CN115266874A - Glucose sensor with low potential and wide detection range and preparation method thereof - Google Patents
Glucose sensor with low potential and wide detection range and preparation method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/301—Reference electrodes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/307—Disposable laminated or multilayered electrodes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention discloses a glucose sensor with low potential and wide detection range and a preparation method thereof. The glucose sensor adopts an Au working electrode, an Au counter electrode and an Ag/AgCl reference electrode three-electrode system, and 2 sensors with the same shape jointly complete the extraction and detection functions of the sensor. The invention takes noninvasive detection of human blood sugar as application requirement, adopts high-sensitivity rhodium redox polymer to modify on a film electrode, and fixes enzyme molecules by a glutaraldehyde crosslinking method to prepare a novel biosensor. The sensor prepared by the invention has the advantages that the service life of the sensor can reach 15 days at the temperature of 4 ℃, and the detection range is wide.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a glucose sensor with low potential and wide detection range and a preparation method thereof.
Background
Diabetes has become one of the health-threatening diseases for humans. Blood glucose monitoring in diabetics is very important to control the condition of the disease. For invasive tests generally adopted at present, patients mostly have fear and discomfort, so the research and development of noninvasive glucometers become key subjects of medical circles of various countries.
Among them, sugar molecules of subcutaneous interstitium are obtained by reverse iontophoresis technology, and glucose exuded subcutaneously by an electrochemical biosensor is tested. Compared with direct blood sampling for detecting blood sugar (mmol/L magnitude) of a human body, the concentration of subcutaneous exuded glucose is very low (mmol/L magnitude), the traditional blood sugar biosensor can hardly meet the requirements on detection range and sensitivity, and the sensor capable of detecting low-concentration glucose can not be put into practical use due to the limitation of electrode manufacturing. At 0.53V operating voltage, noninvasive detection of subcutaneous blood glucose was achieved by using a hydrogen peroxide sensor, however, higher operating potentials tend to cause other electroactive species in the body fluid to interfere with the test.
Disclosure of Invention
In view of the above-mentioned deficiencies in the prior art, the present invention provides a glucose sensor with low potential and wide detection range, and a method for preparing the same, wherein large-scale detection of glucose is necessary in many applications, but is still challenging. The commercial enzyme-based glucose detection test paper cannot be reused, and meanwhile, the current enzyme-free glucose sensor has a narrow detection range and slow glucose oxidation kinetics.
The invention adopts rhodium redox polymer coupled with glucose oxidase as an electron mediator modified film Au electrode, and prepares a novel biosensor by fixing glucose oxidase molecules by a glutaraldehyde method, and the current response characteristics of the biosensor to standard glucose and subcutaneous glucose under 2 conditions can respectively reach 23.955 nA/(mmol L)-1) And 18.941 nA/(mmol L)-1) The service life can reach 15 days in the environment of 4 ℃.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problem is as follows:
a method for preparing a glucose sensor with low potential and wide detection range comprises the step of cross-linking and fixing a redox polymer formed by glucose oxidase and rhodium on a working electrode in a three-electrode system.
Furthermore, in the three-electrode system, the working electrode is an Au working electrode, the counter electrode is an Au counter electrode, and the reference electrode is an Ag/AgCl reference electrode.
Further, the method comprises the following steps:
(1) Etching a protective film on a plastic substrate (130 mm multiplied by 100 mm) into a mask pattern of a strip electrode, then sequentially sputtering a transition metal layer and an Au electrode layer on the protective film, then removing the mask on the substrate, then etching, and introducing oxygen for cleaning to form a working electrode;
(2) Taking stainless steel as a mask plate, and then, silk-screening a mixed solution of silver and silver chloride at intervals on strip-shaped electrodes on a substrate to prepare an Ag/AgCl reference electrode;
(3) Cutting a rectangular groove (100 mm multiplied by 4.0 mm) in the middle of the adhesive tape (130 mm multiplied by 50 mm), and then adhering the adhesive tape on a substrate, wherein the electrodes exposed on the substrate in the groove are a working electrode (2.0 mm multiplied by 4.0 mm) and a reference electrode (20 mm multiplied by 4.0 mm) of the sensor in sequence;
(4) Mixing glucose oxidase, a cross-linking agent and a rhodium-containing redox polymer solution uniformly to form an enzyme solution, dripping 0.5mL of the enzyme solution on the surface of a working electrode of a sensor, naturally airing at room temperature to form a film, washing with water, airing, cutting into single sensors, and storing at room temperature for later use.
