A method of construction of a flexible wearable electrochromic sensing patch for detecting lactic acid in sweat

Abstract

The application belongs to the technical field of wearable sensor, and discloses a construction method of a flexible wearable electrochromic sensing patch for detecting lactic acid in sweat. The method comprises the following steps: preparing a LOx-Ag NDs-C / PET flexible electrode; and constructing a wearable electrochromic sensing patch based on the flexible electrode. The wearable sensing patch constructed by the application has the advantages of flexibility, non-invasiveness, visualization, reusability and the like. The prepared sensing patch relies on polyethylene terephthalate (PET) flexible material, and is based on the electrochromic principle of Prussian blue (PB). The naked eye semi-quantitative detection can be realized through the fading degree of PB, and the quantitative detection can be realized through the color change and current size. After the detection is completed, the color of PB is restored by using an applied voltage, so that the reusability of the sensing patch is realized. The selected flexible electrode substrate has a small volume and high flexibility, so that the influence of bending and environmental factors on the sensor is avoided, the application range and stability of the sensor are improved, and the cost and use conditions are reduced.

Description

Technical Field

[0001] This invention belongs to the field of wearable sensing technology, and provides a method for constructing a flexible wearable electrochromic sensing patch, which is used for rapid and sensitive detection of lactic acid in sweat. Background Technology

[0002] Lactic acid is an important physiological indicator in sweat, and its levels can assess physical condition, fatigue, and metabolic activity. In medicine, excessive lactic acid accumulation can lead to lactic acidosis, damaging the liver and kidneys and disrupting the body's pH balance. Researchers have been working to develop biosensors for lactic acid detection, with current research focusing on urine and blood lactic acid testing. However, the lack of portability and invasiveness limits testing conditions and increases the risk of infection. Therefore, there is an urgent need to develop a portable, highly integrated, non-invasive sensor to monitor human health.

[0003] Wearable sensing technology has been widely applied in fields such as medical diagnostics, motion monitoring, and artificial intelligence. In recent years, the rapid development of flexible electronics technology has provided a new direction for the development of wearable sensors. Flexible wearable sensors are electronic devices developed using flexible electronics technology. They can convert human biosignals into recordable electrical signals through signal transmission, helping humans acquire and analyze various information from the human body. They are characterized by their small size and...

[0004] It boasts advantages such as high integration, good biocompatibility, and low energy consumption. Compared with traditional wearable sensors, flexible wearable sensors combine mechanical flexibility with sensing functions. They can be directly attached to the surface of human skin for non-invasive detection, or implanted inside the human body to collect and monitor human physiological information.

[0005] The wearable electrochromic sensor patch constructed in this invention utilizes the transfer of electrons in a redox reaction to achieve visualization and semi-quantitative detection of lactic acid by measuring the degree of reduction of Prussian blue (PB) within a certain time, i.e., the degree of color change. After detection, the color of PB is restored by applying an external voltage, enabling the sensor to be reused. Summary of the Invention

[0006] This invention aims to provide a portable, integrated, and reusable flexible wearable sensing patch. Based on the catalytic oxidation of lactic acid by lactate oxidase, the generated hydrogen peroxide can undergo a redox reaction with Prussian blue, causing a color change in Prussian blue, thus enabling the visualization and non-invasive, sensitive detection of lactic acid in sweat samples.

[0007] This invention utilizes the hydrophilicity of agarose hydrogel to collect sweat into the area of ​​a lactate oxidase-modified silver nanodendritic-carbon paste / polyethylene terephthalate (LOx-Ag NDs-C / PET) electrode chip. Excess sweat can flow out of the sensing device through a flexible hydrophobic SiO2 membrane. Due to the specific catalysis of lactate oxidase on the circular LOx-Ag NDs-C / PET electrode, lactic acid is oxidized to pyruvate. The hydrogen peroxide generated in the reaction can react with Prussian blue on the Prussian blue-silver paste / polyethylene terephthalate (PB-Ag / PET) electrode, resulting in the blue Prussian blue (PB... red The color change of Prussian white (PB) within a certain time period, i.e., the degree of color change, enables the visual semi-quantitative detection of lactic acid. Simultaneously, electron transfer occurs during the redox reaction, enhancing the current output signal. Based on the change in output current and the blue value of the color-changing area, quantitative detection of lactic acid can be achieved. After detection, an external voltage is applied to revert the Prussian white back to Prussian blue, allowing for the reuse of the sensor chip.

