CN115266874A - A kind of glucose sensor with low potential and wide detection range and preparation method thereof - Google Patents

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

A glucose sensor with low potential and wide detection range and preparation method thereof

technical field

The invention belongs to the technical field of biosensors, in particular to a glucose sensor with low potential and wide detection range and a preparation method thereof.

Background technique

Diabetes has become one of the diseases that threaten human health. Blood sugar monitoring for diabetics is very important to control the disease. For the invasive tests that are generally used now, patients often have a sense of fear and discomfort. Therefore, the research and development of non-invasive blood glucose meters has become a key issue in the medical circles of various countries.

Among them, using reverse iontophoresis technology to obtain sugar molecules in the subcutaneous interstitium, and testing the glucose exuded from the subcutaneous tissue through electrochemical biosensors have been studied abroad. Compared with direct blood collection to detect human blood sugar (mmol/L level), the concentration of subcutaneous exudate glucose is very low (mmol/L level), and traditional blood glucose biosensors can hardly meet the requirements in terms of detection range and sensitivity. However, sensors capable of detecting low-concentration glucose cannot be practical due to limitations in electrode fabrication. Under the working voltage of 0.53V, the non-invasive detection of subcutaneous blood glucose is realized by using the hydrogen peroxide sensor. However, the higher working potential easily causes other electroactive substances in the body fluid to easily interfere with the test.

Contents of the invention

Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a glucose sensor with low potential and wide detection range and its preparation method. A large amount of glucose detection is necessary in many applications, but it is still challenging. Commercial enzyme-based glucose detection test strips cannot be reused, and the current non-enzyme glucose sensor has a narrow detection range and slow glucose oxidation kinetics.

The present invention adopts the rhodium redox polymer coupled with glucose oxidase as the electron mediator to modify the film Au electrode, and fixes the glucose enzyme molecules by the glutaraldehyde method to prepare a novel biosensor, and the sensor is sensitive to standard glucose and subcutaneous glucose. The current response characteristics can reach 23.955 nA/(mmol L ^-1 ) and 18.941 nA/(mmol L ^-1 ), respectively, and the lifetime can reach 15 days at 4°C.

In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:

A preparation method of a glucose sensor with low potential and wide detection range, the redox polymer formed by cross-linking and immobilizing glucose oxidase and rhodium on the working electrode in the three-electrode system is sufficient.

Further, 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 following steps are included:

(1) After engraving the protective film (130mm×100mm) on the plastic substrate into the mask pattern of the strip electrode, then sputtering the transition metal layer and the Au electrode layer on it successively, then removing the mask on the substrate and then Etching and cleaning with oxygen to form a working electrode;

(2) take stainless steel as a mask plate, then on the strip electrode on the substrate Interval screen printing silver and silver chloride mixed solution, prepare Ag/AgCl reference electrode;

(3) Carve out a rectangular groove (100mm×4.0mm) in the middle of the adhesive tape (130mm×50mm), and paste it on the substrate. The electrodes on the substrate exposed in the groove are the working electrode of the sensor (2.0mm×4.0 mm) and Reference electrode (20mm×4.0mm);

(4) Mix glucose oxidase and cross-linking agent with rhodium-containing redox polymer solution to form an enzyme solution, take 0.5mL drop-coated on the surface of the working electrode of the sensor, and dry naturally at room temperature to form a film , washed with water, cut into individual sensors after drying, and stored at room temperature for later use.

Further, the substrate is a plastic substrate.

Further, the thickness of the transition metal layer is 10-30 nm, and the thickness of the Au electrode layer is 100-500 nm.

Further, the transition metal layer is a Cr layer

Further, the flow rate of oxygen is 30-50mL/min, the working pressure is 3-5Pa, and the cleaning time is 30-40s.

Further, the crosslinking agent is an aldehyde crosslinking agent.

Further, the crosslinking agent is glutaraldehyde.

Further, the enzyme solution contains 1% BSA, the concentration of glucose oxidase is 5-10 U/mL, and the dosage of the cross-linking agent is 3.5-5% of the volume of the enzyme solution.