Further, the substrate is a plastic substrate.
Further, the thickness of the transition metal layer is 10-30nm, and the thickness of the Au electrode layer is 100-500 nm.
Further, the transition metal layer is a Cr layer
Furthermore, the flow rate of the oxygen is 30-50 mL/min, the working pressure is 3-5 Pa, and the cleaning time is 30-40 s.
Further, the crosslinking agent is an aldehyde crosslinking agent.
Further, the cross-linking agent is glutaraldehyde.
Further, the enzyme solution contains 1% BSA, the concentration of glucose oxidase is 5-10U/mL, and the dosage of the cross-linking agent is 3.5-5% of the volume of the enzyme solution.
The glucose sensor prepared by the method has low potential and wide detection range, adopts an Au working electrode, an Au counter electrode and an Ag/AgCl reference electrode three-electrode system, has the structure shown in figure 1, consists of 2 sensors with the same shape, and jointly completes the extraction and detection functions of the sensors, wherein W1 and W2 are thin film Au working electrodes, C1 and C2 are Au counter electrodes, and W and R form an extraction electrodeAn electrode loop, wherein R1 and R2 are silk-screen Ag/AgCl reference electrodes for providing a stable reference voltage during sensor test, and the area of the sensitive part of the working electrode is 0.85cm2。
The glucose sensor is applied to noninvasive detection of human blood glucose.
The technical principle of the application is as follows:
glucose oxidase (GOx) immobilized on the working electrode oxidizes glucose, and electrons generated by this process oxidize the reduced dielectric Rh, which is reduced at a lower working voltage, to the oxidized dielectric Rh, which is reduced because the transfer of electrons occurs in the entire three-dimensional network of the redox polymer, so that the reaction current density is large and the sensitivity is high. In addition, redox polymers have high electron transfer efficiency because they do not diffuse freely into the bulk solution. The response current in the reaction is proportional to the glucose concentration. The rhodium polymer can detect glucose at a lower potential, and avoids the interference of other electrochemical active substances. Meanwhile, rhodium can carry out rapid electron transfer between the electrode and the enzyme, so that the dependence on the oxygen concentration in the bulk solution is eliminated to a certain extent.
The invention has the beneficial effects that:
1. the glucose biosensor with specificity is formed by fixing enzyme molecules on the surface of a membrane electrode by adopting a glutaraldehyde crosslinking method. The sensitivity of the sensor is improved by selecting a redox polymer with high sensitivity. The current response characteristics of the sensor to 2 cases of standard glucose and subcutaneous glucose were studied, and the sensitivity was 23.955 nA/(mmol L)-1) And 18.941 nA/(mmol L)-1) The linear correlation reaches above 0.99.
2. The invention adopts an accelerated life test to analyze that the service life of the sensor can reach 15 days at the temperature of 4 ℃. And gives sensor within and between batches of less than 5% accuracy. The designed three-electrode sensor has the advantages of high sensitivity, good stability, low detection limit and the like, integrates the functions of sampling and detection, and combines the anti-iontophoresis technology to realize the extraction and detection of subcutaneous blood sugar.
3. The glutaraldehyde enzyme crosslinking experiment is adopted, the response current of the sensor can be effectively improved in RP, the linear working area is wide, and the sensitivity is high. The response time of the sensor is 10s, the sensor has good consistency and stability, and 90% of the initial activity of the sensor can be still maintained after the sensor is used for one month, which shows that the glutaraldehyde enzyme crosslinking can maintain higher enzyme activity in RP, and the sensor has the advantages of low cost, simple operation and quick detection. Can be expected to be produced in batches for the fields of medical diagnosis, food industry, environmental detection and the like.
