Non-diagonal giant magneto-impedance sensor for detecting biomarkers and preparation method

Abstract

The invention relates to the technical field of magnetic sensors, in particular to a non-diagonal giant magneto-impedance sensor for detecting biomarkers and a preparation method thereof.The non-diagonal giant magneto-impedance sensor comprises an impedance sensor, a PI film, a SiO2 film, a self-assembly monomolecular film and an array microcavity, and the impedance sensor comprises a sensitive element and an induction coil; a-aminopropyltriethoxysilane self-assembled monomolecular film prepared on a SiO2 film can be used for fixing a monoclonal antibody, a microcavity array is placed on the self-assembled monomolecular film and is used for biological immune reaction, the magnetic label adopts streptavidin magnetic beads, the surface of the magnetic bead is modified by streptavidin, and the magnetic label can be used for biological immune reaction. Streptavidin molecules having multiple peptide chains are shown, which can be used to specifically bind biotinylated antibodies. According to the impedance sensor, a thin strip with a parallel structure is adopted as a sensitive element, and high-sensitivity biomarker detection can be realized on the basis of the principle of a double-antibody sandwich method.

Description

Technical Field

[0001] The present invention relates to the technical field of magnetic sensors, and in particular to a non-diagonal giant magneto-impedance sensor for detecting biomarkers and a preparation method thereof. Background Art

[0002] In recent years, biomarker detection has become a key research area in medical diagnosis and biomonitoring. Biomarkers can provide critical information for early disease diagnosis, prognostic assessment, and monitoring of treatment efficacy, and are particularly valuable in the detection of cancer, infectious diseases, and autoimmune diseases. To improve the sensitivity, specificity, and rapid response of biomarker detection, a growing number of sensing technologies are being developed for efficient biomarker analysis.

[0003] Currently, biomarker detection technologies primarily include enzyme-linked immunosorbent assay (ELISA), immunochromatography, and polymerase chain reaction (PCR). Although these traditional techniques have been widely used in biomarker detection, they often suffer from slow detection speeds, complex procedures, limited sensitivity, and expensive equipment. Therefore, the development of novel biosensors with high sensitivity, strong biocompatibility, ease of operation, and cost-effectiveness is crucial.

[0004] The giant magnetoimpedance (GMI) effect, a novel sensing technology, has garnered widespread attention in the field of biosensors in recent years. Compared to traditional magnetoresistive sensors, GMI sensors offer higher sensitivity and are capable of detecting signals at extremely low magnetic fields. Therefore, GMI sensors hold great potential for application in biosensing. However, existing GMI sensors still face challenges in their application, such as insufficient sensitivity, poor selectivity for biological samples, and complex sensor structures. Summary of the Invention

[0005] The purpose of the present invention is to provide a non-diagonal giant magnetoimpedance sensor for detecting biomarkers and a preparation method thereof, aiming to overcome the shortcomings of existing biomarker detection technologies and improve the sensitivity, specificity and convenience of detection.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a non-diagonal giant magnetoimpedance sensor for detecting biomarkers, comprising an impedance sensor, a PI film, a SiO2 film, a self-assembled monolayer and an array microcavity, wherein the impedance sensor comprises a sensitive element and an induction coil; the induction coil is arranged outside the sensitive element, the sensitive element has a sensitive element electrode, the sensitive element electrodes are located on both sides of the sensitive element, the induction coil has coil electrodes, the coil electrodes are located on both sides of the induction coil, the PI film is arranged on the top of the induction coil, the SiO2 film is arranged on the side of the PI film away from the induction coil, the self-assembled monolayer is arranged on the side of the SiO2 film away from the PI film, and the array microcavity is arranged on the side of the self-assembled monolayer away from the SiO2 film.

[0007] The sensitive element is a Co-based or Fe-based amorphous thin strip with a parallel structure and is manufactured using a MEMS process. The induction coil is a three-dimensional solenoid coil structure and is manufactured using a MEMS process.

[0008] During the preparation of the impedance sensor, polyimide is used as an insulating and supporting material to fill all spaces between the sensitive element and the induction coil and protect the device.

