Preparation method and application of tension sensor for monitoring cortisol in sweat

Abstract

The invention discloses a preparation method and application of a tension sensor for monitoring cortisol in sweat. According to the scheme, on the basis of a stretchable electrode (SWNTs / PDMS) which is prepared by patterning in a manner that a single-walled carbon nanotube is subjected to suction filtration and transfer printing on polydimethylsiloxane, cortisol is used as an imprinting molecular template by utilizing an electrochemical deposition technology, and Prussian blue (PB) and pyrrole (Py) are electrically polymerized to prepare an electrochemical sensor of a molecularly imprinted polymer (MIP), so that the change of the concentration of cortisol in sweat can be monitored in real time; a reliable and practical method is provided for rapid and pressure-free pressure monitoring. A scanning electron microscope is used for electrode modification morphology characterization, and an electrochemical workstation is used for electrode sensing performance characterization. The sensor realizes the function of dynamically monitoring the day and night change rule of cortisol in sweat, and has the characteristics of real-time detection, high sensitivity and selectivity and good stability.

Description

Technical Field

[0001] The present invention specifically relates to a method for preparing a flexible wearable sensor based on molecular imprinting for real-time and highly sensitive detection of concentration changes of psychological stress biomarkers in sweat and tensile strain detection, belonging to the field of biosensor applications. Background Art

[0002] As the pace of life and work accelerates, the psychological pressure of the people has greatly increased, and the problem of mental illness has become increasingly prominent. This has brought heavy economic pressure to families and society. Therefore, there is an urgent need to develop an effective means of stress assessment. The latest advances in wearable sensors have made it possible to monitor the body's vital signs non-invasively, in real time, and continuously. Through on-site human sweat analysis, wearable biosensors can provide profound information about personal health at the molecular level.

[0003] Cortisol, also known as the "stress hormone", is secreted by the adrenal glands. 3%-5% of the cortisol in the body circulates as free cortisol, which is biologically active and can bind to glucocorticoid receptors and affect metabolism. Excessive release of cortisol often leads to metabolic disorders, muscle weakness, osteoporosis, anxiety, depression, and schizophrenia. The cortisol concentration in sweat is usually in the range of 23-390nM and shows a strong correlation with serum cortisol. Therefore, non-invasive sweat cortisol monitoring is a feasible method to assess human cortisol levels.

[0004] The current research methods for cortisol include antigen-antibody binding and specific binding of DNA chains, but the detection time is long, it is easily affected by the detection environment, and the cost is high. Molecular imprinting polymer (MIP) is a polymer synthesized by molecular imprinting technology that has the ability to specifically recognize and selectively detect template molecules. MIPs are natural antibody-antigen analogs with high affinity and selectivity, and have the advantages of simple preparation, high cost-effectiveness, and good stability. MIPs are widely used in important fields such as affinity separation, disease diagnosis, drug delivery, targeted bioimaging, and cancer treatment. Combining MIP with electrochemical sensing technology can quickly and conveniently detect targets. The introduction of carbon nanotubes can be tightly combined with PDMS, and the MIP cavity slides with the sliding of the carbon nanotubes, ensuring the stability of the sensing performance under a certain tensile deformation. We used an electrochemical sensor of molecular imprinting polymer (MIP) prepared by electropolymerizing Prussian blue (PB) and pyrrole (Py) with cortisol as an imprinting template. The detection of cortisol relies on Prussian blue, which has a redox signal. After the molecular imprinting membrane selectively captures cortisol, it occupies the pores of the imprinting membrane, thereby hindering the electron conduction during PB redox. The experimental results show that the change in PB signal and the logarithmic value of cortisol concentration show a good linear relationship. Molecular imprinting provides a high sensitivity and selectivity for real-time detection of cortisol, and has good physical and thermal stability, low cost and simple preparation. Combining MIP with electrochemical sensing technology can quickly and conveniently detect targets, which is crucial for diagnosing diseases related to mental health. Summary of the invention

[0005] In order to achieve one-step, rapid, repeatable, highly sensitive and selective cortisol sensing, the present invention uses cortisol as a template, electropolymerizes PPy, PB as an embedded redox probe, synthesizes a MIP electrode layer, and prepares a user-friendly MIP cortisol sensor. A preparation and application of a flexible sensor for monitoring cortisol in sweat, specifically the preparation of a flexible wearable sensor for detecting changes in the concentration of psychological stress biomarkers in sweat based on molecular imprinting and tensile strain detection, the scheme is as follows:

[0006] 1. Preparation steps:

