Organic-electrochemical-transistor-based online sweat cortisol detection sensor, and preparation method therefor

Abstract

An organic-electrochemical-transistor-based online sweat cortisol detection sensor, and a preparation method therefor. The sensor comprises a substrate and a cover sheet, which are arranged in a stacked manner, wherein the substrate is provided with a sweat-inducing electrode, an organic electrochemical transistor and a molecularly imprinted polymer regeneration electrode, the sweat-inducing electrode being used for inducing skin to secrete sweat, a gate electrode of the organic electrochemical transistor being modified to have a molecularly imprinted polymer and used for detecting cortisol in sweat, and the molecularly imprinted polymer regeneration electrode being used for realizing electrochemical regeneration of a molecularly imprinted polymer membrane; and a sweat inflow channel, a reaction cell and a sweat outflow channel are provided in the cover sheet, the sweat inflow channel and the sweat outflow channel both being connected to the reaction cell and used for sweat collection and circulation. The sensor integrates an inherent signal amplification function of an organic electrochemical transistor, so that the sensitivity of detection can be greatly improved, and in-situ, real-time, non-invasive and continuous cortisol detection can be implemented.

Description

Organic electrochemical transistor-based sweat cortisol online detection sensor and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the application of wearable sensors in the field of life monitoring, in particular to an organic electrochemical transistor-based sweat cortisol online detection sensor and a preparation method thereof. BACKGROUND

[0002] Cortisol is a kind of steroid hormone, which is responsible for regulating various physiological functions, including metabolic processes and immune responses. As the main stress hormone, cortisol works in conjunction with the brain to control mood, motivation, and fear responses. When the body is under stress, the adrenal glands release cortisol into the blood, causing cortisol levels to rise. Cortisol levels have adverse effects on various physiological processes, such as blood pressure, blood sugar levels, and carbohydrate metabolism. Persistent stress can disrupt homeostasis of the cardiovascular, immune, renal, skeletal, and endocrine systems, leading to the development of chronic diseases. Therefore, in-situ, real-time, accurate, and dynamic measurement of cortisol levels is very important.

[0003] Cortisol secretion varies among individuals and has a circadian rhythm, and can be found in various body fluids, such as serum, saliva, and sweat. Currently, blood testing is the most mature method for cortisol quantification, which involves venous blood sampling and in-lab quantification of free cortisol in plasma using mass spectrometry or immunoassay. However, this invasive detection method is time-consuming and expensive, and most importantly, it cannot reflect the dynamic changes of cortisol.

[0004] Among various non-invasive available biological fluids (such as urine and saliva), sweat is the only one that can be continuously collected by wearable devices without human intervention. Cortisol has been found to have passive sweat transport, which is independent of sweat rate, and has a good correlation between sweat and serum levels. Existing wearable electrochemical sensors based on sweat immunoassay usually require complex washing steps to regenerate antibody biological receptors, making it difficult to meet the demand for continuous monitoring. In addition, since small molecules such as cortisol hardly produce significant physical and chemical property changes before and after binding with the receptor, it is a major challenge for ultra-sensitive electrochemical detection of cortisol (0.02-0.5 μM in sweat). Therefore, developing an in-situ, real-time, accurate, and continuous cortisol detection sensor to achieve personalized cortisol concentration monitoring and control has become a pressing problem. SUMMARY

[0005] The present application provides an organic electrochemical transistor-based sweat cortisol online detection sensor and a preparation method thereof to solve the problems of accurate detection and dynamic monitoring in the prior art.

[0006] The technical scheme adopted by the present application is as follows:

[0007] (I) Sweat Cortisol online detection sensor based on organic electrochemical transistor

[0008] The sweat Cortisol online detection sensor comprises a substrate, wherein a sweat inducing electrode, an organic electrochemical transistor and a MIP regeneration electrode are arranged in the substrate, the sweat inducing electrode is used to induce the secretion of sweat by the skin, the gate of the organic electrochemical transistor is modified with a molecular imprinting polymer, the molecular imprinting polymer is used for the detection of the concentration of Cortisol in sweat, and the MIP regeneration electrode is used to realize the electrochemical regeneration of the molecular imprinting polymer.

[0009] The sweat Cortisol online detection sensor comprises a cover sheet, wherein the cover sheet is laminated and covers the surface of the substrate, and is used for the dynamic collection and circulation of sweat; the cover sheet is provided with a sweat inflow channel, a reaction cell and a sweat outflow channel, and the sweat inflow channel and the sweat outflow channel are both in communication with the reaction cell.

[0010] The organic electrochemical transistor comprises a gate, a source and a drain; the gate of the organic electrochemical transistor is modified with a Prussian blue nanoparticle film and a molecular imprinting polymer film in sequence, the Prussian blue nanoparticle film is formed by electrodeposition in a Prussian blue precursor solution through cyclic voltammetry, and the molecular imprinting polymer film is formed by electropolymerization in a polymerization precursor solution through cyclic voltammetry; an organic semiconductor film is spin-coated in the channel between the source and the drain of the organic electrochemical transistor.

[0011] Preferably, the Prussian blue precursor solution comprises the following components at the following concentrations: 1-3 mmol / L of ferric chloride, 1-3 mmol / L of potassium ferricyanide, 0.05-0.5 mol / L of potassium chloride and 0.05-0.5 mol / L of hydrochloric acid; and the polymerization precursor solution comprises the following components at the following concentrations: 0.5-50 mmol / L of pyrrole and 0.5-5 mmol / L of Cortisol.

[0012] Preferably, the organic semiconductor film adopts poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (PEDOT:PSS) material, and the ratio between the width and the length of the channel is 5-40. The length of the channel between the source and the drain of the organic electrochemical transistor is the distance between the source and the drain.

[0013] The MIP regeneration electrode comprises a reference electrode and a counter electrode, and the reference electrode, the counter electrode and the gate of the organic electrochemical transistor together constitute a three-electrode system. The reaction cell contains sweat, and the reference electrode and the counter electrode of the MIP regeneration electrode and the gate, the source and the drain of the organic electrochemical transistor are all in contact with and react with the sweat in the reaction cell.

[0014] The sweat-inducing electrode comprises an anodic sweat-inducing electrode and a cathodic sweat-inducing electrode; the anodic sweat-inducing electrode and the cathodic sweat-inducing electrode are arranged at intervals from each other, the anodic sweat-inducing electrode is modified with an anodic hydrogel, the anodic hydrogel is loaded with cholinomimetics, the cathodic sweat-inducing electrode is modified with a cathodic hydrogel, the cathodic hydrogel is loaded with sodium chloride, and the anodic hydrogel and the cathodic hydrogel are both in contact with the skin.

[0015] The substrate comprises a flexible substrate layer, an electrode layer and an insulating layer arranged in sequence; the electrode layer is printed on the flexible substrate layer by a screen printing process, and the insulating layer covers all the conductive traces in the electrode layer.

