Open gold nano cubic material as well as preparation method and application thereof

Abstract

The invention discloses an open gold nano cubic material as well as a preparation method and application thereof. The open-ended gold nanocube material developed by the invention has excellent Raman enhancement performance, and the Raman enhancement factor is as high as 8.99 * 10 < 8 >; and when the kit is used for detecting two AD markers (A beta 42 and MAO-B), the credibility and the accuracy of a detection result are improved. According to the invention, Abeta42 aggregates with different aggregation degrees can be distinguished, and an effective means is provided for early diagnosis and drug screening of AD.

Description

Technical Field

[0001] This invention relates to an open-cell gold nanomaterial, its preparation method and application, belonging to the field of sensor construction and application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss and cognitive impairment. It is currently unpredictable and incurable. Early symptoms of AD mainly include memory loss, difficulty speaking, and impaired judgment, which are often mistaken for normal aging. This leads to AD being diagnosed at a middle or late stage, significantly increasing the difficulty of treatment. Therefore, the development of early diagnostic technologies for AD is of paramount importance.

[0003] It has been reported that Alzheimer's disease (AD) patients have numerous plaques formed by the aggregation of β-amyloid protein (Aβ) in their brains, and the content of Aβ42 in cerebrospinal fluid is significantly reduced. Therefore, quantitative analysis of Aβ42 in the human brain is of great significance for the early diagnosis of AD. Meanwhile, monoamine oxidase B (MAO-B), an endogenous biological enzyme in the central nervous system, is also closely related to the occurrence and development of AD. Studies have shown that the expression level of MAO-B in the brains of AD patients is about three times that of normal individuals. Therefore, monitoring the content of MAO-B is equally important for the early diagnosis of AD.

[0004] Currently, researchers have developed various analytical methods for detecting Aβ42 or MAO-B. While these detection techniques show some progress, they mostly employ antigen-antibody binding methods, resulting in cumbersome detection procedures, long experimental cycles, and high costs. In contrast, surface-enhanced Raman scattering (SERS) spectroscopy is simple to operate, has high detection sensitivity, and has been widely used in bioanalysis and food safety testing. Moreover, SERS spectroscopy has specific "fingerprint peaks," enabling the simultaneous detection of multiple targets. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for preparing open-ended gold nanocube materials and their application for the quantitative detection of two AD markers, β-amyloid protein (Aβ42) and monoamine oxidase B (MAO-B).

[0006] Technical solution: The preparation method of the open-cell gold nanomaterial of the present invention includes the following steps: (1) Mix hexadecyltrialkylammonium bromide solution, chloroauric acid solution and sodium borohydride solution, stir vigorously, and let stand at room temperature to obtain a solution of gold nanoclusters encapsulated by hexadecyltrialkylammonium bromide. (2) The gold nanocluster solution prepared in step (1) is thoroughly mixed with hexadecyltrimethylammonium chloride solution and ascorbic acid solution, and then chloroauric acid solution is slowly added to the mixed solution. The mixture is stirred at room temperature to obtain a gold seed solution encapsulated by hexadecyltrimethylammonium chloride. (3) Mix the gold seed solution prepared in step (2) with hexadecyltrimethylammonium chloride solution and sodium bromide solution evenly, then add ascorbic acid solution and chloroauric acid solution in sequence, stir vigorously at room temperature, collect the precipitate by centrifugation, and redissolve with hexadecyltrimethylammonium bromide solution to obtain a hexadecyltrimethylammonium bromide-encapsulated gold nano cubic solution. (4) Mix the gold nano cubic solution obtained in step (3) with benzyl dimethyl dodecyl ammonium chloride solution, centrifuge to obtain a precipitate, redissolve with sodium dodecyl sulfate to obtain a gold nano cubic solution coated with sodium dodecyl sulfate; (5) Mix the gold nanocube solution obtained in step (4) with polyvinylpyrrolidone solution, chloroauric acid solution and hydroquinone solution evenly, stir at room temperature, let stand in the dark, centrifuge to obtain precipitate, and redissolve with sodium dodecyl sulfate solution to obtain open gold nanocube material.

