A method for preparing a flexible wearable electrochemical sensor for sweat cortisol detection

Abstract

The application discloses a preparation method of a flexible wearable electrochemical sensor for sweat cortisol detection. The method comprises the following steps: firstly, a carbon nanotube / polyurethane (CNT / PU) film with conductivity is prepared by using an improved wet spinning method; then, a carbon nanotube-based composite film electrode is prepared by hot depositing a carbon nanotube / polyvinyl alcohol (CNT / PVA) solution on the CNT / PU film; subsequently, streptavidin functionalized Ni-Co MOF nanomaterial is modified on the surface of the electrode; finally, the flexible stretchable film electrode for cortisol detection is prepared by incubating and combining with biotinylated cortisol aptamer. After that, the film electrode is combined with a sweat collecting cloth, fixed on a film with a polydimethylsiloxane (PDMS) flexible substrate, and covered with a square frame type PDMS waterproof bandage as a sweat collecting channel, so as to prepare a wearable electrochemical sensor for real-time monitoring of cortisol in human sweat.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of an electrochemical sensor, in particular to a preparation method of a flexible wearable electrochemical sensor for sweat cortisol detection. BACKGROUND

[0002] Cortisol, as a hormone released by the adrenal cortex under stress, is a medically proven stress biomarker. When the human body is under continuous and long-term stress for a long time, cortisol will be abnormally released, which may cause metabolic disorders or damage to the immune system. Therefore, detecting cortisol levels will help understand the current physiological state of the body, thereby preventing adverse consequences.

[0003] Cortisol is usually determined by electrochemical and enzyme-linked immunosorbent (ELISA), gas chromatography, analytical-mass spectrometry, radioimmunoassay, surface-enhanced Raman spectroscopy, and ultraviolet spectroscopy. These methods usually require professional technicians to operate, and have characteristics such as large sample consumption and long time consumption, and the accuracy of these methods needs to be further improved. SUMMARY

[0004] The purpose of the present application is to provide a painless and convenient preparation method of a flexible wearable electrochemical sensor that can be applied to continuous monitoring of cortisol in human sweat.

[0005] Technical scheme: The preparation method of the flexible wearable electrochemical sensor for sweat cortisol detection according to the present application comprises the following steps:

[0006] (1) Dissolve polyurethane (PU) particles, then add carbon nanotubes (CNT) and stir to obtain a carbon nanotube / polyurethane (CNT / PU) mixed solution, then spread the mixed solution in a mold, and after standing, peel off to obtain a CNT / PU (CP) film.

[0007] (2) Dissolve polyvinyl alcohol (PVA), then add carbon nanotubes and stir to form a mixed solution, modify the carbon nanotube / polyvinyl alcohol (CNT / PVA) solution on the CP film by thermal deposition method, dry to obtain a carbon nanotube / polyvinyl alcohol / carbon nanotube / polyurethane (CCP) film, and finally cut the film into an electrode shape;

[0008] (3) Drop coat streptavidin (SA) modified nickel-cobalt MOF (Ni-Co MOF) on the electrode, dry, then add biotinylated aptamer on the surface of the material for incubation, and finally add bovine serum albumin solution for blocking, which serves as the working electrode; drop coat Ag / AgCl slurry on the CCP electrode, dry, and serve as the reference electrode; and the unmodified CCP electrode serves as the counter electrode.

[0009] Preferably, in the step (1), the concentration of the polyurethane in N,N-dimethylacetamide is 0.034-0.040 g / mL, the stirring temperature during dissolution is 80-85℃, the stirring time is 30-60 min, the mass ratio of the polyurethane to the carbon nanotube is 10-1:1-2, more preferably, the mass ratio of the polyurethane to the carbon nanotube is 2-1:1, the stirring time is 20-24 h, and the standing time in deionized water is 15-30 min.

[0010] Preferably, in the step (2), the concentration of the polyvinyl alcohol solution is 0.01-0.05 g / mL, the mass ratio of the polyvinyl alcohol to the carbon nanotube is 2-1:1-5, more preferably, the mass ratio of the polyvinyl alcohol to the carbon nanotube is 1:1-2:1, and the stirring temperature in both times is 80-85℃, and the stirring time in both times is 30-60 min.

