An electrochemical microneedle biosensor and a preparation method thereof

Abstract

The invention discloses an electrochemical microneedle biosensor and a preparation method thereof. The biosensor is a three-electrode system, comprising a working electrode, a counter electrode and a reference electrode, wherein the substrate materials of the three electrodes are all silicon-based high-density microneedle arrays; wherein the working electrode is a silicon-based high-density microneedle with a sputtered gold layer, gold nanoparticles with a dendrite structure are deposited on the surface, and a carcinoembryonic antigen (CEA) aptamer with high specificity is combined through an Au-S bond; the electrochemical microneedle biosensor provided by the invention adopts a high-density silicon-based microneedle array to construct a three-electrode system to detect carcinoembryonic antigen in tissue fluid, the working electrode has gold nanoparticles with a dendrite structure, and the active surface area is preferably increased, the patch-type structure with three electrodes in one is easy to operate, can realize non-invasive rapid detection, and has high detection sensitivity, good specificity and low detection limit, thereby providing a simple and quick method for detecting tumor biomarkers such as carcinoembryonic antigen.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and in particular relates to an electrochemical microneedle biosensor and a preparation method thereof. Background Art

[0002] Cancer is a disease that seriously threatens human health and life, and early diagnosis and early intervention of cancer are important ways to improve the survival rate of patients. Relevant studies have shown that the occurrence of malignant tumors is closely related to changes in the expression of related protein genes in cells. Therefore, establishing a method for rapid, accurate and sensitive detection of tumor cells and their gene expression levels is of great significance for the study of the pathogenesis, early diagnosis and treatment of tumors. Among them, tumor markers are active substances closely related to tumors, and their presence or content changes in the human body can serve as an important basis for determining whether a tumor exists or not.

[0003] Carcinoembryonic antigen (CEA) is a widely used tumor marker. Its abnormal content level in serum is often associated with some tumor diseases, such as lung cancer and gastrointestinal cancer. At present, the main methods for clinical detection of CEA include radioimmunoassay, immunochemiluminescence and enzyme-linked immunosorbent assay, which have the disadvantages of complex operation, time-consuming and high cost. Electrochemical biosensors achieve quantitative detection of targets through electrochemical signals such as current, and have the characteristics of low detection cost, short response time and simple operation. However, for the current label-free electrochemical biosensors, although simple to operate, there are certain deficiencies in sensitivity and poor stability. As people pay more attention to their own health, the demand for real-time rapid detection and wearable monitoring is increasing. Therefore, the development of a highly sensitive, highly specific, simple and portable method to detect the concentration of CEA in tissue fluid is of great significance for clinical rapid diagnosis. Summary of the invention

[0004] To solve the above problems, the present invention discloses an electrochemical microneedle biosensor and a preparation method thereof. By designing and preparing a high-density microneedle array electrode with a dendrite structure and combining it with aptamer modification, it is possible to perform real-time detection of carcinoembryonic antigens in tissue fluid with high sensitivity, high specificity and convenient operation. The sensor also has the advantages of miniaturization and low production cost.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] An electrochemical microneedle biosensor is a three-electrode system, including a working electrode, a counter electrode and a reference electrode. The substrate materials of the three electrodes are all high-density silicon-based microneedle arrays. The microneedle array is a close-packed hexagonal structure, and the number of microneedles per square centimeter is 9000-11000. The working electrode is a high-density silicon-based microneedle array electrode with a sputtered gold layer, and gold nanoparticles with a dendrite structure are deposited on the surface and are bound to a carcinoembryonic antigen (CEA) aptamer through an Au-S bond. The counter electrode is a high-density silicon-based microneedle array electrode with a sputtered platinum layer, and the reference electrode is a high-density silicon-based microneedle array electrode of silver / silver chloride.

[0007] Preferably, the carcinoembryonic antigen (CEA) aptamer is a DNA nucleotide sequence; the DNA nucleotide sequence is: 5'-AAAAAAATACCAGCTTATTCAATT-3', and the 5' end of the aptamer is modified with a thiol group to: 5'-HS-C6-AAAAAAATACCAGCTTATTCAATT-3'.

