A sweat glucose detection device and method based on a multi-step voltage step method

Abstract

The application discloses a sweat glucose detection device and method based on a multi-step voltage step method, which is composed of a sweat stimulation module, a sweat collection module, a three-electrode electrochemical detection module and an upper computer. The process of detecting glucose by using the sweat glucose detection device is as follows: (1) the sweat stimulation module is used to stimulate the skin to sweat, and the sweat collection module is used to collect sweat; (2) the collected sweat is mixed with 0.1M NaOH solution at a ratio of 1:1; (3) the three-electrode electrochemical detection module detects the glucose concentration of the mixed solution based on the multi-step voltage step method; and (4) the result is uploaded to the upper computer for display. The application only needs to control four kinds of potentials applied between the working electrode and the reference electrode, so that the sweat stimulation and the sweat glucose detection can be realized, and various interference substances on the surface of the electrode are removed after the detection is completed, which is beneficial to the continuous measurement of glucose. Because there is a glucose selective permeation membrane, the measurement can be carried out in a complex sample solution, and the application has good stability and sensitivity, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a sweat glucose detection device and method based on a multi-step voltage step method, belonging to the field of medical device technology. Background Technology

[0002] Sweat, as a metabolic byproduct, plays a crucial role in regulating body temperature. Furthermore, sweat is rich in biochemical information, such as lactic acid, glucose, and various inorganic ions. These components not only reflect the body's metabolic status but can also be used for disease diagnosis. Detecting glucose in sweat can predict changes in blood sugar levels. Electrochemical biosensors, with their advantages of portability, ease of operation, and high sensitivity, have wide applications in medical and health fields, as well as environmental monitoring. Sweat glucose detection based on electrochemical technology remains a key research focus.

[0003] There are two ways to obtain sweat. One is to raise body temperature through exercise, thereby expelling sweat. This method can obtain a sufficient amount of sweat in a short time, but it is not suitable for people with decreased exercise function. The other is to use electrodialysis to penetrate substances that can stimulate sweating into the skin, thereby stimulating sweat glands to sweat. The stimulating substances can take effect quickly and last for a long time, which can shorten the entire detection time and enable continuous detection.

[0004] Glucose detection can be divided into two methods: enzyme-based and enzyme-free. Enzyme-based detection is the main method for glucose detection. Glucose oxidase has the advantages of high selectivity and sensitivity, and can eliminate the influence of a large number of interfering substances, enabling glucose detection in complex samples, such as glucose in sweat. However, temperature and pH affect the activity and stability of glucose oxidase, and both stability and activity decrease significantly with increasing use time. Even though many methods have been developed to improve enzyme stability, the operation process is complex and cumbersome, and is still affected by temperature and pH. Enzyme-free detection uses materials with electrocatalytic oxidation properties of glucose as the working electrode. During electrochemical detection, electron transfer occurs between glucose and the working electrode. Compared with enzyme-based detection, enzyme-free detection has long-term stability. However, existing enzyme-free glucose electrodes can only detect glucose in alkaline environments and pure solution conditions. In neutral or weakly acidic solutions, or in solutions containing many complex substances, the electrode surface adsorbs charged ions and oxides from the solution, leading to reduced electrode sensitivity and inaccurate detection of the target substance. Therefore, designing suitable enzyme-free glucose electrodes to detect the glucose content of various body fluids in the human body remains a key research focus for researchers. Summary of the Invention

[0005] The purpose of this invention is to provide an enzyme-free electrochemical detection device and method for stimulating skin sweating and detecting the glucose content in sweat. The core of this method is the multi-step voltage step method.

[0006] The present invention discloses a sweat glucose detection device based on a multi-step voltage step method, which collects sweat by stimulating sweating through electrodialysis and detects glucose in the sweat through a multi-step voltage step method in a three-electrode electrochemical detection system.

[0007] The method for stimulating sweating involves attaching a paper battery to the skin and applying a small amount of a sweat-stimulating substance to the positive electrode. This substance can penetrate the stratum corneum through electrophoresis and electroosmosis, thereby stimulating sweat glands to sweat. This phenomenon directly and indirectly constitutes the active transport of substances.