Drawings
FIG. 1 is a three electrode architecture diagram of a sensor;
FIG. 2 is a graph of the CV (cyclic voltammetry) response of the sensor to different glucose concentrations;
FIG. 3 is a graph of the i-t (time-current curve) response of a sensor to different glucose concentrations;
FIG. 4 is a linear plot of sensor versus glucose concentration;
FIG. 5 is a graph of long term stability of the sensor.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and confirmed by the appended claims, and all changes that can be made by the invention using the inventive concept are intended to be protected.
Example 1
A preparation method of a glucose sensor with low potential and wide detection range comprises the following specific steps:
(1) After a protective film on a plastic substrate (130 mm multiplied by 100 mm) is carved into a mask pattern of a strip electrode, a transition layer Cr (10 nm-) and an Au electrode layer (100 nm) are sputtered on the protective film in sequence;
(2) After the mask is removed, the substrate is placed in a plasma etcher, and O is introduced2(working air pressure 3.0 Pa, O2Flow 30 mL/min) "rinse" for 30s.
(3) Taking stainless steel as a mask plate, and carrying out interval silk-screen printing on mixed slurry of silver and silver chloride on a strip electrode on a plastic substrate to form an integrated Ag/AgCl reference electrode;
(4) Cutting a rectangular groove (100 mm multiplied by 4.0 mm) in the middle of the adhesive tape (130 mm multiplied by 50 mm), and then adhering the adhesive tape on a plastic substrate, wherein the electrodes exposed on the substrate in the groove are a working electrode (2.0 mm multiplied by 4.0 mm) and a reference electrode (20 mm multiplied by 4.0 mm) of the sensor in sequence;
(5) Adding GOx and glutaraldehyde into redox polymer solution, mixing thoroughly, dripping 0.5mL of GOx and glutaraldehyde onto the surface of a working electrode of a sensor, naturally drying at room temperature to form a film, washing with water, cutting into individual sensors after drying, and storing at room temperature for later use.
Example 2
A preparation method of a glucose sensor with low potential and wide detection range comprises the following specific steps:
(1) After a protective film on a plastic substrate (130 mm multiplied by 100 mm) is carved into a mask pattern of a strip electrode, a transition layer Cr (30 nm) and an Au electrode layer (300 nm) are sputtered on the protective film in sequence;
(2) After the mask is removed, the substrate is placed in a plasma etcher, and O is introduced2(working air pressure 4.0 Pa, O2Flow rate 35 mL/min) "rinse" for 35s.
(3) Taking stainless steel as a mask plate, and screen-printing mixed slurry of silver and silver chloride on a strip electrode on a plastic substrate at intervals to form an integrated Ag/AgCl reference electrode;
(4) The middle part of the adhesive tape (130 mm multiplied by 50 mm) is carved with a rectangular groove (100 mm multiplied by 4.0 mm) and then is adhered on a plastic substrate, and the electrodes exposed on the substrate in the groove are a working electrode (2.0 mm multiplied by 4.0 mm) and a reference electrode (20 mm multiplied by 4.0 mm) of the sensor in sequence;
(5) Adding GOx and glutaraldehyde into redox polymer solution, mixing completely, dripping 0.5mL of GOx and glutaraldehyde onto the surface of a working electrode of a sensor, naturally drying at room temperature to form a film, washing with water, cutting into single sensors after drying, and storing at room temperature for later use.
Example 3
A preparation method of a glucose sensor with low potential and wide detection range comprises the following specific steps:
(1) After a protective film on a plastic substrate (130 mm multiplied by 100 mm) is carved into a mask pattern of a strip electrode, a transition layer Cr (20 nm) and an Au electrode layer (200 nm) are sputtered on the plastic substrate in sequence;
(2) After the mask is removed, the substrate is placed in a plasma etcher, and O is introduced2(working gas pressure 3.0 Pa, O2Flow rate 35 mL/min) "rinse" for 30s.
(3) Taking stainless steel as a mask plate, and screen-printing mixed slurry of silver and silver chloride on a strip electrode on a plastic substrate at intervals to form an integrated Ag/AgCl reference electrode;
(4) Cutting a rectangular groove (100 mm multiplied by 4.0 mm) in the middle of the adhesive tape (130 mm multiplied by 50 mm), and then adhering the adhesive tape on a plastic substrate, wherein the electrodes exposed on the substrate in the groove are a working electrode (2.0 mm multiplied by 4.0 mm) and a reference electrode (20 mm multiplied by 4.0 mm) of the sensor in sequence;
(5) Adding GOx and glutaraldehyde into redox polymer solution, mixing thoroughly, dripping 0.5mL of GOx and glutaraldehyde onto the surface of a working electrode of a sensor, naturally drying at room temperature to form a film, washing with water, cutting into individual sensors after drying, and storing at room temperature for later use.