[0016] The cover sheet comprises a reaction cell layer, a microchannel layer, an inlet layer and a skin contact layer arranged in sequence on the surface of the substrate; in a thickness direction perpendicular to the surface of the substrate, the reaction cell layer is spaced apart to form reaction cells and sweat outlet channels, the sweat outlet channels are in communication with sweat outlets formed on the substrate; the microchannel layer is formed with sweat discharge grooves, the sweat discharge grooves are in communication with the reaction cells and the sweat outlet channels, thereby forming sweat outflow channels; the microchannel layer is also spaced apart to form 3-10 sweat microchannels, the inlet layer is spaced apart to form 3-10 sweat inlets, and the skin contact layer is spaced apart to form 3-10 through holes; the number and arrangement positions of the through holes, the sweat inlets and the sweat microchannels are the same and are aligned one by one in the thickness direction; the through holes are in communication with the corresponding sweat inlets and sweat microchannels, thereby forming sweat inflow channels; the skin contact layer is in contact with the skin and is bonded to the skin.

[0017] The sensor has a sensitivity of 1.332 mu A dec -1 , and a detection limit of 0.754 nM.

[0018] (II) Preparation method of sweat cortisol online detection sensor based on organic electrochemical transistor

[0019] The preparation method comprises the following steps:

[0020] S1) obtaining a flexible substrate layer by using polyethylene terephthalate material, printing an electrode layer on the flexible substrate layer by using carbon ink through a screen printing process, and obtaining a first substrate.

[0021] S2) spin-coating an organic semiconductor film at a channel between a source electrode and a drain electrode of an organic electrochemical transistor of the first substrate, and obtaining a second substrate.

[0022] S3) depositing an insulating layer on a surface of the electrode layer of the second substrate by using a screen printing process, and obtaining a third substrate.

[0023] S4) immersing the third substrate into a Prussian blue precursor solution, depositing a Prussian blue nanoparticle film on the surface of the organic electrochemical transistor gate by cyclic voltammetry, then taking out the third substrate and immersing it into a polymerization precursor solution, electro-polymerizing a molecularly imprinted polymer film on the surface of the Prussian blue nanoparticle film by cyclic voltammetry, to obtain a fourth substrate. The step S4) is specifically as follows:

[0024] S4.1) irradiating the organic electrochemical transistor gate of the third substrate using a plasma cleaning machine, after irradiating for 5-10 min, then placing the third substrate after plasma cleaning into a sulfuric acid solution with a concentration of 0.5 mol / L, and performing activation treatment by cyclic voltammetry, to obtain the third substrate after gate activation; wherein the activation treatment conditions are as follows: the scanning range is -1.2 to 1 V, the scanning rate is 500 mV / s, and the number of cycles is 30;

[0025] S4.2) placing the third substrate after gate activation into a Prussian blue precursor solution, depositing a Prussian blue nanoparticle film on the surface of the organic electrochemical transistor gate by cyclic voltammetry, taking it out of the Prussian blue precursor solution and washing with water, and placing it again into the Prussian blue precursor solution for secondary deposition, to obtain the third substrate after gate deposition;

[0026] The Prussian blue precursor solution comprises the following components with the following concentrations: 1-3 mmol / L of ferric chloride, 1-3 mmol / L of potassium ferricyanide, 0.05-0.5 mol / L of potassium chloride, and 0.05-0.5 mol / L of hydrochloric acid;

[0027] The deposition conditions and the secondary deposition conditions are as follows: the scanning range is -0.2 to 0.6 V, the scanning rate is 50 mV / s, and the number of cycles is 40;

[0028] S4.3) placing the third substrate after gate deposition into a polymerization precursor solution, electro-polymerizing a molecularly imprinted polymer film on the surface of the Prussian blue nanoparticle film by cyclic voltammetry, to obtain the third substrate after gate electro-polymerization;

[0029] The polymerization precursor solution comprises the following components with the following concentrations: 0.5-50 mmol / L of pyrrole and 0.5-5 mmol / L of cortisol; and the electro-polymerization conditions are as follows: the scanning range is 0-1 V, the scanning rate is 50 mV / s, and the number of cycles is 20;

[0030] S4.4) after soaking the third substrate after gate electro-polymerization in a mixed solution of ethanol and methanol for 1 h, and after PB activation treatment and PB stabilization treatment, obtaining a fourth substrate;

[0031] The process of the PB activation treatment is: Amperometric i-t Curve scanning in a hydrochloric acid solution with a concentration of 0.5 mol / L, the potential is set to 1 V, and the duration is 600 seconds.

[0032] The process of the PB stable treatment is: cyclic voltammetry scanning in a potassium chloride solution with a concentration of 0.1 mol / L, the potential scanning range is-0.2 to 0.6 V, the scanning rate is 50 mV / s, and the number of cycles is 10.

[0033] S5) Submerging the fourth substrate into a silver precursor solution to deposit silver on the reference electrode surface of the MIP regenerated electrode, and then sequentially dropping an iron chloride solution and a reference solution on the silver-deposited reference electrode surface, and obtaining a fifth substrate after drying.

[0034] S6) Using CO2 laser engraving cutting technology to engrave and cut the double-sided insulating tape to obtain a skin contact layer and a reaction pool layer; and using CO2 laser engraving cutting technology to engrave and cut a polydimethylsiloxane material to obtain an inlet layer and a microchannel layer; the conditions of the engraving and cutting treatment are: power of 30% to 90%, speed of 70% to 100%, and point number per inch of 500 to 1000.

[0035] S7) Assembling the fifth substrate obtained in step S5), the reaction pool layer, the microchannel layer, the inlet layer and the skin contact layer obtained in step S6) layer by layer, after the assembly is completed, the anode hydrogel solution and the cathode hydrogel solution are respectively injected into two sweat-inducing electrode contact holes, and after solidification treatment, the sweat cortisol online detection sensor is obtained.

[0036] (Three) Sweat cortisol online detection method of the sweat cortisol online detection sensor based on an organic electrochemical transistor

[0037] The detection method comprises the following steps:

[0038] D1) Attaching the sweat cortisol online detection sensor on the skin of a person to be detected, connecting each electrode connection point to a flexible power supply circuit module through a wire, and communicating the flexible power supply circuit module with a mobile terminal;

[0039] D2) Using the mobile terminal to issue an instruction to activate the sweat-inducing electrode, and the sweat-inducing electrode stimulates the skin to secrete sweat;

[0040] D3) After 5 to 20 minutes, the sweat fills the reaction pool, using the mobile terminal to issue an instruction to activate the organic electrochemical transistor, the organic electrochemical transistor detects the cortisol concentration of the sweat and outputs to the mobile terminal, and the mobile terminal displays the cortisol concentration.

[0041] The detection method further comprises a step D4):

[0042] D4) turn off the sweat inducing electrode and the organic electrochemical transistor, apply a voltage of -0.2V to the working electrode to activate the regeneration electrode, activate the MIP regeneration electrode, after 10-30 minutes, the MIP electric regeneration of the organic electrochemical transistor gate, return to step D2) to continue the detection.

[0043] Compared with the prior art, the present application has the following advantages:

[0044] (1) The sensor of the present application can realize the selective combination of MIP film and cortisol, rapid response, and reliable in-situ regeneration of MIP film, realizing dynamic monitoring and continuous monitoring of cortisol concentration. The sensitivity of the sensor of the present application is 1.332 μA dec -1 , and the detection limit is 0.754 nM.

[0045] (2) The sensor of the present application combines the inherent signal amplification function of OECT, greatly improving the sensitivity of detection.

