specific recognition of biomolecules, and plays a key role in the current laboratory and clinical analysis of various chemical and biological targets. The final aim of the electrochemical sensor is to construct a more simple, sensitive and reliable sensing interface, further amplify the detection signal and improve the sensitivity and accuracy. The development of new functional nano materials and nanotechnology provides new possibility for improving the performance of electrochemical sensors, and the search for high-performance electrode materials is the key for preparing excellent electrochemical sensors and is also an important direction of current research.
With the increasing demand of the nano structure for high-performance devices, the research of the controllable micro-nano structure becomes one of the hot spots of scientific research. The preparation of the controllable micro-nano structure focuses on the application of micro-nano processing technology, so that the preparation technology is developed towards the direction of simplifying preparation process, reducing cost and having high repeatability. The laser beam has the advantages of directional light emission, great energy density, high precision, high machining speed, flexible non-mechanical contact machining and control and the like, so that the laser beam is widely applied to laser micromachining. Especially, the laser direct writing technology can get rid of the constraint of a mask, greatly improves the processing efficiency and the repeatability, and provides a new research idea for the next generation of intelligent health detection.
Graphene is a two-dimensional sp of a single atomic layer 2 The hybrid carbon nanosheet has excellent physical and chemical properties due to large specific surface area, high electron mobility, thermal conductivity, biocompatibility, ultralow density and mechanical flexibility, so that the hybrid carbon nanosheet is widely applied to the fields of sensors, lithium batteries, supercapacitors and the like. The Laser Induced Graphene (LIG) technology can induce a Polyimide (PI) substrate to directly generate graphene with a three-dimensional porous structure, large specific surface area and high conductivity are shown, high temperature and solvent are not needed in the preparation process, and the preparation method is widely applied to preparation of flexible electrodes. In addition, as the most typical embedded system, the microelectrode with smaller volume enables the electrochemical analysis to be portable and rapid, and is more beneficial to practical application. If wearable sweat sensing, need not the pretreatment, can conveniently detect target analyte fast.
Diabetes is a disease that affects millions of people worldwide, and thus there is still a high need for the study of glucose biosensors, and efforts are being made to find cheaper, more accurate, minimal or non-invasive methods to quantify glucose levels in real time. Glucose oxidase (GOx) has been widely studied because of its important catalytic activity. The method has the advantages of low price, good stability and strong practicability, and is an ideal model molecule for preparing the enzyme sensor. In view of the characteristics of high selectivity and high catalytic activity of the enzyme, the glucose oxidase method is the most widely used blood glucose detection method in the market. However, the difficulty in working is to immobilize the enzyme on the desired platform, the enzyme immobilization process should be inexpensive, rapid 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 a desired substrate, such as adsorption, entrapment or covalent cross-linking. However, the adsorption process has dynamic equilibrium and is easy to fall off, so that the detection method is unstable, the embedding method influences the activity of the enzyme, and the covalent crosslinking has better stability and reusability.
Typically, GOx is fixed on a rigid substrate, for example 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 invention adopts the LIG electrode, has good biocompatibility, good environment as enzyme and the effect of promoting electron transfer. The method combining electrochemistry and laser can provide a good environment for covalent bonding of glucose oxidase in a graphene matrix and electron transfer of GOx, and an additional medium is not required to be added to improve the activity of the enzyme. The proposed glucose sensing method, due to its excellent electrochemical properties, biocompatibility and relatively simple manufacturing route, shows potential in glucose monitoring, especially in non-invasive and painless.
Disclosure of Invention
The invention aims to provide a preparation method of a laser direct-writing graphene/enzyme electrode and application of the laser direct-writing graphene/enzyme electrode in glucose sensing.
The invention designs a three-electrode pattern, prepares a micro flexible graphene electrode by combining a Laser Induced Graphene (LIG) technology, utilizes a pi-pi superposition effect of a hydrophobic end benzene ring of 1-pyrenebutyric acid and a six-membered ring structure of graphene to carboxylate a working area on the surface of the LIG electrode so as to covalently cross-link GOx, records that a current signal generated by catalyzing glucose by GOx under constant voltage has a direct relation with glucose concentration by adopting a portable electrochemical workstation and a timing current analysis method, and prepares a portable glucose sensor. Further promotes the development of portable, green and low-cost biosensors.
In order to achieve the purpose, the invention adopts the following technical scheme:
a portable preparation method of a laser-induced graphene/enzyme electrode 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.
Further, 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.
Further, 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.
Further, the specific operation of fixing the working area by polydimethylsiloxane PDMS in the step 2) is as follows: PDMS Silicone Elastomer (SYLGARD @ 184 Silicone Elastomer base) and a curing agent were mixed in 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.