[0008] The substance that stimulates sweating can be one of hyaluronic acid, acetylcholine, or methacholine.

[0009] When hyaluronic acid is used, applying a 1mA current to the electrode for 20 minutes will allow the sweat glands at the negative electrode to continue sweating for several hours. When acetylcholine is used, diluting its concentration by 1-10% and applying a 1mA current to the electrode for 50-100 seconds will allow the sweat glands at the negative electrode to continue sweating for 35-45 minutes. When methacholine is used, diluting its concentration by 1-10% and applying a 1mA current to the electrode for 160-400 seconds will allow the sweat glands at the negative electrode to continue sweating for 60 minutes.

[0010] A microfluidic device for collecting sweat is placed in the negative electrode area. The device is composed of a paper base and a PET film. The paper base actively absorbs sweat and stores it in the PET film.

[0011] The paper base is rectangular, 10mm long and 300μm wide. It is divided into two parts: a hydrophilic layer and a hydrophobic layer. The hydrophilic layer is a large triangle, and the hydrophobic layer is the part outside the hydrophilic layer. The top of the large triangle corresponds to the entrance of the paper base for absorbing sweat, and the base of the large triangle is connected to the PET film. According to the principle of Laplace force, when the liquid is absorbed by the paper base, there is a wedge angle. The width of the contact with the paper base will continuously increase as the liquid moves, thus forming a Laplace force. The sweat will spontaneously flow from the top of the large triangle to the bottom, thereby achieving efficient sweat collection.

[0012] The electrochemical working electrode in this invention is made of gold nanomaterials, which have good electrocatalytic oxidation performance for glucose, with silver / silver chloride as the reference electrode and carbon as the counter electrode.

[0013] To prevent charged ions or other oxides in sweat from affecting glucose detection, the working electrode is modified with a glucose selective permeation membrane, which is a total fluorine solution and a halogenated hydrocarbon oil.

[0014] The multi-step voltage step method described in this invention involves changing the potential between the working electrode and the reference electrode. This potential is the difference between the working electrode voltage and the reference electrode voltage. Each step of the potential change requires a sustained period to stimulate sweating and detect glucose in sweat. The main steps include:

[0015] The first step is to apply a positive potential to stimulate sweating: a positive potential of 2.5 to 3.3V is applied between the working electrode and the reference electrode. Depending on the stimulating drug used, the voltage in this step can last from 50 seconds to 20 minutes.

[0016] The second step is to apply the initial voltage for electrochemical detection: apply a negative potential of -1.5 to -2V between the working electrode and the reference electrode. The initial voltage is used to activate the electrode and lasts for 2 seconds.

[0017] The third step is to apply a glucose oxidation voltage: a positive potential of 0.2V is applied between the working electrode and the reference electrode, the glucose oxidation time is 5s, and the current signal is collected by a three-electrode electrochemical current detection module after the oxidation is completed.

[0018] The fourth step is to apply a positive potential to remove oxides from the electrode surface: A positive potential of 1 to 1.2 V is applied between the working electrode and the reference electrode to remove oxides produced by glucose oxidation and other substances in sweat from the electrode surface. This step lasts for 2 seconds.

[0019] By using the above steps to test standard glucose solutions of different concentrations, a linear formula for the current corresponding to the glucose concentration is obtained. Subsequently, the detected current can be converted into the corresponding sweat glucose concentration using the formula.

[0020] The stimulation of sweat glands involves applying a positive voltage to generate a 1mA current. A pre-applied medication at the positive electrode penetrates the stratum corneum through electrophoresis and electroosmosis, acting on the sweat glands to stimulate sweating. Electrochemical detection uses a negative initial voltage to activate the electrode. Electrochemical oxidation of glucose involves applying a conventional potential for a period of time, oxidizing the glucose, and then detecting the electrochemical signal. The applied conventional potential is the potential required for the oxidation of glucose on the working electrode surface, and its magnitude corresponds to the material of the working electrode. During the oxidation process, a large amount of oxidation products adhere to the electrode; therefore, a positive potential needs to be applied for a period of time to remove these products from the electrode surface for subsequent detection.

