A detection platform for SERS detection and a construction method thereof, and a SERS detection method

Abstract

The present application belongs to the technical field of SERS detection, and particularly relates to a detection platform for SERS detection and a construction method thereof, and a SERS detection method. The present application sets a quartz wafer on the quartz plate cover corresponding to the hole position of the hole plate, and presses the liquid level in the hole to a uniform height, effectively reducing the laser incidence height error caused by the concave liquid surface or the convex liquid surface, thereby improving the stability and repeatability of the SERS signal, and effectively making up for the inconsistency of detection caused by uneven liquid surface. Especially under the premise that the Raman equipment is difficult to automatically adjust the focal length, the structural uniform sample height provides physical guarantee for different sample detection. In addition, the silicon wafer is arranged at the bottom of the quartz wafer at the four corners as a calibration reference point of the laser probe irradiation height, so that the system can accurately focus and keep consistent irradiation conditions in multi-hole scanning, and realize the uniformity of sample detection in different batches.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of SERS detection, and particularly relates to a detection platform for SERS detection and a construction method thereof, and a SERS detection method. BACKGROUND

[0002] In recent years, Raman spectroscopy has been widely used in chemical, environmental and food safety detection fields due to its high sensitivity, strong molecular specificity and fast response capability. Among them, surface-enhanced Raman spectroscopy (SERS) technology is particularly prominent, which can significantly enhance the Raman signal and realize the detection of trace substances. With the continuous improvement of food safety and environmental protection requirements, high-throughput detection has become an important direction of the development of Raman technology.

[0003] High-throughput analysis mainly relies on automated detection systems, and a 96-well plate self-running SERS analysis platform has become an important tool for detecting high-throughput requirements due to its ability to simultaneously process multiple samples. However, in actual detection, the concave / convex liquid surface formed by the capillary action or surface tension of the liquid to be detected in the 96-well plate causes the liquid level of the liquid to be detected in the well to be inconsistent, which further affects the irradiation height of the laser probe, and ultimately limits the stability and reproducibility of the detection results. In addition, since the Raman detection equipment cannot automatically adjust the irradiation height of the laser probe so far, the inconsistency of the laser irradiation height in different detection batches will affect the consistency of the quantitative analysis results. Therefore, improving the consistency of the liquid level and improving the stability between batches have become the key to solving the stability problem of high-throughput detection. SUMMARY

[0004] The application aims to provide a detection platform for SERS detection and a construction method thereof, and a SERS detection method.

[0005] In order to achieve the above-mentioned purpose, the application provides the following technical scheme:

[0006] The application provides a detection platform for SERS detection, which comprises a well plate and a quartz cover plate covering the well plate, the lower surface of the quartz cover plate is provided with a plurality of quartz wafers, the plurality of quartz wafers and the well positions of the well plate are in one-to-one correspondence, and the diameter of the quartz wafer is not greater than the diameter of the well position.

[0007] The lower surface of the quartz wafer located at the four corners is provided with a silicon wafer; the area of the silicon wafer is not greater than the area of the quartz wafer.

[0008] The thickness of the quartz cover plate is 0.45-0.55 mm.

[0009] The thickness of the quartz wafer is 1.4-1.5 mm.

[0010] The thickness of the silicon wafer is 0.5-1mm.

[0011] Preferably, the well plate comprises a 96-well plate, a 24-well plate, a 48-well plate or a 384-well plate.

[0012] Preferably, the diameter of the quartz wafer is 3.9-4.1mm.

[0013] The application also provides a method for constructing the detection platform for SERS detection, comprising the following steps:

[0014] (1) Place the quartz cover plate in the positioning fixture, coat the center point of each predetermined hole with ultraviolet curing glue, then adhere the quartz wafer to the surface of the ultraviolet curing glue, and perform ultraviolet curing to obtain the cover plate with the adhered quartz wafer;

[0015] (2) Coat the surface of the quartz wafer at the four corners with ultraviolet curing glue, then adhere the silicon wafer, and perform ultraviolet curing to obtain the cover plate with the adhered silicon wafer;

[0016] (3) Cover the cover plate with the adhered silicon wafer on the well plate, so that the side with the adhered quartz wafer is in contact with the well plate, to obtain the detection platform for SERS detection.

[0017] Preferably, in step (1), the coating amount of ultraviolet curing glue on each predetermined hole is 0.5-1μL; and the coating diameter of the ultraviolet curing glue is 1mm.

[0018] In the adhering process, the quartz wafer is also subjected to pressure.

[0019] Preferably, after the adhering, another quartz cover plate is arranged on the surface of all the quartz wafers to flatten the quartz wafers, and the flattening pressure is 1kg and the pressure holding time is 10min.