[0007] Further, in step (1), the final concentrations of the cetyltrialkylammonium bromide solution, chloroauric acid solution, and NaBH4 solution in the reaction solution are 97.5 mM, 0.25 mM, and 0.6 mM, respectively. The reaction temperature is 25±2℃, the stirring time is 3-5 min, and the standing time is 2-3 h.

[0008] Further, in step (2), the final concentrations of the hexadecyltrimethylammonium chloride solution, ascorbic acid solution, and chloroauric acid solution are 72 mM, 27 mM, and 0.18 mM, respectively, and the stirring time is 15-30 min, and the stirring temperature is 25±2℃.

[0009] Further, in step (3), the final concentrations of the cetyltrimethylammonium chloride solution, sodium bromide solution, ascorbic acid solution, and chloroauric acid solution in the reaction solution are 48 mM, 4.8 mM, 0.31 mM, and 0.24 mM, respectively; the volume of the gold seed solution is 18 mL; the reaction temperature is 25±2℃; and the reaction time is 25-30 min.

[0010] Furthermore, the concentration of the benzyl dimethyl dodecyl ammonium chloride solution is 50-60 mM.

[0011] Furthermore, the concentration of the sodium dodecyl sulfate solution is 0.01%-0.05% w / w.

[0012] Furthermore, the concentration of the polyvinylpyrrolidone solution is 0.8%-1.2% w / w.

[0013] Furthermore, the final concentrations of the chloroauric acid solution and the hydroquinone solution are 1.07 mM and 21.4 mM, respectively; the stirring and standing temperature is 25±2℃; and the concentration of the SDS solution is 0.01-0.05% (w / w).

[0014] The open-cell gold nanocubic material of the present invention is prepared by the above-described preparation method.

[0015] The application of the open-cell gold nanocubic material described in this invention in the preparation of a sensor for detecting biomarkers of Alzheimer's disease.

[0016] Furthermore, the marker is Aβ42 or MAO-B.

[0017] Furthermore, the application includes detecting the concentration of Aβ42 monomers and the degree of Aβ42 aggregation.

[0018] The present invention discloses a method for quantitatively detecting two AD markers using a Raman sensor, wherein the markers for quantitative detection are selected from any one of the following: A) When the biomarker to be tested is Aβ42: A1) Detection of Aβ42 concentration: The above-mentioned open gold nanocube material, thiosulfate T solution and Aβ42 solution with different concentrations were mixed, dropped onto a silicon wafer, dried and then the Raman signal was measured using a Raman spectrometer. A linear relationship equation between different concentrations of Aβ42 solution and the Raman signal intensity difference was constructed. The above-mentioned open gold nanocube material, thiosulfate T solution and the sample to be tested are mixed, dropped onto a silicon wafer, dried and then the Raman signal is measured using a Raman spectrometer. The Raman signal is then substituted into the above linear relationship equation to obtain the content of Aβ42 in the sample solution. A2) Detection of Aβ42 aggregation degree: Aβ42 monomers were heated in a 37℃ constant temperature water bath for 0 h, 12 h, 24 h, 36 h and 48 h to prepare Aβ42 aggregates with different aggregation degrees. The above-mentioned open gold nanocube material, thiosulfate T solution and Aβ42 aggregates with different aggregation degrees were mixed and subjected to 2D-LDA analysis, hierarchical cluster analysis and heat map analysis to obtain standard results. The above-mentioned open gold nanocube material, thiamine T solution and the sample to be tested are mixed and subjected to 2D-LDA analysis, hierarchical cluster analysis and thermogram analysis. By comparing with the standard results, the degree of aggregation of Aβ42 in the sample to be tested can be obtained. B) When the biomarker to be tested is MAO-B: Phenethylamine and MAO-B containing different concentrations were mixed and reacted at a constant temperature. The resulting mixture was then uniformly mixed with the above-mentioned open gold nanocube material, dropped onto a silicon wafer, dried, and the Raman signal was measured using a Raman spectrometer. A linear relationship equation between different concentrations of MAO-B solution and the Raman signal intensity difference was constructed. Phenethylamine and the sample to be tested are mixed and reacted at a constant temperature. The resulting mixture is then uniformly mixed with the above-mentioned open gold nanocubic material, dropped onto a silicon wafer, dried, and the Raman signal is measured using a Raman spectrometer. The Raman signal is then substituted into the above-mentioned linear relationship equation to obtain the content of MAO-B in the sample solution.