The glucose sensor with low potential and wide detection range prepared by the above method uses a three-electrode system of Au working electrode, Au counter electrode and Ag/AgCl reference electrode. The structure is shown in Figure 1. The sensor consists of two sensors with the same shape , to complete the extraction and detection functions of the sensor together, wherein, W1 and W2 are thin film Au working electrodes, C1 and C2 are Au counter electrodes, W and R form the extraction electrode circuit, R1 and R2 are silk screen Ag/AgCl reference electrodes, and use To provide a stable reference voltage during sensor testing, the area of the sensitive part of the working electrode is 0.85cm ² .

The application of the glucose sensor in the non-invasive detection of human blood glucose.

The technical principle of this application is:

Glucose oxidase (GOx) immobilized on the working electrode oxidizes glucose, and the electrons generated in this process oxidize the reduced electrolyte Rh, and Rh becomes the oxidized Rh, which is reduced at a lower working voltage, because the electrons The transfer occurs in the whole three-dimensional network of the redox polymer, so the reaction current density is high and the sensitivity is high. In addition, since the redox polymer cannot freely diffuse into the bulk solution, it has a high electron transfer efficiency. The response current in the reaction is proportional to the glucose concentration. This rhodium polymer can detect glucose at a lower potential, avoiding the interference of other electrochemically active species. At the same time, since rhodium can transfer electrons quickly between the electrode and the enzyme, the dependence on the oxygen concentration in the bulk solution is eliminated to a certain extent.

Beneficial effects of the present invention:

1. The present invention adopts glutaraldehyde cross-linking method to immobilize enzyme molecules on the surface of membrane electrodes to form a specific glucose biosensor. The sensitivity of the sensor is improved by selecting a highly sensitive redox polymer. The current response characteristics of the sensor to standard glucose and subcutaneous glucose were studied. The sensitivities were 23.955nA/(mmol L ^-1 ) and 18.941nA/(mmol L ^-1 ), respectively, and the linear correlations were above 0.99.

2. According to the accelerated life test of the present invention, the life of the sensor can reach 15 days at 4°C. The intra-assay and inter-assay accuracies of the sensors are both less than 5%. The designed three-electrode sensor has the advantages of high sensitivity, good stability, and low detection limit. It integrates the functions of sampling and detection, and combines the reverse iontophoresis technology to realize the extraction and detection of subcutaneous blood sugar.

3. The present invention adopts the glutaraldehyde enzyme cross-linking experiment, which can effectively improve the response current of the sensor in RP, with wide linear working area and high sensitivity. The response time of the sensor is 10s and has good consistency and stability. After one month of use, it can still maintain 90% of its initial activity, which shows that glutaraldehyde enzyme cross-linking can maintain high enzyme activity in RP. Inexpensive, simple to operate and fast to detect. It is expected to be mass-produced and used in medical diagnosis, food industry, environmental testing and other fields.

Description of drawings

Figure 1 is a three-electrode architecture diagram of the sensor;

Fig. 2 is the CV (cyclic voltammetry) response figure of sensor under different glucose concentrations;

Fig. 3 is the i-t (time-current curve) response figure of sensor under different glucose concentrations;

Fig. 4 is the linear graph of sensor under different glucose concentrations;

Figure 5 is a graph of the long-term stability of the sensor.

Detailed ways

The specific embodiments of the present invention are described below so that those skilled in the art can understand the present invention, but it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes Within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are included in the protection list.

Example 1

A method for preparing a glucose sensor with low potential and wide detection range, the specific process is as follows:

(1) After engraving the protective film (130mm × 100mm) on the plastic substrate into the mask pattern of the strip electrode, sputter the transition layer Cr (10nm-) and Au electrode layer (100nm) successively on it;

(2) After the mask is removed, put the substrate into a plasma etching machine, and pass through O ₂ (working pressure 3.0 Pa, O ₂ flow rate 30mL/min) to "clean" for 30s.

