CN115494130A

CN115494130A - Toothbrush electrochemical sensor construction method

Info

Publication number: CN115494130A
Application number: CN202211136514.0A
Authority: CN
Inventors: 崔悦
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2022-12-20

Abstract

The invention discloses a construction method of an electrochemical sensor of a toothbrush, which comprises the following steps: s1: preparing a two-electrode sensor and a three-electrode sensor on a toothbrush by a screen printing or micro-nano processing method; s2: performing enzyme or ion selective membrane functionalization on a working electrode of a toothbrush sensor; s3: drying a toothbrush sensor sample, drying an enzyme or ion selective membrane on a working electrode, and taking out and soaking the toothbrush for later use before use; s4: measuring, measuring the response of the current or voltage induced by the analyte at different concentrations. The biosensor consists of a working electrode, a reference electrode and a counter electrode on a toothbrush substrate, wherein a specific enzyme or an ion selective membrane is fixed on the working electrode and is used for specifically detecting analytes, the analytes can be glucose, lactic acid, creatinine, uric acid, cholesterol, triglyceride, potassium ions, sodium ions, calcium ions and the like, and the health condition of teeth can be known according to different components of toothbrush and saliva.

Description

Toothbrush electrochemical sensor construction method

Technical Field

The invention relates to the technical field of toothbrush electrochemical sensor construction methods, in particular to a toothbrush electrochemical sensor construction method.

Background

Toothbrushes are used as common tools for daily use and play an important role in human life. In particular, the close contact of the toothbrush with the oral conditions during brushing makes it more accessible to salivary biomarkers and toothpaste residues. Toothbrush heads are usually made of plastic or other hard materials, and sensors on toothbrushes have now built into accelerometers and magnetic sensors.

The health of the mouth determines the health of the person, and saliva or toothpaste contains various analytes which may indicate health, such as glucose, lactic acid, calcium carbonate or fluoride. Therefore, it is necessary to invent a device which can measure and accurately measure the contents of the components of the toothbrush and the saliva so as to know the health condition of the oral cavity of people, so as to facilitate the later nursing of the oral cavity.

Disclosure of Invention

The invention aims to provide a toothbrush electrochemical sensor construction method, which aims to solve the problem that saliva or toothpaste contains various analytes in the background technology, and can indicate health conditions, such as glucose, lactic acid, calcium carbonate or fluoride. Therefore, the invention is needed to invent a device which can measure and accurately measure the contents of the components of the toothbrush and the saliva so as to know the health condition of the oral cavity of people and bring convenience to the later nursing of the oral cavity.

In order to achieve the purpose, the invention provides the following technical scheme: a toothbrush electrochemical sensor construction method comprises the following steps:

s1: preparing two-electrode and three-electrode sensors: carbon, or gold, or platinum is manufactured on the toothbrush head as a working electrode, and Ag/AgCl is used as a reference electrode/counter electrode to serve as a two-electrode sensor. Carbon, or gold, or platinum is manufactured on the toothbrush head as a working electrode and a counter electrode, and Ag/AgCl is used as a reference electrode as a three-electrode sensor.

S2: enzyme functionalization: mixing the fixing agent and specific enzyme, and dripping the mixture on a working electrode to cover the working electrode, wherein the enzyme selects Glucose Oxidase (GOD) for detecting glucose; or, lactate oxidase for detecting lactic acid; or, uricase for detecting uric acid; or, creatine aminohydrolase and creatine oxidase mixture for detecting creatinine; or, cholesterol oxidase for detecting cholesterol; or, a lipase, glycerol kinase, and glycerol phosphate oxidase mixture for triglycerides;

s3: drying the enzyme: drying the enzyme, and storing the sample in a refrigerator at 4 deg.C for more than 8 hr, or drying at about 40 deg.C for 0.5 hr. The toothbrush can be stored in a refrigerator at room temperature or 4 ℃. Before use, the toothbrush is taken out and placed in PBS buffer solution for soaking for standby;

s4: and (3) measurement: the response of the current caused by different concentrations of hydrogen peroxide or specific analytes was measured by using a potentiostat, kept at room temperature, and the data were recorded.

Preferably, the two electrodes and the three electrodes in the step S1 are prepared by a screen printing method or a micro-nano processing method.