[0008] The method for constructing a flexible wearable electrochromic sensor patch includes the following steps:

[0009] Step 1: Preparation of bio-enzyme-modified Ag NDs flexible electrodes:

[0010] (1.1) After cleaning the flexible polyethylene terephthalate (PET) substrate with ethanol, dry it under an infrared lamp; then place the PET substrate under the screen printing template; then uniformly drop conductive carbon paste ink onto the screen printing template, print it evenly with a squeegee, and record it as C / PET after drying.

[0011] (1.2) Dissolve AgNO3 and KNO3 powders in deionized water and stir magnetically until dissolved to obtain a mixture solution Ag + Electrodeposition solution; immersing the conductive carbon paste substrate (C / PET), platinum wire electrode, and Ag / AgCl electrode in Ag... + Ag NDs flexible electrodes, denoted as Ag NDs-C / PET, were prepared by cyclic voltammetry in the electrodeposition solution.

[0012] (1.3) Dissolve Na2HPO4 and NaH2PO4 in deionized water, make up to volume with a volumetric flask, and adjust the pH to obtain a PBS buffer solution; disperse lactate oxidase in PBS buffer containing BSA, incubate for a period of time, then add polyethylene glycol diglycidyl ether (PEGDE) and mix, then uniformly drop-coat Ag NDs-C / PET, and incubate for a period of time to obtain a lactate oxidase-modified Ag NDs flexible electrode, denoted as LOx-Ag NDs-C / PET;

[0013] Step 2, Fabrication of the PB flexible electrode:

[0014] (2.1) After cleaning the flexible PET substrate with ethanol, dry it under an infrared lamp; then place the PET substrate under the screen printing template; evenly drop conductive silver paste ink onto the screen printing template, print it evenly with a squeegee, and record it as Ag / PET after drying.

[0015] (2.2) Dissolve K3[Fe(CN)6] and FeCl3 powder in deionized water, add HCl solution dropwise, and stir magnetically until dissolved to obtain a mixture solution PB electrodeposition solution;

[0016] (2.3) The conductive silver paste substrate (Ag / PET), platinum wire electrode, and Ag / AgCl electrode were immersed in PB electrodeposition solution, and the PB flexible electrode was prepared by step voltage method, which was denoted as PB-Ag / PET.

[0017] Step 3: Preparation of SiO2 flexible hydrophobic film:

[0018] Hydrophobic SiO2 powder was dissolved in anhydrous ethanol solution, then perfluorooctyltriethoxysilane was added, and after magnetic stirring, the mixture was ultrasonicated to obtain a translucent hydrophobic SiO2 suspension.

[0019] Then, a semi-transparent hydrophobic SiO2 suspension was dropped onto a flexible PET substrate and uniformly spin-coated multiple times to obtain a flexible hydrophobic SiO2 film.

[0020] Step 4: Preparation of agarose gel electrolyte:

[0021] Agarose powder was dissolved in PBS buffer, heated in an oil bath and stirred until completely dissolved, and then the solution was dropped onto a circular mold. After cooling to room temperature, a circular agarose gel electrolyte was obtained, denoted as AG-gel.

[0022] Step 5: Construction of the flexible wearable electrochromic sensor patch:

[0023] The LOx-Ag NDs-C / PET obtained in step 1 is punched into a circle using a puncher to obtain a circular LOx-Ag NDs-C / PET.

[0024] Then, cut a circle out of the middle of the PB-Ag / PET obtained in step 2 to obtain a ring-shaped PB-Ag / PET.

[0025] Then, cut the SiO2 flexible hydrophobic film obtained in step 3 into a square, and then cut out a circle in the middle to obtain a square SiO2 flexible hydrophobic film with circular holes.

[0026] Finally, a ring-shaped PB-Ag / PET was placed inside the circular hole of the SiO2 flexible hydrophobic membrane, and LOx-Ag NDs-C / PET was placed in the center of the PB-Ag / PET circular hole. Then, AG-gel was covered on PB-Ag / PET to construct a flexible wearable electrochromic sensor patch.

[0027] In step (1.1),

[0028] The thickness of the flexible PET substrate is 0.05 mm.

[0029] The amount of conductive carbon paste used is 5 mg / cm³. 3 It has a matte black color, a sheet resistance of about 20Ω, a viscosity of 45-50Pa·s, a fineness of 5μm, and a temperature resistance of 180℃.