[0046] (3) The sensor of the present application realizes dynamic collection of sweat by stimulating sweat secretion from human skin through a sweat inducing electrode, and combining the design of microstructure and microchannel in the cover sheet, thereby realizing in-situ, real-time, non-invasive and continuous cortisol detection. BRIEF DESCRIPTION OF DRAWINGS

[0047] Fig. 1 is a schematic diagram of the modification of the organic electrochemical transistor of the present application;

[0048] Fig. 2 is a layout diagram of the electrode layer in the present application;

[0049] Fig. 3 is a view of the layer-by-layer assembly of the substrate and the cover sheet in the present application;

[0050] Fig. 4 is a performance test result diagram of the sensor of the present application; (A) is the LSV curve diagram of PB, PB-MIP electrode before and after demolding; (B) is the LSV curve diagram of PB-MIP electrode before and after combination with cortisol molecules and after electrochemical regeneration; (C) is the transfer characteristic curve (left axis) and transconductance curve (right axis) of the OECT sensor before the gate is modified with PB-MIP; (D) is the output characteristic curve diagram of the OECT sensor before the gate is modified with PB-MIP; (E) is the transfer characteristic curve (left axis) and transconductance curve (right axis) of the OECT sensor after the gate is modified with PB-MIP; (F) is the output characteristic curve diagram of the OECT sensor after the gate is modified with PB-MIP;

[0051] Figure 5 is a result diagram of the response of the sensor to cortisol in the application; (A) is a MIP-OECT channel current response curve diagram when the cortisol concentration in the phosphate buffer solution is 1 nM~10 μM; (B) is a non-imprinted polymer (NIP)-OECT channel current response curve diagram when the cortisol concentration in the phosphate buffer solution is 1 nM~10 μM; (C) is a MIP-OECT channel current response curve diagram when the cortisol concentration in the sweat is 1 nM~10 μM; (D) is a selectivity test of the cortisol sensor in the presence of 1 mM cortisol, structural analogues and potential interfering chemicals;

[0052] Figure 6 is a diagram of the influence of the W / L value of the organic electrochemical transistor on the performance of the sensor in the application;

[0053] Figure 7 is a schematic diagram of the use of the sensor in the application;

[0054] Figure 8 is a cross-sectional view of the use of the sensor in the application. DETAILED DESCRIPTION

[0055] The application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] The sweat cortisol online detection sensor provided by the application comprises a substrate, wherein a sweat inducing electrode, an organic electrochemical transistor and a MIP regeneration electrode are arranged in the substrate; the sweat inducing electrode is used to induce the secretion of sweat from the contacted skin; the gate of the organic electrochemical transistor is modified with a molecularly imprinted polymer (MIP), which is used for the detection of cortisol in sweat; and the MIP regeneration electrode is used to realize the electrochemical regeneration of the molecularly imprinted polymer modified on the gate of the organic electrochemical transistor.

[0057] The sweat cortisol online detection sensor provided by the application further comprises a cover sheet, which is laminated and covers the surface of the substrate and is used for the dynamic collection and circulation of sweat; the cover sheet is provided with a sweat inflow channel, a reaction pool and a sweat outflow channel, and the sweat inflow channel and the sweat outflow channel are both in communication with the reaction pool.

[0058] After the sweat cortisol online detection sensor provided by the application is attached to the skin, the sweat inducing electrode in the substrate stimulates the skin to secrete sweat, the sweat enters the reaction pool through the sweat inflow channel and is contained in the reaction pool. The organic electrochemical transistor on the substrate contacts the sweat in the reaction pool and uses the molecularly imprinted polymer modified on its gate as a sensing element to detect the cortisol concentration in the sweat. After the reaction pool is filled with sweat, the sweat overflowing the reaction SCS pool is discharged from the sensor through the sweat outflow channel. Thus, the dynamic detection is realized through the collection and circulation of sweat.

[0059] The organic electrochemical transistor comprises a gate, a source and a drain. As shown in FIG. 1, the gate of the organic electrochemical transistor is sequentially modified with a Prussian blue nanoparticle film and a molecularly imprinted polymer film. Among them, the molecularly imprinted polymer modified gate is used as a sensing element for detecting the concentration of cortisol in sweat (electrolyte in FIG. 1). The Prussian blue nanoparticle modified gate is used as a redox probe. The source and the drain of the organic electrochemical transistor (OECT) are provided with a channel, and an organic semiconductor film is spin-coated at the channel. The organic semiconductor film includes but is not limited to p(g2T-TT), poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (PEDOT:PSS) and poly(3,4-ethylenedioxythiophene)-toluene sulfonate (PEDOT:TOS), etc.

[0060] Specifically, the Prussian blue nanoparticle film is electrodeposited on the surface of the gate of the organic electrochemical transistor in a Prussian blue precursor solution by cyclic voltammetry, and the molecularly imprinted polymer film is electropolymerized on the surface of the Prussian blue nanoparticle film in a polymerization precursor solution by cyclic voltammetry. The components of the Prussian blue precursor solution include ferric chloride, potassium ferricyanide, potassium chloride and hydrochloric acid, and the concentrations of the components are 2.5 mmol / L, 2.5 mmol / L, 0.1 mol / L and 0.1 mol / L, respectively. The components of the polymerization precursor solution include pyrrole monomer and cortisol template molecule, and the concentrations of pyrrole and cortisol are 40 mmol / L and 1 mmol / L, respectively.

[0061] The organic electrochemical transistor further comprises a gate connection point, a source connection point and a drain connection point, the gate connection point is electrically connected with the gate through a conductive trace, the source connection point is electrically connected with the source through a conductive trace, and the drain connection point is electrically connected with the drain through a conductive trace.

[0062] The MIP regeneration electrode comprises a reference electrode and a counter electrode, and the reference electrode, the counter electrode and the gate of the organic electrochemical transistor together constitute a three-electrode system. The MIP regeneration electrode takes the gate of the organic electrochemical transistor as a working electrode, and together with the reference electrode and the counter electrode forms a three-electrode system to realize the electrochemical regeneration of the sensing element MIP. The reference electrode is used to provide a stable standard potential for the working electrode during MIP regeneration, and to maintain the stability of the detection system.

[0063] The MIP regeneration electrode further comprises a reference electrode connection point and a counter electrode connection point, the reference electrode connection point is electrically connected with the reference electrode through a conductive trace, and the counter electrode connection point is electrically connected with the counter electrode through a conductive trace.

[0064] The sweat-inducing electrode includes an anode sweat-inducing electrode and a cathode sweat-inducing electrode. The sweat-inducing electrode is used to induce local sweating instead of the traditional method of heating or strenuous exercise, specifically: the sweat-inducing electrode uses a water gel loaded with a muscarine agonist, and connects the electrodes (cathode and anode) that stimulate sweat to the skin. By passing a small current through the skin, the agonist molecules are transmitted, stimulating the sweat glands to trigger local sweating. In the sweat-inducing electrode, the anode sweat-inducing electrode and the cathode sweat-inducing electrode are arranged at a distance from each other, the anode sweat-inducing electrode is modified with an anode water gel, the anode water gel is loaded with cholinomimetics, and the cathode sweat-inducing electrode is modified with a cathode water gel, the anode water gel and the cathode water gel are in contact with the skin, and the current flowing through the skin forms a sweat-inducing loop, and then by passing a small current through the skin, the cholinomimetics and other agonist molecules loaded in the anode water gel are transmitted, stimulating the sweat glands to secrete sweat, in order to trigger local sweating. Among them, the cholinomimetics can be at least one of acetylcholine, methacholine, carbachol and bethanechol.