Preferably, the micro-nano processing method of the working electrode and the counter electrode of the three electrodes adopts evaporation or sputtering to obtain gold or platinum in a nano layer, and then a Prussian blue layer is electroplated on the gold or platinum to obtain a gold/Prussian blue electrode or a platinum/Prussian blue electrode.

Preferably, the micro-nano processing method of the reference electrode of the three electrodes adopts sputtering or evaporation to generate a silver electrode, and then partial silver in ferric chloride solution generates silver chloride through chemical reaction to obtain the silver/silver chloride electrode.

Preferably, the micro-nano processing manufacture of the two electrodes adopts evaporation or sputtering to obtain gold or platinum of a nano layer, and then an electronic medium layer, such as a Prussian blue layer, is electroplated on the gold or platinum to obtain a gold/Prussian blue electrode or a platinum/Prussian blue electrode.

Preferably, the micro-nano processing and manufacturing of the reference/counter electrode of the two electrodes adopts sputtering or evaporation to produce a silver electrode, and then part of silver in ferric chloride solution generates silver chloride through chemical reaction to obtain the silver/silver chloride electrode.

Preferably, the working electrode and the counter electrode manufactured by screen printing of the three-electrode system adopt screen printing gold composite slurry, platinum composite slurry and carbon composite slurry, and generally contain an electronic mediator such as Prussian blue, and the reference electrode adopts screen printing silver/silver chloride composite slurry.

Preferably, the working electrode manufactured by screen printing of the two-electrode system adopts screen printing gold composite slurry, platinum composite slurry and carbon composite slurry, and the reference/counter electrode adopts screen printing silver/silver chloride composite slurry.

Preferably, another method for constructing an electrochemical sensor of a toothbrush comprises the following steps:

s1: preparing two-electrode and three-electrode sensors: carbon, or gold, or platinum is manufactured on the toothbrush head as a working electrode, and Ag/AgCl is used as a reference electrode/counter electrode to serve as a two-electrode sensor. Working and counter electrodes based on carbon, or gold, or platinum were fabricated on the toothbrush head, with Ag/AgCl as the reference electrode, as a three-electrode sensor.

S2: selection and immobilization of ion-selective membranes: according to the detection of different ions, selecting corresponding ion selective membranes, wherein the ion selective membranes are used for detecting potassium ions, chloride ions, calcium ions, sodium ions and the like, and the sodium ion liquid membrane selective electrode is as follows: the ion carrier also comprises various carriers such as ETH157, ETH227, ETH2120 and the like, wherein the calcium ion liquid membrane selective electrode comprises: mainly prepared from organic phosphate, non-cyclic compound ETH1001 and crown compound, wherein the potassium ionophore is valinomycin and polyvinyl chloride. The ion selective membrane is immobilized on the working electrode of the sensor.

S3: drying the ion selective membrane: the ion selective membrane of the toothbrush sample was dried, after which the toothbrush was placed in PBS buffer solution for soaking.

S4: measurement: the measurement is performed by open circuit voltage by using a circuit board or an electrochemical workstation in a state of being maintained at room temperature.

Compared with the prior art, the invention has the beneficial effects that: the construction method of the toothbrush electrochemical sensor successfully prepares the biosensor on a toothbrush, the biosensor consists of a working electrode and a reference/counter electrode on a toothbrush substrate, specific enzyme is fixed on the working electrode, or an ion selective membrane which can be a specific ion selective membrane corresponding to potassium ions, calcium ions and sodium ions is used for specifically detecting analytes which can be glucose, lactic acid, creatinine, uric acid, cholesterol, triglyceride, potassium ions, sodium ions, chloride ions, calcium ions and the like, and the health condition of teeth can be known according to different components of the toothbrush and saliva.