[0019] Further, the concentration of thiosulfate T is 2.5-10 mM; the concentration of phenylethylamine is 1.2-2.0 mM.

[0020] Furthermore, in method A2), the isothermal reaction temperature is 37±2℃ and the reaction time is 1.5-2 h.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The open-cell gold nanoparticle material developed in this invention has excellent Raman enhancement properties, with a Raman enhancement factor (EF) as high as 8.99 × 10⁻⁶. 8 When used to detect two AD biomarkers (Aβ42 and MAO-B), this invention improves the reliability and accuracy of the test results. Furthermore, it can distinguish Aβ42 aggregates with different degrees of aggregation, providing an effective means for the early diagnosis and drug screening of AD. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the mechanism for detecting two AD markers using an OX-AuNCs-based Raman sensor. Figure 2 A shows the UV-Vis absorption spectra of SDS-AuNCs and OX-AuNCs (the inset shows the corresponding solution photographs). Figure 2 B is the HRTEM image of SDS-AuNCs. Figure 2 C is the HAADF-STEM image of OX-AuNCs; Figure 3 A represents the SERS spectra of ThT at different Aβ42 concentrations. Figure 3 B represents the ThT value at 1601 cm⁻¹ under different Aβ42 concentrations (66.7 pM - 0.34 mM). -1 SERS intensity changes at the location Figure 3 Inset B shows the calibration curve for the quantitative detection of Aβ42; Figure 4 Graphs showing the analysis of Aβ42 aggregates at different degrees of aggregation: Figure 4A is a 2D-LDA analysis chart. Figure 4 B represents hierarchical cluster analysis. Figure 4 C represents heatmap analysis; Figure 5 A shows the SERS spectra of PEA at different MAO-B concentrations. Figure 5 B represents the PEA concentration at 1003 cm⁻¹ under different MAO-B concentrations (0.01-20 mg / mL). -1 SERS signal variation graph at the location, Figure 5 Inset B shows the calibration curve for MAO-B activity detection; Figure 6 A represents the repeatability of MAO-B activity detection. Figure 6 B represents the stability of MAO-B activity detection. Figure 6 C represents the selectivity for MAO-B activity detection; Figure 7 The Raman spectra of Rh6G on different substrates are shown. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] The reagents and instruments used in this experiment are as follows: Chloroauric acid trihydrate (HAuCl4·3H2O), sodium borohydride (NaBH4), benzyl dimethyl dodecyl ammonium chloride (BDAC), and polyvinylpyrrolidone (PVP, K30) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Hexadecyltrimethylammonium chloride (CTAC), hexadecyltrialkylammonium bromide (CTAB), ascorbic acid, sodium dodecyl sulfate (SDS), hydroquinone, and sodium bromide were purchased from Aladdin (China). Rhodamine 6G and phenethylamine (≥98%) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Aβ42 peptide was purchased from Sangon Biotech (Shanghai) Co., Ltd. Thioflavin T (ThT) and MAO-B were purchased from Sigma-Aldric (Beijing, China). Ultrapure water (25℃, 18.2 MΩ·cm) was obtained from a Millipore Direct-Q 3UV pure water system (Millipore, USA).

