Detection platform for SERS (Surface Enhanced Raman Scattering) detection, construction method of detection platform and SERS detection method

Abstract

The invention belongs to the technical field of SERS (Surface Enhanced Raman Scattering) detection, and particularly relates to a detection platform for SERS detection, a construction method of the detection platform and an SERS detection method. According to the invention, the quartz wafers are arranged on the quartz plate cover corresponding to the hole positions of the hole plate, and the liquid level in the hole is pressed to a uniform height, so that the laser incidence height error caused by a concave liquid level or a convex liquid level is effectively reduced, the stability and repeatability of SERS signals are improved, and the detection inconsistency caused by the uneven liquid level is effectively made up. Particularly, on the premise that the focal length of Raman equipment is difficult to automatically adjust, physical guarantee is provided for detection of different samples by structurally unifying the heights of the samples. Besides, silicon wafers are arranged at the bottoms of the quartz wafers at the four corners and serve as calibration reference points of the irradiation height of the laser probe, so that the system can accurately focus and keep consistent irradiation conditions in porous scanning, and the uniformity of sample detection in different batches is realized.

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 of the liquid to be detected in the 96-well plate due to capillary action or surface tension 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 laser probe of the Raman detection equipment cannot be automatically adjusted, 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 the stability between batches have become the key to solving the stability problem of high-throughput detection. SUMMARY

[0004] The purpose of the present application is 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 purpose, the present application provides the following technical scheme: The present application provides a detection platform for SERS detection, comprising 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 one-to-one correspond, and the diameter of the quartz wafer is not greater than the diameter of the well position. The lower surface of the quartz wafer is provided with a silicon wafer; the area of the silicon wafer is not greater than the area of the quartz wafer. The thickness of the quartz cover plate is 0.45-0.55 mm. The thickness of the quartz wafer is 1.4-1.5 mm. The thickness of the silicon wafer is 0.5-1 mm.

[0006] Preferably, the well plate comprises a 96-well plate, a 24-well plate, a 48-well plate or a 384-well plate.

[0007] Preferably, the diameter of the quartz round piece is 3.9-4.1mm.

[0008] The application further provides a construction method of the detection platform for SERS detection. (1) Place the quartz cover plate in the positioning fixture, coat the center point of each predetermined hole position with ultraviolet curing glue, then adhere the quartz round piece to the surface of the ultraviolet curing glue, and perform ultraviolet curing to obtain the cover plate with the adhered quartz round piece; (2) Coat the surface of the quartz round piece 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; (3) Cover the cover plate with the adhered silicon wafer on the hole plate, so that the side with the adhered quartz round piece is in contact with the hole plate, to obtain the detection platform for SERS detection.

[0009] Preferably, in step (1), the coating amount of ultraviolet curing glue on each predetermined hole position is 0.5-1μL; and the coating diameter of the ultraviolet curing glue is 1mm. In the adhering process, the quartz round piece is also subjected to pressure.

[0010] Preferably, after the adhering, another quartz cover plate is arranged on the surface of all the quartz round pieces, and the quartz round pieces are flattened, the flattening pressure is 1kg, and the pressure holding time is 10min. 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.

[0011] 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.

[0012] The application further provides a SERS detection method, which comprises the following steps: Mix the detection probe and the solution of the to-be-detected substance, place them in the detection platform, and perform SERS detection; 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; The mixed solution obtained by mixing is in contact with the quartz round piece in the detection platform; The detection probe is an antibody-functionalized modified Prussian blue nanoparticle; The antibody is obtained by immunization of the to-be-detected substance. The modified Prussian blue nanoparticles comprise Prussian blue and nanogold wrapped inside the Prussian blue; The SERS detection comprises qualitative detection or quantitative detection.

[0013] Preferably, the to-be-detected substance in the to-be-detected substance solution comprises thiamethoxam, pyridaben, acetamiprid, imidacloprid, malathion or nitenpyram.

