A method for fabricating a highly sensitive microcavity SERS chip for real-time detection of Tau protein.

Abstract

This invention discloses a highly sensitive SERS chip with a microcavity structure for the real-time detection of Tau protein, comprising the following steps: preparation of a composite microcavity substrate; substrate performance evaluation; construction of an immune signal probe; and specific recognition and detection of Tau protein. This invention uses gold nanoparticles (AuNPs) modified PS microspheres immobilized on a silicon substrate using polydopamine (PDA) as an immune capture chip. Utilizing R6G as the Raman signal molecule, the electromagnetic field is enhanced through synergistic coupling between the optical confinement of the PS microcavity and the localized surface plasmon resonance (LSPR) effect of AuNPs, promoting enhanced light-matter interaction and improving the Raman detection signal of the substrate.

Description

Technical Field

[0001] This invention relates to a method for fabricating SERS biosensor chips, and particularly to a method for fabricating a highly sensitive SERS chip with a microcavity structure for real-time detection of Tau protein. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease in clinical practice. Studies have shown that Tau protein is a key driver of Alzheimer's disease. Currently, SERS biosensing technology has become the most promising technique for on-site detection due to its advantages such as high specificity, high sensitivity, non-invasiveness, and simple operation. Typically, SERS enhancement is the result of the combined effects of electromagnetic field enhancement and chemical enhancement. Electromagnetic enhancement is based on the amplification of the surrounding local electromagnetic field through local surface plasmon resonance of nanostructured metals such as gold, silver, and copper. Chemical enhancement originates from the chemical interaction between the SERS substrate and probe molecules. Noble metal nanoparticles, as SERS substrates, exhibit extremely high sensitivity in enhancing the Raman signal of the analyte, amplifying the Raman signal to 10⁻⁶. 15 This technology holds promise as a crucial tool for single-molecule detection. Among these, noble metal gold nanoparticles exhibit high SERS sensitivity due to their ability to confine electromagnetic hotspots below 10 nm within structural gaps. As molecules adsorb at these hotspots, Raman scattering light can couple with surface plasmon resonances, resulting in electromagnetic enhancement. However, developing a SERS sensor with strong enhancement factors, stability, uniformity, and reproducibility requires a highly efficient SERS substrate. The poor uniformity of many state-of-the-art SERS substrates is primarily due to non-uniform hotspot distribution. This irregular distribution of hotspots leads to cluttered and disordered Raman signals, making research particularly challenging.

[0003] In recent years, three-dimensional structures have been used to increase the density of metal nanostructures supporting LSPR, which can not only provide more hot spots and binding sites for probe molecules, but also improve the stability of substrate signals. Among them, optical microcavities, through continuous total internal reflection, confine light within a circular path around the resonant cavity, enhancing the interaction between light and matter, and are used to enhance the electromagnetic field in SERS sensing systems. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a method for preparing a highly sensitive SERS chip with a microcavity structure for real-time detection of Tau protein.

[0005] By uniformly coating high-density AuNPs onto the surface of PS optical microspheres, the overall enhancement of Raman spectra is achieved by synergistic coupling of the optical confinement property of PS microcavities and the LSPR effect of AuNPs.

[0006] This invention enables the modification of PS / AuNPs composite microspheres on a silicon substrate, effectively combining Raman signal probes to achieve real-time detection of Tau protein. The invention uses gold nanoparticles (Au NPs) modified PS microspheres, immobilized on a silicon substrate with polydopamine (PDA), to create an immunocapture chip. Utilizing R6G as the Raman signal molecule, the electromagnetic field is enhanced through synergistic coupling between the optical confinement of the PS microcavity and the localized surface plasmon resonance (LSPR) effect of AuNPs, promoting enhanced light-matter interaction and improving the Raman detection signal on the substrate.

[0007] Technical solution: The present invention provides a method for preparing a highly sensitive SERS chip for real-time detection of Tau protein using a microcavity structure, comprising the following steps:

[0008] (1) Preparation of composite microcavity substrate;

[0009] (2) Substrate performance evaluation;

[0010] (3) Construct immune signal probes;

[0011] (4) Specific recognition and detection of Tau protein.