[0009] Among them, the terminal group of the self-assembled monolayer is amino, which can react with the carboxyl groups in proteins, antibodies and DNA bioactive substances without affecting the biological activity, and can be used to fix antibodies. The microcavity array is prepared by micro-nano engraving technology and is used for biological immune response. A magnetic label is arranged between the self-assembled monolayer and the array microcavity. The magnetic label uses streptavidin-ylated magnetic beads, and the surface is modified with streptavidin to show streptavidin molecules with multiple peptide chains. These molecules can be used to specifically bind to biotinylated antibodies. The target antibody is fixed on the self-assembled monolayer. After the antigen solution and the magnetic bead-antibody solution are mixed, an immune reaction occurs with the antibody on the SAM to form an antibody-antigen-antibody complex. The captured magnetic beads are detected by a non-diagonal GMI sensor.

[0010] In a second aspect, the present invention further provides a method for preparing a non-diagonal giant magneto-impedance sensor for detecting biomarkers, which is applied to the non-diagonal giant magneto-impedance sensor for detecting biomarkers as described in the first aspect above, comprising the following steps:

[0011] Use MEMS technology to make sensitive components and induction coils;

[0012] A layer of SiO2 film is sputtered on the PI film, ultrasonically cleaned, and rinsed with a sulfuric acid / hydrogen peroxide mixed solution to remove surface contaminants and form the required hydroxyl groups;

[0013] Immersing the SiO2 film in a 60°C ethanol solution containing 1% v / v APTES to form an organosilanized self-assembled monolayer on the substrate, which is then attached to the surface of the induction coil.

[0014] preparing an array microcavity and fixing it on the surface of the self-assembled monolayer to immobilize the antibody;

[0015] The streptavidin-labeled immunomagnetic bead solution and the biotinylated monoclonal or polyclonal antibody solution are fully mixed in a centrifuge tube to prepare a magnetic bead-antibody coupling solution, which is then dropped into the array microcavity and washed and dried.

[0016] The present invention relates to a non-diagonal giant magneto-impedance sensor for detecting biomarkers. The self-assembled monolayer of 1,2-aminopropyltriethoxysilane prepared on the SiO2 film can be used to immobilize monoclonal antibodies. The microcavity array is placed on the self-assembled monolayer for bioimmune reactions. The magnetic labels utilize streptavidin-modified magnetic beads, whose surfaces are modified with streptavidin to display streptavidin molecules with multiple peptide chains that can specifically bind to the biotinylated antibodies. A non-diagonal detection method is employed, with current input at the sensing element terminals to detect the signal at both ends of the induction coil. The PI film is a preferred substrate for the sputtered SiO2 due to its high-temperature resistance, chemical corrosion resistance, and ease of peeling. The impedance sensor utilizes parallel thin ribbons as the sensing element. Based on the dual-antibody sandwich principle, it can achieve highly sensitive biomarker detection. Compared to traditional diagonal GMI sensors, this impedance sensor offers higher sensitivity and greater geometric design freedom, while also exhibiting superior noise immunity and temperature stability. In particular, the thin-ribbon non-diagonal GMI sensor provides a larger surface area than the amorphous wire non-diagonal sensor, enabling a more effective immune response. The parallel thin-ribbon design further significantly improves the sensitivity of the sensor. SiO2 is selected as the substrate and modified with the self-assembled monolayer. With its excellent biostability and compatibility, it provides a good environment for biomolecule fixation and immune response, ensuring the stability and reliability of the sensor in biological detection. The array microcavity provides a specific space for biological immune response, allowing antigens, antibodies and magnetic beads to more effectively contact and react, improving reaction efficiency and specificity, and achieving accurate detection of biomarkers. By fixing different target antibodies on the self-assembled monolayer, the sensor can achieve simultaneous detection of multiple biomarkers, providing more comprehensive information for biomedical research, clinical diagnosis and disease monitoring, supporting personalized medicine and precision treatment, overcoming the shortcomings of existing biomarker detection technology, and improving the sensitivity, specificity and convenience of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 1 is an isometric view of a non-diagonal GMI sensor for detecting biomarkers according to the present invention.

[0019] Figure 2 This is a right side view of the non-diagonal GMI sensor for detecting biomarkers according to the present invention.

[0020] Figure 3 1 is a front view of a non-diagonal GMI sensor for detecting biomarkers according to the present invention.

[0021] Figure 4 It is a structural diagram of the sensitive element in the non-diagonal GMI sensor of the present invention.

[0022] Figure 5 This is a process flow chart of preparing a base microcavity array using the micro-nano engraving technology of the present invention.

[0023] Figure 6 It is an immunoreaction diagram of APTES self-assembled monolayer formed on SiO2 film.

[0024] Figure 7 Figure 3 is an immunoreaction diagram of antibody immobilization in a microcavity.

[0025] Figure 8 It is an immunoreaction diagram when magnetic bead-antibody conjugate solution is dropped into the microcavity.