Examples of the experiments
1. Cyclic voltammetry and amperometric detection experiments
Both cyclic voltammetry and amperometric tests were performed on a CHI 760E electrochemical workstation detection system. The specific process is as follows:
in a 25mL beaker, 10mL of phosphate buffer pH =7.4 (0.01M PBS) was added, the stirring magneton was placed in the beaker beforehand, the sensor was inserted, the stirrer was switched on and adjusted to a constant speed, the working potential was 0.13V (Ag/AgCl reference electrode). And stopping stirring when the current response is tested, firstly, detecting the background current of the sensor, when the response is in a stable state, adding the glucose of the solution to be tested, continuing stirring the magnetons for 1min to ensure that the glucose of the solution to be tested is uniformly filled in the whole solution, then, stopping stirring, starting a test program, and recording the oxidation current. The experimental temperatures were both 25 ℃, and the results are shown in fig. 2 and fig. 3.
2. Sensor electrochemical property detection
A thin film Au working electrode was modified with 8mL of rhodium polymer. FIG. 3 shows the peak current change of the redox polymer at the cathode and anode at different scan rates, which are 5, 10, 20, 50, 100, 200, 500mV/s. As can be seen from the figure: oxidation peak current (I) thereofpa) And reduction peak current (I)pc) The ratio of (A) to (B) is Ipa/IpC1 and the peak current is linear with the square root of the scan rate, indicating that the rhodium redox polymer has reversible electrochemical properties.
3. Current response of sensor to standard glucose
FIG. 4 shows the current response curve of the sensor to standard glucose. Wherein, according to the glucose concentration corresponding to the curve, each sample is measured for 3 times, and the working voltage is 0.18V. As can be seen from the figure: after the glucose solution is added, the current response of the sensor is gradually increased along with the increase of the glucose concentration, the increase of the current value and the increase of the glucose concentration form a linear relation, the current response signal quickly tends to be stable, and meanwhile, the current response signal of the glucose solution has a very good linear relation in the concentration range of 5.0-45 mmol/L. Linear correction equation I of sensor in 5.0-45mmol/L rangep=5.6796x-2.4909, the minimum detection limit is 0.3mmol/L, the correlation coefficient R is 0.9979, and the sensitivity of the sensor is 23.955 nA/(mmol L)-1). The results show that: the use of rhodium polymers effectively reduces the operating voltage of the sensor.
4. Sensor repeatability study
The result of repeated testing of 10mmol/L glucose between the single biosensor in the same batch and different sensors in different batches shows that the intra-batch precision refers to the current response result of 10mmol/L glucose tested by 1 sensor repeatedly for 10 times, and the inter-batch precision refers to the current response result of 10mmol/L glucose tested by 10 sensors respectively. The results show that: the accuracy of the sensor in batch and between batches is respectively 4.07 percent and 3.22 percent, and both are less than 5 percent, which shows that the prepared sensor has better repeatability and consistency.
5. Sensor stability detection
The conventional life test evaluates the performance of the sensor by detecting how long it has been stored at 4 ℃. In physicochemical terms, the rate of a chemical reaction increases with increasing reaction temperature. Biological reagents are stored at different temperatures, and the "rate" of reagent failure and the temperature and time of storage are also related to the physicochemical reaction rate. The arrhenius empirical formula summarizes this physicochemical rule. Arrhenius describes the effect of temperature on reaction rate by an empirical formula: lnk = -EaRT + B, wherein k is the reaction rate; t is the absolute temperature of the reaction.
Designing and testing: the preservation time for which the reagent was out of order at multiple temperatures was performed, the reciprocal of which (number of days or hours) was the rate at which the reagent deteriorated. The test temperature was converted to an absolute temperature T, and the reciprocal of the retention time was plotted on semilogarithmic paper to find that lnk is Y1/T is X. An optimal fit line is plotted on the graph, which is extrapolated back to the logarithm of the corresponding reaction rate at T of 277K (4 ℃). The logarithm (exponential) is solved by the logarithm value, and then the reciprocal of the logarithm value is solved, namely the estimated stable time.