[0030] The scraping time is 10 seconds, and the number of scrapings is 5-8 times; the drying conditions are: drying at 60℃ for 2 hours.

[0031] In step (1.2),

[0032] Ag + The electrodeposition solution contained 0.1 mol / L KNO3 and 0.01 mol / L AgNO3; the dissolution temperature was 20℃.

[0033] The cyclic voltammetry method has a voltage range of -1 to 0V, a scan rate of 50mV / s, and a scan cycle of 7.

[0034] In step (1.3),

[0035] The PBS buffer solution has a pH of 7.40 and a concentration of 0.1 mol / L;

[0036] The concentration of lactate oxidase dispersed in PBS was 5 mg / mL;

[0037] The concentration of BSA dispersed in PBS was 2.5 mg / mL;

[0038] The PEDGE concentration was 3.8 mg / mL, the incubation temperature was 37℃, and the incubation time was 2 hours.

[0039] In step (2.1),

[0040] The thickness of the flexible PET substrate is 0.05 mm.

[0041] The conductive silver paste used is a silver-gray paste, and the dosage is 3 mg / cm³. 3 Sheet resistance ≤15mΩ, viscosity 10-15Pa·s, particle size ≤10μm, solid content 57±1wt%, temperature resistance 130℃;

[0042] The scraping time is 10 seconds, and the number of scrapings is 5-8 times; the drying conditions are: drying at 60℃ for 2 hours.

[0043] In step (2.2), the concentration of K3[Fe(CN)6] in the PB electrodeposition solution is 0.005 mol / L; the concentration of FeCl3 is 0.005 mol / L; the concentration of hydrochloric acid is 0.005 mol / L; and the dissolution temperature is 20℃.

[0044] In step (2.3), the step voltage method uses a voltage of 0.3V and a time of 75s.

[0045] In step 3, the concentration of hydrophobic SiO2 powder in the SiO2 suspension is 12 mg / mL, the concentration of perfluorooctyltriethoxysilane is 16 mg / mL, the stirring time is 18 h, the ultrasonic temperature is 20–30 °C, and the ultrasonic time is 20 min.

[0046] In step 4, the mass ratio of agarose powder to PBS buffer is 0.45g:15g; the oil bath temperature is 130℃.

[0047] In step 5,

[0048] The diameter of the cut circular LOx-Ag NDs-C / PET is 0.6 cm, and the area is 9π mm². 2 ;

[0049] The circular PB-Ag / PET ring has an inner diameter of 1 cm, an outer diameter of 2 cm, and a working area of ​​0.75π cm². 2 ;

[0050] The working area of ​​the square-shaped, round-hole SiO2 flexible hydrophobic membrane is (9-π) cm². 2 The diameter of the round hole is 2cm;

[0051] 0cm < inner diameter of PB-Ag / PET ring - diameter of circular LOx-Ag NDs-C / PET < 1cm; the gap between LOx-AgNDs-C / PET and PB-Ag / PET allows for sweat collection; the SiO2 flexible hydrophobic membrane is used to remove excess sweat, and medical double-sided tape is attached to the reverse side to adhere to the skin for actual sweat detection.

[0052] The flexible wearable electrochromic sensor patch prepared according to this invention is used for lactic acid-specific detection. The specific steps are as follows:

[0053] (1) After incubating lactate oxidase on the surface of LOx-Ag NDs-C / PET for a period of time, it was placed together with PB-Ag / PET into an electrolytic cell containing electrolyte. The two electrodes were connected by an electrochemical workstation, and lactic acid of different concentrations was added to the electrolytic cell. The current-time (IT) signal was collected, and standard curves were plotted for current versus time, current versus lactic acid concentration, and lactic acid concentration versus PB-Ag / PET color.

[0054] (2) In actual sweat sample testing, after incubating lactate oxidase on the surface of LOx-Ag NDs-C / PET for a period of time, the lactic acid in the sweat was collected on the surface of LOx-Ag NDs-C / PET, and PB-Ag / PET produced a color change.

[0055] (3) Collect color signals from sweat samples with unknown lactic acid concentration using the method in step (2), and substitute them into the color standard curve of lactic acid concentration and PB-Ag / PET to obtain the lactic acid concentration in the unknown sweat samples.

[0056] In step (1), the lactic acid concentration is 0.25 mmol / L to 35 mmol / L, and the detection volume is 50 μL.