(3) take stainless steel as mask plate, on the strip electrode on the plastic substrate, the mixed paste of screen printing silver and silver chloride at intervals, forms the integrated Ag/AgCl reference electrode;

(4) Carve out a rectangular groove (100mm×4.0mm) in the middle of the tape (130mm×50mm), and paste it on the plastic substrate. The electrodes on the substrate exposed in the groove are the working electrodes of the sensor (2.0mm×4.0 mm) And reference electrode (20mm×4.0mm);

(5) Add GOx and glutaraldehyde into the redox polymer solution, mix well, take 0.5 mL drop-coated on the surface of the working electrode of the sensor, and let it dry naturally at room temperature to form a film, wash it with water, and divide it after drying. Cut into individual sensors and store at room temperature for later use.

Example 2

A method for preparing a glucose sensor with low potential and wide detection range, the specific process is as follows:

(1) After engraving the protective film (130mm × 100mm) on the plastic substrate into the mask pattern of the strip electrodes, sputter the transition layer Cr (30nm) and Au electrode layer (300nm) successively on it;

(2) After removing the mask, put the substrate into a plasma etching machine, and pass through O ₂ (working pressure 4.0 Pa, O ₂ flow rate 35mL/min) to "clean" for 35s.

(3) take stainless steel as mask plate, on the strip electrode on the plastic substrate, the mixed paste of screen printing silver and silver chloride at intervals, forms the integrated Ag/AgCl reference electrode;

(4) Carve out a rectangular groove (100mm×4.0mm) in the middle of the tape (130mm×50mm), and paste it on the plastic substrate. The electrodes on the substrate exposed in the groove are the working electrodes of the sensor (2.0mm×4.0 mm) And reference electrode (20mm×4.0mm);

(5) Add GOx and glutaraldehyde into the redox polymer solution, mix well, take 0.5 mL drop-coated on the surface of the working electrode of the sensor, and let it dry naturally at room temperature to form a film, wash it with water, and divide it after drying. Cut into individual sensors and store at room temperature for later use.

Example 3

A method for preparing a glucose sensor with low potential and wide detection range, the specific process is as follows:

(1) After engraving the protective film (130mm × 100mm) on the plastic substrate into the mask pattern of the strip electrode, sputter the transition layer Cr (20nm) and Au electrode layer (200nm) successively on it;

(2) After the mask is removed, put the substrate into a plasma etching machine, and pass through O ₂ (working pressure 3.0 Pa, O ₂ flow rate 35mL/min) to "clean" for 30s.

(3) take stainless steel as mask plate, on the strip electrode on the plastic substrate, the mixed paste of screen printing silver and silver chloride at intervals, forms the integrated Ag/AgCl reference electrode;

(4) Carve out a rectangular groove (100mm×4.0mm) in the middle of the tape (130mm×50mm), and paste it on the plastic substrate. The electrodes on the substrate exposed in the groove are the working electrodes of the sensor (2.0mm×4.0 mm) And reference electrode (20mm×4.0mm);

(5) Add GOx and glutaraldehyde into the redox polymer solution, mix well, take 0.5 mL drop-coated on the surface of the working electrode of the sensor, and let it dry naturally at room temperature to form a film, wash it with water, and divide it after drying. Cut into individual sensors and store at room temperature for later use.

Experimental example

1. Cyclic voltammetry experiment and current detection experiment

Both cyclic voltammetry experiments and current detection experiments were carried out on a CHI 760E electrochemical workstation detection system. The specific process is as follows:

In a 25mL beaker, add 10mL of pH = 7.4 phosphate buffer solution (0.01M PBS), place a stirring magnet in the beaker in advance, insert a sensor, turn on the stirrer and adjust it to a constant speed, and the working potential is 0.13 V (Ag/AgCl reference electrode). When testing the current response, stop stirring. First, detect the background current of the sensor. When the response is in a steady state, add the glucose in the solution to be tested, and continue to stir for 1 min, so that the glucose to be tested is evenly filled with the entire solution, and then , stop stirring, start the test program, and record the oxidation current. The experimental temperature is 25°C, and the test results are shown in Figure 2 and Figure 3.