[0011] Preferably, in the step (3), the preparation method of the streptavidin-modified Ni-Co MOF is as follows: dissolving amino terephthalic acid, nickel nitrate hexahydrate and cobalt nitrate hexahydrate, adding sodium hydroxide solution, then transferring the mixed solution to a reaction kettle for reaction to obtain a Ni-Co MOF material; dispersing the Ni-Co MOF in a phosphate buffer solution, taking part of the Ni-Co MOF dispersion, adding glutaraldehyde and stirring, centrifuging, and re-dispersing in the phosphate buffer solution, then adding streptavidin and incubating overnight, centrifuging and washing to obtain the streptavidin-modified Ni-Co MOF.

[0012] Preferably, the concentration of the Ni-Co MOF dispersed in the phosphate buffer solution is 5-25 mg / ml.

[0013] Preferably, in the step (3), the concentration of the aptamer is 1-2 μg / mL, the modification volume is 4-5 μL, and the incubation time is 45-50 min.

[0014] Invention principle: MOF material has good catalytic effect on hydrogen peroxide, but due to the coupling of aptamer-cortisol on the surface of MOF to form a complex, the catalytic electrolyte (containing hydroquinone-hydrogen peroxide) current signal of MOF is weakened, thereby realizing quantitative detection of cortisol.

[0015] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: (1) The prepared sensor can be combined with a polydimethylsiloxane (PDMS) flexible film and attached to the skin, and applied to real-time monitoring of human disease signals; (2) The prepared wearable biosensor has excellent detection performance and good stability, has a low detection limit for cortisol detection, and the oxidation peak current can still maintain 89.1% of the original after storage for 7 days in a daily environment; (3) The prepared sensor has good tensile properties and can still exhibit good electrochemical behavior after the fiber is stretched to 20%. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a physical diagram of a wearable biosensor electrode;

[0017] Figure 2 is a differential pulse voltammogram of different concentrations of cortisol added in a phosphate buffer solution containing hydroquinone and hydrogen peroxide;

[0018] Figure 3 is a column chart of the oxidation peak current of the sensor after storage for 7 days in a daily environment. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be further described below in combination with the drawings.

[0020] Example 1

[0021] 1. Preparation of a biosensor

[0022] (1) Preparation of a CP film:

[0023] Dissolve 0.068 g of polyurethane (PU) in 2 mL of N,N-dimethylacetamide (DMAC) and stir at 80°C for 30 min, then add 0.034 g of CNT to it, and continue stirring for 24 h to obtain a CNT / PU homogenate. Then evenly spread the original solution in a square mold, add deionized water, and after standing for 15 min, peel off to obtain a CNT / PU (CP) film.

[0024] (2) Preparation of a CNT / PVA solution and a CCP film electrode:

[0025] Dissolve 0.02 g of polyvinyl alcohol (PVA) in 2 mL of deionized water, stir at 80°C for 30 min, then add 0.02 g of carbon nanotubes (CNT), and continue stirring for 24 h to prepare a CNT / PVA solution. Disperse the CNT / PVA solution on the surface of the CNT / PU film by thermal deposition, and after drying, obtain a CCP film. Cut the prepared CCP film into a specific three-electrode shape.

[0026] (3) Preparation of streptavidin-modified Ni-Co MOF:

[0027] Amino terephthalic acid, nickel nitrate hexahydrate and cobalt nitrate hexahydrate were dissolved in N,N-dimethylformamide, then 2 mL of 0.4 M sodium hydroxide solution was slowly added under stirring; subsequently, the mixed solution was transferred to a reaction kettle and reacted at 100 °C for 10 h, washed with N,N-dimethylformamide and ethanol after cooling to room temperature, and vacuum dried to obtain a Ni-Co MOF material. 2 mL of 2.5 wt% glutaraldehyde was added to 2 mL of Ni-Co MOF (15 mg / mL) dispersed in a phosphate buffer solution, and the product was centrifuged (4000 rpm) and washed after stirring at 37 °C for 3 h, and then redispersed in 1 mL of phosphate buffer solution. Subsequently, 20 μL of 10 μg / mL streptavidin was mixed with 100 μL of the mixture and incubated overnight in a shaker. After centrifugation (3000 rpm) and washing, streptavidin-modified Ni-Co MOF was obtained and stored at 4 °C for later use.