Further, the 1-pyrenebutyric acid solution in the step 3) is specifically 5 mmol/L1-pyrenebutyric acid acetic acid solution, the dosage of the 1-pyrenebutyric acid acetic acid solution is 2-5 mu L, and the standing time is 1 hour.
Further, the mixed solution of the glucose oxidase GOx and the glutaraldehyde in the step 4) is prepared by mixing the GOx solution and the glutaraldehyde solution according to the volume ratio of 7:3(V: V). Wherein the GOx solution is prepared by dissolving GOx with a final concentration of 4mg/mL and bovine serum albumin BSA with a final concentration of 2 mg/mL in 0.1mol/L PBS buffer (pH 7). The concentration of the glutaraldehyde aqueous solution is 0.5% (W/W), and the glutaraldehyde aqueous solution is prepared as it is.
A preparation method of a graphene/enzyme electrode glucose sensor comprises the following steps:
(1) and dropwise adding the sample solution on the surface of the graphene/enzyme electrode, covering the three electrodes, and monitoring the change curve of current along with time by adopting a chronoamperometry so as to construct the glucose sensor.
The volume of the sample solution in the step 1) is 50-100 mu L, the sample solution can be serum, sweat and urine, and the detection limit concentration of glucose is 0.05-5.0 mmol/L; the adopted instrument is a Redtite Sensit BT mini electrochemical analyzer, the detection of the smart phone can be realized by connecting the Bluetooth with the smart phone, and the APP software is PStouch 2.7; the constant voltage of the adopted timing current is 0.3-0.8V, and the timing interval time is 80 s.
The laser direct-writing graphene/enzyme electrode prepared by the method.
The graphene/enzyme electrode is applied to a glucose sensor.
The invention has the following remarkable advantages:
1) the planar microelectrode prepared by the laser direct writing technology is simple and convenient to operate, does not need an organic solvent, is green and environment-friendly, has extremely low cost, can be used for patterning electrodes in a microscale, can be prepared in batches, creates special requirements for an electrode system in a micrometer size range, and is beneficial to designing a miniaturized electrochemical sensor. The LIG technology can induce a Polyimide (PI) substrate to directly generate graphene with a three-dimensional porous structure, shows large specific surface area and high conductivity, and is beneficial to assembling more GOx on the surface of an electrode while maintaining good electrochemical properties of a modified electrode.
2) The enzyme immobilization process should be inexpensive, rapid 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 a desired substrate, such as adsorption, entrapment or covalent cross-linking. However, the adsorption process has dynamic equilibrium and is easy to fall off, so that the detection method is unstable, the embedding method influences the activity of enzyme, and covalent crosslinking has better stability and reusability. According to the invention, a 1-pyrenebutyric acid mediator is selected, the hydrophobic terminal pyrene ring is firmly combined with the pi-pi superposition effect of the benzene ring structure (namely a hexagonal honeycomb structure) of graphene, the hydrophilic terminal carboxyl can be effectively bonded with GOx, the adopted assembly process is simple, convenient and direct, and the assembly of GOx can be realized by two steps.
3) The prepared electrode has good stability and selectivity for sensing glucose, and can be repeatedly used.
4) The sensor developed by the invention can be used for not only serum samples, but also glucose sensing of biological fluids such as human sweat, urine and the like, and can be used for noninvasive monitoring of diabetes. The micro electrode needs a small amount of sample, only about 50-100 mu L, is beneficial to the detection of the biological fluid sample, and does not need pretreatment. Meanwhile, the flexibility and biocompatibility of the electrode have good application prospects in wearable sweat sensing.
Drawings
FIG. 1 is a schematic diagram of the preparation process and glucose sensing analysis of an LIG/enzyme electrode;
FIG. 2 SEM images of LIG (A, B) and LIG/GOx (C, D) electrodes;
FIG. 3 CV diagram of different modified electrodes in potassium ferricyanide solution: 5 mmol/L KCl containing 0.1mol/L, sweep rate: 0.1V/s;
fig. 4 CV graphs of LIG and LIG/GOx electrodes in PBS and glucose solution: 0.1mol/L, PBS: 0.1mol/L, pH = 7;
FIG. 5 is a graph of i-t curves of LIG/GOx electrode at different potentials for glucose detection;
FIG. 6I-t graph of glucose solution with different concentrations for LIG and LIG/GOx electrodes, constant potential: 0.7V;
FIG. 7 is a graph of the working current versus glucose concentration in units of glucose concentration: mol/L, constant potential: 0.7V.