[0020] In step (1), the ultraviolet curing conditions include that the wavelength of the ultraviolet light source is 365nm, the light intensity is 20mW / cm 2 , and the time is 60s.

[0021] Preferably, in step (2), the ultraviolet curing conditions include that the wavelength of the ultraviolet light source is 365nm, the light intensity is 20mW / cm 2 , and the time is 90s.

[0022] The application also provides a SERS detection method, comprising the following steps:

[0023] Mix the detection probe and the solution of the to-be-detected substance, place them in the detection platform, and perform SERS detection.

[0024] The detection platform is the detection platform for SERS detection in the technical solution or the detection platform for SERS detection constructed by the construction method in the technical solution.

[0025] The mixed solution and the quartz wafer in the detection platform are contacted;

[0026] The detection probe is an antibody-functionalized modified Prussian blue nanoparticle;

[0027] The antibody is obtained by immunization of the to-be-detected substance;

[0028] The modified Prussian blue nanoparticle comprises Prussian blue and nanogold wrapped inside the Prussian blue;

[0029] The SERS detection comprises qualitative detection or quantitative detection.

[0030] Preferably, the to-be-detected substance in the to-be-detected substance solution comprises thiamethoxam, pyrifluquinazon, acetamiprid, imidacloprid, malathion or nitenpyram.

[0031] The application effectively reduces the laser incidence height error caused by concave liquid surface or convex liquid surface by setting quartz wafers on the quartz plate cover corresponding to the hole positions of the hole plate, and pressing the liquid level in the hole to a uniform height, thereby improving the stability and repeatability of the SERS signal, and effectively compensating for the inconsistency caused by uneven liquid level. Especially under the premise that the Raman equipment is difficult to automatically adjust the focal length, the structure of the sample height is uniform, which provides physical protection for different sample detection. In addition, the silicon wafer is arranged at the bottom of the quartz wafer at the four corners as a calibration reference point for the irradiation height of the laser probe, so that the system can accurately focus and keep consistent irradiation conditions in multi-hole scanning, and realize the uniformity of sample detection in different batches.

[0032] The application also provides a SERS detection method. The application further enhances the detection performance of the system by introducing an anti-interference SERS probe. The probe uses Prussian blue containing C≡N group as a Raman reporter molecule, and the Raman signal only appears at 2155cm -1 . -1 cm -1 , which can effectively avoid the spectral interference of biological components in complex sample matrix, and at the same time avoid the spectral interference from the columnar concave plate cover, realize accurate and high-throughput SERS analysis, and provide an accurate, stable, sensitive and efficient solution for high-throughput detection of multiple batches of complex samples.

[0033] In combination with the above optimization design, the automatic SERS detection platform based on the hole plate cover assistance can significantly improve the detection efficiency and flux, greatly improve the stability and consistency of the detection results, can be widely applied to the fields of food safety, environmental monitoring and biological sample analysis, and has the technical advantages of high detection efficiency, strong anti-interference ability, excellent result consistency and the like. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 are characterization results of Au@PB NPs, wherein A and B are TEM images, C is an XPS full spectrum scanning image, D is a Raman image, E is an ultraviolet-visible absorption spectrum image, and F is an XRD image;

[0035] Figure 2 are test result images of the anti-interference Raman response verification and the feasibility verification part of the automatic 96-hole plate, wherein A is a test sample schematic diagram of Au@PB NPs of different concentrations, B is an ultraviolet-visible absorption spectrum image of TXM specific antibody modified Au@PB NPs, C is a characteristic peak Raman intensity consistency performance image of Au@PB NPs of different concentrations under the assistance of the plate cover, D is an ultraviolet-visible absorption spectrum image of antigen functionalized magnetic nanoparticles, E is a Raman spectrum comparison image of Au@PB NPs and traditional SERS probes and food samples, F is an EDS element analysis of antigen functionalized magnetic nanoparticles, and G is a zeta potential of AuNPs, Au@PB NPs and TXM specific antibody modified Au@PB NPs;

[0036] Figure 3 are test result images of the probe synthesis optimization part, wherein A is a Raman image under different temperatures in the synthesis of PB NPs, B is a Raman image under different temperatures in the one-pot synthesis of Au@PB NPs, C is a Raman response under different pH levels in the TXM detection, D is a Raman image of a blank sample at 2155 cm -1 , E is a Raman image under different antigen concentrations on MNPs, and F is a Raman image of 100 ng / mL TXM detection under different incubation times;