2. Detection of electrochemical characteristics of the sensor

A thin-film Au working electrode was modified with 8 mL of rhodium polymer. Fig. 3 shows the changes in the cathode and anode peak currents of redox polymers at different scan rates, and the scan rates are 5, 10, 20, 50, 100, 200, and 500 mV/s. It can be seen from the figure that the ratio of the oxidation peak current (I _pa ) to the reduction peak current (I _pc ) is I _pa /I _pC ≈ 1, and the peak current has a linear relationship with the square root of the scan rate, indicating that rhodium redox Polymers have reversible electrochemical properties.

3. The current response of the sensor to standard glucose

Figure 4 shows the current response curve of the sensor to standard glucose. Among them, according to the glucose concentration corresponding to the curve, each sample was measured 3 times, and the working voltage was 0.18V. It can be seen from the figure that when the glucose solution is added, the current response of the sensor increases gradually with the increase of the glucose concentration, and the increase of the current value has a linear relationship with the increase of the glucose concentration, and the current response signal quickly tends to be stable. At the same time, within the concentration range of 5.0-45mmol/L, the current response signal of the glucose solution has a very good linear relationship. In the range of 5.0-45mmol/L, the linear calibration equation I _p of the sensor is 5.6796x-2.4909, the lowest detection limit is 0.3mmol/L, the correlation coefficient R is 0.9979, and the sensitivity of the sensor is 23.955nA/(mmol L ^-1 ). The results show that the use of rhodium polymer effectively reduces the operating voltage of the sensor.

4. Sensor repeatability research

The accuracy of repeated testing of 10 mmol/L glucose between a single biosensor developed in the same batch and different sensors between batches, the results show that the intra-batch accuracy refers to the current response of 1 sensor repeated 10 times to test 10 mmol/L glucose As a result, the inter-batch precision refers to the current response results of 10 mmol/L glucose tested with 10 sensors. It can be seen from the results that the intra-batch and inter-batch accuracies of the sensor are 4.07% and 3.22%, respectively, both less than 5%, indicating that the prepared sensor has good repeatability and consistency.

5. Sensor stability detection

The traditional life test is to evaluate the performance of the sensor by detecting how long the sensor is stored at 4°C. In physical chemistry, the rate of a chemical reaction increases as the reaction temperature increases. Biological reagents are stored at different temperatures, and the "rate" of reagent failure and the temperature and time of storage also conform to the relationship between physical and chemical reaction rates. The Arrhenius empirical formula sums up this law of physical chemistry. Arrhenius described the influence of temperature on the reaction rate with the following empirical formula: lnk=-E _a RT+B, where k is the reaction rate; T is the absolute temperature of the reaction.

Design experiment: Carry out the storage time at which the reagents are stored at multiple temperatures, and the reciprocal of the time (days or hours) is the rate at which the reagents deteriorate at this temperature. Convert the test temperature to absolute temperature T, form each data pair with the reciprocal of storage time, and draw a graph on semi-logarithmic paper, let lnk be Y1/T and X. Draw a best matching line on the graph, and push back from this line to the logarithmic value of the corresponding reaction rate when T is 277K (4°C). Calculate the antilog (exponent) from the logarithmic value, and then calculate the reciprocal of this value, which is the estimated stable time.

Based on the above principles, the lifetime stability performance of the glucose biosensor was observed. Tests were carried out at 20, 35, 55°C. The response of the biosensor at 4°C is taken as the detection result of the control product, and the detection result is 90% or less of the response result of the biosensor at 4°C as invalid.

Plot the paired results of X(1/T) and Y[lg(1/d)] or perform linear regression processing (linear relationship on semi-logarithmic paper), and calculate the absolute temperature of 4°C at 277K from the regression formula , the expected number of days of stability is 15d. Therefore, it is estimated from the experiment that the prepared biosensor can be stored stably for about 15 days at 4°C. (See FIG. 5 ) It can be seen from the above that the sensor prepared by the present 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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