Preparation method and application of biomarker sensor for monitoring sweat gout

Abstract

The invention discloses a preparation method and application of a biomarker sensor for monitoring sweat gout. According to the scheme, an Au sensing electrode is prepared on the surface of a flexible polyethylene glycol terephthalate (PET) thin film in a patterning manner by utilizing an evaporation technology, and a polypyrrole functional layer is prepared on the surface of the electrode by virtue of a molecular imprinting technology; the gout biomarker high-sensitivity detection sensor is constructed in an efficient and rapid manner; the sensor can effectively identify uric acid and xanthine in sweat and display the concentration level of the uric acid and the xanthine, and can provide real-time sensing service for prevention of gout diseases and monitoring of diagnosis and treatment processes of patients with hyperuricemia and yellow urine diseases; a scanning electron microscope is used for carrying out microtopography characterization on the molecularly imprinted functional layer, and an electrochemical workstation is used for carrying out sensing recognition performance characterization. The sensor has the characteristics of rapid identification, convenient sensing and wide detection range for biomarkers of gout, hyperuricemia and yellow urine.

Description

Technical Field

[0001] The present invention specifically relates to a method for preparing a flexible wearable sensor based on molecular imprinting technology for real-time and highly sensitive detection of gout biomarker concentration changes in sweat and its application, belonging to the field of nanomaterial biosensor applications. Background Art

[0002] With the popularity of mobile devices and the rise of digital medicine, wearable sensors with wireless signal transmission have great development potential in special medical equipment development scenarios due to the non-invasive characteristics of the detection method, timely transmission of sensor monitoring information, and great attention has been paid to the development of wearable sensors in areas such as patient symptom monitoring, real-time sensing of drug concentration, and treatment process monitoring. Human sweat is a liquid rich in biomarker substances produced by the metabolism of the human sweat gland secretion system. The biomarker substances include: uric acid, xanthine, K + 、Na + , glucose, lactic acid, amino acids, etc. These biomarkers are closely related to the health status of the human body. Paying attention to the changes in these indicator substances in sweat can achieve the potential risk assessment of the onset of related diseases, disease diagnosis and treatment process regulation by monitoring sweat biological indicator substances. Compared with body fluids such as blood, saliva, and biological interstitial fluid, sweat is easier to obtain, which not only avoids the risk of damage from human invasion, but also avoids the problem of unclean saliva acquisition.

[0003] The accumulation of uric acid in the human body can cause hyperuricemia, which has a high risk of developing into gout. High concentrations of uric acid in the blood can cause sodium urate crystals to accumulate in the joints of patients, forming gout joint swelling. Xanthine can form uric acid under the metabolism of xanthine oxidase and is the main upstream biochemical substance in the uric acid metabolic pathway. Under healthy conditions, the blood uric acid concentration is between 85 and 420 μM, and the xanthine level in urine is about 40 to 160 μM. Uric acid and xanthine are redox active and can be directly detected electrochemically in sweat. By monitoring the uric acid and xanthine content in sweat, real-time and continuous change monitoring of diseases such as gout, hyperuricemia, and xanthine can be achieved, providing biomarker sensing and monitoring convenience for disease prevention and treatment.

[0004] Fluorescence, spectrophotometry, colorimetry, high-performance liquid chromatography and other methods are currently commonly used for gout biomarker detection, but some of these methods require large special equipment to implement, and some lack timely and reliable data reading results, which have certain limitations, especially in the development of miniature real-time data sensors. Electrochemical sensing can quantitatively detect and identify various electrically active chemical substances, and can use electrocatalysis, aptamers and other methods to identify targets.

[0005] Molecular imprinting technology uses various forms of polymers to construct specific cavities that match the target. After the target is eluted, this specific cavity can retain a chamber structure that is highly matched with the target. When this cavity is filled with the target again, the conductivity of the polymer modified layer will be enhanced. If other electrical signal generators are doped into the polymer, the more the target fills the cavity, the more it will hinder the transmission of electrical signals to the electrode. Modifying the molecular imprinting sensing layer on the surface of the electrochemical sensing electrode can give full play to the advantages of both. It can use the specific cavity to quickly identify small molecule targets, and can also use electrochemical sensing to achieve quantitative concentration readings. The constructed sensor can monitor the concentration changes of gout biomarkers in sweat on the surface of human skin. Summary of the invention