[0037] Figure 4 are test result images of the analysis performance part of the SERS analysis platform, wherein A is a TXM test sample schematic diagram in the range of 0~100 ng / mL based on a 96-hole plate, B is a nonlinear fitting curve of the Raman intensity at 2155 cm -1 of the test sample, C is a Raman spectrum image of the test sample, D is a standard curve of the Raman intensity at 2155 cm -1 of the test sample;

[0038] Figure 5 are test result images of the specificity and stability part of the SERS analysis platform. DETAILED DESCRIPTION

[0039] The application provides a detection platform for SERS detection, comprising a well plate and a quartz cover plate covering the well plate, a lower surface of the quartz cover plate is provided with a plurality of quartz wafers, the plurality of quartz wafers and well positions of the well plate are in one-to-one correspondence, and a diameter of the quartz wafer is not greater than that of the well position.

[0040] A lower surface of the quartz wafer at four corners is provided with a silicon wafer; an area of the silicon wafer is not greater than that of the quartz wafer.

[0041] The thickness of the quartz cover plate is 0.45-0.55 mm.

[0042] The thickness of the quartz wafer is 1.4-1.5 mm.

[0043] The thickness of the silicon wafer is 0.5-1 mm.

[0044] In the application, the well plate preferably comprises a 96-well plate, a 24-well plate, a 48-well plate or a 384-well plate.

[0045] In the application, the thickness of the quartz cover plate is 0.4-0.55 mm, preferably 0.5 mm. In the application, the diameter of the quartz wafer is preferably 3.9-4.1 mm, and is further preferably 4 mm; the thickness is 1.4-1.5 mm. In the application, the thickness of the silicon wafer is 0.5-1 mm.

[0046] The application further provides a construction method of the detection platform for SERS detection.

[0047] (1) placing the quartz cover plate in a positioning clamp, coating ultraviolet curing glue at a center point of each predetermined well position, then adhering the quartz wafer to a surface of the ultraviolet curing glue, and performing ultraviolet curing to obtain the cover plate with the adhered quartz wafer;

[0048] (2) coating ultraviolet curing glue on surfaces of the quartz wafers at four corners, then adhering the silicon wafer, and performing ultraviolet curing to obtain the cover plate with the adhered silicon wafer;

[0049] (3) covering the cover plate with the adhered silicon wafer on the well plate, so that the side with the adhered quartz wafer is in contact with the well plate, to obtain the detection platform for SERS detection.

[0050] The application places the quartz cover plate in a positioning clamp, coats ultraviolet curing glue at a center point of each predetermined well position, then adheres the quartz wafer to a surface of the ultraviolet curing glue, and performs ultraviolet curing to obtain the cover plate with the adhered quartz wafer.

[0051] In the present application, before the bonding, the quartz cover plate and the quartz wafer are preferably pretreated; the pretreatment preferably comprises: wiping the sample surface with acetone to remove existing organic impurities; then washing with anhydrous ethanol to remove residual solvents and particulates; and then treating the sample with ultraviolet ozone to enhance the hydrophilicity of the surface, thereby improving the adhesion of the ultraviolet curing adhesive.

[0052] In the present application, the amount of ultraviolet curing adhesive applied to each predetermined hole site is preferably 0.5-1 μL; the application diameter of the ultraviolet curing adhesive is 1 mm. In the present application, the application is preferably performed using a precision dispensing machine. In the present application, the dispensing operation must ensure accurate positioning of each adhesive dot and uniform adhesive amount, and must avoid overflow to ensure consistency of the subsequent bonding effect.

[0053] In the present application, the bonding process preferably comprises: gently sucking the quartz wafer using a vacuum suction pen and placing it vertically on the corresponding adhesive dot. In the present application, the bonding process preferably further comprises pressing the quartz wafer; the pressing force is preferably 10-20 g, and during the pressing process, the ultraviolet curing adhesive is uniformly spread to an area not exceeding the area of the quartz wafer.

[0054] In the present application, after the bonding, another quartz cover plate is preferably arranged on the surface of all the quartz wafers, and the quartz wafers are flattened; the flattening pressure is preferably 1 kg, and the pressure holding time is preferably 10 min.

[0055] In the present application, the ultraviolet curing conditions preferably comprise: the wavelength of the ultraviolet light source is 365 nm, the light intensity is 20 mW / cm 2 , and the time is 60 s. In the present application, the ultraviolet curing process is preferably performed in regions, and the uncured regions are covered with a metal plate; the region-by-region performance is preferably to cure one-fourth of the area each time. In the present application, region-by-region ultraviolet curing can prevent material deformation due to local heating during the curing process.

[0056] After the ultraviolet curing, the present application preferably further comprises allowing the cured sample to stand; the standing time is preferably 10 min. In the present application, standing can fully dissipate heat and stabilize the adhesive layer structure.