[0014] The application effectively reduces the laser incidence height error caused by concave liquid surface or convex liquid surface by setting quartz round plates 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 making up for the inconsistency caused by uneven liquid surface. 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 guarantee for different sample detection. In addition, the silicon wafer at the bottom of the quartz round plate at the four corners is set 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.

[0015] The application also provides a SERS detection method. The application further strengthens the detection performance of the system by introducing an anti-interference SERS probe. The probe uses Prussian blue containing C≡N groups as a Raman reporter molecule, and the Raman signal only appears at 2155cm -1 -1 at the "silent region" (1800~2800cm -1 -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.

[0016] Combined with the above optimization design, the application based on the hole plate cover assisted automatic SERS detection platform can significantly improve the detection efficiency and throughput while greatly improving the stability and consistency of the detection results, and can be widely applied in the fields of food safety, environmental monitoring and biological sample analysis, and has the technical advantages of high detection efficiency, strong anti-interference ability and excellent result consistency. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The characterization results of Au@PB NPs are shown in the figure, wherein A and B are TEM graphs, C is an XPS full spectrum scan graph, D is a Raman graph, E is an ultraviolet-visible absorption spectrum graph, and F is an XRD graph; Figure 2Figure for the results of the anti-interference Raman response verification and the feasibility verification part of the automated 96-well plate; wherein A is a schematic diagram of Au@PB NPs stock solution dilution group test samples of different concentrations, B is an ultraviolet-visible absorption spectrum diagram of TXM specific antibody modified Au@PB NPs, C is a diagram showing the consistent performance of the characteristic peak Raman intensity of Au@PB NPs stock solution of different concentrations under the assistance of a plate cover, D is an ultraviolet-visible absorption spectrum diagram of antigen functionalized magnetic nanoparticles, E is a Raman spectrum comparison diagram of Au@PB NPs and traditional SERS probes and food samples, F is an EDS element analysis of antigen functionalized magnetic nanoparticles, G is the zeta potential of AuNPs, Au@PB NPs and TXM specific antibody modified Au@PB NPs; Figure 3 Figure for the test results of the probe synthesis optimization part; wherein A is a Raman diagram under different temperatures in the synthesis of PBNPs, B is a Raman diagram under different temperatures in the one-pot synthesis of Au@PB NPs, C is the Raman response of TXM detection under different pH levels, D is a Raman diagram of a blank sample at 2155cm -1 E is a Raman diagram of different antigen concentrations on MNPs, F is a Raman diagram of different incubation times for 100ng / mL TXM detection; Figure 4 Figure for the test results of the analysis performance part of the SERS analysis platform; wherein A is a schematic diagram of TXM test samples in the range of 0~100ng / mL based on a 96-well plate, B is a nonlinear fitting curve of the Raman intensity at 2155cm -1 of the test sample, C is a Raman spectrum diagram of the test sample, D is a standard curve of the Raman intensity at 2155cm -1 of the test sample; Figure 5 Figure for the test results of the specificity and stability part of the SERS analysis platform. DETAILED DESCRIPTION

[0018] The application provides a detection platform for SERS detection, which comprises 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 the diameter of the quartz wafer is not greater than the diameter of the well position; A lower surface of the quartz wafer located at four corners is provided with a silicon wafer; the area of the silicon wafer is not greater than the area of the quartz wafer; The thickness of the quartz cover plate is 0.45~0.55mm; The thickness of the quartz wafer is 1.4~1.5mm; The thickness of the silicon wafer is 0.5~1mm.

[0019] In the present application, the well plate preferably comprises a 96-well plate, a 24-well plate, a 48-well plate or a 384-well plate.

[0020] In the present application, the thickness of the quartz cover plate is 0.4-0.55 mm, preferably 0.5 mm. In the present application, the diameter of the quartz wafer is preferably 3.9-4.1 mm, further preferably 4 mm; the thickness is 1.4-1.5 mm. In the present application, the thickness of the silicon wafer is 0.5-1 mm.