Drawings

FIG. 1 is a schematic view of a biosensor set-up process according to the present invention;

FIG. 2 is a perspective view of a dual electrode of the present invention;

FIG. 3 is a schematic perspective view of a three-electrode assembly according to the present invention;

FIG. 4 is a cyclic voltammogram of a sensor of the present invention in a two-electrode configuration and 2mMH2O 2;

FIG. 5 is a cyclic voltammogram of a sensor of the invention in a three electrode configuration and 2mMH2O 2;

FIG. 6 is a cyclic voltammogram of a glucose oxidase immobilized biosensor of the invention with a two-electrode configuration and 2mM glucose;

FIG. 7 is a cyclic voltammogram of a glucose oxidase immobilized biosensor of a three electrode configuration and 2mM glucose in accordance with the present invention;

FIG. 8 shows the final product H of the glucose enzymatic reaction of the present invention ₂ O ₂ A current-time curve diagram and a calibration curve of the two-electrode structure toothbrush sensor of (1);

FIG. 9 is a schematic view of a current-time response curve and a calibration curve of a dual electrode glucose detecting toothbrush sensor of the present invention;

FIG. 10 is a graph of stability of a sensor of the present invention at various temperatures;

FIG. 11 is a graph of the current response of the sensor of the present invention to 4mM glucose at 37 deg.C and room temperature;

FIG. 12 is a graph of the selectivity of the sensor of the present invention for different interferents;

figure 13 is a graph of the effect of a dentifrice of the present invention on a sensor.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The first embodiment is as follows: referring to fig. 1-3, the present invention provides a technical solution: a toothbrush electrochemical sensor construction method comprises the following steps:

s1: preparing a two-electrode sensor and a three-electrode sensor: carbon, or gold, or platinum is manufactured on the toothbrush head as a working electrode, and Ag/AgCl is used as a reference electrode/counter electrode as a two-electrode sensor. A working electrode and a counter electrode based on carbon, or gold, or platinum were fabricated on the toothbrush head, with Ag/AgCl as the reference electrode, as a three-electrode sensor. The two electrodes and the three electrodes are prepared by a micro-nano processing method or a screen printing method. The method comprises the steps of adopting a vapor deposition or sputtering method for micro-nano processing of a working electrode and a counter electrode of the three electrodes to obtain gold or platinum of a nano layer, then electroplating the gold or platinum to generate a Prussian blue layer on the gold or platinum or Prussian blue electrode, adopting a sputtering or vapor deposition method for micro-nano processing of a reference electrode of the three electrodes to generate a silver electrode, then generating silver chloride by partial silver in ferric chloride solution through a chemical reaction, and obtaining the silver/silver chloride electrode. The micro-nano processing and manufacturing of the two electrodes adopts evaporation or sputtering to obtain gold or platinum of a nano layer, then an electronic medium layer such as a Prussian blue layer is electroplated on the gold or platinum to obtain a gold/Prussian blue electrode or a platinum/Prussian blue electrode, the micro-nano processing and manufacturing of the reference/counter electrode of the two electrodes adopts sputtering or evaporation to produce a silver electrode, then partial silver in ferric chloride solution generates silver chloride through chemical reaction, and the silver/silver chloride electrode is obtained. The working electrode and the counter electrode manufactured by the screen printing of the three-electrode system adopt screen printing gold composite slurry, platinum composite slurry and carbon composite slurry, generally contain an electronic mediator such as Prussian blue, and the reference electrode adopts screen printing silver/silver chloride composite slurry. The working electrode manufactured by screen printing of the two-electrode system adopts screen printing gold composite slurry, platinum composite slurry and carbon composite slurry, and the reference/counter electrode adopts screen printing silver/silver chloride composite slurry.

S2: enzyme functionalization: the immobilizing agent and GOD were mixed in the same volume, the immobilizing agent concentration was 2%, the GOD concentration was 10U/μ l, and the immobilizing agent and GOD mixture was 5 μ l, and then the mixture was dropped on the working electrode, covering the working electrode, by immobilizing specific enzymes on the working electrode, the specific enzymes being used for the biological enzymes such as uricase for uric acid sensors, creatine aminohydrolase/sarcosine oxidase mixture for creatinine biosensors, glucolase for glucose biosensors, cholesterol oxidase for cholesterol biosensors, lipase/glycerol kinase/glycerol phosphate oxidase mixture for triglyceride biosensors.

s4: measurement: the response of the current induced by different concentrations of hydrogen peroxide or specific analytes was measured at constant voltage by using a circuit board or electrochemical workstation or potentiostat, kept at room temperature, and the data was recorded.