[0057] After obtaining the cover plate bonded with the quartz wafers, the present application coats ultraviolet curing adhesive on the surface of the quartz wafers at the four corners, bonds a silicon wafer, performs ultraviolet curing, and obtains a cover plate bonded with the silicon wafer.

[0058] In this invention, prior to bonding the silicon wafer, it is preferable to pre-treat the silicon wafer, and the pre-treatment process is preferably the same as the pre-treatment process for the quartz wafer described above. In this invention, the method of applying the UV adhesive is preferably the same as the coating process described in the above technical solutions, and will not be repeated here.

[0059] In this invention, the preferred conditions for ultraviolet curing include: a wavelength of 365 nm for the ultraviolet light source and a light intensity of 20 mW / cm². 2 The curing time is 90 seconds. In this invention, after the ultraviolet curing, it is also preferable to use microscopic focusing to detect the consistency of the height difference on the surface of each silicon wafer (to compare whether the size and shape of the spot after focused laser irradiation are consistent under the same objective lens height).

[0060] In this invention, after obtaining the cover plate with the silicon wafer attached, it is preferable to perform post-processing on the cover plate. The post-processing preferably includes: using a scalpel or scraper to remove excess adhesive from the edge of the quartz disc; then wiping away residual adhesive residue with a cotton swab soaked in acetone; after cleaning, observing each bonding area with a microscope to check whether the adhesive layer is uniform and whether there are defects such as bubbles or cracks.

[0061] After obtaining the cover plate with the silicon wafer attached, the present invention covers the cover plate with the silicon wafer attached onto the perforated plate, so that the side with the quartz disc attached is in contact with the perforated plate, thereby obtaining the detection platform for SERS detection.

[0062] In this invention, after the covering is completed, it is preferable to further include using a white light interferometer to detect the contact consistency and parallelism between the quartz disc and the liquid surface of the orifice plate, so as to confirm that the bonding quality meets the experimental requirements.

[0063] The present invention also provides a SERS detection method, comprising the following steps:

[0064] The detection probe and the solution of the analyte are mixed and placed on the detection platform for SERS detection;

[0065] The detection platform is the detection platform for SERS detection described in the above technical solution or the detection platform for SERS detection constructed by the construction method described in the above technical solution;

[0066] The mixture obtained by mixing comes into contact with the quartz disc in the detection platform;

[0067] The detection probe is an antibody-functionalized modified Prussian blue nanoparticle.

[0068] The antibody is obtained by immunizing the substance to be detected.

[0069] The modified Prussian blue nanoparticles include Prussian blue and gold nanoparticles encapsulated within the Prussian blue.

[0070] The SERS detection includes qualitative detection or quantitative detection.

[0071] The present application does not have special limitations on the specific process of the qualitative detection or quantitative detection, and the process known to those skilled in the art can be used.

[0072] In the present application, the substance to be detected in the solution of the substance to be detected preferably includes thiamethoxam, cyclaniliprole, acetamiprid, imidacloprid, malathion or nitenpyram.

[0073] Unless otherwise specified, the materials and equipment used in the present application are commercially available in the art.

[0074] The technical solutions in the present application will be described clearly and completely in combination with the embodiments in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0075] Example 1

[0076] A 96-well plate is used as an example to construct a detection platform.

[0077] The quartz cover plate (thickness of 0.5 mm), quartz wafer and silicon wafer are pretreated, and the specific operation includes: wiping the surface of the sample with acetone, then cleaning twice with anhydrous ethanol, and treating with ultraviolet ozone to obtain the pretreated quartz cover plate, quartz wafer and silicon wafer.

[0078] The pretreated quartz cover plate is placed in a special positioning fixture, and a precision dispensing machine is used to add 0.5 μL of ultraviolet curing glue at the center of each predetermined hole site, and the glue point diameter is controlled to be about 1 mm.

[0079] After dispensing is completed, the pretreated quartz wafer (thickness of 1.5 mm, diameter of 4 mm) is gently sucked by a vacuum suction pen and vertically placed on the corresponding glue point; during the lamination process, about 10 g of slight pressure needs to be applied to each quartz wafer to promote the glue to spread uniformly to a diameter of about 4 mm; then, with the aid of a level, the other quartz cover plate is horizontally pressed flat on all quartz wafers (pressure of 1 kg, pressure holding time of 10 min);

[0080] After lamination is completed, regional curing is performed, and each time the quarter area of the plate cover is cured (the uncured area is covered with a metal plate), and the curing conditions include: using a ultraviolet light source with a wavelength of 365 nm and an intensity of 20 mW / cm 2 After curing is completed, the sample is left to stand for 10 minutes to obtain the cover plate with the quartz wafer laminated.