[0028] (4) Preparation of flexible biosensor electrode:

[0029] The flexible biosensor electrode is composed of three electrodes: a counter electrode, a reference electrode and a working electrode.

[0030] 5 μL of streptavidin-modified Ni-Co MOF solution was dropped on the CCP electrode, dried at 37 °C, and then 5 μL of 1 μg / mL biotinylated DNA aptamer (the DNA aptamer used is referred to in the literature

Dalirirad, S.; Steckl, A. J., Aptamer-based lateral flow assay for point of care cortisol detection in sweat. Sensor. Actua. B-Chem. 2019, 283, 79-86.

[0031] A commercial Ag / AgCl reference electrode paste was dropped onto the CCP electrode and dried at 60 °C for 30 minutes to form a reference electrode.

[0032] The unmodified CCP electrode is used as the counter electrode.

[0033] The electrolyte is a phosphate buffer solution containing 10 mM hydroquinone and 20 mM hydrogen peroxide, with a pH value of 7.4 and a concentration of 0.1 M.

[0034] The three electrodes are combined with a polydimethylsiloxane (PDMS) flexible substrate, a sweat-absorbing cloth, and a square-shaped waterproof bandage made of PDMS as a sweat channel, to obtain a sensor device that can be attached to the skin, as shown in FIG. 1. Figure 1 The prepared sensor has good stretchability and can still exhibit good electrochemical behavior after the fiber is stretched by 20%.

[0035] 2. Cortisol detection of the sensor

[0036] The three electrodes of the prepared sensor are connected to an electrochemical workstation, and the sensor is placed in a phosphate buffer solution containing 10 mM hydroquinone and 20 mM hydrogen peroxide for detection.

[0037] Example 2

[0038] The preparation method of Example 1 is referred to, except that the mass ratio of CNT and PU is adjusted in the preparation of the CNT / PU film.

[0039] The specific operation is as follows: CNT / PU with a ratio of 1:10, 1:5, 1:2, 1:1, and 2:1 is mixed with 2 mL N.N-dimethylacetamide to obtain CNT / PU homogenate with different ratios.

[0040] Example 3

[0041] The preparation method of Example 1 is referred to, except that the mass ratio of CNT and PVA is adjusted in the preparation of the CNT / PVA solution.

[0042] The specific operation is as follows: CNT / PVA with a ratio of 5:1, 3:1, 2:1, 1:1, and 1:2 is mixed with 2 mL deionized water to obtain a solution with different ratios.

[0043] Example 4

[0044] The preparation method of Example 1 is referred to, except that the concentration of Ni-Co MOF in the functionalized Ni-Co MOF dispersion liquid is adjusted in the preparation of the working electrode.

[0045] The specific operation is as follows: 5 mg, 10 mg, 15 mg, 20 mg, and 25 mg of Ni-Co MOF powder is dispersed in 1 mL of phosphate buffer solution to form a dispersion liquid with different concentrations of Ni-Co MOF.

[0046] The sensor electrodes prepared in the above embodiments were used to detect cortisol. Figure 2 Shown are differential pulse voltammograms of the reaction mixture with different concentrations of cortisol added to a phosphate buffer solution containing 10 mM hydroquinone and 20 mM hydrogen peroxide.

[0047] Depend on Figure 2 As can be seen, the current signal gradually decreases with increasing cortisol concentration, indicating that as cortisol binds to the MOF material through the aptamer, the catalytic effect of Ni-Co MOF on hydrogen peroxide gradually weakens. Furthermore, when the mass ratio of CNT to PVA is 1:1, the mass ratio of CNT to PU is 1:2, and the modification concentration of functionalized Ni-Co MOF on the electrode is 15 mg / mL, the sensor's oxidation peak current is the highest. This indicates that these are the optimal ratios and modification concentrations.