[0065] The sweat-inducing electrode also includes an anode connection point and a cathode connection point, the anode sweat-inducing electrode is electrically connected to the anode connection point through a conductive trace, and the cathode sweat-inducing electrode is electrically connected to the cathode connection point through a conductive trace.

[0066] The cathode water gel is loaded with sodium chloride, and the loading amount of sodium chloride (i.e. the mass percentage of sodium chloride in the cathode water gel) is the same as the loading amount of cholinomimetics (i.e. the mass percentage of cholinomimetics in the anode water gel). The loading amount of cholinomimetics and sodium chloride is preferably 1wt%. The water gel can use an agarose water gel with a mass percentage of 3%.

[0067] The substrate includes a flexible substrate layer, an electrode layer and an insulating layer arranged in sequence. The electrode layer is printed on the flexible substrate layer by a screen printing process, and the insulating layer covers all the conductive traces in the electrode layer. The conductive trace is a line with a conductive function in the electrode layer.

[0068] The sweat inducing electrode, the organic electrochemical transistor and the MIP regeneration electrode are all located in the electrode layer and are spaced apart in the plane where the electrode layer is located, that is, the sweat inducing electrode, the organic electrochemical transistor and the MIP regeneration electrode are spaced apart and printed on the flexible substrate layer. The plane where the electrode layer is located can be divided into a reaction window area, a sweat inducing area, a conductive trace area and an electrode connection point area according to functions. The sweat inducing area is printed with the functional structure of the sweat inducing electrode, and the functional structure of the sweat inducing electrode includes an anode sweat inducing electrode and a cathode sweat inducing electrode. The conductive trace area is printed with the conductive traces of the three electrodes, and the conductive trace area is covered with an insulating layer. The electrode connection point area is printed with the electrode connection points of the three electrodes, and the electrode connection points are used for external connection of a signal detection circuit; the electrode connection points of the sweat inducing electrode include an anode connection point and a cathode connection point. The electrode connection points of the organic electrochemical transistor include a gate connection point, a source connection point and a drain connection point. The electrode connection points of the MIP regeneration electrode include a reference electrode connection point and a counter electrode connection point. The gate connection point of the organic electrochemical transistor simultaneously serves as a working electrode connection point of the MIP regeneration electrode. The reaction window area is printed with the functional structures of the MIP regeneration electrode and the organic electrochemical transistor, and the functional structure of the organic electrochemical transistor includes a gate, a source and a drain, and the functional structure of the MIP regeneration electrode includes a reference electrode and a counter electrode. The reaction window area is arranged corresponding to the center of the reaction cell, so that the gate, the source and the drain of the organic electrochemical transistor and the reference electrode and the counter electrode of the MIP regeneration electrode can all contact the sweat in the reaction cell. The reaction window area is not covered with an insulating layer.

[0069] The cover sheet is made of polydimethylsiloxane (PDMS material) and is used to provide a place for sweat collection and refreshing. As shown in FIG. 3, the cover sheet includes, in sequence, a reaction cell layer, a microchannel layer, an inlet layer and a skin contact layer arranged on the surface of the substrate. In the direction perpendicular to the surface of the substrate, the reaction cell layer is spaced apart to provide reaction cells and a sweat outlet channel, and the sweat outlet channel is in communication with the sweat outlet formed on the substrate. The microchannel layer is formed with a sweat discharge channel, and the sweat discharge channel is in communication with the reaction cell and the sweat outlet channel, thereby forming a sweat outflow channel.

[0070] The microchannel layer is further spaced apart to provide 3-10 sweat microchannels, the inlet layer is spaced apart to provide 3-10 sweat inlets, and the skin contact layer is spaced apart to provide 3-10 through holes. The number and arrangement position of the through holes, the sweat inlets and the sweat microchannels are the same and are aligned one by one in the thickness direction, the through holes are in communication with the corresponding sweat inlets and the corresponding sweat microchannels, and the through holes form a sweat inflow channel. The skin contact layer is in contact with the skin and is bonded to the skin.

[0071] As shown in Figure 8, the main function of the adhesion layer (skin contact layer) is to connect the skin and the PDMS cover sheet. After the sweat is generated near the anode, it flows through the adhesion layer to the inlet layer (including 3-8 circular sweat inlets). In addition to the same number of circular holes as the inlet layer, the microchannel layer also has a sweat discharge channel for discharging excess sweat. After the reaction pool is filled with sweat, the excess sweat will overflow the elliptical reaction area of the reaction pool layer and enter the sweat discharge channel of the microchannel layer. The sweat is discharged from the circular hole on the substrate of the reaction pool layer in Figure 2. The sensor of the present application stimulates the human skin to secrete sweat through the sweat-inducing electrode, and realizes the dynamic collection of sweat by combining the design of the microstructure and the microchannel in the cover sheet, thereby realizing the in-situ, real-time, non-invasive and continuous cortisol detection.

[0072] Two sweat-inducing electrode contact holes are provided on the cover sheet and spaced apart. The two sweat-inducing electrode contact holes each penetrate the cover sheet in the thickness direction. The shapes and positions of the two sweat-inducing electrode contact holes correspond one-to-one to the shapes and positions of the anode sweat-inducing electrode and the cathode sweat-inducing electrode, respectively, so that the anode hydrogel on the anode sweat-inducing electrode and the cathode hydrogel on the cathode sweat-inducing electrode can respectively contact the skin and not contact each other.

[0073] The substrate and the cover sheet are bonded by a physical method (such as thermal bonding, pressure bonding, etc.) or a chemical method (such as chemical adhesive, photoresist bonding, etc.).

[0074] The present application also provides a preparation method of a sweat cortisol online detection sensor, which specifically comprises the following steps:

[0075] S1) A flexible substrate layer is obtained by using polyethylene terephthalate material. A substrate drawing (including an electrode layer and an insulating layer) is obtained by using Auto CAD software. The electrode layer is printed on the flexible substrate layer by screen printing process using carbon ink, and a first substrate is obtained.

[0076] S2) An organic semiconductor film is spin-coated at the channel between the source and the drain of the organic electrochemical transistor in the first substrate, and a second substrate is obtained. Specifically, ethylene glycol, sodium dodecylbenzenesulfonate and 3-glycidyloxypropyltrimethoxysilane are added to poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid), and the coating solution is obtained after stirring for 1 h. The coating solution is spin-coated at the channel between the source and the drain, and the second substrate is obtained after annealing at 120℃ for 1 h.

[0077] S3) The insulating layer is deposited on the surface of the electrode layer of the second substrate by screen printing process, and a third substrate is obtained.

[0078] S4) submerging the third substrate into a Prussian blue precursor solution, depositing a Prussian blue nanoparticle film on the surface of the organic electrochemical transistor gate by cyclic voltammetry, then taking out the third substrate and submerging it into a polymerization precursor solution, electro-polymerizing a molecularly imprinted polymer film on the surface of the Prussian blue nanoparticle film by cyclic voltammetry, to obtain a fourth substrate.