[0021] The multi-step voltage step method involves: first, applying a high-level signal to control the constant current source to output 1mA current; second, applying a negative voltage of -1.5 to -2V for 2s; third, applying a glucose oxidation potential of 0.2V for 5s; and fourth, applying a positive voltage of 1V for 2s.

[0022] Beneficial effects: Compared with the prior art, the method of detecting glucose in the sweat glucose detection device of the present invention only requires controlling four potentials applied between the working electrode and the reference electrode to achieve stimulation of sweating and detection of glucose in sweat. After the detection is completed, various interfering substances on the electrode surface are removed, which is conducive to continuous glucose measurement. Because of the presence of a glucose selectively permeable membrane, it can be measured in complex sample solutions, with good stability and sensitivity, and has broad application prospects. Attached Figure Description

[0023] Figure 1 Functional flowchart of a sweat glucose detection device;

[0024] Figure 2 This is a diagram illustrating the principle of electrodialysis stimulating perspiration.

[0025] Figure 3 This is a schematic diagram of a sweat collection device.

[0026] Figure 4 This is a picture of an actual sweat collection device;

[0027] Figure 5 This is a photograph of a glucose electrode.

[0028] Figure 6 This is a schematic diagram of an electrochemical potentiostat.

[0029] Figure 7 This is a picture of the actual glucose detection device;

[0030] Figure 8 This is a block diagram of the circuit principle of a glucose detection device;

[0031] Figure 9 To detect the linear relationship between different glucose concentrations and detection current using a glucose electrode;

[0032] Figure 10 To investigate the relationship between the concentration of different interfering substances and the detection current using a glucose electrode;

[0033] Figure 11 A comparison graph of sweat glucose concentration and blood glucose levels detected by a glucose electrode; Detailed Implementation

[0034] The embodiments of the present invention will be further described with reference to the accompanying drawings.

[0035] The detection device of the present invention is as follows Figure 1 Perform sweat glucose testing as shown in the steps below:

[0036] (1) The host computer sends a sweat stimulation command, uses the sweat stimulation module to stimulate the skin to sweat, and uses the sweat collection module to collect the sweat.

[0037] (2) The collected sweat was mixed with 0.1M NaOH solution at a ratio of 1:1;

[0038] (3) Use a pipette to draw 100 μL of the mixed sweat and drop it onto the detection electrode;

[0039] (4) The host computer sends a glucose detection command. After waiting for 9 seconds, the host computer displays the data, which is the sweat glucose value.

[0040] After the test is completed, the above procedure can be repeated to perform glucose testing on the sweat sample.

[0041] The aforementioned sweat-stimulating module uses the principle of electrodialysis to stimulate sweating, such as... Figure 2 As shown, the positive and negative electrodes are attached to the skin. A small amount of medication is applied to the positive electrode. When hyaluronic acid is selected as the stimulating drug, applying a 1mA current to the electrode for 20 minutes allows the sweat glands at the negative electrode to continuously sweat for several hours. When acetylcholine is used, its concentration is diluted to 1%. Applying a 1mA current to the electrode for 97 seconds causes the sweat glands at the negative electrode to begin sweating, which can continue for 35 minutes. If its concentration is diluted to 10%, the application time of the 1mA current can be reduced to 52 seconds, but the sweat glands at the negative electrode can still continuously sweat for 45 minutes. When methacholine is used, regardless of whether its concentration is diluted to 1% or 10%, applying a 1mA current to the electrode takes 200 seconds for the sweat glands at the negative electrode to begin sweating, and the sweating process can continue for 60 minutes. Therefore, 10% acetylcholine is preferred as the drug to stimulate sweating. After the stimulating drug has taken effect, the sweat collection module can be used to collect sweat at the negative electrode.