The glucose biosensor is observed for the stability of the service life according to the principle. The tests were carried out at 20, 35, 55 ℃. The response of the biosensor at 4 ℃ is used as the detection result of the control product, and the detection result is invalid when 90% or less of the response result of the biosensor at 4 ℃ is used as the failure result.
The paired results of X (1/T) and Y [ lg (1/d) ] are plotted or subjected to a linear regression (in a linear relationship on a semilogarithmic paper), and the number of days expected to be stable is 15d at an absolute temperature 277K of 4 ℃ as estimated from the regression formula. Therefore, the biosensor prepared was stable at 4 ℃ for about 15 days as predicted by the test. (see fig. 5) in conclusion, it can be seen that the sensor prepared by the invention has a lower oxidation potential.
Claims (10)
1. A preparation method of a glucose sensor with low potential and wide detection range is characterized in that glucose oxidase and rhodium redox polymer are fixed on a working electrode in a three-electrode system in an alternating mode.
2. The preparation method according to claim 1, wherein the working electrode in the three-electrode system is an Au working electrode, the counter electrode is an Au counter electrode, and the reference electrode is an Ag/AgCl reference electrode.
3. The method of claim 2, comprising the steps of:
(1) Preparing a protective film of a substrate into a strip-shaped electrode mask pattern, sputtering a transition metal layer and an Au electrode layer on the electrode mask pattern in sequence, removing the mask on the substrate, etching, and introducing oxygen for cleaning to form a working electrode;
(2) Then, silk-screen printing a mixed solution of silver and silver chloride on the strip-shaped electrode on the substrate at intervals to prepare an Ag/AgCl reference electrode;
(3) Uniformly mixing glucose oxidase, a cross-linking agent and a rhodium redox polymer solution, dripping the mixture on the surface of a working electrode, drying the mixture at room temperature to form a film, coating an enzyme solution on the film, and carrying out cross-linking and fixing at room temperature for 3-5 days.
4. The method of claim 3, wherein the substrate is a plastic substrate.
5. The method according to claim 3, wherein the transition metal layer has a thickness of 10 to 30nm, and the Au electrode layer has a thickness of 100 to 500nm.
6. The production method according to claim 3 or 5, wherein the transition metal layer is a Cr layer.
7. The method according to claim 3, wherein the flow rate of the oxygen gas is 30 to 50mL/min, the working pressure is 3 to 5Pa, and the cleaning time is 30 to 40 seconds.
8. The method according to claim 3, wherein the crosslinking agent is an aldehyde crosslinking agent.
9. The preparation method according to claim 3, wherein the concentration of glucose oxidase in the enzyme solution is 5-10U/mL, and the dosage of the cross-linking agent is 3.5-5% of the volume of the enzyme solution.
10. A glucose sensor having a low potential and a wide detection range, which is produced by the method according to any one of claims 1 to 9.
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Effective date of registration: 20221207 Address after: 210000 building 6, No. 699-10, Xuanwu Avenue, Xuanwu District, Nanjing City, Jiangsu Province Applicant after: Jiangsu YueKai Biotechnology Co.,Ltd. Applicant after: ZHEJIANG POCTECH Corp. Address before: 610044 No.24, 1st South section of 1st ring road, Wuhou District, Chengdu City, Sichuan Province Applicant before: SICHUAN University |
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Effective date of registration: 20221221 Address after: 210000 building 6, No. 699-10, Xuanwu Avenue, Xuanwu District, Nanjing City, Jiangsu Province Applicant after: Jiangsu YueKai Biotechnology Co.,Ltd. Applicant after: ZHEJIANG POCTECH Corp. Applicant after: Jiangsu Yuyue kailite Biotechnology Co.,Ltd. Address before: 210000 building 6, No. 699-10, Xuanwu Avenue, Xuanwu District, Nanjing City, Jiangsu Province Applicant before: Jiangsu YueKai Biotechnology Co.,Ltd. Applicant before: ZHEJIANG POCTECH Corp. |