[0008] The present invention also provides a method for preparing the electrochemical microneedle biosensor, comprising the following steps:

[0009] Step (1): Select a double-polished or single-polished monocrystalline silicon wafer with a thickness of 500-1000 μm;

[0010] Step (2): forming a silicon oxide film with a thickness of 100-300 nm on the surface of the silicon wafer by chemical vapor deposition, and then forming a close-packed hexagonal structure pattern by photoresist, exposure and development; removing the silicon oxide not covered by the photoresist by reactive plasma etching technology, and etching the silicon to form a silicon pillar array by deep silicon etching;

[0011] Step (3): using isotropic dry etching and wet etching (hydrogen fluoride: nitric acid: acetic acid volume ratio of 1:10:25) to etch the silicon pillars in turn, and supplemented with bubbling treatment, so as to make the silicon cylinder array thinner and sharper while maintaining the integrity of the close-packed hexagonal structure pattern;

[0012] Step (4): depositing a titanium / gold film on the surface of the microneedle array by magnetron sputtering, and then growing dendrite-structured gold nanoparticles on the surface of the microneedle electrode by electrodeposition to form a working electrode; depositing a titanium / platinum film on the surface of the microneedle array to form a counter electrode; depositing a silver film on the surface of the microneedle array to obtain a silver microneedle electrode, and then immersing the silver microneedle electrode in silver chloride ink, taking it out and drying it to obtain a silver / silver chloride reference electrode, thereby completing the preparation of the three-electrode system.

[0013] Step (5): Modify the aptamer on the working electrode. The main process includes the following steps:

[0014] a. Place the working electrode microneedle array in a shaker at 150 / min for 10 min, and wash the electrode surface thoroughly with 99.5% acetone, 99.5% alcohol, and deionized water (PCR grade) respectively; take out the nitrogen to dry, and place in a vacuum drying oven at 70°C for 10 min to remove residual moisture;

[0015] b. Prepare 0.5 M sulfuric acid solution and use cyclic voltammetry (CV) with a scan range of -0.2~1.6V and 50 scans to test the working electrode until a stable redox peak is obtained;

[0016] c. Place the aptamer in a centrifuge for centrifugation, prepare the aptamer concentration to 2 μM with 1 mM dithiothreitol and 10 mM phosphate buffered saline (PBS) at pH 7.4, anneal at 90°C for 5 min, then cool to room temperature in the air, drop 150 μL of the above aptamer solution on the surface of the working electrode microneedle treated in step b, and incubate at 37°C in a constant temperature shaker for 2 h; take a PBS buffer solution with a concentration of 10 mM and pH 7.4 to fully wash 5 times, and the aptamer is modified on the working electrode.

[0017] Preferably, in step (3), the isotropic dry etching time is 10-20 min, and the corrosion time is 5-10 min.

[0018] Preferably, the density of the microneedle array in step (3) is 9000 microneedles / cm 2 -11000 / cm 2 , height is 150um-400um, and microneedle diameter is 50um-100um.

[0019] Preferably, the deposition current of the electro-deposition method in step (4) is 0.1-0.3A, and the deposition time is 10-60S.

[0020] The beneficial effects of the present invention are:

[0021] The electrochemical microneedle biosensor of the present invention is designed to prepare a high-density microneedle array, and deposits gold nanoparticles with a dendrite structure on the surface of the microneedle electrode, and then binds the carcinoembryonic antigen (CEA) aptamer through the Au-S bond, and has the advantages of both electrochemical sensors and aptamers, and can have the advantages of high sensitivity and high specificity at the same time;

[0022] The electrochemical microneedle biosensor of the present invention has a three-electrode integrated patch structure, and can realize direct skin contact detection of carcinoembryonic antigen through real-time monitoring of voltammetric current, without the need for blood extraction and processing. The constructed sensor is easy to prepare and operate, and has good specificity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation of the electrochemical microneedle biosensor of the present invention;

[0024] Figure 2 This is a scanning electron microscope image of the working electrode of the electrochemical microneedle biosensor of the present invention;

[0025] Figure 3 This is a schematic diagram of the electrochemical microneedle biosensor for detecting carcinoembryonic antigen according to the present invention;

[0026] Figure 4 The differential pulse voltammogram of the electrochemical microneedle biosensor of the present invention for detecting changes in carcinoembryonic antigen concentration;

[0027] Figure 5 This is a comparison chart of the specificity of the electrochemical microneedle biosensor of the present invention in detecting carcinoembryonic antigen. DETAILED DESCRIPTION

[0028] The present invention is preferably explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0029] As used herein, "biomarker" and "marker" are used interchangeably to refer to an indication of normal or abnormal progression in an individual or an indication of a disease or other condition in an individual or a target molecule that expresses these. In more detail, "biomarker" and "marker" are normal or abnormal, and if abnormal, anatomical, physiological, biochemical or molecular parameters that are associated with the presence of a specific physiological state or progression, whether chronic or acute. Biomarkers can be detected and measured by a variety of methods including laboratory testing and medical imaging.