[0057] The beneficial effects of this invention are:

[0058] This invention prepares LOx-Ag NDs-C / PET and PB-Ag / PET flexible electrodes, constructs a flexible wearable sensing platform, and introduces lactate oxidase to catalyze the oxidation of lactate, causing a color change in the Prussian blue electrode for visual detection. A rapid, sensitive, and accurate method for detecting lactate has been successfully established, and its features and advantages are described below:

[0059] (1) In this invention, LOx-Ag NDs-C / PET and PB-Ag / PET flexible electrodes were prepared and used in the construction of a wearable sensing platform. Using Ag NDs electrodes with excellent conductivity as electrode materials is beneficial for enzyme loading and at the same time, it improves the electron transfer rate in redox reactions.

[0060] (2) The PB-Ag / PET flexible electrode prepared in this invention is applied to the lactic acid sensing system to construct a flexible wearable electrochromic visualization sensing platform, realizing the visualization detection of lactic acid. The current value and the lactic acid concentration show a good linear relationship in the range of 0.25 mmol / L to 35 mmol / L, and the detection limit is as low as 6.3 nM.

[0061] (3) The PB-Ag / PET flexible electrode prepared by the present invention can restore the color to the state before detection by applying an external voltage after detecting lactic acid, so that the sensor can be reused and costs are saved.

[0062] (4) Compared with traditional detection methods, the flexible wearable electrochromic sensor proposed in this invention has the advantages of good specificity, low cost and reusability in detecting lactic acid. Attached Figure Description

[0063] Figure 1 Detection mechanism of flexible wearable electrochromic sensor.

[0064] Figure 2 Scanning electron microscope images of nanomaterials: (A) Ag NDs-C / PET; (B) LOx-Ag NDs-C / PET; (C) AC impedance diagrams of flexible electrodes C / PET (a), Ag NDs-C / PET (b), LOx-Ag NDs-C / PET (c).

[0065] Figure 3 (A) Current versus time at different lactic acid concentrations; (B) Linear relationship between current and lactic acid concentration; (C) Linear relationship between color change of PB-Ag / PETD electrode and lactic acid concentration.

[0066] Figure 4 (A) Sensor patch detection performance graph after storage for different times; (B) Sensor patch detection performance graph after repeated use for different numbers of times. Detailed Implementation

[0067] The present invention will now be described in detail with reference to examples and accompanying drawings, but the present invention is not limited to these embodiments.

[0068] Figure 1 A schematic diagram of the mechanism of the constructed flexible wearable electrochromic sensor.

[0069] Example 1:

[0070] (1) Preparation of bioenzyme-modified AgNDs flexible electrodes:

[0071] First, clean the flexible PET substrate with anhydrous ethanol and dry it under an infrared lamp for 5 minutes. Then, place the PET substrate under the screen printing template. Next, evenly drop 2g of conductive carbon paste ink onto the screen printing template, print it evenly 5-8 times with a squeegee, and dry it at 60°C for 2 hours.

[0072] Dissolve 0.0816 g of silver nitrate and 0.4853 g of potassium nitrate powder in 48 mL of deionized water and stir magnetically until dissolved to obtain a mixture solution Ag. + Electrodeposition solution; then, the conductive carbon paste substrate, platinum wire electrode, and Ag / AgCl electrode are immersed in Ag... +Ag NDs were prepared in an electrodeposition solution using cyclic voltammetry: the voltage range was -1 to 0 V, the scan rate was 50 mV / s, and the scan period was 7 cycles. A flexible conductive carbon paste with an area of ​​3 cm² was used. 2 The actual working area of ​​the circle after cutting is 9π mm. 2 .

[0073] Figure 2 (A) is a scanning electron microscope image of Ag NDs obtained in step (1), with dendrite length of about 0.5 μm.

[0074] 8.95 g of sodium monohydrogen phosphate and 3.90 g of sodium dihydrogen phosphate were dissolved in 100 mL of deionized water, and the volume of each solution was adjusted to 250 mL. The pH was adjusted to 7.40 to obtain a PBS buffer solution. Then, BSA was dispersed in the PBS buffer solution at a concentration of 2.5 mg / mL, and lactate oxidase was dispersed in the above solution at a concentration of 5 mg / mL. After incubation for a period of time, PEGDE at a concentration of 3.8 mg / mL was added, and the mixture was uniformly drop-coated onto Ag NDs-C / PET. After incubation for 2 h, the lactate oxidase-modified Ag NDs flexible electrode LOx-Ag NDs-C / PET was obtained.