[0081] The surface of the quartz round piece located at the four corners (i.e. A1, A12, H1, H12 positions) is coated with ultraviolet curing glue in the above manner, and then the pretreated silicon wafer (1*1 mm in size and 1 mm in thickness) is attached, and ultraviolet curing is performed. The curing conditions include: using a 365 nm wavelength, 20 mW / cm2 light intensity ultraviolet light source for 90 s to obtain a cover plate with a silicon wafer attached; 2

[0082] The cover plate with a silicon wafer attached is covered on a 96-well plate, with the side with the quartz round piece attached in contact with the well plate. The contact consistency and parallelism of the quartz round piece with the liquid surface of the 96-well plate are detected by a white light interferometer to obtain the detection platform for SERS detection.

[0083] Performance test

[0084] Thiamethoxam (TXM) is used as an example to verify the performance of the test platform.

[0085] The TXM antibody (TXM ab ) and TXM-BSA antigen are purchased from Bio-Rad Laboratories.

[0086] I. Preparation of SERS detection probe and capture substrate

[0087] (1) 10 mL of ultrapure water is heated to 40℃, then 200 μL of 10 mM FeCl3 solution and 10 mM K4Fe(CN)6·3H2O solution are added to form a clear blue solution. After vortex mixing for 5 minutes, the obtained Prussian blue nanoparticles (PB NPs) are cooled to room temperature, and then stored at 4℃.

[0088] The above obtained PB NPs are mixed with L-ascorbic acid solution for 1 minute, then 10 mM HAuCl4 solution is added, and Au@PB NPs solution is obtained by stirring at 40℃ for 3 minutes. Then the solution is centrifuged at 6000 rpm for 10 minutes to remove the supernatant, and the precipitate is washed twice with ultrapure water to obtain Au@PB NPs.

[0089] TXM ab is combined with Au@PB NPs through electrostatic interaction to construct an antibody functionalized SERS probe (Au@PB@TXMab) to achieve specific recognition of TXM pesticide. The above obtained Au@PB NPs are adjusted to pH 7.5 with 0.2 mol / L K2CO3 solution, and 5.3 mg / mL TXM ab ​The mixture was incubated at room temperature with shaking for 2 hours; then BSA solution was added to the mixed solution to a final concentration of 1%, and incubated at room temperature for 1 hour to block unbound sites; finally, the SERS probe was centrifuged at 7500 rpm for 5 minutes to remove unbound TXM. ab After resuspending in PBS solution, store at 4°C for later use.

[0090] (2) Preparation of MNPs@TXM-BSA antigen (capture substrate)

[0091] Take 100 μL of MNPs (carboxylated Fe3O4 magnetic nanoparticles purchased from Sigma-Aldrich) with a concentration of 10 mg / mL and place them in a centrifuge tube. Wash twice with PBS buffer under magnetic separation conditions, and then add 15 mM MES (2-morpholinoethanesulfonic acid pH 5.5). 6.0) Mix the solution thoroughly, wash twice, and resuspend in PBS buffer. Then add 200 μL of EDC (20 mg / mL, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 200 μL of NHS (10 mg / mL, N-hydroxysuccinimide), and shake for 30 minutes to activate the carboxyl groups on the surface of the magnetic nanoparticles. After removing the supernatant by magnetic separation, wash three times with PBS buffer, and then resuspend in PBS buffer. After adding TXM antigen, shake at room temperature for 16 hours, add BSA to a final concentration of 1%, and incubate at room temperature for 1 hour to block the remaining non-specific adsorption sites. After coupling, wash the MNPs-TXM antigen complex (capture substrate) 2-3 times with PBS solution under the action of a magnet, and store at 4°C.

[0092] II. Establishment of an anti-interference immune analysis platform

[0093] Add the SERS probe to a 96-well plate, then add TXM standard solution of different concentrations and the capture substrate to each well sequentially. Mix thoroughly and react with shaking at room temperature for 30 minutes. During this process, the antigen (i.e., TXM) on the capture substrate and the TXM in the TXM standard solution react with the TXM on the detection probe. ab Competitive binding was employed to capture and immobilize the antigen on the substrate and the antibody on the probe. Unbound material was washed away, and after magnetic separation, the mixture was washed twice with ultrapure water. Finally, the mixture was resuspended in 330 μL of ultrapure water and placed in a well plate. The slightly convex liquid surface was flattened by a quartz disc on a quartz cover plate. The height of the laser probe was calibrated by the silicon wafer under the quartz disc, and the 96-well plate was horizontally corrected before rapid Raman spectroscopy detection was performed. Each sample was measured in triplicate to establish a standard curve, where the concentration of TXM was the independent variable and the Raman intensity was the dependent variable.