[0042] The principle of the sweat collection module is as follows: Figure 3 As shown, according to the Laplace force in fluid mechanics, liquid will spontaneously flow from the wedge angle inwards, achieving efficient liquid collection. However, both excessively large and small wedge angles will affect the Laplace force. When the wedge angle α is 3.4°, the liquid velocity at the wedge angle position is the fastest. Below this angle, the liquid will remain stationary under the influence of gravity. Above this angle, the width of the liquid contacting the hydrophilic portion of the paper base increases rapidly during movement, the surface curvature of the liquid decreases rapidly, and the Laplace force decreases accordingly. Therefore, a 3.4° wedge angle is formed on the paper base using both hydrophilic and hydrophobic materials as the sweat collection module of this invention. A physical image is shown below. Figure 4 As shown.

[0043] After collecting sweat using the sweat collection module, the sweat was mixed with 0.1M NaOH solution at a 1:1 ratio to obtain alkaline sweat. 100 μL of this alkaline sweat was then added dropwise to the detection electrode. The detection electrode is shown below. Figure 5As shown, a three-electrode electrochemical system is constructed using a nano-gold electrode as the working electrode 1, silver / silver chloride as the reference electrode 2, and carbon as the counter electrode 3. Furthermore, since sweat contains a wide variety of complex components, many of which can interfere with glucose detection, a glucose-selective permeable membrane needs to be modified on the surface of the working electrode to improve electrode selectivity. 10 μL of a 5% total fluorocarbon solution was pipetted onto the working electrode surface and dried in a 60°C oven for 10 min. After drying, a 1:2 mixture of 5% total fluorocarbon solution and 8% halogenated hydrocarbon oil was mixed, and 10 μL of this solution was pipetted onto the working electrode and dried in a 60°C oven for 5 h. This yields a working electrode modified with a glucose-selective permeable membrane, which is then connected to the detection circuit for glucose detection.

[0044] The detection circuit is designed using the constant potential principle to achieve the multi-step voltage step method. For example... Figure 6 The diagram illustrates the principle of an electrochemical potentiostat. The potentiostat applies a potential between the working electrode and the reference electrode, and this potential does not change due to chemical reactions. Simultaneously, it measures the current between the working electrode and the counter electrode.

[0045] Based on the principle of potentiostat, a device was designed as follows: Figure 7 The glucose detection device shown has the following circuit block diagram: Figure 8 As shown, a microcontroller with built-in AD function is selected as the main control chip for electrochemical signal acquisition. By controlling the conduction of different channels of the analog switch, the potential of the working electrode and the reference electrode is controlled to realize the multi-step voltage step method for glucose concentration detection. The electrochemical current signal between the working electrode and the counter electrode is converted into a voltage signal by the back-end processing circuit. The ADC acquires the signal and realizes the glucose concentration detection according to the relationship formula between glucose concentration and current. The detection results are transmitted to the host computer wirelessly.

[0046] The formula relating glucose concentration to current requires the use of a modified electrode and detection circuit to detect standard glucose solutions. First, using 0.1M NaOH solution as a glucose dilution solution, glucose solutions of different concentrations between 50 μM / L and 1000 μM / L were prepared. The glucose in the solutions was detected using an electrochemical workstation and the glucose detection device of this invention. In the technical menu of the electrochemical workstation's host computer settings, the multipotential step method was selected, and the voltage and duration of each step were set in the parameters. In this test, the first step stimulating sweating was skipped; therefore, the voltage and duration for the last three steps were: applying -1.5V for 2 seconds, applying 0.2V for 5 seconds, and applying 1V for 2 seconds. The detection device was set consistent with the electrochemical workstation. Glucose concentrations were detected sequentially from 50 μM / L to 1000 μM / L, from lowest to highest, to obtain the relationship between glucose oxidation current and concentration, as follows: Figure 9 As shown, the detection current and glucose concentration exhibit a linear relationship within the range of 50 μM / L to 1000 μM / L, and the detection current error between the detection device of this invention and the electrochemical workstation is less than 2 μA. The formula for the relationship between glucose concentration and current is derived after data processing.