Example two: another method for constructing an electrochemical sensor for toothbrushes, comprising the steps of:

s1: preparing two-electrode and three-electrode sensors: carbon, or gold, or platinum is manufactured on the toothbrush head as a working electrode, and Ag/AgCl is used as a reference electrode/counter electrode as a two-electrode sensor. Manufacturing a working electrode and a counter electrode based on carbon, gold or platinum on a toothbrush head, and taking Ag/AgCl as a reference electrode and a three-electrode sensor;

s2: selection and immobilization of ion-selective membranes: according to the detection of different ions, selecting corresponding ion selective membranes, wherein the ion selective membranes are used for detecting potassium ions, chloride ions, calcium ions, sodium ions and the like, and the sodium ion liquid membrane selective electrode is as follows: the ion carrier also comprises ETH157, ETH227, ETH2120 and the like, wherein the calcium ion liquid membrane selective electrode: mainly prepared from organic phosphate, non-cyclic compound ETH1001 and crown compound, wherein the potassium ionophore is valinomycin and polyvinyl chloride. The ion-selective membrane is immobilized on the working electrode of the sensor.

S3: drying the ion selective membrane: the ion selective membrane of the toothbrush sample was dried. Before use, the toothbrush is placed in a PBS buffer solution for soaking for standby.

S4: and (3) measurement: the measurement is performed by open circuit voltage by using a circuit board or an electrochemical workstation in a state of being maintained at room temperature.

In summary, the following steps: as shown in FIGS. 1 to 3, in the construction method of the electrochemical sensor using the toothbrush, firstly, carbon, gold, platinum pastes are printed on the toothbrush head as the working electrode as the counter/reference electrode and the working electrode, respectively, then the toothbrush is placed in an oven and dried in an environment of 80 ℃ for 30min, at which time the distance between the centers of the two electrodes is about 8mm, then the toothbrush is placed at room temperature and cooled for 20min at room temperature, and silver paste is deposited at the 3mm end of each electrode, copper wires can be fixed, finally walls are built on the toothbrush head to create a liquid-containing region, the walls constitute a hollow rectangular chamber, the chamber has a length of 2cm, a width of 1.2cm, a height of 5mm and a wall thickness of 2mm, then a fixative having a concentration of 2% and GOD having a concentration of 10U/. Mu.l are mixed in the same volume, then 5. Mu.l of the mixture is dropped on the working electrode to cover about half of the area, storing the toothbrush in a refrigerator at 4 ℃ for more than 8H, taking out the toothbrush, soaking the toothbrush in PBS solution for about 60min for later use, taking out a potentiostat, connecting two electrodes to the potentiostat through electric wires, setting a constant potential of 600mV between a working electrode and a counter electrode, firstly adding 400 μ l of PBS solution to the toothbrush head to completely cover the electrodes, then opening the potentiostat for several minutes and keeping the signals stable, pouring a certain amount of analyte (H2O 2 or glucose) into the liquid, when the current signal is stable again, adding the same type of analyte with different volumes or concentrations, keeping the same work until enough statistical data is obtained, finally drawing a response curve of the current signal along with time according to the statistical data, drawing a calibration curve of the current response and the H2O2 or glucose concentration, and the cyclic voltammograms of the sensors on the toothbrush were studied in PBS solution containing 2ml of H2O2 or glucose (50ml, pH 7.0), maintained at room temperature below 25 ℃, at scanning speeds of 30, 50, 70 and 100mV/s, to achieve the construction of biosensors on the toothbrush to measure the composition of the toothbrush and saliva.

Example three:

fig. 4-7 show cyclic voltammograms of a toothbrush sensor. FIG. 4 shows a cyclic voltammogram of a two-electrode configured sensor in PBS solution with 2mMH2O2, with four different scan rates. H2O2 participates in the oxidation and reduction of the surface of the toothbrush head, and the potential is changed from-1V to 1V. At a scan rate of 100mV/s, the potential at the peak was 0.393V and the current at the peak was 309 μ A. FIG. 5 shows a cyclic voltammogram of a sensor in a three-electrode configuration in PBS solution containing 2mMH2O 2. The shape of the curve is somewhat different from that of the two-electrode configuration and the peak current is higher. At a scan rate of 100mV/s, the potential at the peak was 0.656V and the current at the peak was 529. Mu.A. FIG. 6 shows cyclic voltammograms of a two-electrode configuration of a GOD-immobilized toothbrush sensor in PBS containing 2mM glucose, with four different scan rates. Glucose can be catalyzed by GOD to produce H2O2, which is why the shape of the curve in FIG. 6 is similar to the curve in FIG. 4. The configuration of the curve depends mainly on the characteristics of the electrodes. The reaction in fig. 6 is weaker than that in fig. 4, probably because H2O2 in fig. 4 is added directly to the solution and oxidation occurs directly, while H2O2 in fig. 6 is a product of an enzymatic reaction, which may limit the reaction rate. At a scan rate of 100mV/s, the potential at the peak is 0.524V and the current at the peak is 267 μ A. When there is no GOD on the electrodes or no glucose in the solution, there is no distinct peak in these curves, demonstrating the catalytic function of GOD on glucose. FIG. 7 shows a cyclic voltammogram of a biosensor in a three-electrode configuration in PBS solution containing 2mM glucose. The curve shape in fig. 7 is also similar to the curve in fig. 5, with a higher peak current than the sensor in the two-electrode configuration. At a scan rate of 100mV/s, the potential at the peak is 0.621V and the current at the peak is 501 μ A. Likewise, there was no distinct peak when there was no GOD on the electrodes or no glucose in the solution. These curves show that the sensor with the two-electrode configuration or the three-electrode configuration can have good sensing performance, and the potential of 0.4 to 1V can generate obvious signal response. The difference in CV curve shapes between the two-electrode configuration and the three-electrode configuration may be due to the potential change in the two-electrode configuration when current is passed through the silver/silver chloride electrode. While the three-electrode configuration is more accurate in amperometric measurements, the two-electrode configuration of the sensor also allows for relatively accurate detection of H2O2 and glucose.

Example four: a toothbrush electrochemical sensor construction method comprises the following steps:

s1: preparing the sensor: printing carbon, gold and platinum slurry on a toothbrush head as a working electrode, wherein the toothbrush head is different from the first embodiment in that a three-electrode structure is adopted and comprises a carbon graphite working electrode, a carbon graphite counter electrode and an Ag/AgCl reference electrode, the size of each electrode is the same as that of the double-electrode configuration, the distance between the centers of the electrodes is reduced to 4mm, the toothbrush is placed into an oven for drying, one of the three-electrode structure is the working electrode, the other one of the three-electrode structure is the counter electrode, the working electrode and the counter electrode are made of silk-screen printing gold composite slurry, platinum composite slurry and carbon composite slurry and generally contain an electronic medium, and the reference electrode is made of silk-screen printing silver/silver chloride composite slurry.

S2: and (3) cooling: the toothbrush is placed at room temperature for cooling, and after the toothbrush is cooled, silver paste is deposited at the end part of each electrode, so that the copper wire for experiments can be fixed.

S3: establishing a reaction cavity: the glue gun creates walls on the toothbrush head that create a liquid containing area that form a hollow rectangular chamber that can be used to limit the reaction area of the sensor during analyte detection.

S4: enzyme functionalization: the fixative and GOD were mixed in the same volume, the concentration of the fixative was 2%, the concentration of GOD was 10U/μ l, and the mixture of the fixative and GOD was 5 μ l, and then the mixture was dropped on the working electrode to cover the working electrode.

S5: drying the enzyme: the samples were stored in a refrigerator for more than 8 hours, after which the toothbrush was removed and placed in PBS buffer for immersion.

S6: and (3) measurement: the response of different concentrations of H2O2 and glucose to the current was measured by using a potentiostat, maintained at room temperature, and the data was recorded.

In the step S1, the working environment of the oven is kept at 80 ℃ and continuously works for 30min, so that the enzyme on the toothbrush cannot be killed, and the oven can be kept to be in a complete drying state.

And in the step S2, the cooling time at room temperature is 20min, the end part of the electrode is positioned at the end of 3mm of the electrode, and the toothbrush is placed at room temperature and cooled for 20min, so that the toothbrush can be kept completely cooled, and the situation that the deposited silver paste is melted is avoided.

The dimensions of the rectangular chamber in step S3 are 2cm long, 1.2cm wide, 5mm high and 2mm thick, thereby constructing an area for accommodating a liquid, by which the reaction area of the sensor can be limited.

In step S5, the refrigerator is stably kept at 4 ℃, and the enzyme can be completely fixed after the refrigerator is placed in the refrigerator for at least more than 8 hours.