[0026] Figure 9 This is a flow chart of a method for preparing a non-diagonal giant magneto-impedance sensor for detecting biomarkers provided by the present invention.

[0027] In the figure: 1-sensitive element, 2-induction coil, 3-sensitive element electrode, 4-coil electrode, 5-PI film, 6-SiO2 film, 7-self-assembled monolayer, 8-array microcavity, 9-monoclonal antibody, 10-antigen, 11-polyclonal antibody, 12-biotin, 13-magnetic beads, 14-writing end mill. DETAILED DESCRIPTION

[0028] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0029] See also Figures 1 to 9 In the first aspect, the present invention provides a non-diagonal giant magneto-impedance sensor for detecting biomarkers, comprising an impedance sensor, a PI film 5, a SiO2 film 6, a self-assembled monolayer 7 and an array microcavity 8, wherein the impedance sensor comprises a sensitive element 1 and an induction coil 2; the induction coil 2 is arranged outside the sensitive element 1, the sensitive element 1 has a sensitive element electrode 3, and the sensitive element electrode 3 is located on both sides of the sensitive element 1, the induction coil 2 has a coil electrode 4, and the coil electrode 4 is located on both sides of the induction coil 2, the PI film 5 is arranged on the top of the induction coil 2, the SiO2 film 6 is arranged on the side of the PI film 5 away from the induction coil 2, the self-assembled monolayer 7 is arranged on the side of the SiO2 film 6 away from the PI film 5, and the array microcavity 8 is arranged on the side of the self-assembled monolayer 7 away from the SiO2 film 6.

[0030] In an embodiment of the present invention, the self-assembled monolayer 7 of aminopropyltriethoxysilane prepared on the SiO2 film 6 can be used to immobilize monoclonal antibodies 9. The microcavity array is placed on the self-assembled monolayer 7 for biological immune response. The magnetic label utilizes streptavidin-modified magnetic beads 13, whose surface is modified with streptavidin to display streptavidin molecules with multiple peptide chains, which can be used to specifically bind to the biotinylated antibody 12. A non-diagonal detection method is used, with current input to the sensing element 1 to detect the signal at both ends of the induction coil 2. The PI film 5 is a substrate chosen for the sputtered SiO2 due to its high temperature resistance, chemical corrosion resistance, and ease of peeling. The impedance sensor uses parallel thin strips as the sensing element 1. Based on the principle of a double antibody sandwich method, it can achieve highly sensitive biomarker detection. Compared with traditional diagonal GMI sensors, this impedance sensor has higher sensitivity and greater geometric design freedom, while also exhibiting superior noise immunity and temperature stability. In particular, the thin-strip non-diagonal GMI sensor provides a larger surface area than the amorphous wire non-diagonal sensor, enabling a more effective immune response. The parallel thin-strip design further significantly improves the sensitivity of the sensor. SiO2 is selected as the substrate and modified with the self-assembled monolayer 7. With its excellent biostability and compatibility, it provides a good environment for biomolecule fixation and immune response, ensuring the stability and reliability of the sensor in biological detection. The array microcavity 8 provides a specific space for biological immune response, allowing antigens 10, antibodies and magnetic beads 13 to more effectively contact and react, improving reaction efficiency and specificity, and achieving accurate detection of biomarkers. By fixing different target antibodies on the self-assembled monolayer, the sensor can achieve simultaneous detection of multiple biomarkers, providing more comprehensive information for biomedical research, clinical diagnosis and disease monitoring, supporting personalized medicine and precision treatment, overcoming the shortcomings of existing biomarker detection technology, and improving the sensitivity, specificity and convenience of detection.

[0031] Furthermore, during the preparation of the impedance sensor, polyimide is used as an insulating and supporting material to fill all spaces between the sensitive element 1 and the induction coil 2 and protect the device.

[0032] Furthermore, the terminal group of the self-assembled monolayer 7 is an amino group, which can react with the carboxyl groups in proteins, antibodies and DNA bioactive substances without affecting the biological activity, and can be used to fix antibodies. The microcavity array is prepared by micro-nano engraving technology for biological immune response. A magnetic label is arranged between the self-assembled monolayer 7 and the array microcavity 8. The magnetic label adopts streptavidin-ylated magnetic beads 13, and the surface is modified with streptavidin to show streptavidin molecules with multiple peptide chains. These molecules can be used to specifically bind to biotinylated antibodies. The target antibody is fixed on the self-assembled monolayer 7, and the antigen 10 solution and the magnetic bead 13-antibody solution are mixed to react with the antibody on the SAM to form an antibody-antigen 10-antibody complex. The captured magnetic beads 13 are detected by the non-diagonal GMI sensor.