FIG. 8 is a graph of the i-t curves of the LIG/GOx electrode for the selectivity tests of different small molecule interfering substances, for each sample concentration: 1 mmol/L, potential: 0.7V.
FIG. 9 is a graph of the effect of repeated use of LIG/GOx electrodes i-t;
FIG. 10 is a graph of the effect of precision i-t of LIG/GOx electrodes;
FIG. 11 is a pictorial view of a flexible electrode in a rolled state;
FIG. 12 is a graph of i-t curves before and after the LIG/GOx electrode is crimped;
FIG. 13 LIG/GOx electrode real sample addition recovery test i-t plot.
Detailed Description
For a better understanding of the present invention, reference is made to the following examples and accompanying drawings which are set forth to illustrate, but are not to be construed as the limit of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
The instrument comprises the following steps: nano Pro-III type laser Printer (Tianjin Jia silver nanotechnology Co., Ltd.).
The preparation of LIG/GOx electrode and glucose sensing analysis process are shown in FIG. 1: the specific process comprises the steps of a) laser direct writing three-electrode patterned graphene; b) preparing an LIG electrode; c) self-assembling 1-pyrenebutyric acid; d) glutaraldehyde covalent bonding GOx; e) and (6) timing current analysis.
Example 1
Designing a microelectrode pattern of a three-electrode system, adopting a 450 nm laser, outputting power of 5.5W, printing relative depth of 15%, and laser relative intensity of 30%, printing a LIG electrode graphene pattern on a high-insulation PI film, washing the electrode with water and ethanol in sequence, and drying. Ag/AgCl paste is coated on a reference electrode, conductive silver paste is coated at the tail end of the three electrodes to serve as a signal output connector, and the curing time is 2 hours at 80 ℃. Mixing polymer and curing agent in SYLGARD 184 PDMS according to the mass ratio of 10: 1, fully stirring and uniformly mixing, semi-solidifying at 85 ℃ for 3min, taking a small amount of syringe needles for fixing the circular area of a working area and preventing the dropwise added test sample solution from diffusing, thereby preparing the LIG electrode.
Example 2
In the working area of the LIG electrode prepared in example 1, 3 μ L of 5 mmol/L acetic acid solution of 1-pyrenebutanoic acid was dropped, uniformly dispersed, left to stand for 1 hour, the electrode was rinsed with acetic acid, ethanol, and water in order, and dried at room temperature to obtain the LIG/pyrenebutanoic acid electrode, and the surface of the electrode was carboxylated.
Example 3
Preparing a GOx solution: PBS solution (0.1mol/L, pH 7) containing 4mg/mL GOx and 2 mg/mL BSA.
In the working area of the LIG/pyrenebutyric acid electrode prepared in example 2, the surface was first soaked with 2. mu.L of ethanol, air-dried to be half-dry, the surface tension was reduced, then 10. mu.L of a mixed solution prepared by mixing GOx solution and 0.5wt% of glutaraldehyde solution in a volume ratio of 7:3 was added dropwise, activated overnight at 4 ℃, rinsed with pure water, and air-dried for use, so as to prepare the LIG/GOx electrode.
Example 4
The surface morphology of the LIG and LIG/GOx composite electrodes was characterized using Scanning Electron Microscopy (SEM) (fig. 2). Fig. 2 (A, B) is a Scanning Electron Microscope (SEM) image of the graphene electrode, and it can be seen that the surface of the graphene electrode has a spatial network structure, which shows a uniform layered structure, uniform dispersion and compact texture, indicating that the graphene electrode has a large specific surface area. The LIG/GOx composite electrode (fig. C, D) still retains a complete mesoporous structure, which indicates that the electrode surface morphology is not damaged during the GOx modification process, and the graphene membrane layer is thickened, indicating that carboxyl groups rich on the graphene surface are covalently crosslinked with glutaraldehyde, and glucose oxidase is successfully fixed on the electrode.