In the step S5, the soaking time of the PBS buffer solution is kept about 1 hour, and the pH value of the sample on the toothbrush can be adjusted through the PBS buffer solution, so that the sample can be protected.

And S6, connecting the electrochemical workstation with the working electrode and the reference electrode/counter electrode through a lead, and detecting the current between the working electrode and the counter electrode under the constant potential of the working electrode vs.

The current-time curve and the calibration curve of the double-electrode structure toothbrush sensor for detecting the final product H2O2 of the glucose enzymatic reaction are shown in figure 8, and the operation steps are as follows:

after the signals are stable, sequentially adding H2O2 with different concentrations into the solution, recording the change of the concentration of the H2O2 after each addition of the buffer solution, after the analyte is added, because the concentration of the H2O2 near the working electrode is increased rapidly, the H2O2 is oxidized to increase the current, leading the current to increase rapidly, then, the H2O2 in the PBS solution diffuses rapidly, a new balance point is reached within 4min, and the total concentration of the H2O2 in the solution after each addition is recorded. The results show that there is a strong correlation between the current response and the H2O2 concentration. The current increased linearly with H2O2 concentration, the curve showed a wide range of linearization from 0.12mM to 13.1mM, a slope of 0.1214. Mu.A/mM, R2 of 0.9884, and a detection limit of 5.0. Mu.M (signal-to-noise ratio of 3). The change in current due to the change in concentration is consistent with the slope of the calibration curve. C7 The current response (3.58 mM) was 0.405. Mu.A, and the current response/concentration change was 0.1130. Mu.A/mM. This is very small (6.92%) from the slope of the calibration curve.

Two additional sensors were fabricated to check for reproducibility, one with a slope of 0.1185 μ A/mM, R2 of 0.9911, and a limit of detection of 6.6 μ M, and the other with a slope of 0.1344 μ A/mM, R2 of 0.9977, and a limit of detection of 4.5 μ M.

The current-time response curve and the calibration curve of the glucose detection toothbrush sensor with the dual-electrode structure are shown in figure 9, and the operation steps are as follows:

the PBS solution is added on the electrode, after the current signal is stable, glucose is dripped into the PBS solution, the glucose generates H2O2 under the catalysis of GOD, the oxidation reaction and the electron transfer on the surface of the electrode are promoted, so that the current is improved. After only a few seconds, the current reaches a peak and begins to drop at a decreasing rate. Glucose diffuses in PBS and the glucose concentration at the working electrode decreases, resulting in a decrease in the current signal. Within 4 minutes, the current again stabilized and another portion of the glucose was poured into PBS. Similar to the detection of H2O2, the change in glucose concentration in the buffer solution after each addition. A total of 7 different glucose solutions were added to make one round of measurements and a calibration curve of the toothbrush sensor to glucose was recorded, the concentration representing the total concentration of glucose in the buffer after each addition, there was a strong correlation between the steady current response and the glucose concentration, the current response increasing proportionally with the glucose concentration. Linearization was in the range of 0.18mM to 5.22mM, slope was 0.0817 μ a/mM (R2 = 0.9929), detection limit was calculated to be 5.0 μ M (signal-to-noise ratio was 3), which was completely satisfactory for detection of glucose in saliva, current change due to change in glucose concentration was consistent with the slope of the calibration curve, current response due to C4 (1.02 mM) was 0.089 μ a, and current response/concentration change was 0.0868 μ a/mM. This is very small (6.27%) from the slope of the calibration curve. Two other sensors fixed to the GOD were set up to check reproducibility. The last point is that the current response is saturated at high concentrations due to the saturation reaction of the enzyme, and the results obtained from the above experimental procedure demonstrate that a glucose amperometric biosensor on a toothbrush can be successfully constructed.