[0006] In order to obtain highly sensitive uric acid and xanthine biosensors, the present invention is based on a dual-channel flexible patterned Au electrode, and its working area is molecularly imprinted and functionalized. The constructed sensor can detect uric acid and xanthine with high sensitivity. The sensor has the advantages of fast detection speed, high sensitivity, and wide detection range for gout biomarkers. A method for preparing a nanoenzyme flexible sensor for monitoring gout, the technical scheme is as follows:

[0007] A method for preparing a sensor for monitoring gout biomarkers in sweat based on molecular imprinting technology, the steps are as follows:

[0008] 1) The patterned electrode is prepared in the following manner:

[0009] Double-channel Au patterned electrodes were prepared on flexible PET film using an evaporation deposition instrument.

[0010] 2) The molecular imprinting electrodeposition modification of the electrode working area is carried out in the following manner:

[0011] A 0.2M pyrrole monomer solution, a 50mM FeCl3 solution, a 50mM K3[Fe(CN)6] solution, a 1.0M HCl solution, and a 50mM imprinted molecule solution were prepared using phosphate buffer solution (pH=7.4). The above solutions were diluted and mixed using phosphate buffer solution so that the concentration of each component was diluted to 1 / 10 of the original concentration. The pH of the solution needs to be adjusted to 2-4 during the dilution process. The acidic environment is conducive to the polymerization of pyrrole monomers. A three-electrode system was established using a patterned electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The molecular imprinting layer was prepared using electrochemical cyclic voltammetry. The cycle potential was set to -0.2V~0.9V, the scan rate was set to 50mV / s, and the number of scan cycles was selected in the range of 10-50. The uric acid molecular imprinted polypyrrole functional layer on the electrode surface prepared when the number of scan cycles was 30 is as shown in the attached figure. Figure 1As shown. After the cyclic modification, the molecular imprinting electropolymerization residue was rinsed with deionized water and then dried with nitrogen. Cyclic voltammetry was used to elute the imprinted molecules in a phosphate buffer solution at pH = 7.4 to reveal the molecular imprinting cavity. The elution potential range was set to -0.2V to 0.8V, the cyclic scanning rate was set to 50mV / s, and the number of elution cycles was selected in the range of 15 to 25.

[0012] An application of a nanoenzyme flexible sensor for monitoring gout, specifically the detection of uric acid and xanthine in human sweat:

[0013] The electrochemical cyclic voltammetry method was used in a phosphate buffer solution of pH = 7.4 to complete the elution of the imprinted molecules in the potential range of -0.2V to 0.8V. After elution, 300 μL of uric acid solutions of different concentrations were dripped onto the nitrogen-dried electrodes, and the electrode current signal was read using the chronoamperometry method to obtain the chronoamperometric curves of the current of the sensing electrode in the presence of different concentrations of uric acid (see Figure 2). Figure 2 ), and the relationship curve between the current density value and the uric acid concentration at a specific timing point (as shown in the attached Figure 3 The detectable concentration range of uric acid is 0.1μM~10mM. The current density at a specific timing point changes linearly with the concentration. The higher the concentration, the smaller the current density value at the specific timing point of 120s. The minimum detection limit of the sensor can reach 0.05μM. 300μL of xanthine solution of different concentrations was added to the eluted electrode respectively. The electrode current signal was read by the chronoamperometry to obtain the chronoamperometry curve of the current of the sensor electrode in the presence of different concentrations of xanthine (as shown in the attached figure). Figure 4 ), and the relationship curve between the current density value and the xanthine concentration at a specific timing point (as shown in the attached Figure 5 The detectable concentration range of xanthine is 0.1μM~5mM. The current density at a specific timing point changes linearly with the concentration. The higher the concentration, the smaller the current density value at a specific timing point. The minimum detection limit of the sensor can reach 0.05μM.

[0014] Detection of uric acid in human sweat environment:

[0015] 1) Using the successfully prepared uric acid sensor, the electrochemical signal data of the sweat collected from volunteers during 0-140 min of exercise were collected, and the curves of the change of uric acid concentration in sweat with the passage of exercise time were obtained (see Figure 2). Figure 6 The results showed that the concentration of uric acid gradually decreased from 87.1 μM at 20 minutes to 23.6 μM at 140 minutes as the exercise time went by.