[0047] Before conducting sweat testing, it was necessary to verify the modified electrode's resistance to interference from various oxides in sweat. Therefore, different concentrations of ascorbic acid, lactic acid, and uric acid were prepared, and a multi-step voltage step method with the same parameters was used for detection. The results are as follows: Figure 10 As shown, the oxidation current of these interfering substances does not increase with increasing concentration and remains at a very low level. Therefore, the detection of glucose concentration in sweat using the modified electrode will not be affected by these substances.

[0048] Figure 11 This invention continuously detects the glucose content in the sweat of the same person before and 2 hours after meals for 10 days. The concentration before and after meals shows a clear trend, and the relationship between the concentration and blood sugar is compared. The trends of the two are similar.

[0049] The enzyme-free sweat glucose detection device of the present invention can be used to detect glucose content in complex solutions, and can easily collect sweat without exercise. It can also be applied to other solutions containing glucose, such as tissue fluid and saliva. The measured values ​​are transmitted to a mobile phone or computer for viewing via wireless transmission technology.

Claims

1. A sweat glucose detection device based on a multi-step voltage step method, characterized in that: It includes a sweat stimulation module, a sweat collection module, and a three-electrode electrochemical current detection module; The sweat-stimulating module uses a paper battery attached to the skin, with a sweat-stimulating substance pre-coated on the positive electrode. A constant current of 1 mA is generated by applying a positive potential of 2.5~3.3V. Electrophoresis and electroosmosis are used to allow the stimulating substance to penetrate the stratum corneum and stimulate the sweat glands to sweat. The sweat-stimulating substance is one of hyaluronic acid, acetylcholine, and methacholine. When the concentration of acetylcholine is diluted to 1~10%, the current is applied for 50~100s; when the concentration of methacholine is diluted to 1~10%, the current is applied for 160~400s; and when the concentration of hyaluronic acid is diluted to 20min. The sweat collection module is a microfluidic device combining a paper base and a PET membrane. The paper base is rectangular (10 mm long and 300 μm wide) and consists of a hydrophilic layer and a hydrophobic layer. The hydrophilic layer has a large triangular structure with the sweat inlet at the top and the PET membrane at the bottom. The hydrophilic and hydrophobic layers form a 3.4° wedge angle, allowing sweat to spontaneously flow to the PET membrane for storage via Laplace force. The three-electrode electrochemical current detection module includes a working electrode, a counter electrode, and a reference electrode. The working electrode is made of gold nanomaterials and its surface is modified with a glucose selective permeable membrane (prepared by mixing 5% perfluorinated solution and 8% haloalkanes oil in a 1:2 ratio; during preparation, 10 μL of 5% perfluorinated solution is first drop-coated onto the working electrode surface and dried at 60°C for 10 min, then 10 μL of the mixture of 5% perfluorinated solution and 8% haloalkanes oil in a 1:2 ratio is drop-coated and dried at 60°C for 5 h). The counter electrode is a carbon electrode, and the reference electrode is a silver / silver chloride electrode. During the test, the sweating was first stimulated by electrodialysis through the sweat stimulation module, and then the sweat was collected by the sweat collection module. The sweat was mixed with 0.1M NaOH solution at a ratio of 1:1, and the glucose concentration was detected by a three-electrode system based on the multi-step voltage step method.

2. The apparatus according to claim 1, characterized in that: The preferred substance for stimulating sweating is acetylcholine at a concentration of 10%. Applying a 1mA current for 52-97 seconds can cause the sweat glands at the negative electrode to continuously sweat for 35-45 minutes.

3. The apparatus according to claim 1, characterized in that: The multi-step voltage step method specifically includes four steps, where the potential in each step is the working electrode voltage minus the reference electrode voltage: The first step is to apply a positive electric potential of 2.5~3.3V to stimulate sweating for 50s~20min (depending on the stimulant). The second step is to apply a negative potential of -1.5 to -2V to activate the electrode for 2 seconds. The third step is to apply a positive potential of 0.2V to oxidize glucose for 5 seconds, and then collect the current signal after the oxidation is completed. The fourth step is to apply a positive potential of 1~1.2V to remove oxides and interfering substances from the electrode surface for 2 seconds.

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