[0048] Depend on Figure 3 As shown, after the sensor prepared in Example 1 was stored in an everyday environment for 7 days, cyclic voltammetry (CV) was used to measure the current response of the sensor in a phosphate buffer solution containing 10 mM hydroquinone, 20 mM hydrogen peroxide, and 10 ng / mL cortisol. The results showed that after 7 days, the oxidation peak current response still maintained 89.1% of the original value, demonstrating the sensor's excellent long-term stability.

[0049] This sensor demonstrates excellent cortisol detection performance. Highly flexible and integrated electrochemical aptamer sensors have broad application prospects in real-time monitoring and management. They can be applied to wearable cortisol detection in human sweat.

Claims

1. A method for preparing a flexible wearable electrochemical sensor for detecting cortisol in sweat, characterized in that: The following steps are involved: (1) dissolving polyurethane particles, adding carbon nanotubes and stirring to obtain a CNT / PU mixed solution, spreading the mixed solution in a mold, and peeling it off after standing to obtain a CNT / PU film, wherein the mass ratio of the carbon nanotubes to the polyurethane is 1:2-1:1; (2) dissolving polyvinyl alcohol, adding carbon nanotubes and stirring to form a mixed solution, modifying the CNT / PVA solution on the CNT / PU film by thermal deposition, and preparing a CCP film after drying, and cutting the film into an electrode shape, wherein the mass ratio of polyvinyl alcohol to carbon nanotubes is 1:1-2:1; (3) Streptavidin-modified Ni-Co MOF was drop-coated on the electrode. After drying, biotinylated aptamer was added to the surface of the material for incubation. Finally, bovine serum albumin solution was added for blocking, and this was used as the working electrode. The Ag / AgCl slurry was drop-coated on the CCP electrode and used as the reference electrode after drying; the unmodified CCP electrode was used as the counter electrode.

2. The method for preparing a flexible wearable electrochemical sensor for detecting sweat cortisol according to claim 1, characterized in that: The concentration of the solution after the polyurethane particles are dissolved in step (1) is 0.034-0.040 g / mL, the stirring temperature during dissolution is 80-85° C., and the stirring time is 30-60 min.

3. The method for preparing the flexible wearable electrochemical sensor for detecting sweat cortisol according to claim 1, characterized in that: The stirring time in step (1) is 20-24h.

4. The method for preparing the flexible wearable electrochemical sensor for detecting sweat cortisol according to claim 1, characterized in that: The standing time in step (1) is 15-30 minutes.

5. The method for preparing a flexible wearable electrochemical sensor for detecting sweat cortisol according to claim 1, wherein: The concentration of the solution after the polyvinyl alcohol is dissolved in step (2) is 0.01-0.05 g / mL.

6. The method for preparing the flexible wearable electrochemical sensor for detecting sweat cortisol according to claim 1, characterized in that: The stirring temperature in step (2) is 80-85° C., and the stirring time is 30-60 min.

7. The method for preparing a flexible wearable electrochemical sensor for detecting sweat cortisol according to claim 1, wherein: In the step (3), the preparation method of streptavidin-modified Ni-Co MOF is as follows: aminoterephthalic acid, nickel nitrate hexahydrate and cobalt nitrate hexahydrate are stirred and dissolved, sodium hydroxide solution is added, and the mixed solution is transferred to a reactor for reaction to obtain Ni-Co MOF material; Ni-Co MOF is dispersed in a phosphate buffer solution, a portion of the Ni-Co MOF dispersion is taken, glutaraldehyde is added, stirred, centrifuged, and redispersed in a phosphate buffer solution, and then streptavidin is added and incubated overnight. After centrifugation and washing, streptavidin-modified Ni-Co MOF is obtained.

8. The method for preparing a flexible wearable electrochemical sensor for detecting sweat cortisol according to claim 1, wherein: The concentration of the aptamer in step (3) is 1-2 μg / mL, the modification volume is 4-5 μL, and the incubation time is 45-50 min.

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