阅读说明：本技术 一种葡萄糖电极、微流控芯片、微流控无源汗液贴片及其制备方法和应用 (Glucose electrode, microfluidic chip, microfluidic passive sweat patch and preparation method and application thereof ) 是由 蒋兴宇 牟磊 于 2020-12-04 设计创作，主要内容包括：本发明涉及一种葡萄糖电极、微流控芯片、微流控无源汗液贴片及其制备方法和应用。所述葡萄糖电极包括基体电极以及依次包覆在所述基体电极表面的聚苯胺膜和葡萄糖氧化酶膜。本发明通过在基体电极上修饰离子选择性膜,形成离子选择性葡萄糖电极,首次实现了利用离子选择电极来进行汗液中葡萄糖的无源检测,其原理是基于葡萄糖氧化酶催化葡萄糖生成氢离子,然后用氢离子选择电极检测生成的氢离子,进而计算出葡萄糖浓度,且不会对被测者造成创伤。采用该葡萄糖电极制备而成的微流控芯片以及微流控无源汗液贴片均具有优异的性能和广阔的应用前景。(The invention relates to a glucose electrode, a microfluidic chip, a microfluidic passive sweat patch, and a preparation method and application thereof. The glucose electrode comprises a matrix electrode, and a polyaniline film and a glucose oxidase film which are sequentially coated on the surface of the matrix electrode. The ion selective membrane is modified on the substrate electrode to form the ion selective glucose electrode, so that the passive detection of glucose in sweat by using the ion selective electrode is realized for the first time. The microfluidic chip and the microfluidic passive sweat patch prepared by the glucose electrode have excellent performance and wide application prospect.)

1. The glucose electrode is characterized by comprising a matrix electrode, and a polyaniline film and a glucose oxidase film which are sequentially coated on the surface of the matrix electrode.

2. The glucose electrode of claim 1, wherein the base electrode comprises a carbon ink electrode, a carbon electrode, or a carbon ink printed electrode;

preferably, the glucose oxidase membrane contains a combination of glucose oxidase, chitosan and acetic acid;

preferably, the mass ratio of the glucose oxidase to the chitosan to the acetic acid is (0.2-1):1 (1-3), preferably 0.5:1: 2.

3. A method of manufacturing a glucose electrode according to claim 1 or 2, comprising the steps of:

(1) polymerizing an aniline monomer onto a substrate electrode to obtain an electrode coated with a polyaniline film;

(2) and dropping the glucose oxidase membrane solution on the electrode coated with the polyaniline membrane, and drying to obtain the glucose electrode.

4. The method according to claim 3, wherein in the step (1), the polymerization method is cyclic voltammetry;

preferably, in the cyclic voltammetry, the voltage is-0.2V-1.0V;

preferably, in the cyclic voltammetry, the number of cycles is 20 to 30, preferably 25;

preferably, in the cyclic voltammetry, the voltage rate is 0.05-0.2V/s, preferably 0.1V/s;

preferably, in the cyclic voltammetry, the reference electrode is an Ag/AgCl electrode;

preferably, in the cyclic voltammetry, the electrolyte is a hydrochloric acid solution of aniline monomer;

preferably, the concentration of the aniline monomer in the electrolyte is 0.05-2mol/L, preferably 0.1 mol/L;

preferably, the concentration of HCl in the electrolyte is 0.5-2mol/L, preferably 1 mol/L;

preferably, step (1) specifically comprises: electropolymerizing an aniline monomer onto a carbon ink electrode by a cyclic voltammetry method to obtain an electrode coated with a polyaniline film; the cyclic voltammetry conditions include: the voltage is-0.2V-1.0V, the cycle time is 20-30 times, the voltage rate is 0.05-0.2V/s, the reference electrode is an Ag/AgCl electrode, and the electrolyte is hydrochloric acid solution of aniline;

preferably, in the step (2), the glucose oxidase membrane solution is a PBS buffer solution containing glucose oxidase, chitosan and acetic acid;

preferably, the concentration of the glucose oxidase in the glucose oxidase membrane solution is 8-12mg/mL, preferably 10 mg/mL;

preferably, in step (2), the temperature of the drying is 3-6 ℃, preferably 4 ℃;

preferably, in the step (2), the drying time is 10-18 h.