[0025] Artificial cerebrospinal fluid (ACSF): 124 mM NaCl, 26 mM NaHCO3, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 10 mM D -glucose, pH 7.4.

[0026] Talos F200X high-resolution transmission electron microscope (Thermo Fisher Scientific, USA), HH.S21-4 constant temperature water bath (Shenglan, Jiangsu, China), Cary 100 UV-Vis spectrophotometer (Agilent Technologies, Singapore), magnetic stirrer (IKA, Germany), InVia RamanMicroscopy confocal micro Raman spectrometer (Renishaw, UK), 5424R high-speed refrigerated centrifuge (Eppendorf, Germany).

[0027] Example 1: Preparation and Characterization of OX-AuNCs (1) Preparation of OX-AuNCs The CTAB solution (9.75 mL, 100 mM), HAuCl4·3H2O solution (250 mL, 10 mM), and NaBH4 solution (600 mL, 10 mM) were thoroughly mixed and stirred vigorously for 3 min. The mixture was then allowed to stand at 25 °C for 2 h to obtain CTAB-encapsulated gold nanoclusters.

[0028] The gold nanoclusters coated with CTAB (50 mL) were mixed with CTAC solution (2 mL, 2 mM) and ascorbic acid solution (1.5 mL, 100 mM) until homogeneous. Then, HAuCl4·3H2O solution (2 mL, 0.5 mM) was added while stirring, and stirring was continued for 15 min to obtain the gold seed solution coated with CTAC.

[0029] The gold seed solution (18 mL) was mixed thoroughly with CTAC solution (6 mL, 100 mM) and sodium bromide solution (30 mL, 2 mM). Ascorbic acid solution (390 mL, 10 mM) and HAuCl4·3H2O solution (6 mL, 0.5 mM) were then added sequentially to the mixture, and the mixture was stirred at 25 °C for 25 min. The precipitate was then collected by centrifugation (8000 rpm, 5 min) and reconstituted with CTAB solution (500 mL, 1 mM) to obtain a CTAB-encapsulated gold nanocube solution.

[0030] The CTAB-coated gold nanocube solution (150 mL) was mixed with BDAC solution (82.5 mL, 200 mM) and ultrapure water (67.5 mL), and the precipitate was collected by centrifugation (8000 rpm, 5 min). The precipitate was then reconstituted with SDS solution (0.01%, w / w) to obtain SDS-coated gold nanocubes (SDS-AuNCs).

[0031] SDS-coated gold nanocubes (40 mL) were mixed thoroughly with PVP solution (40 mL, 1%, w / w), HAuCl4·3H2O solution (30 mL, 5 mM), and hydroquinone solution (30 mL, 100 mM). The mixture was stirred for 30 s and then allowed to stand in the dark for 30 min. The precipitate was collected by centrifugation (6000 rpm, 3 min) and redissolved with SDS solution (0.01%, w / w) to obtain OX-AuNCs.

[0032] (2) Characterization of OX-AuNCs The prepared SDS-AuNCs and OX-AuNCs were characterized using a UV-Vis spectrophotometer. Figure 2 A). Compared to SDS-AuNCs, the UV absorption peak of OX-AuNCs showed a significant red shift, and the solution color changed from rose pink to grayish blue. The morphology of SDS-AuNCs and OX-AuNCs was characterized using high-resolution transmission electron microscopy (HRTEM). Figure 2 (B and C). SDS-AuNCs are regular cubic structures, while OX-AuNCs are irregular structures with numerous grooves, with SDS-AuNCs located at the center of OX-AuNCs.