[0007] 1. Preparation of single-walled carbon nanotube / polydimethylsiloxane (SWNTs / PDMS) stretchable electrodes

[0008] (1-1) Preparation of polydimethylsiloxane elastic substrate

[0009] Prepare the polydimethylsiloxane (PDMS) elastic substrate. First, mix the elastic agent and the curing agent at a mass ratio of 10:1 to form PDMS. Stir it evenly with a glass rod. After there are no bubbles, pour it on a clean silicon wafer and spin it on a glue machine at a low speed of 500 rpm, then spin it at 800 rpm for 60 seconds. Put the PDMS film in an oven, set the oven temperature to 65 degrees, and cure it for 35 minutes.

[0010] (1-2) Preparation of SWNTs solution and transfer of CNT electrodes onto PDMS

[0011] Weigh 140 mg of sodium dodecyl sulfate (SDS) on a balance and put it into a beaker, add 20 mL of ultrapure water to make a solution, and sonicate until SDS is completely dissolved. Then weigh 4 mg of SWNT and add it to the above solution. Put all the above solutions in an ultrasonic instrument, and sonicate for 90 minutes at 45W power and 25Hz frequency in an ice water bath. Ensure that the single-walled carbon nanotubes are evenly dispersed. It can be used as an experimental SWNTs solution, and then disperse it by sonication for 30 minutes before each use. First print the filter membrane with an electrode pattern, filter it through a suction filtration device, first drip water to wet the filter membrane, and then add 125 μL of CNT solution (drip according to the electrode pattern. After drying, add water and ethanol in turn. Transfer the filtered electrode to the semi-cured PDMS membrane, place it in an oven, set the oven temperature to 65 degrees, and cure it for 6 hours to complete.

[0012] 2. Preparation of molecular imprinted working electrode: deposition modification

[0013] The CNT / PDMS electrode was placed in a PBS solution (containing 0.02 mol / L pyrrole, 5 mmol / LFeCl3, 5 mmol / L K3[Fe(CN6)], 6 mmol / L cortisol and 0.1 mol / L HCl) at pH = 7.4. A three-electrode system was used, with the patterned circular electrode as the working electrode, the platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The MIP membrane was prepared by electropolymerization for 5-30 cycles at a scan rate of 50 mV s-1 in the potential range of -0.2-0.9 V using an electrochemical workstation cyclic voltammetry. Then, PPy-PB was over-oxidized in PBS for 15 cycles (50 mV s-1) in the potential range of -0.2-0.8 V by CV to elute the embedded cortisol molecules from the PPy-PB matrix to produce molecular cavities. The polypyrrole functional layer of cortisol molecules imprinted on the surface of the stretchable electrode is shown in the attached figure. Figure 1As shown. For the preparation of NIP, the same preparation method as MIP was adopted, without using cortisol molecules as templates in the polymerization step. Although the polymerization layer did not contain templates, the PPy peroxidation step was still performed to ensure that other experimental conditions were the same as those of the MIP sensor. Finally, the prepared NIP-based electrode was washed twice with deionized water and dried at room temperature until use.

[0014] 2. Detection application, specifically the tensile strain detection of flexible wearable sensors based on molecular imprinting to detect changes in concentration of psychological stress biomarkers in sweat, the steps are as follows:

[0015] 1. Detection of cortisol in the unstretched and stretched states of the electrode

[0016] 300 μL of cortisol solution of different concentrations was dripped onto the prepared sensor electrode probe, and the biological signal of cortisol concentration was converted by the three-electrode system of the electrochemical workstation to acquire the electrode, so that it has a specific recognition function for the cavity and is converted into an electrical signal. After 60 seconds, different concentrations of cortisol correspond to different electrical signal curves. The MIP-modified flexible electrode detects cortisol in the concentration range of 1nM to 10μM in the unstretched state. The MIP-modified sensor shows a good response signal as shown in the attached figure. Figure 2 As shown in the figure, the current value changes from 0.982μA to 0.3208μA. The linear relationship between the current value and the cortisol concentration is shown in the attached figure. Figure 3 As shown, the minimum detection limit reaches 1nM, and the linear equation is y=-0.15075Cc+0.99619. In order to verify the stability of MIP for cortisol under 50% stretching, we conducted the following experiments to characterize the performance of the sensor under stretching. In the experiment, different concentrations of cortisol 1nM-10μM were selected to test the MIP-modified flexible electrode under stretching. Figure 4 In the experimental data, the measured current value changes from 1.076μA to 0.3816μA, and the current value is linearly related to the cortisol concentration, as shown in the attached Figure 5 As shown, the minimum detection limit reaches 1nM, and the linear equation is: y = -0.16376Cc + 1.05119