5. A microfluidic chip comprising a polydimethylsiloxane membrane having a microfluidic channel and an ion-selective sensor attached to the polydimethylsiloxane membrane, the ion-selective sensor comprising the glucose electrode of claim 1 or 2;

preferably, the ion selective sensor further comprises Na⁺Electrode and/or K⁺An electrode;

preferably, the Na⁺The electrode is coated with Na on the surface⁺A carbon ink electrode of an ion selective membrane;

preferably, said K⁺The electrode is coated with K⁺A carbon ink electrode of an ion selective membrane;

preferably, said K⁺The ion selective membrane contains a combination of validamycin, sodium tetraphenylborate, polyvinyl chloride and bis (2-ethyl) sebacate;

preferably, the ion selective sensor further comprises a reference electrode;

preferably, the reference electrode is an Ag/AgCl ink electrode with the surface coated with a polyvinyl butyral film;

preferably, the polyvinyl butyral film contains a combination of polyvinyl butyral, polyoxyethylene-polyoxypropylene-polyoxyethylene block polymer, multi-walled carbon nanotubes and NaCl;

preferably, the microfluidic chip further comprises a urea detection strip and a pH detection strip which are arranged in the microfluidic channel of the polydimethylsiloxane membrane;

preferably, the urea strip comprises a pH paper coated with urease;

preferably, the pH test strip comprises a pH paper.

6. A microfluidic passive sweat patch, comprising the glucose electrode of claim 1 or 2 or the microfluidic chip of claim 5.

7. The microfluidic passive sweat patch of claim 6, wherein the microfluidic passive sweat patch includes the microfluidic chip of claim 5, a metal polymer conductor wire layer, a metal polymer conductor antenna layer, and an electronics layer in a stacked arrangement;

preferably, the microfluidic passive sweat patch further comprises a double-sided adhesive layer adhered to the microfluidic chip;

preferably, the microfluidic chip is connected with the metal polymer conductor lead layer through a silicon-oxygen chemical bond;

preferably, the metal polymer conductor wire layer is connected with the metal polymer conductor antenna layer through a cross-linking product of polydimethylsiloxane;

preferably, the metal polymer conductor antenna layer is connected with the electronic device layer through a conductive adhesive;

preferably, the double-sided adhesive layer contains a microfluidic channel;

preferably, the metal polymer conductor wire layer comprises a liquid metal wire and a stripping layer coated on the surface of the liquid metal wire;

preferably, the liquid metal comprises any one or a combination of at least two of gallium, mercury, gallium-indium alloy, gallium-indium-tin alloy, gallium-zinc alloy or bismuth-tin-lead-indium-cadmium alloy, preferably gallium-indium alloy;

preferably, the material of the release layer comprises any one or a combination of at least two of polydimethylsiloxane, Smooth-on series material, rubber, plastic film, resin, polyurethane, polyethylene, polyvinyl alcohol, polytetrafluoroethylene, polyimide, epoxy resin, polystyrene, PET, polylactic acid, polyglycolic acid, polylactic-glycolic acid copolymer or polylactic-caprolactone, preferably polydimethylsiloxane.

8. A method of manufacturing a microfluidic passive sweat patch according to claim 6 or 7, wherein the method of manufacturing includes the steps of:

(1) activating the polydimethylsiloxane film and the metal polymer conductor wire layer by using oxygen plasma, and then bonding the polydimethylsiloxane film and the metal polymer conductor wire layer together;

(2) connecting the electronic device layer with the metal polymer conductor antenna layer through conductive adhesive;

(3) activating the metal polymer conductor antenna layer and the metal polymer conductor lead layer by using oxygen plasma, and then bonding the two together;

(4) activating the surface by oxygen plasma, and then connecting the ion sensor with a metal polymer conductor lead layer;

(5) activating the microfluidic channel layer on the polydimethylsiloxane membrane through oxygen plasma, placing a urea detection strip and a pH detection strip in the microfluidic channel, and then attaching the urea detection strip and the pH detection strip to the polydimethylsiloxane membrane;

(6) coating a mixture of polydimethylsiloxane prepolymer and a curing agent on the surface of the microfluidic chip, and curing to obtain a microfluidic passive sweat patch;

preferably, in the step (2), the electronic device layer comprises an MLX 90129 chip;

preferably, the power of the oxygen plasma is 50-70W, preferably 60W;

preferably, the time of activation is 0.5-2min, preferably 1 min;

preferably, step (6) further comprises: and adhering a double-sided adhesive layer after the curing.

9. Use of a microfluidic passive sweat patch according to claim 6 or 7 for detecting metabolite content, electrolyte content, urea content or pH in sweat;

preferably, the metabolite comprises glucose;

preferably, the electrolyte comprises potassium ions or sodium ions.

10. A wearable device, characterized in that it comprises the microfluidic passive sweat patch of claim 6 or 7.

Technical Field

The invention relates to the technical field of sensors, in particular to a glucose electrode, a microfluidic chip, a microfluidic passive sweat patch, and a preparation method and application thereof.