[0016] 2) Using the successfully prepared uric acid sensor, the electrochemical signal data of the sweat collected from volunteers within 8 hours after the intake of high-purine food was collected, and the curves of the change of uric acid concentration in sweat with the passage of time after the diet were obtained (see attached). Figure 7 The results showed that the uric acid concentration reached a peak of 110.7 μM 2 hours after the meal, from 60.1 μM when fasting, and then gradually decreased to 63.6 μM at 8 hours.

[0017] Beneficial Effects

[0018] The electrocatalytic oxidation process of uric acid and xanthine by the sensor of the present invention can be characterized by the electrochemical workstation chronoamperometry. The electrochemical signal collected in the test sweat environment is converted into the uric acid and xanthine concentration results in the sweat environment through the linear relationship curve between the current density and the concentration at a specific timing point. This sensor has the characteristics of real-time transmission of detection data, and can detect quickly, with high sensitivity and a wide range. In the sweat environment, the corresponding uric acid and xanthine concentrations can be identified with the help of the current density value at a specific timing point, thereby realizing the concentration sensing of uric acid and xanthine in sweat. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of the uric acid molecular imprinted polypyrrole functional layer on the surface of the flexible electrode of the present invention.

[0020] Figure 2 This is a chronoamperometric current density variation curve of the molecular imprinting electropolymerization electrode of the present invention in the presence of uric acid at different concentrations.

[0021] Figure 3 This is a relationship curve between current density and concentration at a specific timing point in the presence of different concentrations of uric acid in the solution of the present invention.

[0022] Figure 4 The present invention discloses a chronoamperometric current density variation curve of the molecular imprinting electropolymerization electrode in the presence of xanthine at different concentrations.

[0023] Figure 5 It is a relationship curve between current density and concentration at a specific timing point in the presence of different concentrations of xanthine in the scheme of the present invention.

[0024] Figure 6 This is the curve of uric acid concentration change in sweat when the sensor of the present invention monitors the human body during exercise.

[0025] Figure 7 The sensor of the present invention monitors the change curve of uric acid concentration in sweat after a person has a purine diet. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with embodiments, so that those skilled in the art can refer to the description to implement preparation and application.

[0027] 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.

[0028] Preparation of the sensor of the present invention;

[0029] Example 1: A 0.2M pyrrole monomer solution, a 50mM FeCl3 solution, a 50mM K3[Fe(CN)6] solution, a 1.0M HCl solution, and a 50mM imprinted molecule solution were prepared using a phosphate buffer solution (pH = 7.4). The above solutions were diluted and mixed using a phosphate buffer solution so that the concentration of each component was diluted to 1 / 10 of the original concentration. The pH of the solution needs to be adjusted to 2-4 during the dilution process, and the acidic environment is conducive to the polymerization of pyrrole monomers. A three-electrode system was established using a patterned electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The molecular imprinted layer was prepared using electrochemical cyclic voltammetry, the cycle potential was set to -0.2V-0.9V, the scan rate was set to 50mV / s, and the number of scan cycles was 10. After the cyclic modification, the molecular imprinted electropolymerization residue was rinsed with deionized water and then dried with nitrogen. Cyclic voltammetry was used to elute the imprinted molecules in a phosphate buffer solution at pH 7.4 to reveal the molecular imprinted cavity. The elution potential range was set to -0.2 V to 0.8 V, the cycle scan rate was set to 50 mV / s, and the number of elution cycles was 20.

[0030] Example 2, using phosphate buffer solution (pH = 7.4) to prepare a 0.2M pyrrole monomer solution, a 50mMFeCl3 solution, a 50mM K3[Fe(CN)6] solution, a 1.0M HCl solution, and a 50mM imprinted molecule solution. The above solutions were diluted and mixed with phosphate buffer solution so that the concentration of each component was diluted to 1 / 10 of the original concentration. The pH of the solution needs to be adjusted to 2-4 during the dilution process, and the acidic environment is conducive to the polymerization of pyrrole monomers. A three-electrode system was established with a patterned electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The molecular imprinting layer was prepared by electrochemical cyclic voltammetry, the cycle potential was set to -0.2V-0.9V, the scan rate was set to 50mV / s, and the number of scan cycles was 20. After cyclic modification, the molecular imprinting electropolymerization residue was rinsed with deionized water and then dried with nitrogen. Cyclic voltammetry was used to elute the imprinted molecules in a phosphate buffer solution at pH 7.4 to reveal the molecular imprinted cavity. The elution potential range was set to -0.2 V to 0.8 V, the cycle scan rate was set to 50 mV / s, and the number of elution cycles was 20.