[0079] Step S4) is specifically:

[0080] S4.1) irradiating the organic electrochemical transistor gate of the third substrate using a plasma cleaning machine, after irradiating for 5 min, placing the third substrate after plasma cleaning into a sulfuric acid solution with a concentration of 0.5 mol / L, and performing activation treatment by cyclic voltammetry, to obtain the third substrate after gate activation, the activation treatment conditions being: a scanning range of -1.2 to 1 V, a scanning rate of 500 mV / s, and 30 cycles;

[0081] S4.2) placing the third substrate after gate activation into a Prussian blue precursor solution, depositing a Prussian blue nanoparticle film on the surface of the organic electrochemical transistor gate by cyclic voltammetry, taking out from the Prussian blue precursor solution and washing with water, and then placing it into the Prussian blue precursor solution again for secondary deposition, to obtain the third substrate after gate deposition;

[0082] The Prussian blue precursor solution includes the following components with the following concentrations: 2.5 mmol / L of ferric chloride, 2.5 mmol / L of potassium ferricyanide, 0.1 mol / L of potassium chloride, and 0.1 mol / L of hydrochloric acid;

[0083] The deposition conditions and the secondary deposition conditions are both: a scanning range of -0.2~0.6 V, a scanning rate of 50 mV / s, and 40 cycles;

[0084] S4.3) adding a pyrrole solution and a cortisol solution into a phosphate buffer solution to obtain a polymerization precursor solution; placing the third substrate after gate deposition into the polymerization precursor solution, electro-polymerizing a molecularly imprinted polymer film on the surface of the Prussian blue nanoparticle film by cyclic voltammetry, to obtain the third substrate after gate electro-polymerization;

[0085] The pH of the phosphate buffer solution is 6.5, and the concentration is 0.1M; the polymerization precursor solution includes the following components with the following concentrations: 3.5 mmol / L of pyrrole and 0.008 mmol / L of cortisol; the electro-polymerization conditions being: a scanning range of 0~1 V, a scanning rate of 50 mV / s, and 20 cycles;

[0086] S4.4) after soaking the third substrate after gate electro-polymerization in a mixed solution of ethanol and methanol for 1 h, and performing PB activation treatment and PB stabilization treatment, obtaining a fourth substrate;

[0087] The process of the PB activation treatment is: performing an amperometric i-t curve scan in a hydrochloric acid solution with a concentration of 0.5 mol / L, the scan potential is 1 V, and the duration is 600 seconds.

[0088] The process of the PB stabilization treatment is: performing a cyclic voltammetry scan in a potassium chloride solution with a concentration of 0.1 mol / L, the scan range is-0.2 to 0.6 V, the scan rate is 50 mV / s, and the cycle number is 10.

[0089] S5) Submerging the fourth substrate into a silver precursor solution to deposit silver on the reference electrode surface of the MIP regenerated electrode, and then sequentially dropping an iron chloride solution and a reference solution on the silver-deposited reference electrode surface, and obtaining a fifth substrate after drying; the silver precursor solution comprises the following components with the following concentrations: 0.5 mol / L silver nitrate, 1 mol / L sodium thiosulfate and 0.5 mol / L sodium bisulfite; the solvent of the reference solution is methanol, and the reference solution comprises the following components with the following concentrations: 60 mg / mL poly(vinyl butyral) (PVB) and 60 mg / mL sodium chloride.

[0090] S6) According to the preset microstructure patterns of the skin contact layer, the inlet layer, the reaction cell layer and the microchannel layer, performing engraving and cutting treatment on the double-sided insulating tape by using a CO2 laser engraving and cutting technology to obtain the skin contact layer and the reaction cell layer; and performing engraving and cutting treatment on a polydimethylsiloxane material by using the CO2 laser engraving and cutting technology to obtain the inlet layer and the microchannel layer; in step S6), the conditions for the engraving and cutting treatment are: the power is 30% to 90%, the speed is 70% to 100%, and the point number per inch is 500 to 1000; the power and the speed are percentages of the maximum setting parameters.

[0091] S7) Assembling the fifth substrate obtained in step S5), the reaction cell layer, the microchannel layer, the inlet layer and the skin contact layer obtained in step S6) layer by layer, after the assembly is completed, injecting an anode hydrogel solution and a cathode hydrogel solution into two sweat-induced electrode contact holes respectively, and obtaining a sweat cortisol online detection sensor after solidification treatment; the anode hydrogel solution comprises the following components with the following concentrations: 1wt% carbachol and 3wt% agarose; and the cathode hydrogel solution comprises the following components with the following concentrations: 1wt% sodium chloride and 3wt% agarose.

[0092] The application provides a sweat cortisol online detection method using the sweat cortisol online detection sensor.

[0093] D1) attaching the sweat cortisol online detection sensor on the skin of the person to be detected, connecting each electrode connection point to the flexible power supply circuit module through a wire, and connecting the flexible power supply circuit module with the mobile terminal in communication;

[0094] D2) issuing an instruction by using the mobile terminal to activate the sweat inducing electrode, and stimulating the local skin to continuously secrete sweat by the sweat inducing electrode;

[0095] D3) after 5-20 min, the sweat fills the reaction pool, issuing an instruction by using the mobile terminal to activate the organic electrochemical transistor, and the organic electrochemical transistor detects the cortisol concentration of the sweat in real time and outputs to the mobile terminal, and the mobile terminal displays the dynamic cortisol concentration;

[0096] D4) after a period of detection, closing the sweat inducing electrode and the organic electrochemical transistor, applying a constant negative voltage to the working electrode of the MIP regeneration electrode to activate the MIP regeneration electrode, and after 10-30 min, the MIP of the gate of the organic electrochemical transistor is regenerated, if the detection needs to continue, then it can return to step D2) to continue the next round of sweat cortisol online detection, if the detection does not need to continue, then the sensor is closed to stop the detection.

[0097] The specific implementation of the present application is as follows:

[0098] Example 1

[0099] In this embodiment, a sweat cortisol online detection sensor is prepared. In this embodiment, a commercially available double-sided insulating tape with a model number of 3M 468MP is used. The specific steps include the following steps:

[0100] S1) using computer drawing software (Auto CAD) to design a substrate drawing (including an electrode layer and an insulating layer), and customizing a silk screen printing screen. Then, a flexible substrate layer is prepared, the flexible substrate layer is made of polyethylene terephthalate (PET) with a thickness of 188 μm; then, according to the electrode layer drawing, carbon ink is printed on the flexible substrate layer as the electrode layer by a high-precision silk screen printing machine to obtain a first substrate.

[0101] S2) 250 μL of ethylene glycol, 5 mg of sodium dodecylbenzenesulfonate and 50 μL of 3-glycidyloxypropyltrimethoxysilane are sequentially added to 4.7 mL of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid), and stirred for 1 h to improve the conductivity and stability of the semiconductor film to obtain a coating solution. The coating solution is used for spin coating between the source electrode and the drain electrode, and annealing at 120℃ for 1 h to obtain a second substrate.

[0102] S3) According to the insulating layer drawing, the insulating layer is deposited on the surface of the electrode layer of the second substrate by a high-precision screen printing machine and is ultraviolet cured to obtain a third substrate.

[0103] S4) Functional modification of the gate: the gate is functionally modified using Prussian blue nanoparticles and molecularly imprinted polymers. As shown in FIG. 1, the specific process is as follows:

[0104] S4.1) First, the gate is irradiated in a plasma cleaning machine for 5 min and activated in a 0.5 mol / L sulfuric acid solution (the scan range of cyclic voltammetry is -1.2-1 V, and the scan rate is 500 mV s -1 , 30 cycles).