[0033] Furthermore, the signal acquisition system of the impedance sensor is an SR844 DSP lock-in amplifier.

[0034] See also Figure 9 In a second aspect, the present invention further provides a method for preparing a non-diagonal giant magneto-impedance sensor for detecting biomarkers, which is applied to the non-diagonal giant magneto-impedance sensor for detecting biomarkers as described in the first aspect above, comprising the following steps:

[0035] S1 uses MEMS technology to manufacture the sensitive element 1 and the induction coil 2;

[0036] In this embodiment of the present invention, the parallel thin-strip sensor element 1 is fabricated using a MEMS process, including photolithography and etching techniques. The structure of the sensor element 1 is shown in Figure 4. A microscopic three-dimensional solenoid coil structure (induction coil 2) is fabricated around the sensor element 1 using a MEMS process, including sputtering, photolithography, etching, electroplating, and polyimide technology.

[0037] S2 sputters a layer of SiO2 film 6 on the PI film 5, performs ultrasonic cleaning, and rinses with a sulfuric acid / hydrogen peroxide mixed solution to remove surface contaminants and form the required hydroxyl groups;

[0038] In an embodiment of the present invention, a 200 nm thick SiO2 film 6 is sputtered on the PI film 5, and the prepared SiO2 film 6 is placed in deionized water for ultrasonic cleaning for 10 minutes; and rinsed with a sulfuric acid / hydrogen peroxide mixed solution (H2SO4:H2O2=4:1, SPA) at 60°C for 1 hour to remove surface pollutants and form the required hydroxyl groups (-OH).

[0039] S3: immersing the SiO2 film in a 60°C ethanol solution containing 1% v / v APTES to form an organic silanized self-assembled monolayer 7 on the substrate, and then pasting it onto the surface of the induction coil 2;

[0040] Specifically, the polymethyl methacrylate plate was fixed just below the writing end mill 14 (DGK 60) in the micro-nano engraving machine (Roland GX-350), and after setting the engraving parameters, including engraving depth (1.8 mm), circular trajectory, initial position, rotation speed (1780 revolutions per minute), and feed distance (0.05 mm), the writing end mill started to engrave the structure designed before.

[0041] In an embodiment of the present invention, the SiO2 film 6 is immersed in a 60°C ethanol solution containing 1% v / v APTES for 3.5 hours to form the organosilanized self-assembled monolayer 7 on the substrate. After preparation, the self-assembled monolayer is attached to the surface of the induction coil 2 of the impedance sensor.

[0042] S4: preparing an array microcavity 8 and fixing it on the surface of the self-assembled monolayer 7 to fix the antibody;

[0043] In an embodiment of the present invention, the microcavity array with a substrate thickness of 0.2 mm is prepared by micro-nano engraving technology and fixed on the surface of the self-assembled monomolecular film 7 of the impedance sensor for biological immune reaction. The biomarker antibody solution is dropped into the microcavity array and blocked at 37°C for 1 hour; washed with PBS solution and blown dry with nitrogen; BSA solution is dropped into it and blocked at 4°C for 2 hours, unreacted BSA is washed with PBS solution, and dried at room temperature.

[0044] S5: Thoroughly mix the streptavidin-labeled immunomagnetic beads 13 solution and the biotin-labeled monoclonal or polyclonal antibody 11 solution in a centrifuge tube to prepare a magnetic bead 13-antibody coupling solution, and drip it into the array microcavity 8, and then wash and dry it.

[0045] In an embodiment of the present invention, a solution of streptavidin-labeled immunomagnetic beads 13 and a solution of biotin-labeled polyclonal antibody 11 were thoroughly mixed in a centrifuge tube and kept in a constant temperature water bath at 37°C for 40 minutes. Magnetic separation was performed using a spin magnet. 500 μL of PBS solution was added to the centrifuge tube and shaken to form a suspension. The centrifuge tube containing the mixed solution was then placed in a spin magnet and allowed to stand for 5 minutes. After that, 400 μL of the supernatant was removed. The above steps were repeated 3-5 times to obtain a magnetic bead 13-antibody conjugate solution.