[0031] Example 3, using phosphate buffer solution (pH = 7.4) to prepare 0.2M pyrrole monomer solution, 50mM FeCl3 solution, 50mM K3[Fe(CN)6] solution, 1.0M HCl solution, and 50mM imprinted molecule solution. The above solutions were diluted and mixed with phosphate buffer solution so that the concentration of each component was diluted to 1 / 10 of the original concentration. The pH of the solution needs to be adjusted to 2-4 during the dilution process. The acidic environment is conducive to the polymerization of pyrrole monomers. A three-electrode system was established with a patterned electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The molecular imprinting layer was prepared by electrochemical cyclic voltammetry. The cycle potential was set to -0.2V~0.9V, the scanning rate was set to 50mV / s, and the number of scanning cycles was 30. The uric acid molecular imprinted polypyrrole functional layer on the prepared electrode surface is as shown in the attached Figure 1 As shown. After the cyclic modification, the molecular imprinting electropolymerization residue was rinsed with deionized water and then dried with nitrogen. Cyclic voltammetry was used to elute the imprinted molecules in a phosphate buffer solution at pH = 7.4 to reveal the molecular imprinting cavity. The elution potential range was set to -0.2V to 0.8V, the cyclic scan rate was set to 50mV / s, and the number of elution cycles was 20.

[0032] Example 4, using phosphate buffer solution (pH = 7.4) to prepare a 0.2M pyrrole monomer solution, a 50mMFeCl3 solution, a 50mM K3[Fe(CN)6] solution, a 1.0M HCl solution, and a 50mM imprinted molecule solution. The above solutions were diluted and mixed with phosphate buffer solution so that the concentration of each component was diluted to 1 / 10 of the original concentration. The pH of the solution needs to be adjusted to 2-4 during the dilution process, and the acidic environment is conducive to the polymerization of pyrrole monomers. A three-electrode system was established with a patterned electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The molecular imprinting layer was prepared by electrochemical cyclic voltammetry, the cycle potential was set to -0.2V~0.9V, the scan rate was set to 50mV / s, and the number of scan cycles was 40. After cyclic modification, the molecular imprinting electropolymerization residue was rinsed with deionized water and then dried with nitrogen. Cyclic voltammetry was used to elute the imprinted molecules in a phosphate buffer solution at pH 7.4 to reveal the molecular imprinted cavity. The elution potential range was set to -0.2 V to 0.8 V, the cycle scan rate was set to 50 mV / s, and the number of elution cycles was 20.

[0033] Example 5, using phosphate buffer solution (pH = 7.4) to prepare a 0.2M pyrrole monomer solution, a 50mMFeCl3 solution, a 50mM K3[Fe(CN)6] solution, a 1.0M HCl solution, and a 50mM imprinted molecule solution. The above solutions were diluted and mixed with phosphate buffer solution so that the concentration of each component was diluted to 1 / 10 of the original concentration. The pH of the solution needs to be adjusted to 2-4 during the dilution process, and the acidic environment is conducive to the polymerization of pyrrole monomers. A three-electrode system was established with a patterned electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The molecular imprinting layer was prepared by electrochemical cyclic voltammetry, the cycle potential was set to -0.2V~0.9V, the scan rate was set to 50mV / s, and the number of scan cycles was 50. After cyclic modification, the molecular imprinting electropolymerization residue was rinsed with deionized water and then dried with nitrogen. Cyclic voltammetry was used to elute the imprinted molecules in a phosphate buffer solution at pH 7.4 to reveal the molecular imprinted cavity. The elution potential range was set to -0.2 V to 0.8 V, the cycle scan rate was set to 50 mV / s, and the number of elution cycles was 20.

