Preparation method and application of electrochemical sensor for detecting inflammatory factors

Abstract

The invention relates to the technical field of electrochemical sensors, in particular to a preparation method and application of an electrochemical sensor for detecting inflammatory factors. The method comprises the following steps: printing a patterned laser-induced graphene electrode by using a carbon dioxide laser, modifying gold nanoparticles on the surface of graphene by using an electrochemical deposition method, and finally performing specific modification by using an aptamer. An electrochemical deposition method is adopted for the AuNPs / LIG composite electrode, the preparation process is simple and easy to operate, compared with a traditional precious metal aptamer detection electrode, the preparation cost is lower, and the biosensor for providing wound healing state information can better display wound inflammation and healing progress.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and in particular to a preparation method and application of an electrochemical sensor for detecting inflammatory factors. Background Art

[0002] The skin is the outermost organ of the human body and the most vulnerable part of the human body. Patients with diabetes or other underlying diseases may have wounds that develop into chronic wounds. Chronic wounds cause pain and suffering to patients worldwide, and bring an increasing economic burden to patients' families and medical systems. The healing process of chronic wounds is disturbed by the imbalance of multiple microenvironmental and physiological factors, which will remain in the wound exudate. At present, how to quantify the biochemical parameters in the wound microenvironment and apply it to clinical treatment has not been widely promoted. Without on-site diagnosis, neither patients nor doctors can accurately determine the progression of chronic wounds.

[0003] Cytokines and growth factors show dynamic changes in the process of wound healing. They are recognized and important indicators of inflammation in the process of chronic wound formation. It has been proposed that electrochemical sensors can quantify inflammatory factors in wounds. This electrochemical biosensor that provides information on the state of wound healing can better display wound inflammation and healing progress. We chose interleukin-6 (IL-6), a biomarker in chronic wounds. The content of this inflammatory factor is higher in unhealed chronic wounds. We can use electrochemical sensors to quantify IL-6 to determine the specific situation of chronic wound inflammation. The existing preparation methods of electrochemical sensors are complex, costly, and the accuracy of the test results is not high enough. Therefore, it is urgently needed to be solved. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a preparation method and application of an electrochemical sensor for detecting inflammatory factors. The preparation process of this method is simple and easy to operate, and the preparation cost is lower than that of traditional precious metal aptamer detection electrodes. This biosensor that provides wound healing status information can better display wound inflammation and healing progress.

[0005] To achieve the above object, the technical solution of the present invention is implemented as follows: a preparation method and application of an electrochemical sensor for detecting inflammatory factors; the preparation method comprises the following steps:

[0006] S1, laser-induced printing of graphene electrodes on polyimide film using a carbon dioxide laser;

[0007] S2. Preparation of gold nanoparticle / laser-induced graphene composite electrodes using electrochemical deposition on the printed electrodes:

[0008] S3, modifying the IL-6 aptamer on the prepared gold nanoparticle / laser-induced graphene composite electrode by using a dropwise functionalization modification method;

[0009] S4, performing MCH modification on the electrode modified with the IL-6 aptamer;

[0010] S5. Perform analyte modification on the MCH-modified electrode to obtain an electrochemical sensor.

[0011] Preferably, in step S1, the process of printing laser-induced graphene on the polyimide film using a carbon dioxide laser comprises:

[0012] S11, washing the polyimide film with acetone, ethanol and deionized water respectively, and then drying it with nitrogen;

[0013] S12. Fix the clean polyimide film on the acrylic plate with tape, and then use a carbon dioxide laser instrument to print electrodes on the polyimide film according to the computer preset pattern.

[0014] Preferably, in step S2, the process of preparing the gold nanoparticle / laser induced graphene composite electrode by electrochemical deposition comprises:

[0015] S21, preparing a deposition solution,

[0016] S22. In a deposition solution, electrochemically deposit gold nanoparticles onto the laser-induced graphene using cyclic voltammetry until a layer of gold nanoparticles is modified on the surface of the laser-induced graphene.