[0105] S4.2) The deposition of Prussian blue on the gate is completed by cyclic voltammetry scanning (the scan range is -0.2-0.6 V, the scan rate is 50 mV / s, and the scanning number is 40) in a Prussian blue precursor solution containing 2.5 mmol / L FeCl3, 2.5 mmol / L K3Fe(CN)6, 0.1 mol / L KCl, and 0.1 mol / L HCl. In order to obtain a relatively high redox signal, the gate is washed with distilled water and the electrodeposition step is repeated again. Subsequently, the gate is immersed in a mixture of 0.1 mol / L HCl and 0.1 mol / L KCl for cyclic voltammetry scanning to obtain a stable redox signal.

[0106] S4.3) 26.2 μL of pyrrole (final concentration: 40 mmol / L) and 0.25 mL of a cortisone stock solution (final concentration: 1 mmol / L) are dissolved in 30 mL of a phosphate buffer solution (pH=6.5, 0.1 mol / L) to prepare a MIP polymerization precursor solution. The gate on which Prussian blue is deposited is placed in the MIP polymerization precursor solution for electro-polymerization, and cyclic voltammetry scanning (the scan range is 0-1 V, the scan rate is 50 mV / s, and the scanning number is 20) is performed.

[0107] S4.4) The electrode is immersed in a mixed solution of ethanol and methanol for 1 h to extract the cortisone template molecule. Then, the Prussian blue activation process (600 s of constant potential scanning at a scan potential of 1 V in a 0.5 mol / L hydrochloric acid solution) and the Prussian blue stabilization process (cyclic voltammetry scanning with a scan range of -0.2-0.6 V at a scan rate of 50 mV / s for 10 times in a 0.1 mol / L potassium chloride solution) are immediately performed to obtain a fourth substrate.

[0108] S5) Fabrication of the reference electrode in the MIP regenerated electrode: Silver was deposited in a silver precursor solution containing 0.5 mol / L silver nitrate (AgNO3), 1 mol / L sodium thiosulfate (Na2S2O3) and 0.5 mol / L sodium bisulfite (NaHSO3). After adding 0.1 mol / L ferric chloride solution to the surface of the silver-deposited reference electrode, 60 mg of polyvinyl butyral and 60 mg of NaCl were dissolved in 1 mL of methanol by strong stirring to obtain a reference solution, and 6 μL of the prepared reference solution was added to the electrode. After drying overnight, the fifth substrate was obtained.

[0109] S6) Design and preparation of the PDMS cover: The microfluidic channel was fabricated using a 50 W CO2 laser cutting machine (PLS6MW, Universal Laser Systems). The skin contact layer was in direct contact with the skin and served as the accumulation layer (3M 468MP, power 45%, speed 100%, pixel density 1000), followed by the inlet layer containing 7 circular inlets (200 μm thick PDMS, power 90%, speed 100%, pixel density 1000), and then the microchannel layer (300 μm thick PDMS, power 70%, speed 100%, pixel density 1000). The reaction cell layer included a reservoir that completely surrounded the reaction window area, and was fabricated using 3M 468MP (power 45%, speed 100%, pixel density 1000). The area of the reservoir layer was 94 mm 2 , and the volume could be estimated by multiplying the area by the thickness of 3M 468MP (0.1 mm), resulting in 9.4 μL.

[0110] S7) The fifth substrate obtained in step S5), the reaction cell layer, the microchannel layer, the inlet layer and the skin contact layer obtained in step S6) were assembled in the order shown in FIG. 3 to obtain an assembled sensor.

[0111] S8) Functional modification of the sweat-inducing electrode: 3% (w / w) agarose was added to deionized water and heated to 250℃ under constant stirring. When the mixture was completely boiled and homogeneous without any agarose particles, it was cooled to 165℃, and 1% (w / w) carbachol was added. Then the cooled mixture was slowly poured into the assembled sensor and solidified at 4℃ for 10 min.

[0112] A cathode hydrogel was prepared using 1% (w / w) NaCl instead of carbachol in the same way to obtain a sweat cortisol online detection sensor.

[0113] The width / length (W / L) ratio of the channel between the source electrode and the drain electrode in the sweat cortisol online detection sensor prepared in this example was 40. W is the channel width, and L is the channel length, i.e., the distance between the source electrode and the drain electrode.

[0114] Comparative Example 1

[0115] A NIP polymer precursor solution was prepared by dissolving 26.2 μL of pyrrole (final concentration of 40 mmol / L) in 30 mL of a phosphate buffer solution (pH = 6.5, 0.1 mol / L). The Prussian blue-deposited gate was placed in the polymer precursor solution for electro-polymerization, and cyclic voltammetry scanning (scanning range of 0-1 V, scanning rate of 50 mV / s, 20 cycles) was performed.

[0116] A sensor with a PB-NIP film modified gate of an organic electrochemical transistor was prepared in this comparative example. The sensor was prepared in the same manner as in the above examples, except that no cortisol was added.

[0117] Comparative Example 2

[0118] A sensor with a channel between the source and the drain of an organic electrochemical transistor having a width / length (W / L) ratio of 5 was prepared in this comparative example in the same manner as in Example 1.

[0119] Comparative Example 3

[0120] A sensor with a channel between the source and the drain of an organic electrochemical transistor having a width / length (W / L) ratio of 20 was prepared in this comparative example in the same manner as in Example 1.

[0121] The performance of the sweat cortisol on-line detection sensor obtained in the above examples and comparative examples will be detected as follows:

[0122] The MIP regenerated electrode obtained in Example 1 was connected to an electrochemical workstation, as shown in (A) of FIG. 4. The linear sweep voltammetry (LSV) curve obtained at the PB-MIP film modified working electrode (gate of the organic electrochemical transistor) showed that the peak current of PB decreased after the cortisol was extracted from the recognition site. It can be seen that the sensor of the present application can realize selective binding of the MIP film to cortisol and rapid response.

[0123] The regeneration of the sensitive element was evaluated by testing the LSV curves of the PB-MIP before and after the binding of the cortisol molecules and after electrochemical regeneration. The results are shown in Figure 4(B). The LSV curves obtained at the PB-MIP film show that the peak current of the LSV decreases after the binding of the cortisol molecules to the MIP. Since the PB-MIP can be regenerated in situ by applying a constant negative potential to the working electrode, after applying a constant potential of -0.2 V to the working electrode of the MIP-regenerated electrode for 1200 s, the peak current of the LSV almost returns to the level before the binding of the cortisol molecules (electrochemical regeneration in Figure 4(B)). It can be seen that the sensor of the present application can realize reliable in-situ regeneration of the MIP film.

[0124] The OECT sensor electrode obtained in Example 1 was connected to a semiconductor parameter analyzer Keithley 4200A-SCS. Specifically, the gate, source and drain were sequentially connected to the semiconductor parameter analyzer on the probe station. The performance of the OECT device was evaluated by measuring the transfer characteristic curve and the output characteristic curve of the device. The transconductance g m of the OECT biosensor is a key parameter, reflecting its ability to effectively amplify electrical signals, especially for detecting biomarkers with extremely low concentrations in vivo. The higher the transconductance g m , the higher the amplification of weak signals, the higher the signal-to-noise ratio, and the higher the detection sensitivity.