[0094] III. Selectivity Analysis

[0095] To verify the specificity of the analysis platform for TXM, acetamiprid, imidacloprid, malathion and nitenpyram were selected as control samples. The Raman intensity changes of TXM and other control samples were observed at 2155 cm -1 after the completion of the competition reaction.

[0096] Four, the analysis process of TXM in actual samples

[0097] Purchased pears, leeks and celery as test samples, after homogenization, 10g of sample was weighed, different concentrations of TXM standard solution (10, 30 and 50ng / mL) were added, and it was placed at room temperature for 2 hours; then 10mL of acetonitrile solution was added, and it was shaken for 10 minutes; then 1g of NaCl was added to the sample, and after vigorous shaking for 1 minute, it was centrifuged at 4200rpm for 5 minutes. The supernatant was filtered through a 0.22μm filter membrane, and the TXM content was analyzed by the detection platform and HPLC / MS-MS.

[0098] Five, result analysis

[0099] 1. Synthesis and characterization of Au@PB NPs

[0100] After the synthesis of PB NPs suspension by constant temperature and humidity method, L-ascorbic acid and HAuCl4 were added in turn, and Au@PB NPs were quickly formed in the process of PB NPs disintegration and gold core formation. Transmission electron microscopy (TEM) images (A and B of Figure 1 clearly showed Au core (Au@PB NPs) wrapped by thin layer PB, with PB shell thickness of about 3nm and total particle size of about 35nm.

[0101] X-ray diffraction (XRD) results (F of Figure 1 showed that the diffraction pattern of Au@PB NPs simultaneously appeared the characteristic peaks of Au NPs (JCPDS No. 04-0784) and PB NPs (JCPDS No. 07-0784). X-ray photoelectron spectroscopy (XPS) analysis confirmed the electronic structure and surface composition of Au@PB NPs. The full spectrum scan (C of Figure 1 showed that C, N, O and Fe elements existed in Au@PB NPs and PB NPs, while Au only existed in Au@PB NPs. The synthesized core-shell nanostructure showed two absorption peaks at about 524nm and 680nm (E of Figure 1 , which were the characteristic peaks of Au NPs and PB NPs, respectively, further confirming the successful synthesis of Au@PB NPs. As expected, Au@PB NPs showed a Raman signal at 2155cm -1The presence of a strong singlet indicates that the -CN- group exhibits Raman activity in the silent region. This property makes Au@PB NPs a promising probe for food detection, exhibiting lower endogenous and exogenous interference compared to traditional Raman reporter molecules. Figure 1 (D).

[0102] 2. Verification of anti-interference Raman response and feasibility of automated 96-well plate testing

[0103] The feasibility of high-throughput operation of interference-resistant Raman analysis in an automated 96-well plate system was verified by using Au@PB nanoparticles with unique Raman peaks in the silent region.

[0104] To systematically evaluate signal stability, different concentrations (12.5%, 25%, 50%, and 100%) of Au@PB NPs stock solution were prepared in 96-well plates for dilution. Figure 2 A). For example Figure 2 As shown in Figure C, the Raman signal intensity of each well in the microplate exhibits extremely high consistency and increases proportionally with the increase of Au@PB NPs concentration, indicating consistency and reliability in detecting Au@PBNPs signal intensity regulated by sample concentration. This observation supports the potential for stable and interference-resistant SERS analysis in automated 96-well plates.

[0105] To detect TXM, a TXM-specific antibody (TXM) is used. ab Au@PBNPs were modified with ) UV-Vis spectroscopy confirmed this coupling, showing a redshift of the absorption peak from 526 nm to 546 nm, a redshift of 680 nm to 700 nm, and a protein characteristic peak at 280 nm. Figure 2 B). Accompanied by a significant change in zeta potential ( Figure 2 (G), Au@PB NPs via TXM ab After modification, the Zeta potential increased significantly to -30 mV, which is TXM. ab Successful coupling to the Au@PB NP surface provides clear evidence.

[0106] Furthermore, compared to traditional SERS probes and various real samples, the Raman spectra of individual Au@PB NPs show distinct peaks in the Raman silent region. Figure 2 The E value indicates that it is very suitable for anti-interference detection applications.

[0107] To detect TXM using a competitive binding method, magnetic nanoparticles (MNPs) were functionalized with the antigen. Successful coupling of the MNPs to the antigen resulted in a red shift of the absorption peak from 604 nm to 624 nm. Figure 2 (D) and EDS elemental analysis ( Figure 2The presence of antigenic elements (Cl, S) was confirmed by the F-test. These results validate the feasibility of anti-interference TXM detection in an automated high-throughput 96-well plate system.