Example 5
The electrochemical activity of the LIG, LIG/pyrenebutyric acid, LIG/GOx electrodes prepared in examples 1, 2, 3 was tested using cyclic voltammetry. Preparing a potassium ferricyanide solution with the concentration of 5 mmol/L and containing 0.1mol/L KCl. Taking out the LIG, LIG/pyrenebutyric acid and LIG/GOx electrodes, transferring 100 mu L of potassium ferricyanide solution, dropwise adding the potassium ferricyanide solution on the surface of the electrodes, and performing electrochemical scanning by adopting a cyclic voltammetry method, wherein the scanning range is as follows: -0.4-0.5V, sweep speed: 0.1V/s. As can be seen from fig. 3, the LIG electrode has a relatively distinct redox peak. After the pyrenebutyric acid modifies the bare electrode, the response current is obviously reduced because the pyrenebutyric acid belongs to a small molecular group, the conductivity is poor, the pyrenebutyric acid has obvious barrier effect on the electron transfer on the surface of the electrode, and the pyrenebutyric acid still shows better oxidation peak and reduction peak. The peak current of the LIG/GOx electrode is further reduced, which shows that GOx is successfully modified on the surface of the electrode, and the LIG/pyrenebutyric acid electrode sufficiently fixes GOx, so that the electron transfer between the GOx electroactive center and the surface of the electrode is well promoted, the bioactivity of GOx is kept, and the GOx is provided with a microenvironment suitable for directly performing electrochemical behaviors. The CV curve of the GOx modified LIG electrode still has a more obvious oxidation reduction peak, which indicates that the LIG/GOx electrode surface still has a better electrochemical response.
Example 6
The LIG electrode prepared in example 1 and the LIG/GOx electrode prepared in example 3 were taken, 100 μ L of PBS solution or 0.1mol/L of glucose solution was respectively pipetted onto the electrodes using a pipette gun, and electrochemical scanning was performed using cyclic voltammetry for each of the two electrodes, the scanning range: 0.0-0.8V, sweeping speed: 0.1V/s. As can be seen in fig. 4, the LIG/GOx electrode has a more sensitive current signal to glucose than the LIG electrode, but no current peak, so the chronoamperometry was used for the later analysis.
Example 7
PBS is used as a solvent to prepare a series of concentrations of 0, 5.0 multiplied by 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 A glucose solution of mol/L.
A series of LIG/GOx electrodes prepared in example 3 were removed, 100. mu.L of the series concentration glucose solutions were removed from low to high, and the electrodes were i-t scanned. And (3) rinsing the electrode for 3 times by using a solution with a higher first-order concentration every time of replacing the solution, and respectively performing i-t scanning at a potential of 0.5V, wherein each concentration is timed for 80 s to obtain an i-t curve. A series of glucose concentration scans were performed at varying potentials of 0.6V and 0.7V using different LIG/GOx electrodes, the results are shown in FIG. 5. When the potential is 0.5V, 0.6V and 0.7V, the sensitivity of the LIG/GOx electrode to the glucose is obviously detected, and the sensitivity of the LIG/GOx electrode to the glucose is increased along with the increase of the voltage. The LIG/GOx electrode has the most obvious response to the i-t current of the glucose at 0.7V and the highest sensitivity response to the glucose; the superiority of the method can be related to the catalytic activity and stability of the glucose oxidase. The influence of different potentials is combined, 0.7V with a larger current signal is selected as the optimal working potential of the LIG/GOx electrode on the glucose detection timing current, the catalytic activity of GOx can be effectively excited at the potential, and the current response to glucose is better.
Example 8
Using the LIG electrode prepared in example 1 and the LIG/GOx electrode prepared in example 3, 100. mu.L series of 0, 5.0X 10 concentrations were pipetted from low to high using a pipette gun -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 And dropwise adding a mol/L glucose solution on the electrode, rinsing the electrode for 3 times by using a solution with a higher first-order concentration every time the solution is replaced, and respectively performing i-t scanning at a potential of 0.7V, wherein each concentration is timed for 80 s to obtain an i-t curve. The result is shown in fig. 6, compared with the LIG electrode, the LIG/GOx electrode has a rapid response to glucose, and the response current gradually increases in a stepwise manner as the concentration of glucose in the system increases, which indicates that the LIG/GOx electrode has a very high affinity and enzyme catalytic activity to glucose.
The current result changes with the concentration, as shown in fig. 7, when the potential is 0.7V, the current response of the LIG/GOx electrode is exponential to the logarithm of the glucose concentration, which indicates that the LIG/GOx electrode has a more sensitive current response to glucose detection, while the LIG electrode has no response to glucose, thus proving that GOx plays a directional catalytic role in the electrochemical reaction on glucose. This shows that the LIG/GOx electrode prepared by the present invention can measure the glucose concentration by the change value of the timing current generated by GOx catalytic reaction, and the minimum detection concentration is 5.0 × 10 -5 mol/L, and the sensitivity is much higher than that of the conventional blood glucose meter (Roche excellence blood glucose meter, the detection range is 0.6-33.3 mM; and the detection range of the Sanno GA-3 blood glucose meter is 1.1-33.3mM）。
Example 9
The LIG/GOx electrode prepared in example 3 was removed, 100. mu.L of 1 mmol/L tartaric acid, sucrose, galactose, lactobionic acid and glucose solution were removed in sequence and the electrode was i-t scanned. And (3) rinsing with water every time when the solution is replaced, rinsing the electrode for 3 times with the next test solution, performing i-t scanning at a potential of 0.7V, and timing for 80 s to obtain an i-t curve. As can be seen from fig. 8, LIG/GOx electrodes showed a significant galvanic response to glucose, whereas sucrose, galactose, lactobionic acid had little effect on glucose detection and tartaric acid had little effect. Therefore, sugar micromolecules such as galactose and the like hardly cause interference on glucose detection, and the interference of the active substances can be well eliminated by the prepared LIG/GOx electrode, so that the LIG/GOx electrode has good selectivity on glucose, strong affinity with glucose and strong anti-interference performance.