[0125] The transconductance g m of the organic electrochemical transistor is related to the material of the conductive polymer and the width-length ratio W / L of the channel, i.e. the ratio between the width W and the length L of the channel: W / L. Example 1 provides a preferred combination: the conductive polymer is poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (PEDOT:PSS), and W / L = 40. The performance comparison chart of the sensors obtained in Example 1 and Comparative Examples 2-3 is shown in Figure 6. As shown in Figure 6(A), all the PEDOT:PSS-based OECTs work in depletion mode, in which a positive gate voltage triggers the decrease of the channel current (I ds As shown in Figure 6(B), when the W / L ratio is maximum, i.e. 40, the maximum value of the transconductance is maximum, and when V g = 0.16 V, the transconductance is about 1.8 mS.

[0126] As shown in Figure 4(C) and Figure 4(E), in the depletion mode, a positive gate bias triggers the de-doping of the channel, which reduces the drain current until it reaches the off state. The addition of the PB-MIP layer on the gate reduces the g m of the OECT by about 1 times, while V g ​The sensor of the present application can maintain high performance when the gate voltage is kept at 0.18 V. The decrease in transconductance can be due to the modification of PM-MIP increasing the impedance between the gate and the channel. Figure 4(D) and Figure 4(F) are respectively the output characteristic curves of the OECT sensor before and after the modification of the gate.

[0127] As shown in Figure 5, the detection performance of the sensor obtained in the present application and the comparative example was evaluated in phosphate buffer solution and sweat respectively. Among them, Figure 5(A) is the detection performance of the sensor MIP-OECT obtained in the present application in phosphate buffer solution, Figure 5(B) is the detection performance of the sensor NIP-OECT obtained in the comparative example in phosphate buffer solution, Figure 5(C) is the detection performance of the sensor MIP-OECT obtained in the present application in sweat, and Figure 5(D) is the selective recognition result of the sensor MIP-OECT obtained in the present application to cortisol.

[0128] As can be seen from Figure 5(A), when the concentration of cortisol increases from 1 nM to 10 μM, a significant decrease in current reading can be detected, and thus the sensor obtained in the present application is responsive to the concentration of cortisol. As can be seen from Figure 5(B), as a control, the sensor NIP-OECT obtained in the comparative example has little response to similar concentrations of cortisol.

[0129] As can be seen from Figure 5(A) and Figure 5(C), the relationship between the current change value -ΔI ds and the concentration of cortisol was linearly fitted, and the sensitivity of the sensor obtained in the present application was 1.332 μA dec -1 , and the detection limit was 0.754 nM. It can be seen that the sensor of the present application combines the inherent signal amplification function of OECT, greatly improving the sensitivity of detection.

[0130] In addition, as shown in Figure 5(D), the sensor MIP-OECT obtained in the present application exhibits selective recognition to the cortisol target and effectively distinguishes other structural analogues and conventional biochemical molecules.

[0131] Example 2

[0132] The present embodiment provides an online detection method for sweat cortisol based on an organic electrochemical transistor sensor, which specifically comprises the following steps:

[0133] (1) Attach the wearable patch for sweat cortisol to the skin of the person to be detected, and connect each electrode to a portable electrochemical workstation;

[0134] (2) Stimulate the human skin to induce sweat by the sweat-inducing electrode;

[0135] (3) Detect and record the cortisol concentration of the induced sweat by the OECT sensor;

[0136] (4) Regenerate the sensitive element MIP of the gate electrode by MIP regeneration electrode, realize real-time and continuous monitoring of cortisol.

[0137] As shown in Figure 7, for wireless measurement, the remotely controlled battery-powered circuit module is integrated with a customized mobile application for Android devices, thus realizing Bluetooth data transmission of the wearable patch. The wearable sensor patch and user interface are shown in the figure, including three functional buttons of Sweat Induction, OECT and MIP. Before activating the OECT biosensor module, sweat induction can be performed first for 10 minutes. Once the sweat fills the reaction cell, the OECT biosensing can be started. After about 150 s, the OECT current stabilizes, allowing the recording of I ds After OECT biosensing, the MIP regeneration process usually takes about 1200 s, during which a voltage of-0.2 V is applied to the MIP regeneration electrode, regenerating the MIP on the gate electrode, and then 10 minutes of sweat induction is performed to prepare for subsequent detection.

[0138] The above detailed description is used to explain and illustrate the present application, rather than limit the present application, and any modification and change made to the present application within the spirit and protection scope of the claims, falls within the protection scope of the present application.

[0139] The above description is only the preferred embodiment of the present application, and any equivalent changes or modifications made to the structure, features and principles described in the scope of the present application patent application, are included in the scope of the present application patent application.

Claims

1. An organic electrochemical transistor-based sweat cortisol on-line detection sensor, characterized in that: The device comprises: a substrate, which is provided with a sweat-inducing electrode for inducing the skin to secrete sweat, an organic electrochemical transistor, and a molecularly imprinted polymer (MIP) regeneration electrode, the gate of the organic electrochemical transistor being modified with a MIP, the MIP regeneration electrode being used to realize electrochemical regeneration of the MIP; a cover sheet, which is laminated on the surface of the substrate to collect and circulate sweat, and is provided with a sweat inflow channel, a reaction cell, and a sweat outflow channel, the sweat inflow channel and the sweat outflow channel both being in communication with the reaction cell.

2. The organic electrochemical transistor-based sweat cortisol online detection sensor according to claim 1, characterized in that: The organic electrochemical transistor comprises a gate, a source, and a drain; the gate of the organic electrochemical transistor is successively modified with a Prussian blue nanoparticle film and a MIP film, the Prussian blue nanoparticle film being formed by electrodeposition in a Prussian blue precursor solution through cyclic voltammetry, and the MIP film being formed by electropolymerization in a polymerization precursor solution through cyclic voltammetry; an organic semiconductor film is spin-coated in the channel between the source and the drain of the organic electrochemical transistor; the Prussian blue precursor solution comprises the following components at the following concentrations: 1-3 mmol / L of ferric chloride, 1-3 mmol / L of potassium ferricyanide, 0.05-0.5 mol / L of potassium chloride, and 0.05-0.5 mol / L of hydrochloric acid; the polymerization precursor solution comprises the following components at the following concentrations: 0.5-50 mmol / L of pyrrole and 0.5-5 mmol / L of cortisol; the MIP regeneration electrode comprises a reference electrode and a counter electrode, the reference electrode, the counter electrode, and the gate of the organic electrochemical transistor together forming a three-electrode system; the reaction cell contains sweat, and the reference electrode and the counter electrode of the MIP regeneration electrode, as well as the gate, the source, and the drain of the organic electrochemical transistor, are all in contact with the sweat in the reaction cell.

3. The organic electrochemical transistor-based on-line sweat cortisol detection sensor according to claim 2, characterized in that: The organic semiconductor film comprises poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid), and the ratio between the width and the length of the channel between the source and the drain of the organic electrochemical transistor is 5-40. The substrate comprises a flexible substrate layer, an electrode layer, and an insulating layer arranged in sequence; the electrode layer is printed on the flexible substrate layer by a screen printing process, and the insulating layer covers all conductive traces in the electrode layer.