[0017] 2. Detection of 10μM cortisol when the electrode is stretched 0%-50%

[0018] 300 μL of 10 μM cortisol solution was dripped onto the prepared sensor electrode probe, and the electrode was stretched from 0% to 50% for a test. The biological signal of cortisol concentration was converted by the three-electrode system of the electrochemical workstation to enable it to have a specific recognition function for the cavity and converted into an electrical signal. The relationship between the current value and the different stretching states is shown in the attached figure. Figure 6As shown, the current value does not change significantly, and the stability of the MIP-modified flexible electrode is tested under 0%-50% stretching state.

[0019] 3. A molecular imprinting method for detecting actual sweat samples at different time periods within a day

[0020] 40 μL of actual sweat sample was dripped onto the prepared sensor electrode probe, and the electrode was tested every two hours from 7:00 to 19:00. The biological signal of cortisol concentration in sweat was converted by the three-electrode system of the electrochemical workstation to acquire the electrode, so that it has a specific recognition function for the cavity and converted into an electrical signal. The calibration curve of cortisol in artificial sweat was tested using the MIP-modified wearable electrode. The corresponding amperometric response signal and calibration curve were calculated and we obtained the cortisol content tested at different times as shown in the attached figure. Figure 7 shown.

[0021] Beneficial Effects

[0022] The selective binding of cortisol in the imprint cavity leads to the blocking of the PB electron transfer pathway, thereby reducing the PB oxidation current. The degree of this current reduction reflects the cortisol concentration in sweat and can therefore be used as an analytical signal. Characterization was performed using an electrochemical workstation chronoamperometry three-electrode system. The electrochemical signal collected in the test sweat environment was converted into the cortisol concentration result in the sweat environment through a linear relationship curve. In the sweat environment, the sensor can display a current signal after 60 seconds of testing, and identify the corresponding cortisol concentration through the current signal value, thereby realizing the concentration sensing of cortisol in sweat. Stability sensing under stretching is also achieved, and it is shown that the MIP wearable sensor monitors the circadian rhythm of cortisol. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of the cortisol molecular imprinted polypyrrole functional layer on the surface of the stretchable electrode of the present invention.

[0024] Figure 2 This is the current change curve of the flexible MIP electrode in the present invention in the unstretched state with different cortisol in artificial sweat.

[0025] Figure 3 It is a relationship curve between the current value and concentration of different cortisols in artificial sweat of the flexible MIP electrode in the unstretched state of the present invention.

[0026] Figure 4 It is the chronoamperometric current change curve of the flexible MIP electrode of the present invention under the state of being stretched by 50% with different concentrations of cortisol.

[0027] Figure 5It is a relationship curve between the current value and the concentration of cortisol of different concentrations when the flexible MIP electrode of the present invention is stretched by 50%.

[0028] Figure 6 This is the current change curve of the flexible MIP electrode of the present invention when stretched 0%-50% and 10 μM cortisol.

[0029] Figure 7 It is the actual sweat sample detection of different time periods by the flexible MIP electrode in the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, so that those skilled in the art can refer to the description to implement the preparation.

[0031] It should be understood that the terms “comprising”, “including” and “having” used herein do not exclude the use or addition of one or more other device elements, and combinations thereof.

[0032] Example 1

[0033] This embodiment provides a flexible wearable sensor based on molecular imprinting for monitoring cortisol in sweat. The electrode molecular imprinting modification preparation method is as follows: 0.02mol / L pyrrole, 5mmol / LFeCl3, 5mmol / L K3[Fe(CN)6], 6mmol / L cortisol and 0.1mol / L HCl are added to a PBS solution with a pH of 7.4, and a three-electrode system is used, with a patterned circular electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The electrochemical workstation cyclic voltammetry working mode is used with a scanning rate of 50mV s in the potential range of -0.2-0.9V. -1 The MIP membrane was prepared by electropolymerization for 10 cycles. After the electropolymerization process, the electrode was washed twice with deionized water to remove the remaining compounds. Then, PPy-PB was over-oxidized in PBS by CV in the potential range of -0.2-0.8 V for 15 cycles (50 mV s -1 ), the embedded cortisol molecules were eluted from the PPy-PB matrix to generate molecular cavities.