[0034] Detection applications in human sweat:

[0035] Example 6, uric acid detection, in a phosphate buffer solution of pH = 7.4, the molecular imprinted modified electrode was eluted by -0.2V ~ 0.8V electrochemical cyclic voltammetry, and 300μL of 100μM uric acid solution was added to the electrode after elution and dried by nitrogen. The current time change curve of the sensing electrode within 120s in the presence of uric acid at this concentration was obtained, as shown in the attached figure. Figure 2 As shown in Figure 2, the current density at a specific timing point (such as 120s) is 1.98uA cm -2 , as attached Figure 3 Shown

[0036] Example 7, xanthine detection, using pH = 7.4 phosphate buffer solution to complete the elution of the molecular imprinted modified electrode, 300 μL of 200 μM xanthine solution was added to the electrode to obtain the current timing change curve of the sensing electrode within 120 s in the presence of xanthine at this concentration, as shown in the attached figure. Figure 4 As shown in the figure, the current density value at a specific timing point (such as 120s) is 2.48uAcm -2 , as attached Figure 5 shown.

Claims

1. A method for preparing a sensor for monitoring sweat gout biomarkers, characterized in that: The preparation steps are as follows: (1) a patterned dual-channel Au electrode is prepared on the surface of a PET substrate material by evaporation deposition; (2) pyrrole monomers are polymerized on the electrode surface by molecular imprinting technology, and after eluting the template, a polymer imprinted cavity matching the structure and functional groups of the gout biomarker is formed.

2. The preparation method according to claim 1, characterized in that The (2) step is specifically as follows: (2-1) using phosphate buffer solution to prepare pyrrole monomer solution, concentrated FeCl3 solution, K3[Fe(CN)6] solution, imprinting molecule solution, and using deionized water to prepare HCl solution; (2-2) diluting and mixing the above solution in a phosphate buffer solution so that the concentration of each component is diluted to 1 / 10 of the original concentration; during the dilution process, the pH of the solution needs to be adjusted to 2-4, as an acidic environment is conducive to the polymerization of pyrrole monomers; (2-3) A three-electrode system was established with a patterned circular electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The molecular imprinting layer was deposited and prepared by electrochemical cyclic voltammetry. The cycle potential was set to -0.2 V to 0.9 V, the scan rate was set to 50 mV / s, and the number of scan cycles was selected in the range of 10 to 50; (2-4) After the cyclic modification, the molecular imprinting electropolymerization residues were rinsed with deionized water and then dried with nitrogen gas; Cyclic voltammetry is used to elute the imprinted molecules in a phosphate buffer solution to reveal the molecular imprinted cavity. The elution potential range is set to -0.2V to 0.8V, the cycle scan rate is set to 50mV / s, and the number of elution cycles is selected within the range of 10 to 20.

3. The preparation method according to claim 2, characterized in that: The pH of the phosphate buffer solution is 7.

4.

4. The preparation method according to claim 2, characterized in that: The concentrations of the FeCl3 solution, K3[Fe(CN)6] solution, imprinting molecule solution, and HCl solution are 0.2M, 50mM, 50mM, and 1.0M, respectively.

5. The preparation method according to claim 1, characterized in that: The molecular imprinting layer is prepared by electropolymerization on the electrode surface, the cavities are evenly distributed, and the size of the polymer cluster dendritic pyrrole structure is in the range of 100 to 400 nm.

6. An application of a sensor for monitoring sweat gout biomarkers, characterized in that: Detection of uric acid in human sweat: The molecular imprinted modified electrode is used to elute the imprinted molecules in a phosphate buffer solution with the help of -0.2V~0.8V electrochemical cyclic voltammetry. After elution, uric acid solutions of different concentrations are dripped onto the electrode that has been dried by nitrogen. The electrode current signal is read by the chronoamperometry to obtain the chronoelectric change curve of the sensing electrode current in the presence of different concentrations of uric acid, as well as the relationship curve between the current density value at a specific timing point and the uric acid concentration. The uric acid detection concentration range is 0.1μM~10mM, and the current density at a specific timing point changes linearly with the concentration. The higher the concentration, the smaller the current density value at the specific timing point. The minimum detection limit of the sensor is 0.05μM.

7. The use according to claim 6, characterized in that Detection of xanthine in human sweat: Use a phosphate buffer solution with pH = 7.4 to elute the imprinted molecules from the molecularly imprinted modified electrode, add xanthine solutions of different concentrations onto the electrode, and read the electrode current signal using the chronoamperometry method to obtain the chronoamperometric curve of the sensing electrode current in the presence of different concentrations of xanthine, as well as the relationship curve between the current density value and the xanthine concentration at a specific timing point. The xanthine detection concentration range is 0.1μM to 5μM.

Images