Background

The wearable sensor can monitor signals of motion, electrophysiology, temperature and the like of a human body in real time. Under the support of technologies such as the Internet of things, flexible sensors and big data, wearable sensors are widely applied to the biomedical fields such as motion monitoring, precise medicine and chronic disease protection. By integrating sensors into wearable products such as tattoos, patches, wristbands, watches, and clothing, physical, biological, and chemical signals of a patient can be monitored in real time. The data may be wirelessly transmitted to a data center to implement a telemedicine system. Most wearable body fluid tests today are health monitoring via sweat, tears, urine, and the like. The sweat component is stable, a stable monitoring data source can be provided for health protection, and the diagnostic value is good. Therefore, wearable sweat detection is particularly attractive.

Few products are currently being developed for wearable sweat sensors, but many relevant literature reports exist. In 2017 Javey reported a Fully integrated wearable sweat sensor, and since that, research in this area entered a new stage (full integrated wearable sensor array for multiplexed in situ fertilization analysis. DOI:10.1038/nature 16521). The sensor simultaneously and selectively measures sweat metabolites (such as glucose and lactate) and electrolytes (such as sodium and potassium ions), calibrates the sensor's response to skin temperature, and connects to a small wearable on-board processor unit through which the conversion, conditioning, processing of signals is performed and the results can be wirelessly communicated to a smartphone. But this sensor picture does not show that both activation and sensing of the sensor require a battery. Because of the large power consumption, a large battery needs to be connected behind the sensor. This completely defeats the purpose of wearable sensors.

Conventional wearable sensors do not enable accurate capture, storage, volume measurement, and chemical analysis of fluids, thereby limiting the ability to isolate body fluids from contaminants and limiting the ability to subsequently extract, sample, and chemically analyze. In addition, the traditional glucose detection device often needs to generate a wound, is inconvenient to use, cannot realize glucose detection in sweat, and is limited in application.

Therefore, there is a need in the art to develop new sensors and wearable devices that combine the functions of collection, transmission, storage, and chemical analysis.

Disclosure of Invention

In view of the shortcomings of the prior art, it is an object of the present invention to provide a glucose electrode, and more particularly, to provide a glucose electrode capable of passive detection. The glucose electrolysis can accurately analyze metabolites in sweat on the premise of no wound.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention aims to provide a glucose electrode, which comprises a matrix electrode, and a polyaniline film and a glucose oxidase film which are sequentially coated on the surface of the matrix electrode.

According to the invention, the ion selective membrane (polyaniline membrane and glucose oxidase membrane) is modified on the substrate electrode to form the ion selective glucose electrode, so that the passive detection of glucose in sweat by using the ion selective electrode is realized for the first time.

Preferably, the substrate electrode comprises a carbon ink electrode, a carbon electrode or a carbon ink printed electrode.

Preferably, the glucose oxidase membrane contains a combination of glucose oxidase, chitosan and acetic acid.

According to the invention, the three substances are preferably selected to form the glucose oxidase membrane, the acetic acid is helpful for dissolving chitosan, the chitosan can stabilize and protect the glucose oxidase and the glucose oxidase from reacting with glucose in sweat, and the test accuracy can be further improved.

Preferably, the mass ratio of the glucose oxidase, chitosan and acetic acid is (0.2-1):1 (1-3), wherein, (0.2-1) includes but is not limited to 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, etc., and (1-3) includes but is not limited to 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, etc., preferably 0.5:1: 2.

Another object of the present invention is to provide a method for producing a glucose electrode according to the first object, the method comprising the steps of:

(1) polymerizing an aniline monomer onto a substrate electrode to obtain an electrode coated with a polyaniline film;

(2) and dropping the glucose oxidase membrane solution on the electrode coated with the polyaniline membrane, and drying to obtain the glucose electrode.

Preferably, in step (1), the method of polymerization is cyclic voltammetry.

Preferably, in the cyclic voltammetry, the voltage is-0.2V-1.0V, such as-0.1V, 0.2V, 0.3V, 0.4V, 0.5V, 0.6V, 0.7V, 0.8V, 0.9V, and the like.

Preferably, in the cyclic voltammetry, the number of cycles is 20-30, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, etc., preferably 25.

Preferably, in the cyclic voltammetry, the voltage rate is 0.05-0.2V/s, such as 0.1V/s, 0.12V/s, 0.14V/s, 0.16V/s, 0.18V/s, etc., preferably 0.1V/s. Since cyclic voltammograms are the current values measured at cyclic voltages, the voltage rate refers to the rate of change of the voltage.

Preferably, in the cyclic voltammetry, the reference electrode is an Ag/AgCl electrode.

Preferably, in the cyclic voltammetry, the electrolyte is a hydrochloric acid solution of aniline monomer.

Preferably, the concentration of aniline monomer in the electrolyte is 0.05-2mol/L, such as 0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L, 1mol/L, 1.2mol/L, 1.4mol/L, 1.6mol/L, 1.8mol/L, etc., preferably 0.1 mol/L.