[0033] Example 2: Determination of OX-AuNCs Enhancer Factor Rhodamine 6G (Rh6G) was used as the Raman reporter molecule, and Raman signals were measured by dropping it onto silicon (Si) substrates and OX-AuNCs substrates, respectively. Figure 7 (The Si substrate used has a concentration of 10). -4 M Rh6G solution, OX-AuNCs substrate with a concentration of 10 -12 Rh6G solution of M. Rh6G at 1509 cm⁻¹ was recorded under both substrates. -1 The SERS signal at the location is used to calculate the Raman enhancement factor of OX-AuNCs according to formula (1). In formula (1), C OX-AuNCs and I OX-AuNCs Represents the concentration and corresponding intensity of Rh6G on OX-AuNCs substrates, respectively. Si and I Si The values ​​represent the concentration and corresponding intensity of Rh6G on the Si substrate, respectively. The Raman enhancement factor EF of OX-AuNCs was calculated to be 8.99 × 10⁻⁶. 8 .

[0034] (1) Example 3: Quantitative detection of Aβ42 and differentiation of Aβ42 aggregates (1) Quantitative detection of different concentrations of Aβ42 Different concentrations of Aβ42 monomer solutions were mixed thoroughly with ThT solution (5 mL, 5 mM) and ultrapure water to prepare a 30 mL mixed solution. 10 mL of OX-AuNCs solution was taken, centrifuged to collect the OX-AuNCs precipitate, and then reconstituted with the above mixed solution. After thorough mixing, 15 mL was dropped onto a clean silicon wafer. After drying, the Raman spectrum of ThT was recorded using a micro Raman spectrometer. Figure 3 As shown in Figure A, the Raman signal of ThT decreases with increasing Aβ42 concentration. When the Aβ42 concentration is in the range of 66.7 pM to 0.2 mM, the decrease in the ThT Raman signal (ΔI) is... ThT The concentration of Aβ42 was linearly correlated with the concentration of Aβ42. Figure 3 B), the linear relationship equation is ΔI ThT =165353.45C Aβ42 +1809.76 (R) 2 =0.995), ΔI ThT Let ΔI, C be the value of ThT. Aβ42 The concentration of the Aβ42 solution is given.

[0035] (2) Differentiation of Aβ42 aggregates with different degrees of aggregation Aβ42 monomers of the same concentration (2 μM) were placed in a 37°C constant temperature water bath to prepare Aβ42 aggregates with different degrees of aggregation. The aggregation times were 0 h, 12 h, 24 h, 36 h, and 48 h, respectively. The prepared Aβ42 aggregates were used to replace the Aβ42 solution in (1) for Raman signal detection. Then, ThT was measured at 1601 cm⁻¹. -1 and 1328 cm -1 The Raman signals at the location were subjected to 2D-LDA analysis, hierarchical cluster analysis, and heatmap analysis, respectively. Figure 4 As can be seen from the figure, Aβ42 aggregates with the same degree of aggregation form a single cluster, and different clusters are distributed in different locations, indicating that there are obvious differences between various Aβ42 aggregates due to their different degrees of aggregation.

[0036] Example 4: Quantitative Detection of MAO-B Activity PEA (1.5 mL, 80 mM) and MAO-B of different concentrations were mixed thoroughly and incubated in a 37°C water bath for 2 hours. After the reaction, 30 mL of the mixed solution was taken to redissolve the OX-AuNCs precipitate in Example 3 (1), and after mixing thoroughly, 15 mL was dropped onto a clean silicon wafer. After drying, the Raman spectrum of PEA was recorded using a micro Raman spectrometer. Figure 5As shown in Figure A, the Raman signal intensity of PEA gradually decreases with increasing MAO-B concentration. When the MAO-B concentration is in the range of 0-20 mg / mL, the decrease in PEA Raman signal intensity (ΔI) is... PEA The correlation between the concentration of MAO-B and the logarithm of MAO-B concentration is linear. Figure 5 B), the linear relationship equation is ΔI PEA =11649.42lgC MAO-B +24757.97 (R) 2 =0.997), where ΔI PEA Let ΔI, C be the value of PEA. MAO-B This refers to the concentration of MAO-B.