[0034] Example 2

[0035] 0.02 mol / L pyrrole, 5 mmol / L FeCl3, 5 mmol / L K3[Fe(CN)6], 6 mmol / L cortisol and 0.1 mol / L HCl were added to the PBS solution at pH = 7.4. A three-electrode system was used, with the patterned circular electrode as the working electrode, the platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The electrochemical workstation was used for cyclic voltammetry with a scan rate of 50 mV s in the potential range of -0.2-0.9 V. -1 The MIP membrane was prepared by electropolymerization for 15 cycles. After the electropolymerization process, the electrode was washed twice with deionized water to remove the remaining compounds. Then, PPy-PB was over-oxidized in PBS by CV in the potential range of -0.2-0.8 V for 15 cycles (50 mV s -1 ), the embedded cortisol molecules were eluted from the PPy-PB matrix to produce molecular cavities, and the cortisol molecular imprinted polypyrrole functional layer on the stretchable electrode surface was shown in the attached Figure 1 shown.

[0036] Example 3

[0037] 0.02 mol / L pyrrole, 5 mmol / L FeCl3, 5 mmol / L K3[Fe(CN)6], 6 mmol / L cortisol and 0.1 mol / L HCl were added to the PBS solution at pH = 7.4. A three-electrode system was used, with the patterned circular electrode as the working electrode, the platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The electrochemical workstation was used for cyclic voltammetry with a scan rate of 50 mV s in the potential range of -0.2-0.9 V. -1 The MIP membrane was prepared by electropolymerization for 20 cycles. After the electropolymerization process, the electrode was washed twice with deionized water to remove the remaining compounds. Then, PPy-PB was over-oxidized in PBS by CV in the potential range of -0.2-0.8 V for 15 cycles (50 mV s -1 ), the embedded cortisol molecules were eluted from the PPy-PB matrix to generate molecular cavities.

[0038] Example 4

[0039] 0.02 mol / L pyrrole, 5 mmol / L FeCl3, 5 mmol / L K3[Fe(CN)6], 6 mmol / L cortisol and 0.1 mol / L HCl were added to the PBS solution at pH = 7.4. A three-electrode system was used, with the patterned circular electrode as the working electrode, the platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The electrochemical workstation was used for cyclic voltammetry with a scan rate of 50 mV s in the potential range of -0.2-0.9 V. -1The MIP membrane was prepared by electropolymerization for 25 cycles. After the electropolymerization process, the electrode was washed twice with deionized water to remove the remaining compounds. Then, PPy-PB was over-oxidized in PBS by CV in the potential range of -0.2-0.8 V for 15 cycles (50 mV s -1 ), the embedded cortisol molecules were eluted from the PPy-PB matrix to generate molecular cavities.

[0040] Example 5

[0041] This embodiment provides a flexible wearable sensor for monitoring cortisol in sweat based on molecular imprinting. The specific method for detecting cortisol is as follows: using electrochemical cyclic voltammetry in artificial sweat at pH = 7.4, the imprinted molecules are eluted in the potential range of -0.2V to 0.8V, and 300μL of 1μM cortisol solution is dripped on the electrodes dried by nitrogen after elution, and the sensor current change curve in the presence of cortisol at this concentration is obtained as shown in the attached figure. Figure 2 As shown in the figure, the electrode current is 0.545μA. Figure 3 shown.

[0042] Example 6

[0043] This embodiment provides a flexible wearable sensor for monitoring cortisol in sweat based on molecular imprinting. The specific method for detecting cortisol is as follows: using electrochemical cyclic voltammetry in artificial sweat at pH = 7.4, the imprinted molecules are eluted in the potential range of -0.2V to 0.8V, and the electrodes are dried with nitrogen after elution. When the electrodes are stretched 50%, 300 μL of 1 μM cortisol solution are added dropwise to obtain the change curve of the sensing current in the presence of cortisol at this concentration as shown in the attached figure. Figure 2 As shown in the attached figure, the electrode current is 0.557μA. Figure 3 shown.

[0044] Example 7

[0045] This embodiment provides a flexible wearable sensor for monitoring cortisol in sweat based on molecular imprinting. The specific method for detecting cortisol is as follows: using electrochemical cyclic voltammetry in artificial sweat, eluting the imprinted molecules in a potential range of -0.2V to 0.8V, dripping 300 μL of 100 nM cortisol solution on the electrodes dried by nitrogen after elution, and obtaining a sensor current change curve in the presence of cortisol at this concentration as shown in the attached figure. Figure 2 As shown in the attached figure, the electrode current is 0.322μA. Figure 3 shown.