Preferably, the concentration of HCl in the electrolyte is 0.5-2mol/L, such as 0.6mol/L, 0.8mol/L, 1mol/L, 1.2mol/L, 1.4mol/L, 1.6mol/L, 1.8mol/L, and the like, preferably 1 mol/L.

Preferably, step (1) specifically comprises: electropolymerizing an aniline monomer onto a carbon ink electrode by a cyclic voltammetry method to obtain an electrode coated with a polyaniline film; the cyclic voltammetry conditions include: the voltage is-0.2V-1.0V, the cycle time is 20-30 times, the voltage rate is 0.05-0.2V/s, the reference electrode is an Ag/AgCl electrode, and the electrolyte is hydrochloric acid solution of aniline.

Preferably, in the step (2), the glucose oxidase membrane solution is a PBS buffer solution containing glucose oxidase, chitosan and acetic acid.

Preferably, the concentration of glucose oxidase in the glucose oxidase membrane solution is 8-12mg/mL, such as 8.2mg/mL, 8.4mg/mL, 8.6mg/mL, 8.8mg/mL, 9mg/mL, 9.2mg/mL, 9.4mg/mL, 9.6mg/mL, 9.8mg/mL, 10mg/mL, 10.2mg/mL, 10.4mg/mL, 10.6mg/mL, 10.8mg/mL, 11mg/mL, 11.2mg/mL, 11.4mg/mL, 11.6mg/mL, 11.8mg/mL, and the like, preferably 10 mg/mL.

Preferably, in step (2), the temperature of the drying is 3-6 ℃, e.g., 4 ℃,5 ℃, etc., preferably 4 ℃.

Preferably, in step (2), the drying time is 10-18h, such as 11h, 12h, 13h, 14h, 15h, 16h, 17h, and the like.

Preferably, the preparation of the glucose oxidase membrane solution comprises: dissolving 1% chitosan into 2% acetic acid, and mixing with glucose oxidase solution (10 mg/mL concentration, PBS buffer solution) at a volume ratio of 2: 1. The 1% chitosan refers to a 1% chitosan aqueous solution by mass, and the 2% acetic acid refers to a 2% acetic acid aqueous solution by mass.

The third objective of the present invention is to provide a microfluidic chip, which includes a Polydimethylsiloxane (PDMS) membrane having a microfluidic channel and an Ion Selection (ISE) sensor attached to the PDMS membrane, wherein the ion selection sensor includes a glucose electrode according to one of the objectives.

The microfluidic chip provided by the invention can realize passive detection and analysis of glucose in sweat, and the specific working principle and the glucose electrode are the same as one of the purposes.

Preferably, the ion selective sensor further comprises Na⁺Electrode and/or K⁺And an electrode.

Preferably, the Na⁺The electrode is coated with Na on the surface⁺A carbon ink electrode of an ion selective membrane.

PreferablySaid Na⁺The ion selective membrane contains Na⁺Vector X, tetrakis [3, 5-bis (trifluoromethyl) phenyl]Sodium borate, polyvinyl chloride and bis (ethyl 2-sebacate). Wherein Na⁺The carrier X is a substance known in the art and may be, for example, Na⁺An ionophore III.

Preferably, the preparation method of the Na electrode comprises: na (Na)⁺Drop-casting the mixed solution of selective membrane on carbon ink electrode, drying to obtain Na⁺And an electrode.

Preferably, the Na⁺The solute of the mixed solution of the selective membrane is 1 percent of Na⁺Ionophore X, 0.5% of tetrakis [3, 5-bis (trifluoromethyl) phenyl]Sodium borate, 33% polyvinyl chloride and 65.5% bis (ethyl 2-sebacate), the solvent being tetrahydrofuran.

Preferably, said K⁺The electrode is coated with K⁺A carbon ink electrode of an ion selective membrane.

Preferably, said K⁺The ion selective membrane contains a combination of validamycin, sodium tetraphenylborate, polyvinyl chloride and bis (2-ethyl) sebacate.

Preferably, said K⁺The preparation method of the electrode comprises the following steps: k⁺Drop-casting the selective membrane mixed solution on a carbon ink electrode, and drying to obtain K⁺And an electrode.

Preferably, said K⁺The solute of the selective membrane mixed solution is 2% of validamycin, 0.55% of sodium tetraphenylborate, 33% of polyvinyl chloride and 64.45% of bis (2-ethyl) sebacate, and the solvent is cyclohexanone.

In the preferred technical scheme of the invention, inorganic salt ion Na⁺And K⁺The detection principle of (2) is based on ion selective electrodes. The ion selective electrode is based on the ion selective permeation of the surface modification membrane to change the surface potential of the electrode, and the concentration of ions in sweat is analyzed through analyzing the variation of the potential.