Example four:

FIG. 10 shows the stability of the sensor for detecting glucose at different temperatures. The relative response was highest at 30 ℃ (103.29%) and lowest at 20 ℃ (85.92%), indicating that the sensor was very stable at different temperatures. FIG. 11 shows the i-t of the sensor at 37 ℃ and room temperature for 4mM glucose. The current responses are similar, and temperature does not differ greatly from glucose detection. Fig. 12 shows the selectivity of the sensor. The effect of different interferents (ascorbic acid, uric acid and dopamine) was negligible compared to the galvanic response to 4mM glucose. Figure 13 shows the effect of toothpaste samples. The response caused by different volumes of sample was also much smaller than the response to 4mM glucose. Therefore, the effect of the toothpaste samples was also negligible.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A toothbrush electrochemical sensor construction method is characterized by comprising the following steps:

s1: preparing two-electrode and three-electrode sensors: carbon, or gold, or platinum is manufactured on the toothbrush head as a working electrode, and Ag/AgCl is used as a reference electrode/counter electrode as a two-electrode sensor. Working and counter electrodes based on carbon, or gold, or platinum were fabricated on the toothbrush head, with Ag/AgCl as the reference electrode, as a three-electrode sensor.

S2: enzyme functionalization: mixing the fixing agent and specific enzyme, then dripping the mixture on a working electrode to cover the working electrode, wherein the enzyme selects glucose oxidase for detecting glucose; or, lactate oxidase for detecting lactic acid; or, uricase for detecting uric acid; or, creatine aminohydrolase and sarcosine oxidase mixture for detecting creatinine; or, cholesterol oxidase for detecting cholesterol; or, a lipase, glycerol kinase, and glycerol phosphate oxidase mixture for triglycerides;

s3: drying the enzyme: the samples were stored in a refrigerator at 4 ℃ for more than 8 hours or dried at about 40 ℃ for 0.5 hour. The toothbrush can be stored in a refrigerator at room temperature or 4 ℃. Before use, the toothbrush is taken out and placed in PBS buffer solution for soaking for standby;

s4: measurement: the response of the current caused by different concentrations of hydrogen peroxide or specific analytes was measured and the data recorded by circuit board, electrochemical workstation, or potentiostat, maintained at room temperature.

2. The method for constructing an electrochemical sensor of a toothbrush according to claim 1, wherein: the two electrodes and the three electrodes in the step S1 are prepared by adopting a screen printing method or a micro-nano processing method.

3. The method for constructing an electrochemical sensor of a toothbrush as claimed in claim 2, wherein: the micro-nano processing method of the working electrode and the counter electrode of the three electrodes adopts evaporation or sputtering to obtain gold or platinum of a nano layer, and then a Prussian blue layer is electroplated on the gold or platinum to obtain a gold/Prussian blue electrode or a platinum/Prussian blue electrode.

4. The method for constructing an electrochemical sensor of a toothbrush as claimed in claim 2, wherein: the micro-nano processing method of the reference electrode of the three electrodes adopts sputtering or evaporation to generate a silver electrode, and then partial silver in ferric chloride solution generates silver chloride through chemical reaction to obtain the silver/silver chloride electrode.

5. The method for constructing an electrochemical sensor of a toothbrush as claimed in claim 2, wherein: the micro-nano processing and manufacturing of the two electrodes adopts evaporation or sputtering to obtain gold or platinum of a nano layer, and then an electronic medium layer, such as a Prussian blue layer, is electroplated on the gold or platinum to obtain a gold/Prussian blue electrode or a platinum/Prussian blue electrode.

6. The method for constructing an electrochemical sensor of a toothbrush as claimed in claim 2, wherein: the micro-nano processing and manufacturing of the reference/counter electrode of the two electrodes adopts sputtering or evaporation to generate a silver electrode, and then partial silver in ferric chloride solution generates silver chloride through chemical reaction to obtain the silver/silver chloride electrode.

7. The method for constructing an electrochemical sensor of a toothbrush as claimed in claim 2, wherein: the working electrode and the counter electrode manufactured by the three-electrode system through screen printing adopt screen printing gold composite slurry, platinum composite slurry and carbon composite slurry, generally contain an electronic mediator such as Prussian blue, and the reference electrode adopts screen printing silver/silver chloride composite slurry.

8. The method for constructing an electrochemical sensor of a toothbrush as claimed in claim 2, wherein: the working electrode manufactured by screen printing of the two-electrode system adopts screen printing gold composite slurry, platinum composite slurry and carbon composite slurry, and the reference/counter electrode adopts screen printing silver/silver chloride composite slurry.

9. Another method for constructing an electrochemical sensor for a toothbrush, comprising the steps of:

s2: selection and immobilization of ion-selective membranes: selecting a corresponding ion selective membrane according to the detection of different ions, and fixing the ion selective membrane on a working electrode of the sensor;