[0037] Example 5: Evaluation of the repeatability, stability, and selectivity of MAO-B activity detection (1) Repeatability assessment of MAO-B activity detection To evaluate the repeatability of MAO-B activity detection, three parallel samples with the same MAO-B concentration were prepared according to Example 4. Five points were randomly selected on each sample for Raman spectroscopy acquisition, with a focal length of 1003 cm⁻¹. -1 The signal at the location was plotted, and the result is as follows: Figure 6 As shown in Figure A, the OX-AuNCs Raman sensor exhibits good repeatability in MAO-B activity detection.

[0038] (2) Stability assessment of MAO-B activity assay After preparing the sample according to Example 4 and testing it, the sample was stored at room temperature in the dark. The signal was collected once a week for one month, and the collected PEA Raman signals were compared. The results are as follows. Figure 6 As shown in B.

[0039] (3) Selectivity assessment of MAO-B activity detection To evaluate the selectivity of the OX-AuNCs Raman sensor in detecting MAO-B activity, bovine serum albumin (BSA), human serum albumin (HSA), glucose oxidase (GOx), horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-secretase (BACE1), and acetylcholinesterase (AChE) were selected as interfering substances to replace MAO-B, and PEA Raman signals were acquired according to the steps in Example 4. Figure 6 C). Among them, the concentration of MAO-B was 5 μg / mL, the concentrations of BSA, HSA, GOx, HRP and ALP were 100 μg / mL, and the concentrations of BACE1 and AChE were 50 μg / mL.

[0040] Example 6 Performance Evaluation of Actual Sample Testing (1) Recovery test of Aβ42 in ACSF A mixed solution with a total volume of 30 mL was prepared by replacing the ultrapure water in Example 3(1) with an equal volume of ACSF. Raman signal testing was performed by repeating the procedure in Example 3(1), and the Aβ42 concentration was calculated to be 6.67 × 10⁻⁶. -9 M, 3.33´10 -8 M and 1.33´10 -7 Recovery rate at time M.

[0041] (2) Recovery rate test of MAO-B in human serum PEA (1.5 mL, 80 mM), different concentrations of MAO-B, and human serum (10 mL) were mixed thoroughly and incubated in a 37°C water bath for 2 h. The procedure in Example 4 was repeated for Raman signal testing, and the recoveries of MAO-B at concentrations of 0.10 μg / mL, 1.00 μg / mL, and 5.00 μg / mL were calculated. The results are shown in Table 1. The open-cell gold nanoparticles of this application showed high recoveries and RSDs (less than 6%) for the detection of Aβ42 and MAO-B, indicating good repeatability.

[0042] Table 1. Recovery rates of Aβ42 in artificial cerebrospinal fluid (ACSF) and MAO-B in human serum. .

Claims

1. A method for preparing open-cell gold nanomaterials, characterized in that, Includes the following steps: (1) Mix hexadecyltrialkylammonium bromide solution, chloroauric acid solution and sodium borohydride solution, stir vigorously, and let stand at room temperature to obtain a solution of gold nanoclusters encapsulated by hexadecyltrialkylammonium bromide. (2) The gold nanocluster solution prepared in step (1) is thoroughly mixed with hexadecyltrimethylammonium chloride solution and ascorbic acid solution, and then chloroauric acid solution is slowly added to the mixed solution. The mixture is stirred at room temperature to obtain a gold seed solution encapsulated by hexadecyltrimethylammonium chloride. (3) Mix the gold seed solution prepared in step (2) with hexadecyltrimethylammonium chloride solution and sodium bromide solution evenly, then add ascorbic acid solution and chloroauric acid solution in sequence, stir vigorously at room temperature, collect the precipitate by centrifugation, and redissolve with hexadecyltrimethylammonium bromide solution to obtain a hexadecyltrimethylammonium bromide-encapsulated gold nano cubic solution. (4) Mix the gold nano cubic solution obtained in step (3) with benzyl dimethyl dodecyl ammonium chloride solution, centrifuge to obtain a precipitate, redissolve with sodium dodecyl sulfate to obtain a gold nano cubic solution coated with sodium dodecyl sulfate; (5) Mix the gold nanocube solution obtained in step (4) with polyvinylpyrrolidone solution, chloroauric acid solution and hydroquinone solution evenly, stir at room temperature, let stand in the dark, centrifuge to obtain precipitate, and redissolve with sodium dodecyl sulfate solution to obtain open gold nanocube material.