[0021] The present application also provides a construction method of the detection platform for SERS detection as described in the above technical solutions, comprising the following steps: (1) placing the quartz cover plate in a positioning fixture, coating ultraviolet curing glue at the center point of each predetermined hole position, then adhering the quartz wafer to the surface of the ultraviolet curing glue, and performing ultraviolet curing to obtain the cover plate with the adhered quartz wafer; (2) coating ultraviolet curing glue on the surface of the quartz wafer at the four corners, then adhering the silicon wafer, and performing ultraviolet curing to obtain the cover plate with the adhered silicon wafer; (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.

[0022] In the present application, the quartz cover plate is placed in a positioning fixture, ultraviolet curing glue is coated at the center point of each predetermined hole position, then the quartz wafer is adhered to the surface of the ultraviolet curing glue, and ultraviolet curing is performed to obtain the cover plate with the adhered quartz wafer.

[0023] In the present application, before the adhering, it is also preferably to perform pretreatment on the quartz cover plate and the quartz wafer; 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 ability of the ultraviolet curing glue.

[0024] In the present application, the coating amount of ultraviolet curing glue on each predetermined hole position is preferably 0.5-1 μL; the coating diameter of the ultraviolet curing glue is 1 mm. In the present application, the coating is preferably performed by a precision dispensing machine. In the present application, the dispensing operation needs to ensure accurate position of each glue point and uniform glue amount, to avoid overflow phenomenon, so as to ensure consistency of the subsequent adhering effect.

[0025] In the application, the process of the bonding preferably comprises: using a vacuum suction pen to gently suck the quartz wafer and vertically placing the quartz wafer on the corresponding glue point.

[0026] In the application, the process of the bonding preferably further comprises: pressing the quartz wafer; the pressure of the pressing preferably is 10-20 g, and the pressing facilitates the uniform spreading of the ultraviolet curing glue to an area not exceeding the area of the quartz wafer.

[0027] In the application, the conditions of the ultraviolet curing 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 application, the process of the ultraviolet curing is preferably performed in a region-by-region manner, and a metal plate is used to cover the uncured region; the region-by-region manner preferably comprises curing one quarter of the area each time. In the application, the region-by-region ultraviolet curing can prevent the material from deforming due to local heating during the curing.

[0028] After the ultraviolet curing, the application further preferably comprises: allowing the cured sample to stand for 10 min. In the application, the standing can facilitate heat dissipation and stabilize the structure of the glue layer.

[0029] After obtaining the cover plate bonded with the quartz wafer, the application coats ultraviolet curing glue on the surface of the quartz wafer at the four corners, bonds the silicon wafer, performs ultraviolet curing, and obtains a cover plate bonded with the silicon wafer.

[0030] In the application, before the bonding of the silicon wafer, the application further preferably comprises: pre-treating the silicon wafer; the process of the pre-treating preferably refers to the pre-treating process of the quartz wafer described above. In the application, the coating manner of the ultraviolet glue preferably refers to the coating process described in the above technical solution, which will not be described here.

[0031] In the application, the conditions of the ultraviolet curing preferably comprise: the wavelength of the ultraviolet light source is 365 nm, the light intensity is 20 mW / cm 2 , and the time is 90 s. In the application, after the ultraviolet curing, the application further preferably comprises: using microscopic focusing to detect the consistency of the height difference of the surface of each silicon wafer (comparing the size and shape of the focused laser irradiation spot under the same objective height).

[0032] In the present application, after the cover plate with the silicon wafer is obtained, the cover plate is preferably subjected to post-treatment, which preferably comprises: removing the excess glue present at the edge of the quartz wafer using a scalpel or a spatula; then, wiping the residual glue marks with a cotton swab soaked with acetone; after the cleaning is completed, using a microscope to observe each bonding area to check whether the glue layer is uniform, whether there are bubbles or cracks and other defects.