[0075] Figure 2 (B) is a scanning electron microscope image of LOx-Ag NDs-C / PET obtained in step (1), showing that the enzyme was successfully modified on Ag NDs.

[0076] Figure 2 (C) is the AC impedance diagram of LOx-Ag NDs-C / PET obtained in step (1). The impedance increases after the enzyme is modified on Ag NDs-C / PET.

[0077] (2) Fabrication of PB flexible electrode:

[0078] First, clean the flexible PET substrate with anhydrous ethanol and dry it under an infrared lamp for 5 minutes. Then, place the PET substrate under the screen printing template. Next, evenly drop 1.5g of conductive silver paste ink onto the screen printing template, print it evenly 5-8 times with a squeegee, and dry it at 60℃ for 2 hours.

[0079] Then, 0.1646 g of potassium ferricyanide and 0.1351 g of ferric chloride powder were dissolved in 50 mL of deionized water, and 0.5 mL of 0.05 mol / L HCl solution was added dropwise to dilute the mixture to 100 mL. The mixture was magnetically stirred for 20 min until dissolved, at a reaction temperature of 20 °C for 30 min, to obtain a PB electrodeposition solution. Finally, a PB-Ag / PET flexible electrode was prepared by immersing a conductive silver paste substrate, a platinum wire electrode, and an Ag / AgCl electrode in the PB electrodeposition solution using a stepwise voltage method: the voltage was 0.3 V and the time was 75 s. A πcm flexible conductive silver paste substrate was selected. 2 The actual working area of ​​the annular shape after cutting is 0.75π cm². 2 .

[0080] (3) Preparation of flexible hydrophobic SiO2 films:

[0081] 0.3 g of hydrophobic SiO2 powder was dissolved in 25 mL of anhydrous ethanol solution, and then 0.4 g of perfluorooctyltriethoxysilane was added. After magnetic stirring for 18 h, the mixture was sonicated for 20 min (temperature 20-30℃) to obtain a semi-transparent hydrophobic SiO2 suspension. Then, 0.5 mL of the semi-transparent hydrophobic SiO2 suspension was dropped onto a flexible PET substrate and uniformly spin-coated 3 times to obtain a flexible hydrophobic SiO2 film.

[0082] (4) Preparation of agarose gel electrolyte:

[0083] Dissolve 0.45g of agarose powder in 15g of PBS, heat in an oil bath at 130℃ and stir for 30min until completely dissolved. Then drop the solution onto a circular mold with a diameter of 2cm. After cooling to room temperature, a circular agarose gel electrolyte AG-gel is obtained.

[0084] (5) Construction of flexible wearable electrochromic sensor patch

[0085] First, the obtained bio-enzyme modified electrode was punched into a circle using a punch with a 6mm diameter hole to obtain a circular LOx-Ag NDs-C / PET.

[0086] Then, a circle with a diameter of 1 cm is cut out from the middle of the PB flexible electrode to obtain a ring-shaped PB-Ag / PET.

[0087] Then, the SiO2 flexible hydrophobic membrane is cut into a square with a side length of 3cm, and a circle with a diameter of 2cm is cut out in the middle to obtain a square SiO2 flexible hydrophobic membrane with circular holes.

[0088] Then, a ring-shaped PB-Ag / PET was placed inside the circular hole of the SiO2 flexible hydrophobic membrane, and LOx-Ag NDs-C / PET was placed in the center of the PB-Ag / PET circular hole. Finally, AG-gel was covered on PB-Ag / PET to construct a flexible wearable electrochromic sensor patch.

[0089] (6) Detection of lactic acid using flexible wearable electrochromic sensor patch

[0090] After incubating lactate oxidase on the surface of LOx-Ag NDs-C / PET for 2 hours, it was placed together with PB-Ag / PET into an electrolytic cell containing electrolyte. The two electrodes were connected using an electrochemical workstation, and different concentrations of lactic acid were added dropwise into the electrolytic cell. The current-time (IT) signal was collected, and standard curves were plotted for current versus time, current versus lactic acid concentration, and lactic acid concentration versus PB-Ag / PET color. After the detection was completed, the color of the PB-Ag / PET electrode could be restored by applying an external voltage, enabling multiple reuses.