[0017] Preferably, in step S3, the process of modifying the IL-6 aptamer by the dropwise functionalization modification method comprises:

[0018] S31, cleaning the gold nanoparticle / laser-induced graphene composite electrode with deionized water, and then drying it with nitrogen gas;

[0019] S32, preparing an aptamer solution;

[0020] S33, 10 μL of the aptamer solution was dropped onto the working electrode and allowed to stand at 4°C for 12 h to complete the modification of the IL-6 aptamer.

[0021] S34. Wash the electrode modified with IL-6 aptamer with PBS.

[0022] Preferably, in step S4, the process of performing MCH modification on the electrode modified with IL-6 aptamer comprises:

[0023] S41, prepare MCH solution 1mM;

[0024] S42, take 10 μL of MCH solution and drop it on the electrode modified with IL-6 aptamer, and let it stand at 37°C for 1.5 hours to complete the MCH modification;

[0025] S43. Wash the MCH-modified electrode with PBS.

[0026] Preferably, in step S5, the process of modifying the electrode modified with MCH with an analyte comprises:

[0027] S51. The analyte solution was diluted to different concentrations (0-50 ng / mL) in sequence, and 10 μL was taken and dropped on the electrode modified with IL-6 aptamer and MCH according to the concentration gradient from low to high; the concentrations of the diluted analyte solution were 100 pg / mL, 1 ng / mL, 10 ng / mL, 25 ng / mL, and 50 ng / mL, respectively, and the analyte was connected with the IL-6 aptamer.

[0028] The reason for selecting these concentrations of analytes is that the concentration range of the inflammatory factor IL-6 in human wound exudate is within 50 ng / mL, so the analyte concentration selected for the experimental operation must be included in this range. The test shows that the redox current height of these analytes decreases regularly, which can achieve the quantitative concentration of IL-6 in wound exudate and ensure the feasibility of this sensor in practical applications.

[0029] S52. After standing at 37°C for 1.5 hours, the electrode is washed with PBS. The electrode preparation is completed.

[0030] Preferably, the thickness of the polyimide film in step S11 is 75 μm; in step S12, the operating parameters of the carbon dioxide laser instrument are power 7%, speed 7%, and Z axis is set to -0.1 mm; and the sensing area of ​​the patterned laser induced graphene working electrode is a circle with a diameter of 3 mm.

[0031] Preferably, in step S21, the preparation process of the deposition solution is as follows: 5 mM chloroauric acid and 0.1 M nitric acid are dissolved in deionized water; in step S22, the parameters of the cyclic voltammetry electrochemical deposition are as follows: the deposition potential is: -0.9 V to -0.1 V, and the number of deposition cycles is 10-40 cycles.

[0032] Preferably, in step 32, the preparation process of the aptamer solution is as follows: IL-6 aptamer powder is first dissolved in TE buffer to form a 100 μM solution, and then TM buffer is used as a solution to prepare an aptamer and TECP solution (2-10 μM, 5 mM).

[0033] IL-6 aptamer powder can be purchased directly. It is generally a base sequence. One end of the base sequence is modified with methylene blue and the other end is connected to nanogold particles. The base sequence can bind to the analyte exuded from the wound.

[0034] Preferably, the electrochemical sensor is used to detect inflammatory factors in chronic wound exudate.

[0035] The beneficial effects of the present invention are embodied in:

[0036] (1) The electrochemical sensor prepared by the method provided by the present invention has lower cost and simpler preparation process than the traditional noble metal aptamer sensor. It can be used to accurately detect inflammatory factors in chronic wounds. This sensor is expected to become a companion doctor for patients at home, reminding patients of wound deterioration and taking action when treatment is needed.

[0037] (2) The electrochemical sensor prepared by the method provided by the present invention uses graphene materials. Graphene materials have unique physical and chemical properties, such as high specific surface area and electrical conductivity, making graphene a particularly attractive biosensing material. The emergence of laser-induced graphene (LIG) technology makes it possible to prepare graphene materials with precise patterns on a large scale. In addition, LIG ​​technology is based on laser engraving of polyimide films to achieve one-step production of patterned graphene. Therefore, the advantages of using laser-induced graphene electrochemical biosensors for the development of aptamer-based biosensors are: laser-induced graphene can be prepared on a large scale, has good uniformity, and has high electrical conductivity.