[0012] Utilizing the adhesive properties of polydopamine, AuNP-modified PS microspheres are uniformly arranged on a silicon substrate. Then, biomodification of the substrate is performed, first coating the surface of the composite microspheres with Tau protein monoclonal antibody I to construct an immunosensing chip. Next, Raman signal probes are constructed, and antigen-antibody reactions are completed through specific recognition, allowing the Raman signal probes to bind to the chip. Finally, Raman spectroscopy is used to detect the signal of the resulting sandwich immunosensing chip. The sandwich immunosensing chip constructed in this invention fully utilizes the optical confinement of the PS microcavity and the LSPR effect of AuNPs to synergistically enhance the electromagnetic field, promote the enhanced interaction between light and matter, and improve the Raman detection signal of the substrate.

[0013] Further, in step (1), the silicon wafer substrate is washed and dried, and the cleaned silicon wafer is bombarded with oxygen plasma to perform hydrophilic treatment. The hydrophilic silicon wafer is soaked in hydrochloric acid dopamine solution, and then an equal volume of Tris-HCl buffer solution is added. After the reaction, a polydopamine film is formed on the substrate. The PS / AuNPs composite microsphere solution synthesized by shaking reaction is directly added to the treated PDA silicon wafer to obtain the composite microcavity substrate.

[0014] Furthermore, in step (2), the Raman signal molecule R6G is selected to be located at 1361 cm⁻¹. -1 The strongest Raman peak at the point of origin is used to evaluate the chip's enhancement effect.

[0015] Further, in step (3), R6G solution is added to colloidal gold, and the mixture is reacted on a shaker. The solution is centrifuged to remove excess R6G, and the precipitate is resuspended in pure water to obtain the R6G / AuNPs complex. Then, Tau protein monoclonal antibody II is added to the R6G / AuNPs complex, and the mixture is reacted fully. The mixture is centrifuged to remove free Tau protein monoclonal antibody II. BSA solution is added for blocking, and the mixture is centrifuged to remove free BSA. The mixture is washed and centrifuged three times with ultrapure water under the same conditions. Finally, the precipitate is resuspended in pure water to obtain R6G / AuNPs-labeled Tau monoclonal antibody II, thus obtaining the immune signal probe.

[0016] Further, in step (4), Tau protein monoclonal antibody I is dropped onto the chip, incubated, and non-specific binding sites are deactivated. The target Tau protein stock solution antigen is diluted to different concentrations and dropped onto the chip respectively, and incubated. The same volume of signal probe solution is dropped onto the chip and incubated to complete the recognition of the target Tau protein. The signal is detected using a Raman spectrometer.

[0017] The above detection method mainly involves the preparation of microcavity SERS substrates, construction of signal probes, biomolecule modification, and specific recognition reactions. Specifically,

[0018] The SERS substrate preparation process includes the following steps: First, the silicon wafer substrate is cleaned to remove organic contaminants from the surface, then rinsed thoroughly with ultrapure water and dried. The cleaned silicon wafer is then bombarded with oxygen plasma for hydrophilic treatment. Next, the hydrophilic silicon wafer is immersed in a dopamine hydrochloride solution to form a polydopamine (PDA) film on the substrate. Finally, PS / AuNPs composite microspheres are synthesized via a shaker reaction.

[0019] The signal probe construction process includes the following steps: colloidal gold is added to R6G solution and dissolved in PBS solution to obtain monodisperse R6G-labeled AuNPs. Tau protein monoclonal antibody II is immobilized on R6G-labeled AuNPs through hydrophobic and electrostatic interactions. After thorough mixing, the binding sites on the nanoparticle surface are blocked with BSA. After centrifugation and resuspending, AuNPs / R6G-labeled Tau protein monoclonal antibody II is obtained, thus obtaining the immune signal probe.