Example 10
The LIG/GOx electrode prepared in example 3 was used to sequentially transfer 100. mu.L of the series of 0, 1.0X 10 concentrations from low to high using a pipette -4 、1.0×10 -3 、5.0×10 -3 Dropping a mol/L glucose solution on the electrode, rinsing the electrode 3 times by using a solution with a higher first-order concentration every time of solution replacement, performing i-t scanning at a potential of 0.7V respectively, timing 80 s for each concentration to obtain an i-t curve, washing the electrode with water, drying the electrode in the air, and repeatedly using the electrode for 5 times, wherein the process is shown in figure 9. From fig. 9, it can be seen that after 5 times of continuous repeated use, the current curve is slightly decreased with the increase of the number of scanning times, but still has better current performance, indicating that the LIG/GOx electrode has good reusability.
Example 11
A batch of the LIG/GOx electrodes prepared in example 3 was sampled and transferred by a pipette gun from low to high in the order of 100. mu.L series of concentrations of 0, 1.0X 10 -4 、1.0×10 -3 、5.0×10 -3 And dropwise adding a mol/L glucose solution on the electrode, rinsing the electrode for 3 times by using a solution with a higher first-order concentration every time the solution is replaced, and respectively performing i-t scanning at a potential of 0.7V, wherein each concentration is timed for 80 s to obtain an i-t curve. As can be seen from FIG. 10, the same batch of 5 electrodes, pair seriesAfter glucose concentration is continuously measured, current curves almost coincide, the current performance is good, the response performance is basically stable, and the concentration is 1.0 multiplied by 10 -4 、1.0×10 -3 、5.0×10 -3 The Relative Standard Deviation (RSD) of the glucose solution in mol/L is 7.69%, 6.78% and 5.05%, respectively, which indicates that the LIG/GOx electrode has good precision.
Example 12
The LIG/GOx electrode prepared in example 3 was used to sequentially transfer 100. mu.L of the series of 0, 1.0X 10 concentrations from low to high using a pipette -4 、1.0×10 -3 、5.0×10 -3 And dropwise adding a mol/L glucose solution on the electrode, rinsing the electrode for 3 times by using a solution with a higher first-order concentration every time the solution is replaced, and respectively performing i-t scanning at a potential of 0.7V, wherein each concentration is timed for 80 s to obtain an i-t curve. Another LIG/GOx electrode prepared in example 3 was taken and placed in a rolled state according to fig. 11, i-t scanning was performed in the same manner as described above, and the obtained i-t curve is shown in fig. 12.
Example 13
Selecting 20 times Fetal Bovine Serum (FBS) diluted by PBS buffer solution, 1 times urine diluted by PBS buffer solution and 1 times sweat as biological samples, respectively adding glucose solution to make the final concentration of the biological samples to be 0, 0.1, 1.0 and 5.0 mmol/L in sequence, according to the step of the embodiment 11, different biological samples adopt different electrodes in the same batch, adopting a timing current method to detect the glucose solution with different concentrations added in serum, sweat and urine, an i-t curve is shown in figure 13, and the graph shows that the glucose solution with different concentrations presents obvious step change in different samples, the current value of electrode response is gradually increased along with the increase of the concentration of the glucose solution, and the biological samples have good current response. The recorded current signal values are shown in table 1. The recovery rate of adding glucose with different concentrations into urine, serum and sweat samples is 69.29-97.77%, 105.28-161.72% and 79.09-103.78% in sequence, wherein the urine and sweat have small interference on glucose detection, the serum samples have slight interference, the recovery rate is high, but the detected concentration is considered to be low, and the method is still suitable for serum sample detection. The prepared LIG/GOx electrode can be well applied to glucose detection of biological samples such as urine, serum, sweat and the like.
TABLE 1 LIG/GOx electrode test results in samples
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by 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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