[0038] (3) The electrochemical sensor prepared by the present invention can be used for electrochemical detection of inflammatory factor IL-6 in chronic wounds. It can quantify the concentration of IL-6 in wound exudate according to the redox current of analytes of different concentrations, and has the advantages of low detection limit, fast detection speed, and no need for professional instruments. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a Raman spectrum diagram of laser-induced graphene of the present invention;

[0040] Figure 2 The scanning electron microscope images of the composite electrodes deposited with different numbers of gold nanoparticles of the present invention;

[0041] Figure 3 This is a graph showing the SWV current variation when the composite electrode of gold nanoparticles with different numbers of circles prepared by the present invention detects the inflammatory factor IL-6;

[0042] Figure 4 The SWV current change and linear fitting diagram of the electrochemical sensor prepared in the present invention when detecting different concentrations of inflammatory factor IL-6;

[0043] Figure 5 This is a diagram showing the anti-interference performance of the electrochemical sensor prepared by the present invention. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] like Figure 1-5 As shown:

[0046] Example 1

[0047] This example provides a method for preparing an electrochemical sensor for detecting inflammatory factors:

[0048] The gold nanoparticle precursor solution was prepared with deionized water to a concentration of 5mM chloroauric acid and 0.1M nitric acid. A three-electrode system was used for electrochemical deposition of gold nanoparticles. The reference electrode and the counter electrode were commercial Ag / AgCl electrode and platinum wire electrode, respectively. The working electrode was a patterned laser-induced graphene electrode with a diameter of 3mm (laser printing parameters were power 7%, speed 7%, Z axis -0.1mm). Cyclic voltammetry was used for electrochemical deposition of gold nanoparticles. The deposition potential was -0.9V to -0.1V, and the number of deposition cycles was 30. The deposited electrode was cleaned and dried with nitrogen for later use. IL-6 aptamer powder was first prepared into a 100μM solution with TE buffer, and then aptamer and TECP (10μM, 5mM) solution were prepared with TM buffer as the solution. 10μL of this solution was dropped on the working electrode. After standing at 4℃ for 12h, the IL-6 aptamer was successfully modified and then washed with PBS for later use. The MCH solution was prepared to be 1mM, and 10μL was taken and dropped on the electrode after the aptamer was modified. It was left at 37℃ for 1.5h. MCH was successfully modified. After the electrode was washed with PBS, the analyte solution was prepared and the analyte solution was diluted to different concentrations (0-50ng / mL) in sequence. The analyte concentration gradients were 100pg / mL, 1ng / mL, 10ng / mL, 25ng / mL, and 50ng / mL. 10μL was taken and dropped on the working electrode modified with the aptamer and MCH according to the gradient from low to high. It was left at 37℃ for 1.5h. After washing with PBS, the electrochemical sensor for detecting the inflammatory factor IL-6 was prepared.

[0049] Example 2

[0050] This example provides a method for preparing an electrochemical sensor for detecting inflammatory factors.

[0051] The gold nanoparticle precursor solution was prepared with deionized water to a concentration of 5mM chloroauric acid and 0.1M nitric acid. A three-electrode system was used for electrochemical deposition of gold nanoparticles. The reference electrode and the counter electrode were commercial Ag / AgCl electrode and platinum wire electrode, respectively. The working electrode was a patterned laser-induced graphene electrode with a diameter of 3mm (laser printing parameters were power 7%, speed 7%, Z axis -0.1mm). Cyclic voltammetry was used for electrochemical deposition of gold nanoparticles. The deposition potential was -0.9V to -0.1V, and the number of deposition cycles was 20. The deposited electrode was cleaned and dried with nitrogen for later use. IL-6 aptamer powder was first prepared into a 100μM solution with TE buffer, and then aptamer and TECP (10μM, 5mM) solution were prepared with TM buffer as the solution. 10μL of this solution was dropped on the working electrode. After standing at 4℃ for 12h, the IL-6 aptamer was successfully modified and then washed with PBS for later use. The MCH solution was prepared to be 1mM, and 10μL was dropped on the electrode after the aptamer was modified. It was left at 37℃ for 1.5h. MCH was successfully modified. After the electrode was washed with PBS, the analyte solution was prepared and the analyte solution was diluted to different concentrations (0-50ng / mL) in sequence. 10μL was dropped on the working electrode modified with the aptamer and MCH according to the gradient from low to high, and it was left at 37℃ for 1.5h. After washing with PBS, the electrochemical sensor for detecting the inflammatory factor IL-6 was prepared.