[0030] The room temperature in the present invention is 25°C.

[0031] The electrochemical microneedle biosensor of the present invention is a three-electrode system, including a working electrode, a counter electrode and a reference electrode. The substrate materials of the three electrodes are all high-density silicon-based microneedle arrays. Dendrite-structured gold nanoparticles are deposited on the surface of the microneedle electrode, and then the aptamer is modified on the electrode surface. Specifically, the process includes: forming a silicon oxide film on the surface of a silicon wafer by chemical vapor deposition, forming a silicon column pattern by photolithography and reactive plasma etching processes, deep silicon etching to form a silicon column array, forming a microneedle array by isotropic dry and wet etching processes, and then respectively manufacturing a working electrode, a counter electrode and a reference electrode, wherein the surface of the microneedle working electrode has dendrite-structured gold nanoparticles, and then the electrode surface is combined with a carcinoembryonic antigen (CEA) aptamer with high specificity through an Au-S bond, which can be used for rapid detection of carcinoembryonic antigen expression.

[0032] Example 1 is a specific embodiment of the present invention. Figure 1 As shown, this embodiment provides a method for preparing an electrochemical microneedle biosensor, and the specific steps are:

[0033] Step (1): Select a single-throw (100) crystal orientation silicon wafer with a thickness of 600um;

[0034] Step (2): using chemical vapor deposition to form a silicon oxide film with a thickness of 200 nm on the surface of the silicon wafer, and then forming an array pattern by coating, exposing, and developing; using reactive plasma etching technology to remove the silicon oxide not covered by the photoresist, and using deep silicon etching to etch the silicon to form a silicon pillar array;

[0035] Step (3): firstly adopt isotropic dry etching for 12 minutes, prepare a solution of hydrogen fluoride: nitric acid: acetic acid in a volume ratio of 1:10:25, and etch the silicon pillars for 7 minutes, and supplement with bubbling treatment to make the silicon pillar array thinner and sharper while maintaining the integrity of the close-packed structure pattern;

[0036] Step (4): A titanium / gold film is deposited on the surface of the microneedle array by magnetron sputtering, and then dendrite-structured gold nanoparticles are grown on the surface of the microneedle electrode by electroplating with a deposition current of 0.2 A and a deposition time of 30 s to form a working electrode, such as Figure 2 As shown; a titanium / platinum film is deposited on the surface of the microneedle array to form a counter electrode; a silver film is deposited on the surface of the microneedle array to obtain a silver microneedle electrode, and then the silver microneedle electrode is immersed in silver chloride ink, and then taken out and dried in an oven to obtain a silver / silver chloride reference electrode, thereby completing the preparation of a three-electrode system;

[0037] Step (5) modifying the aptamer on the working electrode, the main process includes the following steps:

[0038] a. Place the working electrode microneedle array in a shaker at 150 R / min for 10 min, and wash the electrode surface thoroughly with 99.5% acetone, 99.5% alcohol, and deionized water (PCR grade), respectively; then dry with nitrogen, and place in a vacuum drying oven at 70°C for 10 min to remove residual moisture;

[0039] b. Prepare 0.5 M sulfuric acid solution and use cyclic voltammetry (CV) with a scan range of -0.2~1.6V and 50 scans to test the working electrode until a stable redox peak is obtained;

[0040] c. Place the aptamer in a centrifuge for centrifugation, prepare the aptamer concentration to 2 μM with 1 mM DTT, 10 mM PBS buffer solution, pH 7.4, anneal at 90 ° C for 5 min, then cool to room temperature in the air, drop 150 μL of the above aptamer solution on the surface of the gold electrode treated in step b, incubate at 37 ° C in a constant temperature shaker for 2 h, and wash it thoroughly 5 times with 10 mM PBS buffer solution at pH 7.4 to obtain the modified working electrode. The electrochemical microneedle biosensor prepared in this embodiment, in which the density of the microneedle array is 10,000 / cm 2 The height of the microneedle is 250um, the diameter of the microneedle is 60um, the prepared microneedle array is complete, and the tip of the microneedle is sharp.