2. The preparation method according to claim 1, characterized in that, The concentration of the benzyl dimethyl dodecyl ammonium chloride solution is 50-60 mM.

3. The preparation method according to claim 1, characterized in that, The concentration of the sodium dodecyl sulfate solution is 0.01%-0.05% w / w.

4. The preparation method according to claim 1, characterized in that, The concentration of the polyvinylpyrrolidone solution is 0.8%-1.2% w / w.

5. An open-cell gold nanocubic material, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the open-cell gold nanocubic material of claim 5 in the preparation of a sensor for detecting biomarkers of Alzheimer's disease.

7. The application according to claim 6, characterized in that, The markers are β-amyloid protein or monoamine oxidase B.

8. The application according to claim 7, characterized in that, The applications include detecting the concentration of β-amyloid monomers and the degree of β-amyloid aggregation.

9. A method for quantitative detection of two AD markers using a Raman sensor, characterized in that, The biomarker for quantitative detection is selected from any one of the following: A) When the biomarker to be tested is β-amyloid: A1) Detection of β-amyloid protein concentration: The open gold nanocube material described in claim 5, thiamine T solution and β-amyloid protein solution containing different concentrations are mixed, dropped onto a silicon wafer, dried and then the Raman signal is measured using a Raman spectrometer to construct a linear relationship equation between β-amyloid protein solution of different concentrations and the Raman signal intensity difference. The open-cell gold nanocube material described in claim 5, thiamine T solution, and the sample to be tested are mixed, dropped onto a silicon wafer, dried, and then the Raman signal is measured using a Raman spectrometer. The Raman signal is then substituted into the above linear relationship equation to obtain the content of β-amyloid protein in the sample solution. A2) Detection of β-amyloid aggregation degree: β-amyloid monomers were heated in a 37℃ constant temperature water bath for 0 h, 12 h, 24 h, 36 h and 48 h to prepare β-amyloid aggregates with different aggregation degrees. The open gold nanocubic material described in claim 5, thiamine T solution and β-amyloid aggregates with different aggregation degrees were mixed and subjected to 2D-LDA analysis, hierarchical cluster analysis and heat map analysis to obtain standard results; The open gold nanocube material described in claim 5, thiamine T solution and the sample to be tested are mixed, and 2D-LDA analysis, hierarchical cluster analysis and thermogram analysis are performed. The degree of aggregation of Aβ42 in the sample to be tested can be obtained by comparing with the standard results. B) When the target biomarker is monoamine oxidase B: Phenethylamine and monoamine oxidase B containing different concentrations were mixed and reacted at a constant temperature. The resulting mixture was then uniformly mixed with the open gold nanocube material described in claim 5, dropped onto a silicon wafer, dried, and the Raman signal was measured using a Raman spectrometer. A linear relationship equation between monoamine oxidase B solutions of different concentrations and the Raman signal intensity difference was constructed. Phenethylamine and the sample to be tested are mixed and reacted at a constant temperature. The resulting mixture is then uniformly mixed with the open gold nanocubic material described in claim 5, dropped onto a silicon wafer, dried, and the Raman signal is measured using a Raman spectrometer. The content of monoamine oxidase B in the sample to be tested is obtained by substituting the Raman signal into the linear relationship equation described above.

10. The method according to claim 9, characterized in that, The concentration of thiosulfate T is 2.5-10 mM; the concentration of phenylethylamine is 1.2-2.0 mM.

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