Preferably, the ion selective sensor further comprises a reference electrode.

Preferably, the reference electrode is an Ag/AgCl ink electrode with a polyvinyl butyral (PVB) film coated on the surface.

Preferably, the polyvinyl butyral film contains a combination of polyvinyl butyral, polyethylene oxide-polypropylene oxide-polyethylene oxide block polymer (PEO-PPO-PEO), multiwalled carbon nanotubes and NaCl.

Preferably, the reference electrode is prepared by a method comprising: and (3) dripping the PVB film mixed solution on an Ag/AgCl ink electrode, and drying to obtain the reference electrode.

Preferably, the PVB film mixed solution comprises PVB, PEO-PPO-PEO, multi-walled carbon nanotubes and NaCl in methanol.

Preferably, the microfluidic chip further comprises a urea detection strip and a pH detection strip disposed in the microfluidic channel of the polydimethylsiloxane membrane.

Preferably, the urea strip comprises a pH paper coated with urease.

The operating principle of the urea detection strip is based on the urea catalysis, and then a colorimetric result is generated on pH test paper. The concentration of urea was calculated from the color change of the urea and pH test strips.

Preferably, the pH test strip comprises a pH paper.

It is a fourth object of the present invention to provide a microfluidic passive sweat patch comprising a glucose electrode according to one of the objects or a microfluidic chip according to the third object.

The invention integrates the micro-fluidic chip into the micro-fluidic passive sweat patch and has the following advantages: first, the microstructure of the microfluidic chip that stores, transports and handles liquids forms the core component of the sensor. By manipulating the precise amount of liquid, sensing accuracy and reliability can be significantly improved. This feature is very useful for wearable devices, since the secretion of body fluids is very small and uncontrollable. Secondly, the microstructure can also form physical content to store and distribute solute at controlled intervals, thereby realizing controllable drug release. Finally, the micro-patterned structures can be used as conduits for electronic devices and to make electrical connections in flexible substrates. Excellent flexibility and tensile properties are provided while maintaining excellent electrical conductivity between micro-electromechanical systems (MEMS).

In addition, since the entire sensor uses near field sensing (RFID) technology, the sensor portion does not require a power source, and the microfluidic passive sweat patch provided by the present invention does not require a power source.

Preferably, the microfluidic passive sweat patch comprises a microfluidic chip, a Metal Polymer Conductor (MPC) wire layer, a Metal Polymer Conductor (MPC) antenna layer, and an electronics layer, all of which are stacked.

In the preferred technical scheme of the invention, the components are combined to form the microfluidic passive sweat patch, firstly, sweat is transmitted to the surface of the sensor by utilizing the driving of the capillary force of the microfluidic chip, and the ISE electrode collects the concentration signals of various analytes and converts the concentration signals into surface potential. Three ISE electrodes (glucose electrode, Na) in the preferred technical scheme⁺Electrode and K⁺Electrodes) share a common reference electrode. After the MLX 90129 chip collects signals, data are transmitted out through the MPC antenna, when the portable reader and the smart phone are close to the sensor, the generated signals are transmitted to the reader through signal transmission and Radio Frequency Identification (RFID), and a power supply of the microfluidic passive sweat patch is provided by the RFID.

The MPC wire layer and MPC antenna layer of the present invention are prepared according to the method of preparing flexible and stretchable conductive traces and circuits described in patent application CN 108668431A.

Preferably, the microfluidic passive sweat patch further comprises a double-sided adhesive layer adhered to the microfluidic chip.

Preferably, the microfluidic chip is connected with the metal polymer conductor lead layer through a silicon-oxygen chemical bond.

Preferably, the metal polymer conductor wire layer and the metal polymer conductor antenna layer are connected by a cross-linked product of polydimethylsiloxane.

Preferably, the metal polymer conductor antenna layer and the electronic device layer are connected through a conductive adhesive.

Preferably, the double-sided adhesive layer contains a microfluidic channel thereon. The microfluidic channel is used for leading in sweat.

Preferably, the metal polymer conductor wire layer comprises a liquid metal wire and a stripping layer coated on the surface of the liquid metal wire.

Preferably, the liquid metal comprises any one or at least two of gallium, mercury, gallium-indium alloy, gallium-indium-tin alloy, gallium-zinc alloy or bismuth-tin-lead-indium-cadmium alloy, preferably gallium-indium alloy.

Preferably, the material of the release layer comprises any one or a combination of at least two of Polydimethylsiloxane (PDMS), a Smooth-on series material, rubber, a plastic film, a resin, polyurethane, polyethylene, polyvinyl alcohol, polytetrafluoroethylene, polyimide, an epoxy resin, polystyrene, PET, polylactic acid, polyglycolic acid, polylactic-glycolic acid copolymer, or polylactic-caprolactone, preferably polydimethylsiloxane.