[0041] Example 2: Based on Example 1, an electrochemical microneedle biosensor was prepared. The difference from Example 1 is that the thickness of the single-polished (100) crystal orientation silicon wafer in step (1) is 500 um, the isotropic dry etching time in step (3) is 10 min, the corrosion time is 8 min, the deposition current in step 4 is 0.3 A, and the deposition time is 50 s. The rest is the same as Example 1.

[0042] The electrochemical microneedle biosensor prepared in this example has a microneedle array density of 11000 / cm 2 , the height of the microneedle is 150um, the diameter of the microneedle is 50um, the height of the prepared microneedle array is inconsistent, and the height of some parts is reduced.

[0043] Example 3: Based on Example 1, an electrochemical microneedle biosensor was prepared. The difference from Example 1 is that the thickness of the single-polished (100) crystal orientation silicon wafer in step (1) is 700 um, the isotropic dry etching time in step (3) is 20 min, the corrosion time is 5 min, the deposition current in step 4 is 0.2 A, and the deposition time is 30 s. The rest is the same as Example 1.

[0044] The electrochemical microneedle biosensor prepared in this example has a microneedle array density of 9000 / cm 2 The height of the microneedle is 350um, the diameter of the microneedle is 90um, the prepared microneedle array is complete, and there is a platform on the top of the microneedle.

[0045] Example 4: The electrochemical microneedle biosensor prepared in this example has a microneedle array density of 9000 / cm 2 -11000 / cm 2 , preferably 10000 roots / cm in this embodiment 2The height of the microneedle is 150um-400um, and the preferred height of the microneedle in this embodiment is 250um. The diameter of the microneedle is 50um-100um, and the preferred diameter of the microneedle in this embodiment is 60um.

[0046] The specific steps of the electrochemical microneedle biosensor prepared in this example for detecting the concentration of carcinoembryonic antigen are as follows:

[0047] (1) Prepare a 0.1 M KCl solution containing 5.0 mM [(FeCN)6] 3- / 4- as the dielectric for the electrochemical test. Use a 10 mM PBS buffer solution with a pH of 7.4 to prepare a series of carcinoembryonic antigen (CEA) solutions with concentrations of 0.5 ng / ml, 5 ng / ml, 50 ng / ml, 500 ng / ml, and 5 μg / ml;

[0048] (2) 0.5 ng / ml, 5 ng / ml, 50 ng / ml, 500 ng / ml, and 5 μg / ml carcinoembryonic antigen (CEA) solutions were dripped onto the surface of the gold electrode modified with the aptamer in sequence, and incubated at 37°C in a constant temperature shaker for 2 h. The electrode was then tested using differential pulse voltammetry (DPV) in a 0.1 M KCl solution containing 5.0 mM [(FeCN)6] 3- / 4-. The differential pulse voltammograms at different CEA concentrations were measured in sequence, as shown in Figure 2. Figure 3 The results show that the electrochemical microneedle biosensor has high sensitivity.

[0049] Figure 4 Shown is the structural change after the aptamer binds to carcinoembryonic antigen on the working electrode of the electrochemical microneedle sensor.

[0050] In order to detect the specificity of the electrochemical microneedle biosensor, the microneedle biosensor prepared above was incubated with 50 ng / ml CEA, 50 ng / ml BSA, 50 ng / ml IL-6, 50 ng / ml HER2, and 50 ng / ml APTAMER in a constant temperature shaker for 2 h, and tested by differential pulse voltammetry to measure the peak current. At the same time, it was compared with an unmodified bare electrode. The test results are shown in Figure 5 .

[0051] Depend on Figure 5 It can be seen that the electrochemical microneedle sensor has high specificity for CEA and high detection sensitivity. BSA, IL-6, HER2, APTAMER, etc. present in the blood will not interfere with its detection of CEA.