[0052] Example 3

[0053] This example provides a method for preparing an electrochemical sensor for detecting inflammatory factors, wherein the concentration of a gold nanoparticle precursor solution is 5mM chloroauric acid and 0.1M nitric acid, prepared with deionized water. A three-electrode system is used to electrochemically deposit gold nanoparticles, wherein the reference electrode and the counter electrode are commercially available Ag / AgCl electrodes and platinum wire electrodes, respectively, and the working electrode is a patterned laser-induced graphene electrode with a diameter of 3mm (laser printing parameters are power 7%, speed 7%, Z axis -0.1mm). Cyclic voltammetry is used to electrochemically deposit gold nanoparticles, and the deposition potential is -0.9V to -0.1V, and the number of deposition turns is 10 turns. The deposited electrode is cleaned and dried with nitrogen for later use. The IL-6 aptamer powder is first prepared into a 100μM solution with TE buffer, and then the aptamer and TECP (10μM, 5mM) solution are prepared with TM buffer as the solution. 10μL of this solution is dropped on the working electrode, and the IL-6 aptamer is successfully modified after standing at 4°C for 12h, and then washed with PBS for later use. The MCH solution was prepared to be 1mM, and 10μL was dropped on the electrode after the aptamer was modified. It was left at 37℃ for 1.5h. MCH was successfully modified. After the electrode was washed with PBS, the analyte solution was prepared and the analyte solution was diluted to different concentrations (0-50ng / mL) in sequence. 10μL was dropped on the working electrode modified with the aptamer and MCH according to the gradient from low to high, and it was left at 37℃ for 1.5h. After washing with PBS, the electrochemical sensor for detecting the inflammatory factor IL-6 was prepared.

[0054] The principle of using electrochemical sensors to detect inflammatory factors in chronic wound exudate: In the specific implementation, due to the selection of a specific IL-6 aptamer sequence, one end is modified with methylene blue (redox probe) and the other end is modified with thiol (thiol is covalently bound to gold nanoparticles). The blank group is the one without modified analyte. The methylene blue is close to the gold nanoparticles, allowing electron transfer, so the redox current peak is higher.

[0055] After analyte modification, the methylene blue probe leaves the electrode and the redox current decreases. The more analyte modification, the lower the redox current.

[0056] By linearly fitting the redox signal and calculating the IL-6 concentration value, the content of inflammatory factor IL-6 in chronic wounds can be calculated, thereby analyzing the healing of chronic wounds.

[0057] Figure 1 The Raman spectrum of the laser-induced graphene electrode of Example 1 includes 1350 cm -1 The D peak and 1590cm -1 The G peak and the typical 2710cm -1 The 2d peak of α indicates the laser-induced formation of graphene.

[0058] Figure 2 The following are microscopic scanning electron microscope images of laser-induced graphene modified with different numbers of gold nanoparticles in the embodiment. The laser-induced graphene is a fibrous structure, and the gold nanoparticles are distributed between the laser-induced graphene layers. The number of gold nanoparticles increases as the number of modified circles increases.

[0059] Figure 3 SWV curves of laser-induced graphene modified with gold nanoparticles with different circles. We compared the current changes of the unmodified analyte and the analyte modified with 50 ng / mL, and finally chose 30 circles for the modification of gold nanoparticles.

[0060] Figure 4 The SWV curves of the prepared electrochemical sensor in detecting analytes of different concentrations. As can be seen from the figure, when the electrochemical sensor of the present invention is used to analyze the concentration of inflammatory factors, the oxidation current gradually decreases with the increase of the aptamer concentration, and the concentration of IL-6 in the range of 0-50ng / mL (including the concentration of IL-6 inflammatory factors in the human body) is linearly related to the decrease in relative peak height.