[0020] The biomolecular modification and specific recognition reaction include the following steps: A certain amount of Tau protein monoclonal antibody I is dropped onto the chip, incubated for a period of time, and then rinsed. BSA is dropped onto the chip to passivate non-specific binding sites and ensure the accuracy of the detection results. The chip is then rinsed sequentially with PBS and ultrapure water. Finally, the prepared composite microcavity immunocapture chip is placed in a 4°C refrigerator for later use. Based on the specific recognition between the target antigen and the corresponding antibody, the target Tau protein stock antigen is diluted to different concentrations, dropped onto the chip separately, incubated, and rinsed with PBS and ultrapure water; then, the same volume of the solution prepared in the above step (signal probe construction) is dropped onto the chip, incubated, and rinsed with PBS and ultrapure water to complete the recognition of the target Tau protein. Finally, the signal is detected using Raman spectroscopy.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] (1) The present invention fully utilizes the optical confinement of the microcavity structure and the LSPR effect of AuNPs to enhance the electromagnetic field, promote the enhancement of the interaction between light and matter, and improve the Raman signal of the substrate.

[0023] (2) At the same time, a sandwich-type immune chip based on microcavity structure was developed, which combines SERS detection technology and biological technology and assembles it into a portable microchip to achieve trace and highly sensitive real-time detection of Tau protein in blood samples of Alzheimer's patients.

[0024] (3) The method of the present invention enables the enhancement factor to reach 3.8 × 10⁻⁶. 9 This significantly enhances the SERS sensing performance. Furthermore, the method exhibits good specificity and stability, making it an effective diagnostic tool for ultrasensitive detection of AD biomarkers. Attached Figure Description

[0025] Figure 1 SEM image of the PS / AuNPs composite microcavity substrate chip in Example 1;

[0026] Figure 2 SERS signals of different substrate chips in Example 1;

[0027] Figure 3 For Example 1, (a) SERS spectra of different concentrations of R6G and (b) 1361 cm⁻¹ -1 The curve showing the relationship between SERS intensity and the logarithm of R6G concentration at a given location. Detailed Implementation

[0028] This embodiment describes a method for preparing a highly sensitive SERS chip with a microcavity structure capable of real-time detection of Tau protein.

[0029] (1) Preparation of composite microcavity substrate: A silicon wafer substrate (0.5cm × 0.5cm) was sequentially immersed in acetone and ethanol, and ultrasonically cleaned for 10 min each to remove organic contaminants from the substrate surface. It was then rinsed with ultrapure water and dried. The cleaned silicon wafer was bombarded with oxygen plasma for hydrophilic treatment at 60W for 5 min. The hydrophilic silicon wafer was then immersed in a 5 mg / mL dopamine hydrochloride solution for 1 h, followed by the addition of an equal volume of 10 mM Tris-HCl buffer. After thorough mixing and reaction at room temperature for 1 h, it was rinsed with ultrapure water and dried, forming a polydopamine (PDA) film on the substrate. 15 μL of a PS / AuNPs composite microsphere solution synthesized via a shaker reaction was directly added to the treated PDA silicon wafer to obtain the composite microcavity substrate. Figure 1 As shown, AuNPs are uniformly coated on the surface of PS optical microcavities, and the PS / AuNPs composite microspheres are densely fixed on the silicon wafer surface, providing an excellent SERS substrate.

[0030] (2) Substrate performance evaluation: To verify the Raman signal enhancement effect of the SERS active chip based on optical microcavity, the Raman signal molecule R6G is located at 1361 cm⁻¹. -1 The strongest Raman peak at a given location was selected to evaluate the chip's enhancement effect. For example... Figure 2 As shown, R6G was detected using three different substrates—Si, Si-Au, and Si-PS-Au—as SERS platforms. R6G(10) -3 The SERS signal of R6G on a smooth Si substrate is almost invisible. However, by modifying the Si wafer with AuNPs, the SERS signal is enhanced to some extent. Further introduction of PS microspheres results in a significant signal enhancement; the Raman intensity of R6G on the Si-PS-Au substrate is approximately 10 times that on the Si-Au substrate. Simultaneously, experimental and computational characterization of the enhancement factor of the substrate were performed. Figure 3 As shown, the enhancement factor is 3.8 × 10⁻⁶. 9 .