Claims

1. A method for preparing a stretch sensor for monitoring cortisol in sweat, characterized in that: Here are the steps: (1) Preparation of single-walled carbon nanotube / polydimethylsiloxane (SWNTs / PDMS) stretchable electrodes: (1-1) Preparation of polydimethylsiloxane elastic substrate Prepare polydimethylsiloxane (PDMS) elastic substrate, mix the elastic agent and curing agent to make PDMS, stir evenly with a glass rod, and pour it on a clean silicon wafer after there are no bubbles. Unscrew it at a low speed on a glue roller and then speed it up, and put the PDMS film in an oven; (1-2) Preparation of SWNTs solution and transfer of CNT electrodes onto PDMS A certain amount of sodium dodecyl sulfate SDS was placed in a beaker, and 20 mL of ultrapure water was added to form a solution. Ultrasonication was performed until the SDS was completely dissolved, and then SWNTs were added to the above solution. All the above solutions were placed in an ultrasonic instrument, and the ultrasonication was performed under an ice-water bath to ensure that the single-walled carbon nanotubes were evenly dispersed, that is, as the experimental SWNTs solution. Subsequently, ultrasonication was performed for 30 minutes before each use to disperse the solution. A filter membrane with an electrode pattern was first printed, and then filtered through a suction filtration device. Water was first added to wet the filter membrane, and then the CNT solution was added. After drying, water and ethanol were added in sequence, and the filtered electrode was transferred to the semi-cured PDMS membrane, and then cured in an oven; (2) Preparation of molecular imprinted working electrode: deposition modification The CNT / PDMS electrode was placed in a PBS solution with a pH of 7.4, using a three-electrode system, with a patterned circular electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode; the MIP membrane was prepared using an electrochemical workstation cyclic voltammetry method; PPy-PB was over-oxidized in PBS by CV to elute the embedded cortisol molecules from the PPy-PB matrix to produce a molecular cavity; For the preparation of NIPs, the same preparation method as that of MIPs was adopted, except that cortisol molecules were not used as templates in the polymerization step; finally, the prepared NIP-based electrodes were washed twice with deionized water and dried at room temperature until use.

2. The preparation method according to claim 1, characterized in that In the above (1-1), the mass ratio of the elastic agent to the curing agent is 10:

1. The glue spreader is first turned on at a low speed of 500 rpm, and then turned at 800 rpm for 60 seconds. The oven temperature is set at 65 degrees and the curing is carried out for 35 minutes.

3. The preparation method according to claim 1, characterized in that: In the above (1-2), sodium dodecyl sulfate is 140 mg, ultrapure water is 20 mL, and SWNT is 4 mg; ultrasonic treatment is performed in an ice-water bath for 90 minutes at a power of 45 W and a frequency of 25 Hz; and CNT solution is 125 μL.

4. The preparation method according to claim 1, characterized in that: In the above (1-2), the oven temperature is set at 65 degrees and the curing time is 6 hours.

5. The preparation method according to claim 1, characterized in that: In (2), the PBS solution contains 0.02 mol / L pyrrole, 5 mmol / L FeCl3, 5 mmol / L K3Fe(CN6), 6 mmol / L cortisol and 0.1 mol / L HCl, and the electrochemical workstation cyclic voltammetry operation mode is a scan rate of 50 mV s in the potential range of -0.2-0.9 V. -1 , electropolymerization for 5-30 cycles; PPy-PB was over-oxidized in PBS for 15 cycles in the potential range of -0.2-0.8 V, i.e., 50 mV s -1 .

6. A detection application of a flexible sensor for monitoring cortisol in sweat, characterized in that: Specifically, the tensile strain detection of the flexible wearable sensor based on molecular imprinting to detect the concentration changes of psychological stress biomarkers in sweat, the detection steps of cortisol are as follows: Different concentrations of cortisol solutions were added to the sensor electrode in the unstretched and stretched states, and the electrode current signal of the chronoamperometry was collected. Under the two states, the current signal change curves of PB oxidized and reduced on the electrode surface with different concentrations of cortisol were obtained, as well as the relationship curve between the current value and the cortisol concentration. The current value changes linearly with the concentration, and the higher the concentration, the smaller the current value.

7. The detection application according to claim 6, characterized in that: The volume of cortisol solution with different concentrations was 300 μL, and the electrode current signal of the chronoamperometry was collected for 60 s.

8. The detection application as claimed in claim 6, characterized in that: The cortisol detection concentration is 0.1 nM-10 μM, and the minimum detection limit of the sensor reaches 0.05 nM.

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