[0108] 3. Optimization of probe synthesis

[0109] The synthesis of Au@PB NPs is highly temperature-dependent, directly affecting the Raman signal intensity of the probe. To optimize the synthesis conditions, a temperature range (20~50℃) was selected for two key steps: (1) PBNPs synthesis; (2) one-pot Au@PB NPs synthesis. The Raman signal intensity at each temperature was evaluated. Figure 3 Based on A and B, the optimal synthesis temperature for PB NPs was determined to be 35℃, and the optimal synthesis temperature for one-pot Au@PB NPs was determined to be 40℃. Au@PB NPs synthesized under these conditions exhibited the highest Raman intensity, making them highly suitable for interference-resistant and high-sensitivity Raman detection.

[0110] During the detection process, the amount of antigen bound to the surface of MNPs and the amount of TXM on Au@PBNPs were measured. ab The amount of surface modification is a key factor determining the sensitivity of a SERS analysis platform. To evaluate TXM... ab The effect of concentration on the surface of SERS probes was investigated by reacting Au@PBNPs with different concentrations (0.15 to 0.9 μg / mL) of TXM. ab Mixed preparation containing different TXM ab A large amount of SERS probe was used. A blank sample was detected at 2155 cm⁻¹. -1 Raman intensity at the location determines the optimal TXM ab Concentration. For example... Figure 3 As shown in D, the Raman intensity varies with TXM ab The effect increased with increasing concentration, reaching a maximum at 0.6 μg / mL, and then decreased, thus determining the optimal TXM. ab The density was 0.6 μg / mL. Similarly, the antigen concentration on MNPs was optimized by testing a concentration range of 0.4 to 2.4 μg / mL. Figure 3 As shown in E, the Raman intensity peaks at 1.6 μg / mL and then decreases. Therefore, the optimal condition for determining maximum sensitivity is TXM. ab The concentrations were 0.6 μg / mL and 1.6 μg / mL.

[0111] To improve the performance of the SERS analysis platform, key reaction parameters, including pH and competing reaction time, were optimized. This was achieved by comparing the reaction time at 2155 cm⁻¹. -1The Raman signal at the pH was evaluated to assess the Raman response of TXM (blank and 100 ng / mL) at different pH levels (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0). Figure 3 As shown in Figure C, the presence of TXM led to more significant dissociation of the SERS probe from the capture probe, resulting in a reduced signal intensity compared to blank detection. The largest signal difference between TXM and blank analysis occurred at pH 7.5, thus determining the optimal competitive reaction pH to be 7.5. Incubation times (10, 20, 30, 40, 50, and 60 minutes) for 100 ng / mL TXM detection were tested. Figure 3 As shown in Figure F, the Raman intensity increased with time, reaching a plateau at 30 minutes, thus determining the optimal incubation time to be 30 minutes. Sensitive and reliable SERS detection was achieved by optimizing the pH and detection reaction time.

[0112] 4. Analytical performance of the SERS analysis platform

[0113] Under optimized conditions, TXM concentrations in the range of 0 to 100 ng / mL were detected using an automated 96-well plate-based SERS analysis platform. Figure 4 A). For example Figure 4 As shown in Figure C, the Raman spectrum of Au@PBNPs shows that the signal intensity gradually decreases with increasing TXM concentration, which is attributed to the reduced competition of the SERS probe during detection. 2155 cm⁻¹ -1 Raman signal at ( Figure 4 B) decreased gradually with increasing TXM concentration. A nonlinear model “y=7548.9-6874.4 / (1+(75.4 / x)2.1)” was used, and a high correlation coefficient (R²) was obtained across the entire concentration range (0~100 ng / mL). 2 =0.986). Within the linear range of 1~100 ng / mL ( Figure 4 The standard curve, defined as y = 7412.64 - 558.67 log₂x, exhibits excellent correlation coefficients (R²). 2 =0.987), LOD was 0.19 ng / mL.

[0114] The above results highlight the system's high sensitivity and anti-interference capabilities, making it well-suited for high-throughput, quantitative SERS analysis over a wide concentration range.

[0115] 5. Specificity and stability of the SERS analysis platform

[0116] Specificity is a key parameter for the reliability of the automated 96-well plate SERS analysis platform. To evaluate the specificity, the responses of TXM and control samples (including cyantraniliprol, imidacloprid, acetamiprid, malathion and nitenpyram) at the same concentrations (0, 30, 50, 100, 300 and 500 ng / mL) were tested using the system under the same experimental conditions. As shown in Fig. 2, the SERS intensity at 2155 cm Figure 5 -1 decreased significantly with the increase of TXM concentration. In contrast, the SERS responses of other pesticides remained constant over the whole range of tested concentrations, confirming the high specificity of the platform for TXM detection.