[0091] Test results as follows Figure 3 :

[0092] Figure 3 When the lactic acid concentration is in the range of 0.25 mM to 35 mM, the signal response of the wearable sensor increases with increasing lactic acid concentration, and the change in current exhibits a good linear relationship with the lactic acid concentration, with the linear equation being I = -1.6424 C. LAC -14.44 (mmol / L), with a correlation coefficient of 0.996 and a detection limit of 6.3 nM; the color change of the PB-Ag / PET electrode showed a good linear relationship with the lactic acid concentration, with the linear equation being B = 1.0143C. LAC +155.27, with a correlation coefficient of 0.997.

[0093] Figure 4 Figure A shows the sensor performance test graph after storage for different times. After ten days of storage, the performance did not decrease significantly.

[0094] Figure 4 Figure B shows the sensor performance test results after being repeated a different number of times. After being used ten times, the performance remained stable.

[0095] Example 2:

[0096] (1) Preparation of bioenzyme-modified AgNDs flexible electrodes:

[0097] First, clean the flexible PET substrate with anhydrous ethanol and dry it under an infrared lamp for 5 minutes. Then, place the PET substrate under the screen printing template. Next, evenly drop 2g of conductive carbon paste ink onto the screen printing template, print it evenly 5-8 times with a squeegee, and dry it at 60°C for 2 hours.

[0098] Dissolve 0.0408 g of silver nitrate and 0.2426 g of potassium nitrate powder in 24 mL of deionized water and stir magnetically until dissolved to obtain a mixture solution Ag. + Electrodeposition solution; then, the conductive carbon paste substrate, platinum wire electrode, and Ag / AgCl electrode are immersed in Ag... + Ag NDs were prepared in an electrodeposition solution using cyclic voltammetry: the voltage range was -1 to 0 V, the scan rate was 50 mV / s, and the scan period was 5 cycles. A flexible conductive carbon paste with an area of ​​3 cm² was used. 2 The actual working area of ​​the circle after cutting is 9π mm. 2 .

[0099] 8.95 g of sodium monohydrogen phosphate and 3.90 g of sodium dihydrogen phosphate were dissolved in 100 mL of deionized water, and the volume of each was adjusted to 250 mL. The pH was adjusted to 7.40 to obtain a PBS buffer solution. Then, BSA was dispersed in the PBS buffer at a concentration of 2.5 mg / mL, and lactate oxidase was dispersed in the above solution at a concentration of 5 mg / mL. After incubation for a period of time, PEGDE was dispersed in the above solution at a concentration of 3.8 mg / mL. The mixture was then uniformly drop-coated onto Ag NDs-C / PET. After incubation for 2 h, the lactate oxidase-modified Ag NDs flexible electrode LOx-Ag NDs-C / PET was obtained.

[0100] Steps (2), (3), (4), (5) and (6) are the same as steps (2), (3), (4), (5) and (6) in Example 1.

[0101] Example 3:

[0102] (1) Preparation of bioenzyme-modified AgNDs flexible electrodes:

[0103] First, clean the flexible PET substrate with anhydrous ethanol and dry it under an infrared lamp for 5 minutes. Then, place the PET substrate under the screen printing template. Next, evenly drop 2g of conductive carbon paste ink onto the screen printing template, print it evenly 5-8 times with a squeegee, and dry it at 60°C for 2 hours.

[0104] Dissolve 0.1224 g of silver nitrate and 0.7278 g of potassium nitrate powder in 72 mL of deionized water and stir magnetically until dissolved to obtain a mixture solution Ag. + Electrodeposition solution; then, the conductive carbon paste substrate, platinum wire electrode, and Ag / AgCl electrode are immersed in Ag... + Ag NDs were prepared in an electrodeposition solution using cyclic voltammetry: the voltage range was -1 to 0 V, the scan rate was 50 mV / s, and the scan period was 10 cycles. A flexible conductive carbon paste with an area of ​​3 cm² was used. 2The actual working area of ​​the circle after cutting is 9π mm. 2 .

[0105] 8.95 g of sodium monohydrogen phosphate and 3.90 g of sodium dihydrogen phosphate were dissolved in 100 mL of deionized water, and the volume of each solution was adjusted to 250 mL. The pH was adjusted to 7.40 to obtain a PBS buffer solution. Then, BSA was dispersed in the PBS buffer solution at a concentration of 2.5 mg / mL, and lactate oxidase was dispersed in the above solution at a concentration of 5 mg / mL. After incubation for a period of time, PEGDE was dispersed in the above solution at a concentration of 3.8 mg / mL. The mixture was then uniformly drop-coated onto Ag NDs-C / PET. After incubation for 2 h, the lactate oxidase-modified Ag NDs flexible electrode LOx-Ag NDs-C / PET was obtained.