[0052] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing an electrochemical microneedle biosensor, characterized in that: The electrochemical microneedle biosensor is a three-electrode system, including a working electrode, a counter electrode and a reference electrode. The substrate material of the three-electrode system is a high-density silicon-based microneedle array. The microneedle array is a close-packed hexagonal structure, and the number of microneedles per square centimeter is 9000-11000. The working electrode is a high-density silicon-based microneedle array electrode with a sputtered titanium-gold layer, and gold nanoparticles with a dendrite structure are deposited on the surface, and the carcinoembryonic antigen aptamer is bound by an Au-S bond. The counter electrode is a high-density silicon-based microneedle array electrode with a sputtered titanium-platinum layer, and the reference electrode is a high-density silicon-based microneedle array electrode of silver / silver chloride; The carcinoembryonic antigen aptamer is a DNA nucleotide sequence; the DNA nucleotide sequence is: 5'-AAAAAAATACCAGCTTATTCAATT-3', and the 5' end of the aptamer is modified with a thiol group to be: 5'-HS-C6-AAAAAAATACCAGCTTATTCAATT-3'; The preparation method comprises the following steps: Step (1): Select a double-polished or single-polished monocrystalline silicon wafer with a thickness of 500-1000 μm; Step (2): forming a silicon oxide film with a thickness of 100-300 nm on the surface of the silicon wafer by chemical vapor deposition, and then forming a close-packed hexagonal structure pattern by photoresist, exposure and development; removing the silicon oxide not covered by the photoresist by reactive plasma etching technology, and etching the silicon to form a silicon column array by deep silicon etching; Step (3): isotropic dry etching and wet etching are performed on the silicon cylinders, supplemented by bubbling treatment, so as to make the silicon cylinder array thinner and sharper while maintaining the integrity of the close-packed hexagonal structure pattern; Step (4): using a magnetron sputtering method to deposit a titanium / gold film on the surface of the microneedle array, and then using an electrodeposition method to grow dendrite-structured gold nanoparticles on the surface of the microneedle electrode to form a working electrode; depositing a titanium / platinum film on the surface of the microneedle array to form a counter electrode; depositing a silver film on the surface of the microneedle array to obtain a silver microneedle electrode, and then immersing the silver microneedle electrode in silver chloride ink, taking it out and drying it to obtain a silver / silver chloride reference electrode, thereby completing the preparation of a three-electrode system; Step (5): Modifying the aptamer on the working electrode, comprising the following steps: a. Place the working electrode microneedle array in a shaker at 150 r / min for 10 min, and wash the electrode surface with acetone, alcohol, and deionized water respectively; take out the nitrogen to dry, and place it in a vacuum drying oven at 70°C for 10 min to remove residual moisture; b. Prepare 0.5 M sulfuric acid solution and use cyclic voltammetry with a scan range of -0.2~1.6V and 50 scans to test the working electrode until a stable redox peak is obtained; c. Place the aptamer in a centrifuge for centrifugation, prepare an aptamer solution with an aptamer concentration of 2 μM using 1 mM dithiothreitol and 10 mM, pH 7.4 phosphate buffer, anneal at 90°C for 5 min, then cool to room temperature in the air, drop 150 μL of the above aptamer solution on the surface of the working electrode microneedle treated in step b, incubate in a constant temperature shaker, and wash thoroughly 5 times with PBS buffer to obtain the modified aptamer on the working electrode.

2. The method for preparing the electrochemical microneedle biosensor according to claim 1, characterized in that: The wet etching solution includes hydrogen fluoride, nitric acid and acetic acid.

3. The method for preparing the electrochemical microneedle biosensor according to claim 2, characterized in that: The volume ratio of the hydrogen fluoride, nitric acid and acetic acid is 1:10:

25.

4. The method for preparing the electrochemical microneedle biosensor according to claim 1, characterized in that: The isotropic dry etching time in step (3) is 10-20 minutes, and the corrosion time is 5-10 minutes.

5. The method for preparing the electrochemical microneedle biosensor according to claim 1, characterized in that: The density of the microneedle array in step (3) is 9000 / cm 2 -11000 / cm 2 , height is 150μm-400μm, and microneedle diameter is 50μm-100μm.

6. The method for preparing an electrochemical microneedle biosensor according to claim 1, characterized in that: The deposition current of the electrodeposition in step (4) is 0.1-0.3A, and the deposition time is 10-60S.

7. The method for preparing an electrochemical microneedle biosensor according to claim 1, characterized in that: The constant temperature shaker in step (5) is set at 37°C and the incubation time is 2 hours.

8. The method for preparing an electrochemical microneedle biosensor according to claim 1, characterized in that: The concentration of the PBS buffer in step (5) is 10 mM and the pH is 7.4.

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