[0031] (3) Construction of immune signal probe: 200 μL of 1 mM R6G was added to 10 mL of colloidal gold solution and reacted at 37 °C and 200 rpm for 2 h on a shaker. The solution was centrifuged at 5000 rpm for 10 min to remove excess R6G and the precipitate was resuspended in pure water to obtain the R6G / AuNPs complex. 100 μL of 10 mg / mL Tau protein monoclonal antibody II was added to 1 mL of the R6G / AuNPs complex and reacted fully for 2 h. The solution was centrifuged at 5000 rpm for 10 min to remove free Tau protein monoclonal antibody II. 1 mL of 1% BSA solution was added to block the reaction for 20 min and centrifuged at 5000 rpm for 10 min to remove free BSA. The solution was washed and centrifuged 3 times with ultrapure water under the same conditions. Finally, the precipitate was resuspended in pure water to obtain R6G / AuNPs-labeled Tau monoclonal antibody II.

[0032] (4) Specific recognition and detection of Tau protein: 15 μL of 0.2 mg / mL Tau protein monoclonal antibody I was added to the chip and incubated at 4 °C for 48 h, then rinsed. 20 μL of BSA was added to the chip to passivate non-specific binding sites and ensure the accuracy of the detection results. The chip was then rinsed with PBS and ultrapure water. Based on the specific interaction between the target antigen and the corresponding antibody, the target Tau protein stock solution antigen was diluted to different concentrations: 10000 pg / mL, 1000 pg / mL, 100 pg / mL, 10 pg / mL, 5 pg / mL, 1 pg / mL, and 0.1 pg / mL. 15 μL of each solution was added to the chip and incubated for 20 min, then rinsed with PBS and ultrapure water. Subsequently, the same volume of the signal probe solution prepared in step (3) was added to the chip and incubated for 20 min, then rinsed with PBS and ultrapure water to complete the specific recognition of the target Tau protein. Finally, the signal was detected using a Raman spectrometer (785 nm; 10 s; 20 x; 1.0% power).

Claims

1. A method for preparing a highly sensitive SERS chip with a microcavity structure capable of real-time detection of Tau protein, characterized in that, The following steps are involved: (1) Preparation of composite microcavity substrate: The silicon wafer substrate is washed and dried. The silicon wafer is bombarded with oxygen plasma and then hydrophilically treated. The hydrophilic silicon wafer is soaked in hydrochloric acid dopamine solution and then an equal volume of Tris-HCl buffer is added. After the reaction, a polydopamine film is formed on the substrate. The PS / AuNPs composite microsphere solution synthesized by shaking reaction is directly dropped onto the treated PDA silicon wafer to obtain the composite microcavity substrate. (2) Substrate performance evaluation: The Raman signal molecule R6G was selected at 1361 cm⁻¹. -1 The strongest Raman peak at the point of origin was used to evaluate the chip's enhancement effect; (3) Construction of immune signal probe: R6G solution was added to colloidal gold and reacted on a shaker. The solution was centrifuged to remove excess R6G and the precipitate was resuspended in pure water to obtain R6G / AuNPs complex. Then, Tau protein monoclonal antibody II was added to the R6G / AuNPs complex and reacted fully. The mixture was centrifuged to remove free Tau protein monoclonal antibody II. BSA solution was added for blocking and centrifuged to remove free BSA. The mixture was washed and centrifuged 3 times with ultrapure water under the same conditions. Finally, the precipitate was resuspended in pure water to obtain R6G / AuNPs-labeled Tau monoclonal antibody II, thus obtaining the immune signal probe. (4) Specific recognition and detection of Tau protein: Tau protein monoclonal antibody I was added to the chip and incubated to passivate non-specific binding sites. The target Tau protein antigen was diluted to different concentrations and added to the chip respectively and incubated. The same volume of signal probe solution was added to the chip and incubated to complete the recognition of the target Tau protein. The signal was detected by Raman spectroscopy.

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