The fifth purpose of the present invention is to provide a method for preparing a microfluidic passive sweat patch described in the fourth purpose, wherein the method for preparing the microfluidic passive sweat patch comprises the following steps:

(1) activating the polydimethylsiloxane film and the metal polymer conductor wire layer by using oxygen plasma, and then bonding the polydimethylsiloxane film and the metal polymer conductor wire layer together;

(2) connecting the electronic device layer with the metal polymer conductor antenna layer through conductive adhesive;

(3) activating the metal polymer conductor antenna layer and the metal polymer conductor lead layer by using oxygen plasma, and then bonding the two together;

(4) activating the surface by oxygen plasma, and then connecting the ion sensor with a metal polymer conductor lead layer;

(5) activating the microfluidic channel layer on the polydimethylsiloxane membrane through oxygen plasma, placing a urea detection strip and a pH detection strip in the microfluidic channel, and then attaching the urea detection strip and the pH detection strip to the polydimethylsiloxane membrane;

(6) and coating a mixture of polydimethylsiloxane prepolymer and a curing agent on the surface of the microfluidic chip, and curing to obtain the microfluidic passive sweat patch.

In the above step, the oxygen plasma can activate crosslinking between the MPC conductive layer and the PDMS on the surface of the MPC antenna layer, thereby connecting the two together. In the step (6), the cured product is sealed to isolate external interference.

Preferably, in the step (2), the electronic device layer includes an MLX 90129 chip.

Preferably, the power of the oxygen plasma is 50-70W, such as 55W, 60W, 65W, 68W, etc., preferably 60W.

Preferably, the time of activation is 0.5-2min, such as 0.6min, 0.8min, 1min, 1.2min, 1.4min, 1.6min, 1.8min, etc., preferably 1 min.

Preferably, step (6) further comprises: and adhering a double-sided adhesive layer after the curing.

The sixth purpose of the present invention is to provide an application of the microfluidic passive sweat patch described in the fourth purpose, wherein the microfluidic passive sweat patch is used for detecting the metabolite content, electrolyte content, urea content or pH in sweat.

Preferably, the metabolite comprises glucose.

Preferably, the electrolyte comprises potassium ions or sodium ions.

It is a seventh object of the present invention to provide a wearable device comprising a microfluidic passive sweat patch as described in the fourth object.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention realizes the passive detection of glucose in sweat by using the ion selective electrode for the first time by modifying the ion selective membrane on the carbon ink electrode, and the principle is based on the principle that glucose oxidase catalyzes glucose to generate hydrogen ions, and then the hydrogen ions generated are detected by using the hydrogen ion selective electrode, so that the glucose concentration is calculated, and no wound is caused to a tested person.

(2) The integration of the microfluidic chip into the microfluidic passive sweat patch of the present invention has the following advantages: first, the microstructure of the microfluidic chip that stores, transports and handles liquids forms the core component of the sensor. By manipulating the precise amount of liquid, sensing accuracy and reliability can be significantly improved. This feature is very useful for wearable devices, since the secretion of body fluids is very small and uncontrollable. Secondly, the microstructure can also form physical content to store and distribute solute at controlled intervals, thereby realizing controllable drug release. Finally, the micro-patterned structures can be used as conduits for electronic devices and to make electrical connections in flexible substrates. Excellent flexibility and tensile properties are provided while maintaining excellent conductivity between MEMS.

Drawings

Fig. 1 is a schematic view of an ion selective sensor in embodiment 1 of the present invention.

FIG. 2 is a schematic view of a urea test strip and a pH test strip in example 1 of the present invention.

Fig. 3 is a schematic structural diagram of a microfluidic passive sweat patch in example 2 of the present invention.

Fig. 4 is a schematic diagram of the fabrication process of the microfluidic passive sweat patch of example 2 of the present invention.

Fig. 5 is a schematic diagram of the MPC antenna in embodiment 2 of the present invention.

FIG. 6a is Na for microfluidic passive sweat patch of example 2 of the invention⁺And (5) a sensor performance test chart.

FIG. 6b is Na for microfluidic passive sweat patch of example 2 of the invention⁺Sensor concentration versus potential.

FIG. 7a is K for microfluidic passive sweat patch of example 2 of the present invention⁺And (5) a sensor performance test chart.

FIG. 7b is K for microfluidic passive sweat patch of example 2 of the present invention⁺Sensor concentration versus potential.

FIG. 8a is H of microfluidic passive sweat patch of example 2 of the present invention⁺And (5) a sensor performance test chart.

FIG. 8b is H of microfluidic passive sweat patch of example 2 of the present invention⁺Graph of sensor pH versus potential.

Fig. 9a is a graph of glucose sensor performance testing of the microfluidic passive sweat patch of example 2 of the present invention.