[0117] To evaluate the stability of the platform, the performance of three batches of SERS probes with surface-bound TXMab was verified over 60 days. Figure 5 It was shown that the SERS intensity at 2155 cm -1 remained stable over time with a relative standard deviation (RSD) less than 4.2%. These results indicated the excellent long-term reproducibility of the platform, confirming its reliability for TXM detection.

[0118] 6. Analysis of real samples

[0119] To evaluate the application of the SERS analysis platform for TXM detection in real samples, TXM solutions at different concentrations (10, 30 and 50 ng / mL) were added to pear, leek and celery samples, respectively, and each experiment was determined in triplicate. As shown in Table 1, the recovery of TXM in the samples was between 91.9 and 108.2% with a relative standard deviation (RSD) of 1.1-4.6%. In addition, the results obtained by this method were similar to those determined by HPLC-MS / MS, indicating that the SERS analysis platform could be used for high-accuracy quantitative determination of TXM in real samples.

[0120] Table 1. Detection results of TXM content in real samples

[0121]

[0122] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.​

Claims

1. A detection platform for SERS detection, characterized in that, It consists of a perforated plate and a quartz cover plate covering the perforated plate. The lower surface of the quartz cover plate is provided with a plurality of quartz discs, and the plurality of quartz discs correspond one-to-one with the holes in the perforated plate. The diameter of the quartz discs is not greater than the diameter of the holes. Silicon wafers are disposed on the lower surface of the quartz discs located at the four corners; the area of ​​the silicon wafers is not greater than the area of ​​the quartz discs. The thickness of the quartz cover plate is 0.45~0.55mm; The thickness of the quartz disc is 1.4~1.5mm; The thickness of the silicon wafer is 0.5~1mm.

2. The detection platform for SERS detection according to claim 1, characterized in that, The perforated plate includes a 96-well plate, a 24-well plate, a 48-well plate, or a 384-well plate.

3. The detection platform for SERS detection according to claim 1, characterized in that, The diameter of the quartz disc is 3.9~4.1mm.

4. The method for constructing the detection platform for SERS detection according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Place the quartz cover plate in the positioning fixture, apply UV curing adhesive to the center point of each predetermined hole, attach a quartz disc to the surface of the UV curing adhesive, and perform UV curing to obtain a cover plate with a quartz disc attached. (2) After coating the surface of the quartz discs located at the four corners with UV-curing adhesive, the silicon wafers are attached and UV-cured to obtain a cover plate with the silicon wafers attached. (3) Cover the cover plate with the silicon wafer attached onto the perforated plate, so that the side with the quartz disc attached is in contact with the perforated plate, to obtain the detection platform for SERS detection.

5. The construction method according to claim 4, characterized in that, In step (1), the amount of UV-curable adhesive applied to each predetermined hole is 0.5~1μL; the coating diameter of the UV-curable adhesive is 1mm. The bonding process also includes applying pressure to the quartz disc.

6. The construction method according to claim 4, characterized in that, After bonding, another quartz cover plate is placed on the surface of all the quartz discs to flatten the quartz discs. The flattening pressure is 1 kg and the pressure holding time is 10 min. In step (1), the conditions for UV curing include: the wavelength of the UV light source is 365nm and the light intensity is 20mW / cm². 2 The time is 60 seconds.

7. The construction method according to claim 4, characterized in that, In step (2), the conditions for UV curing include: a UV light source wavelength of 365 nm and a light intensity of 20 mW / cm². 2 The duration is 90 seconds.

8. A SERS detection method, characterized in that, Includes the following steps: The detection probe and the solution of the analyte are mixed and placed on the detection platform for SERS detection; The detection platform is the detection platform for SERS detection as described in any one of claims 1 to 3 or the detection platform for SERS detection constructed by the construction method described in any one of claims 4 to 7; The mixture obtained by mixing comes into contact with the quartz disc in the detection platform; The detection probe is an antibody-functionalized modified Prussian blue nanoparticle. The antibody is obtained by immunizing the substance to be detected. The modified Prussian blue nanoparticles include Prussian blue and gold nanoparticles encapsulated within the Prussian blue. The SERS detection includes qualitative or quantitative detection.

9. The SERS detection method according to claim 8, characterized in that, The substances to be tested in the solution include thiamethoxam, cyclopyr, acetamiprid, imidacloprid, malathion, or acetamiprid.

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