[0106] Steps (2), (3), (4), (5) and (6) are the same as steps (2), (3), (4), (5) and (6) in Example 1.

Claims

1. A method for constructing a flexible wearable electrochromic sensor patch for detecting lactic acid in sweat, characterized in that, Includes the following steps: Step 1: Preparation of bio-enzyme-modified Ag NDs flexible electrodes: (1.1) After cleaning the flexible polyethylene terephthalate (PET) substrate with ethanol, dry it under an infrared lamp; then place the PET substrate under the screen printing template; then uniformly drop conductive carbon paste ink onto the screen printing template, print it evenly with a squeegee, and after drying, record it as conductive carbon paste substrate C / PET. (1.2) Dissolve AgNO3 and KNO3 powders in deionized water and stir magnetically until dissolved to obtain a mixture solution Ag + Electrodeposition solution; immersing conductive carbon paste substrate C / PET, platinum wire electrode, and Ag / AgCl electrode in Ag... + Ag NDs flexible electrodes, denoted as Ag NDs-C / PET, were prepared by cyclic voltammetry in the electrodeposition solution. (1.3) Dissolve Na2HPO4 and NaH2PO4 in deionized water, make up to volume with a volumetric flask, and adjust the pH to obtain PBS buffer solution; Lactate oxidase was dispersed in PBS buffer containing BSA and incubated for a period of time. Then, polyethylene glycol diglycidyl ether (PEGDE) was added and mixed. The mixture was then uniformly drop-coated onto Ag NDs-C / PET and incubated for a period of time to obtain a lactate oxidase-modified Ag NDs flexible electrode, denoted as LOx-Ag NDs-C / PET. Step 2, Fabrication of the PB flexible electrode: (2.1) After cleaning the flexible PET substrate with ethanol, dry it under an infrared lamp; then place the PET substrate under the screen printing template; evenly drop conductive silver paste ink onto the screen printing template, print it evenly with a squeegee, and after drying, record it as conductive silver paste substrate Ag / PET. (2.2) Dissolve K3[Fe(CN)6] and FeCl3 powder in deionized water, add HCl solution dropwise, and stir magnetically until dissolved to obtain a mixture solution PB electrodeposition solution; (2.3) The conductive silver paste substrate Ag / PET, platinum wire electrode, and Ag / AgCl electrode were immersed in PB electrodeposition solution, and the PB flexible electrode was prepared by step voltage method, which is denoted as PB-Ag / PET. Step 3: Preparation of SiO2 flexible hydrophobic film: Hydrophobic SiO2 powder was dissolved in anhydrous ethanol solution, then perfluorooctyltriethoxysilane was added, and after magnetic stirring, the mixture was ultrasonicated to obtain a translucent hydrophobic SiO2 suspension. Then, a semi-transparent hydrophobic SiO2 suspension was dropped onto a flexible PET substrate and uniformly spin-coated multiple times to obtain a flexible hydrophobic SiO2 film. Step 4: Preparation of agarose gel electrolyte: Agarose powder was dissolved in PBS buffer, heated in an oil bath and stirred until completely dissolved, and then the solution was dropped onto a circular mold. After cooling to room temperature, a circular agarose gel electrolyte was obtained, denoted as AG-gel. Step 5: Construction of the flexible wearable electrochromic sensor patch: The LOx-Ag NDs-C / PET obtained in step 1 is punched into a circle using a puncher to obtain a circular LOx-Ag NDs-C / PET. Then, cut a circle out of the middle of the PB-Ag / PET obtained in step 2 to obtain a ring-shaped PB-Ag / PET. Then, cut the SiO2 flexible hydrophobic film obtained in step 3 into a square, and then cut out a circle in the middle to obtain a square SiO2 flexible hydrophobic film with circular holes. Finally, a ring-shaped PB-Ag / PET was placed inside the circular hole of the SiO2 flexible hydrophobic membrane, and LOx-Ag NDs-C / PET was placed in the center of the PB-Ag / PET circular hole. Then, AG-gel was covered on PB-Ag / PET to construct a flexible wearable electrochromic sensor patch.