Fig. 9b is a graph of glucose sensor concentration versus potential for the microfluidic passive sweat patch of example 2 of the present invention.

Fig. 10 is a urea sensor performance test chart for the microfluidic passive sweat patch of example 2 of the present invention.

Fig. 11 is a graph of urea sensor concentration versus GR value for the microfluidic passive sweat patch of example 2 of the present invention.

Detailed Description

For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

This example provides a glucose electrode, which is prepared as follows:

(1) electropolymerizing an aniline monomer onto a carbon ink electrode by a cyclic voltammetry method to obtain an electrode coated with a polyaniline film; the cyclic voltammetry conditions include: the voltage is-0.2V-1.0V, the cycle number is 25, the voltage rate is 0.1V/s, the reference electrode is an Ag/AgCl electrode, and the electrolyte is a hydrochloric acid solution of aniline monomer (the concentration of the aniline monomer is 0.1mol/L, and the concentration of HCl is 1 mol/L);

(2) dissolving 1% chitosan aqueous solution into 2% acetic acid aqueous solution, then mixing with glucose oxidase solution (the concentration is 10mg/mL, the solution is PBS buffer solution) according to the volume ratio of 2:1 to obtain glucose oxidase membrane solution, dripping the mixed solution on the electrode coated with the polyaniline membrane, and drying for 12h at 4 ℃ to obtain the glucose electrode.

Example 2

The embodiment provides a microfluidic chip and a microfluidic passive sweat patch;

the microfluidic core comprises a PDMS membrane with a microfluidic channel, an ISE sensor (shown in figure 1) attached to the PDMS membrane, and a urea detection strip and a pH detection strip (shown in figure 2) disposed in the microfluidic channel, wherein the ion selective sensor comprises a glucose electrode (example 1) and Na⁺Electrode, K⁺An electrode and a reference electrode.

The microfluidic passive sweat patch comprises a double-sided adhesive layer, a microfluidic chip, an MPC wire layer, an MPC antenna layer and an electronic device layer which are arranged in a stacked mode, as shown in figure 3.

The specific preparation method of the microfluidic passive sweat patch is as follows (the preparation process is shown in fig. 4):

(1) activating the PDMS film and the metal polymer conductor wire layer by using oxygen plasma, and then bonding the PDMS film and the metal polymer conductor wire layer together;

(2) connecting the electronic device layer (MLX 90129 chip) with the MPC antenna layer through conductive adhesive (Osbang 529);

(3) activating the MPC antenna layer and the MPC wire layer by using oxygen plasma at the power of 60W for 1 minute, crosslinking PDMS on the surfaces, and then bonding the two together;

(4) activating the surface by oxygen plasma, and connecting the ion sensor with the metal polymer conductor lead layer;

(5) activating a microfluidic channel layer on the PDMS membrane through oxygen plasma, placing a urea detection strip and a pH detection strip in the microfluidic channel, and then attaching the urea detection strip and the pH detection strip to the polydimethylsiloxane membrane to obtain a microfluidic chip;

(6) and coating a mixture (mass ratio is 10:1) of the PDMS prepolymer and the curing agent on the surface of the microfluidic chip, curing, and adhering a double-sided adhesive layer to obtain the microfluidic passive sweat patch.

The preparation method of the MPC wire layer and the MPC antenna layer comprises the following steps:

1g of liquid metal (gallium indium alloy, EGaIn) was added to a 5mL centrifuge tube containing 1mL of n-decanol and sonicated with a sonicator at 300W for 2 minutes. Ultrasonic treating EGaIn to obtain core containing EGaIn and Ga₂O₃Liquid Metal (LMs) particulate ink that is a shell. The resulting LMs ink was screen printed onto a polyethylene terephthalate (PET) substrate using a 200 mesh screen printing plate to obtain various patterns of LMs particles. After evaporation of the solvent, a mixture of PDMS prepolymer and curing agent (mass ratio 10:1) was poured onto the LMs particle pattern. After curing by baking at 80 ℃ for 1 hour, the PDMS film was peeled off from the PET substrate. Ga₂O₃The outer casing is due toThe shear stress breaks, thereby forming a conductive MPC antenna and wire. The MPC antenna profile is shown in fig. 5.

The preparation method of the urea test strip and the pH test strip comprises the following steps:

(1) and dissolving urease in deionized water to obtain a urease solution with the concentration of 0.01 mg/mL. The pH paper was cut into a circle having a diameter of 2mm, 5. mu.L of urease solution was dropped on the pH paper, and vacuum-dried in a desiccator for 60 minutes to obtain urea detection paper.

(2) And cutting the pH test paper into a circle with the diameter of 2mm to obtain a pH detection strip.