[0046] The antigen 10 solution and the magnetic bead 13-antibody coupling solution were specifically immunized and incubated at room temperature for 2 hours. After the incubation, the same magnetic separation operation was performed to remove the excess liquid. Figure 8 As shown, the magnetic bead 13-antibody-antigen 10 coupling solution was dropped into the microcavity, incubated at 37° C. for 1 h, washed with PBS solution, and dried at room temperature.

[0047] Biomarker detection is based on the principle of a double antibody sandwich method, where biological samples are magnetically labeled, allowing the magnetic sensor to respond to them. First, monoclonal antibody 9 is immobilized on an APTES SAM. Then, an antigen 10 solution and a magnetic bead 13-antibody solution are mixed to react with the monoclonal antibody 9 immobilized on the APTES SAM, specifically capturing different types of biomarkers. Finally, a non-diagonal GMI sensor is used to detect the magnetic beads 13 attached to the captured biomarkers, achieving indirect biomarker detection.

[0048] The above disclosure is merely a preferred embodiment of a non-diagonal giant magnetoimpedance sensor and preparation method for detecting biomarkers according to the present invention. This is certainly not intended to limit the scope of the present invention. A person skilled in the art will understand that any equivalent changes made by implementing all or part of the processes of the above embodiment in accordance with the claims of the present invention still fall within the scope of the invention.

Claims

1. A non-diagonal giant magnetoimpedance sensor for detecting biomarkers, characterized in that ； It includes an impedance sensor, a PI film, a SiO2 film, a self-assembled monomolecular film and an array microcavity, wherein the impedance sensor includes a sensitive element and an induction coil; The induction coil is arranged outside the sensitive element, the sensitive element has a sensitive element electrode, and the sensitive element electrodes are located on both sides of the sensitive element. The induction coil has coil electrodes, and the coil electrodes are located on both sides of the induction coil. The PI film is arranged on the top of the induction coil, the SiO2 film is arranged on the side of the PI film away from the induction coil, the self-assembled monolayer is arranged on the side of the SiO2 film away from the PI film, and the array microcavity is arranged on the side of the self-assembled monolayer away from the SiO2 film.

2. The off-diagonal giant magnetoimpedance sensor for detecting biomarkers according to claim 1, It is characterized by: The sensitive element is a Co-based or Fe-based amorphous thin strip, which has a parallel structure and is manufactured using a MEMS process. The induction coil is a three-dimensional solenoid coil structure and is manufactured using a MEMS process.

3. The non-diagonal giant magnetoimpedance sensor for detecting biomarkers according to claim 1, wherein ； During the preparation process of the impedance sensor, polyimide is used as an insulating and supporting material to fill all spaces between the sensitive element and the induction coil and protect the device.

4. The off-diagonal giant magneto-impedance sensor for detecting biomarkers according to claim 1, It is characterized by: The terminal groups of the self-assembled monolayer are amino groups, which can react with carboxyl groups in proteins, antibodies and DNA bioactive substances without affecting biological activity, and can be used to fix antibodies. The microcavity array is prepared using micro-nano engraving technology and is used for biological immune reactions. A magnetic label is arranged between the self-assembled monolayer and the array microcavity. The magnetic label uses streptavidin-labeled magnetic beads, and the surface is modified with streptavidin to exhibit streptavidin molecules with multiple peptide chains. These molecules can be used to specifically bind to biotinylated antibodies. The target antibody is fixed on the self-assembled monolayer. After the antigen solution and the magnetic bead-antibody solution are mixed, an immune reaction occurs with the antibody on the SAM to form an antibody-antigen-antibody complex. The captured magnetic beads are detected by a non-diagonal GMI sensor.

5. A method for preparing a non-diagonal giant magneto-impedance sensor for detecting biomarkers, applied to the non-diagonal giant magneto-impedance sensor for detecting biomarkers according to any one of claims 1 to 4, characterized in that: The following steps are involved: Use MEMS technology to make sensitive components and induction coils; A layer of SiO2 film is sputtered on the PI film, ultrasonically cleaned, and rinsed with a sulfuric acid / hydrogen peroxide mixed solution to remove surface contaminants and form the required hydroxyl groups; Immersing the SiO2 film in a 60°C ethanol solution containing 1% v / v APTES to form an organosilanized self-assembled monolayer on the substrate, which is then attached to the surface of the induction coil. preparing an array microcavity and fixing it on the surface of the self-assembled monolayer to immobilize the antibody; The streptavidin-labeled immunomagnetic bead solution and the biotinylated monoclonal or polyclonal antibody solution are fully mixed in a centrifuge tube to prepare a magnetic bead-antibody coupling solution, which is then dropped into the array microcavity and washed and dried.

Images