[0061] Figure 5 The figure is a graph of the anti-interference performance of the electrochemical sensor prepared in Example 1. The anti-interference performance was tested using other cytokines in wound exudate. As can be seen from the figure, the electrochemical sensor based on the inflammatory factor IL-6 aptamer-modified gold nanoparticle-laser induced graphite prepared by the present invention has a small response to other inflammatory factors, indicating that the sensor has relatively good anti-interference performance when detecting IL-6.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical sensor for detecting inflammatory factors, characterized in that: The following steps are involved: S1, laser-induced printing of graphene electrodes on polyimide film using a carbon dioxide laser; S2. Preparation of gold nanoparticle / laser-induced graphene composite electrode using electrochemical deposition on the printed graphene electrode: S3, modifying the IL-6 aptamer on the prepared gold nanoparticle / laser-induced graphene composite electrode by using a dropwise functionalization modification method; S4, performing MCH modification on the electrode modified with the IL-6 aptamer; S5. Perform analyte modification on the MCH-modified electrode to obtain an electrochemical sensor.

2. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 1, characterized in that: In step S1, the process of printing laser-induced graphene on a polyimide film using a carbon dioxide laser includes: S11, washing the polyimide film with acetone, ethanol and deionized water respectively, and then drying it with nitrogen; S12. Fix the clean polyimide film on the acrylic plate with tape, and then use a carbon dioxide laser instrument to print electrodes on the polyimide film according to the computer preset pattern.

3. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 1, characterized in that: In step S2, the process of preparing the gold nanoparticle / laser induced graphene composite electrode by electrochemical deposition includes: S21, preparing a deposition solution; S22. In a deposition solution, electrochemically deposit gold nanoparticles onto the laser-induced graphene using cyclic voltammetry until a layer of gold nanoparticles is modified on the surface of the laser-induced graphene.

4. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 3, characterized in that: In step S3, the process of modifying the IL-6 aptamer by the dropwise functionalization modification method includes: S31, cleaning the gold nanoparticle / laser-induced graphene composite electrode with deionized water, and then drying it with nitrogen gas; S32, preparing an aptamer solution; S33, dropping the aptamer solution on the working electrode and leaving it at 4°C for 12 hours to complete the modification of the IL-6 aptamer; S34. Wash the electrode modified with IL-6 aptamer with PBS.

5. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 1, characterized in that: In step S4, the process of performing MCH modification on the electrode modified with the IL-6 aptamer includes: S41, preparing MCH solution; S42, dropping the MCH solution onto the electrode modified with the IL-6 aptamer, and leaving it at 37° C. for 1.5 h to complete the MCH modification; S43. Wash the MCH-modified electrode with PBS.

6. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 1, characterized in that: In step S5, the process of modifying the electrode modified with MCH with an analyte includes: S51, dilute the analyte solution to different concentrations in sequence, and take 10 μL of each solution and drop it on the electrode modified with IL-6 aptamer and MCH according to the concentration gradient from low to high; S52. After standing at 37°C for 1.5 hours, the electrode is washed with PBS. The electrode preparation is completed.

7. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 2, characterized in that: The thickness of the polyimide film in step S11 is 75 μm; in step S12, the operating parameters of the carbon dioxide laser instrument are power 7%, speed 7%, and Z axis is set to -0.1 mm; the sensing area of ​​the patterned laser induced graphene working electrode is a circle with a diameter of 3 mm.

8. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 3, characterized in that: In step S21, the preparation process of the deposition solution is: dissolving chloroauric acid and nitric acid in deionized water; in step S22, the parameters of the cyclic voltammetry electrochemical deposition are: deposition potential: -0.9V to -0.1V, and the number of deposition cycles is 10-40 cycles.

9. The method for preparing an electrochemical sensor for detecting inflammatory factors according to claim 4, characterized in that: In step S32, the preparation process of the aptamer solution is as follows: the aptamer powder is first dissolved into a solution using TE buffer, and then the aptamer and TECP solutions are prepared using TM buffer as a solution.

10. Use of an electrochemical sensor for detecting inflammatory factors according to any one of claims 1 to 9, characterized in that: The electrochemical sensor is used to detect inflammatory factors in chronic wound exudate.

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