Above Na⁺Electrode, K⁺The electrode and reference electrode were prepared as follows:

(1)Na⁺an electrode: mixing 1% of Na⁺Ionophore III, 0.5% of tetrakis [3, 5-bis (trifluoromethyl) phenyl]Sodium borate, 33% polyvinyl chloride and bis (2-ethyl sebacate) dissolved in tetrahydrofuran to form a mixed solution, and the mixed solution is dripped on a carbon ink electrode and dried to obtain Na⁺And an electrode.

(2)K⁺An electrode: dripping a mixed solution formed by dissolving 2% of validamycin, 0.55% of sodium tetraphenylborate, 33% of polyvinyl chloride and bis (2-ethyl) sebacate in cyclohexanone onto a carbon ink electrode, and drying to obtain K⁺And an electrode.

(3) Reference electrode: and (3) dripping and casting a methanol solution of PVB, PEO-PPO-PEO, multi-walled carbon nanotubes and NaCl on the Ag/AgCl ink electrode, and drying to obtain the reference electrode.

The raw material sources used in this example were: polyvinyl chloride was purchased from Macklin under the designation P815910; PVB was purchased from Macklin under the designation P815776; PEO-PPO-PEO is available from Macklin under the brand number P822487; multiwall carbon nanotubes were purchased from aladdin under the designation C139823.

Performance test 1

The following tests were performed on the microfluidic passive sweat patch obtained in example 2:

1M NaCl and KCl solution was serially diluted 1024 times to obtain 1000, 500, 250, 125, 62.5, 31,25, 15.63, 7.81, 3.90, 1.95, 0.98 and 0mM of diluted standard solution. 50 μ L of solution was added to a microfluidic passive sweat patch and its signal monitored using an open circuit potential-time (OCPT) model. As the sodium and potassium ion concentrations increased, the open circuit potential of each sensor increased and exhibited near nernst behavior (fig. 6a, 6b, 7a, 7 b). Open circuit potential is proportional to logarithm of concentration, and standard curve of sodium ion is Y ═ 0.0196 × log₂(X)+0.1695，R²0.9984, standard curve of potassium ion is Y0.0146 × log²(X)+0.1773，R²0.9957. The physiologically relevant concentrations of sodium and potassium ions were approximately 66.3 + -46.0 mM and 9.0 + -4.8 mM, respectively. Phosphate buffered solutions with different pH values (5.91, 6.24, 6.47, 6.64, 6.81, 6.98, 7.17, 7.38, 7.73 and 8.04) were prepared to characterize the microfluidic passive sweat patch. It was observed that the response sensitivity was 55.50 + -6.31 mV/pH (FIG. 8a, FIG. 8b) at room temperature in the pH range of 5.91-8.04. Fig. 9a and 9b show the open circuit potential of the sensor in the microfluidic passive sweat patch at concentrations of 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.020 and 0.010 mM. Within this concentration range, the standard curve for the sensor in the microfluidic passive sweat patch is Y ═ 0.0151 × log₂(X)+0.2071，R²The natural concentration range of 0.33-0.65mM was also covered with 0.9862. Fig. 10 and 11 show the color response of the microfluidic passive sweat patch with different concentrations of uric acid. By analyzing the RGB values of the photographs, we found that the green (G) and red (R) values showed good correlation with the concentration of uric acid.

Example 3

The difference from example 1 is that no chitosan was added.

Example 4

The difference from example 2 is that the glucose electrode of example 1 was replaced with the glucose electrode of example 3.

Performance test 2

The following tests were performed on the microfluidic passive sweat patch obtained in example 4:

standard solution glucose solutions were prepared at concentrations of 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.020 and 0.010 mM. The prepared microfluidic passive sweat patch sensor was placed in solution and its open circuit potential measured. In this concentration range, the sensitivity of the glucose microfluidic passive sensor is not much different from example 2, but the sensor stability is relatively poor.

Comparative example 1

The difference from example 1 is that the polyaniline membrane was replaced with a commercial hydrogen ion permselective membrane.

Comparative example 2

The difference from example 2 is that the glucose electrode of example 1 was replaced with the glucose electrode of comparative example 1.

Performance test 3

The following tests were performed on the microfluidic passive sweat patch obtained in comparative example 2:

standard solution glucose solutions were prepared at concentrations of 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.020 and 0.010 mM. The prepared microfluidic passive sweat patch sensor was placed in solution and its open circuit potential measured. Within this concentration range, the sensitivity of the glucose microfluidic passive sensor is worse than example 2, and cannot cover the concentration range of glucose in sweat. Meanwhile, the voltage difference between different concentrations of the microfluidic passive sweat patch obtained in comparative example 1 is smaller than that of example 2. The standard curve of the sensor is Y ═ 0.0121 × log₂(X)+0.1201，R²0.9464. Both sensitivity and stability were poor compared to example 2.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.