2. The construction method as described in claim 1, characterized in that, In step (1.1), The thickness of the flexible PET substrate is 0.05 mm. The amount of conductive carbon paste used is 5 mg / cm. 3 It has a matte black color, a sheet resistance of 20 Ω, a viscosity of 45-50 Pa·s, a fineness of 5 μm, and a temperature resistance of 180 ℃. The scraping time is 10 seconds, and the number of times is 5-8; the drying conditions are: drying at 60 °C for 2 hours.

3. The construction method as described in claim 1, characterized in that, In step (1.2), Ag + The electrodeposition solution contained 0.1 mol / L KNO3 and 0.01 mol / L AgNO3. The dissolution temperature was 20 °C. The cyclic voltammetry voltage range was -1 to 0 V, the scan rate was 50 mV / s, and the scan period was 7.

4. The construction method as described in claim 1, characterized in that, In step (1.3), The PBS buffer solution has a pH of 7.40 and a concentration of 0.1 mol / L; The concentration of lactate oxidase dispersed in PBS was 5 mg / mL; The concentration of BSA dispersed in PBS was 2.5 mg / mL; The PEDGE concentration was 3.8 mg / mL, the incubation temperature was 37 ℃, and the incubation time was 2 h.

5. The construction method as described in claim 1, characterized in that, In step (2.1), The thickness of the flexible PET substrate is 0.05 mm. The conductive silver paste used is a silver-gray paste, and the dosage is 3 mg / cm³. 3 Sheet resistance ≤15 mΩ, viscosity 10-15 Pa·s, particle size ≤ 10 μm, solid content 57±1 wt%, temperature resistance 130 ℃; The scraping time is 10 seconds, and the number of times is 5-8; the drying conditions are: drying at 60 °C for 2 hours. In step (2.2), the concentration of K3[Fe(CN)6] in the PB electrodeposition solution is 0.005 mol / L; the concentration of FeCl3 is 0.005 mol / L; the concentration of hydrochloric acid is 0.005 mol / L; and the dissolution temperature is 20 ℃. In step (2.3), the step voltage method uses a voltage of 0.3 V for 75 s.

6. The construction method as described in claim 1, characterized in that, In step 3, the concentration of hydrophobic SiO2 powder in the SiO2 suspension is 12 mg / mL, and the concentration of perfluorooctyltriethoxysilane is 16 mg / mL; the stirring time is 18 h, the ultrasonic temperature is 20~30 ℃, and the ultrasonic time is 20 min.

7. The construction method as described in claim 1, characterized in that, In step 4, the mass ratio of agarose powder to PBS buffer is 0.45 g: 15 g; the oil bath temperature is 130 ℃.

8. The construction method as described in claim 1, characterized in that, In step 5, The diameter of the cut circular LOx-Ag NDs-C / PET is 0.6 cm, and the area is 9π mm². 2 ; The circular PB-Ag / PET ring has an inner diameter of 1 cm, an outer diameter of 2 cm, and a working area of ​​0.75π cm². 2 ; The working area of ​​the square-shaped, round-hole SiO2 flexible hydrophobic membrane is (9-π) cm². 2 The diameter of the round hole is 2cm; 0cm < inner diameter of PB-Ag / PET ring - diameter of circular LOx-Ag NDs-C / PET < 1cm.

9. The use of a flexible wearable electrochromic sensor patch constructed by the construction method according to any one of claims 1 to 8 for detecting lactic acid in sweat.

10. The use as described in claim 9, characterized in that, The steps are as follows: (1) After incubating lactate oxidase on the surface of LOx-Ag NDs-C / PET for a period of time, it was placed together with PB-Ag / PET into an electrolytic cell containing electrolyte. The two electrodes were connected by an electrochemical workstation, and lactic acid of different concentrations was added to the electrolytic cell. The current-time (IT) signal was collected, and standard curves were plotted for current versus time, current versus lactic acid concentration, and lactic acid concentration versus PB-Ag / PET color. The lactic acid concentration ranged from 0.25 mmol / L to 35 mmol / L, and the detection volume was 50 µL. (2) In the actual sweat sample test, after incubating lactate oxidase on the surface of LOx-Ag NDs-C / PET for a period of time, the lactic acid in the sweat was collected on the surface of LOx-Ag NDs-C / PET, and PB-Ag / PET produced a color change. (3) Collect color signals from sweat samples with unknown lactic acid concentration using the method in step (2), and substitute them into the color standard curve of lactic acid concentration and PB-Ag / PET to obtain the lactic acid concentration in the unknown sweat samples.

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