4. The organic electrochemical transistor-based on-line sweat cortisol detection sensor of claim 1, wherein: The sweat-inducing electrode comprises an anodic sweat-inducing electrode and a cathodic sweat-inducing electrode; the anodic sweat-inducing electrode and the cathodic sweat-inducing electrode are arranged at a distance from each other, the anodic sweat-inducing electrode is modified with an anodic hydrogel, the anodic hydrogel is loaded with cholinomimetics, the cathodic sweat-inducing electrode is modified with a cathodic hydrogel, the cathodic hydrogel is loaded with sodium chloride, and the anodic hydrogel and the cathodic hydrogel are both in contact with the skin.

5. The organic electrochemical transistor-based on-line sweat cortisol detection sensor of claim 1, wherein: The substrate comprises a flexible substrate layer, an electrode layer, and an insulating layer arranged in sequence; the electrode layer is printed on the flexible substrate layer by a screen printing process, and the insulating layer covers all conductive traces in the electrode layer.

6. The organic electrochemical transistor-based on-line sweat cortisol detection sensor of claim 1, wherein: The cover sheet comprises a reaction cell layer, a micro-channel layer, an inlet layer and a skin contact layer arranged in sequence on the surface of the substrate; the reaction cell layer is provided with reaction cells and sweat outlet channels at intervals, and the sweat outlet channels are in communication with sweat outlets provided on the substrate; the micro-channel layer is provided with sweat discharge channels, which communicate the reaction cells with the sweat outlet channels, thereby forming sweat outflow channels; the micro-channel layer is further provided with 3-10 sweat micro-channels at intervals, the inlet layer is provided with 3-10 sweat inlets at intervals, and the skin contact layer is provided with 3-10 through holes at intervals; the number and arrangement position of the through holes, the sweat inlets and the sweat micro-channels are the same and aligned, the through holes are in communication with the corresponding sweat inlets and sweat micro-channels, forming sweat inflow channels; the skin contact layer is in contact with the skin and is bonded to the skin.

7. A method of manufacturing a sweat cortisol on-line detection sensor according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: S1) obtaining a flexible substrate layer by using polyethylene terephthalate material, printing an electrode layer on the flexible substrate layer by using carbon ink through a screen printing process to obtain a first substrate; S2) spin-coating an organic semiconductor film at the channel between the source and the drain of the organic electrochemical transistor of the first substrate to obtain a second substrate; S3) depositing an insulating layer on the surface of the electrode layer of the second substrate by using a screen printing process to obtain a third substrate; S4) immersing the third substrate in a Prussian blue precursor solution, depositing a Prussian blue nanoparticle film on the surface of the gate of the organic electrochemical transistor by cyclic voltammetry, then taking out the third substrate and immersing it in a polymerization precursor solution, and generating a molecularly imprinted polymer film on the surface of the Prussian blue nanoparticle film by electro-polymerization through cyclic voltammetry to obtain a fourth substrate; S5) immersing the fourth substrate in a silver precursor solution to deposit silver on the surface of the reference electrode of the MIP regenerated electrode, then sequentially adding an iron chloride solution and a reference solution to the surface of the silver-deposited reference electrode, and drying to obtain a fifth substrate; S6) performing engraving and cutting treatment on the double-sided insulating tape by using a CO2 laser engraving and cutting technology to obtain a skin contact layer and a reaction cell layer; performing engraving and cutting treatment on a polydimethylsiloxane material by using a CO2 laser engraving and cutting technology to obtain an inlet layer and a micro-channel layer; the engraving and cutting treatment conditions are as follows: power is 30%-90%, speed is 70%-100%, and the number of points per inch is 500-1000; S7) assembling the fifth substrate obtained in step S5), the reaction cell layer, the micro-channel layer, the inlet layer and the skin contact layer obtained in step S6) layer by layer, injecting an anode hydrogel solution and a cathode hydrogel solution after the assembly is completed, and performing solidification treatment to obtain the sweat cortisol online detection sensor.

8. The method of claim 7, wherein the sensor is prepared by: The step S4) is specifically: S4.1) irradiating the organic electrochemical transistor gate of the third substrate using a plasma cleaner, then placing the third substrate after plasma cleaning in a sulfuric acid solution, and performing activation treatment by cyclic voltammetry to obtain the third substrate after gate activation; wherein the activation treatment conditions are: the scanning range is -1.2 to 1 V, the scanning rate is 500 mV / s, and the number of cycles is 30; S4.2) placing the third substrate after gate activation in a Prussian blue precursor solution, depositing a Prussian blue nanoparticle film on the surface of the organic electrochemical transistor gate by cyclic voltammetry, taking out from the Prussian blue precursor solution and washing with water, and then placing again in the Prussian blue precursor solution for secondary deposition to obtain the third substrate after gate deposition; The Prussian blue precursor solution comprises the following components at the following concentrations: 1-3 mmol / L of iron chloride, 1-3 mmol / L of potassium ferricyanide, 0.05-0.5 mol / L of potassium chloride, and 0.05-0.5 mol / L of hydrochloric acid; The deposition conditions and the secondary deposition conditions are both: the scanning range is -0.2-0.6 V, the scanning rate is 50 mV / s, and the number of cycles is 40; S4.3) placing the third substrate after gate deposition in a polymerization precursor solution, and generating a molecularly imprinted polymer film on the surface of the Prussian blue nanoparticle film by electro-polymerization by cyclic voltammetry to obtain the third substrate after gate electro-polymerization; The polymerization precursor solution comprises the following components at the following concentrations: 0.5-50 mmol / L of pyrrole and 0.5-5 mmol / L of cortisol; and the electro-polymerization conditions are: the scanning range is 0-1 V, the scanning rate is 50 mV / s, and the number of cycles is 20; S4.4) after the third substrate after gate electro-polymerization is immersed in a mixed solution of ethanol and methanol, and after PB activation treatment and PB stabilization treatment, a fourth substrate is obtained; The process of the PB activation treatment is: constant potential scanning in a hydrochloric acid solution with a concentration of 0.5 mol / L, with a potential setting of 1 V and a duration of 600 seconds; The process of the PB stabilization treatment is: cyclic voltammetry scanning in a potassium chloride solution with a concentration of 0.1 mol / L, with a scanning potential range of -0.2 to 0.6 V, a scanning rate of 50 mV / s, and a number of cycles of 10.

9. A method for on-line detection of sweat cortisol using the sweat cortisol on-line detection sensor according to any one of claims 1 to 6, characterized in that: The following steps are included: D1) attaching the sweat cortisol online detection sensor to the skin of a person to be detected, connecting each electrode connection point to a flexible power supply circuit module through a wire, and communicating the flexible power supply circuit module with a mobile terminal; D2) using the mobile terminal to issue an instruction to activate the sweat inducing electrode, and the sweat inducing electrode stimulates the skin to secrete sweat; D3) after 5-20 minutes, using the mobile terminal to issue an instruction to activate the organic electrochemical transistor, and the organic electrochemical transistor detects the cortisol concentration of the sweat and outputs to the mobile terminal, and the mobile terminal displays the cortisol concentration.

10. The on-line sweat Cortisol detection method according to claim 9, characterized in that: The detection method further comprises the following step: D4) applying a voltage of -0.2V to the working electrode activating the regeneration electrode, and activating the MIP regeneration electrode, and after 10-30 minutes, returning to step D2) to continue the detection.

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