[0033] After the cover plate with the silicon wafer is obtained, the cover plate with the silicon wafer is covered on the aperture plate, so that the side with the quartz wafer is in contact with the aperture plate, and the detection platform for SERS detection is obtained.

[0034] In the present application, after the covering, the contact consistency and parallelism of the quartz wafer and the liquid level of the aperture plate are preferably detected by a white light interferometer to confirm that the bonding quality meets the experimental requirements.

[0035] The present application also provides a SERS detection method, comprising the following steps: Mixing the detection probe and the solution of the substance to be detected, and placing them in the detection platform to perform SERS detection; 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; The mixed solution obtained by mixing is in contact with the quartz wafer in the detection platform; The detection probe is an antibody-functionalized modified Prussian blue nanoparticle; The antibody is obtained by immunization of the substance to be detected; The modified Prussian blue nanoparticle comprises Prussian blue and nanogold wrapped inside the Prussian blue; The SERS detection comprises qualitative detection or quantitative detection.

[0036] 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.

[0037] In the present application, the substance to be detected in the solution of the substance to be detected preferably comprises thiamethoxam, cycloxaprid, acetamiprid, imidacloprid, malathion or nitenpyram.

[0038] Unless otherwise specified, the materials and equipment used in the present application are commercially available in the art.

[0039] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Apparently, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of the present application.

[0040] Example 1 A detection platform was constructed by taking a 96-well plate as an example; The quartz cover plate (thickness of 0.5 mm), quartz wafer and silicon wafer were pretreated, and the specific operation included: the sample surface was wiped with acetone, then cleaned twice with anhydrous ethanol, and treated with ultraviolet ozone to obtain the pretreated quartz cover plate, quartz wafer and silicon wafer; The pretreated quartz cover plate was placed in a special positioning fixture, and 0.5 μL of ultraviolet curing glue was added at the center of each predetermined hole site using a precision dispensing machine, and the glue point diameter was controlled to be about 1 mm; After dispensing was completed, the pretreated quartz wafer (thickness of 1.5 mm, diameter of 4 mm) was gently sucked using a vacuum suction pen and vertically placed on the corresponding glue point; during the lamination process, about 10 g of slight pressure was 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 was horizontally pressed flat on all the quartz wafers (pressure of 1 kg, pressure holding time of 10 min); After lamination was completed, the regions were cured, and each time a quarter of the region of the plate cover was cured (the uncured region was covered with a metal plate), and the curing conditions included: the quartz wafer was irradiated with a ultraviolet light source with a wavelength of 365 nm and an intensity of 20 mW / cm 2 for 60 s; after curing was completed, the sample was left to stand for 10 minutes to obtain the cover plate with the quartz wafer laminated thereon; After the quartz wafer at the four corners (i.e. A1, A12, H1, H12 positions) was coated with ultraviolet curing glue in the above manner, the pretreated silicon wafer (size of 1*1 mm, thickness of 1 mm) was laminated thereon, and ultraviolet curing was performed, and the curing conditions included: the quartz wafer was irradiated with a ultraviolet light source with a wavelength of 365 nm and an intensity of 20 mW / cm 2 for 90 s to obtain the cover plate with the silicon wafer laminated thereon; The cover plate with the silicon wafer laminated thereon was covered on the 96-well plate, and the side with the quartz wafer laminated thereon was in contact with the well plate, and the contact consistency and parallelism of the quartz wafer and the liquid surface of the 96-well plate were detected by a white light interferometer to obtain the detection platform for SERS detection.

[0041] Performance test Thiamethoxam (TXM) was taken as an example to verify the performance of the test platform; Wherein TXM antibody (TXM ab ) and TXM-BSA antigen were purchased from Bio-Rad Laboratories.

[0042] I. Preparation of SERS detection probe and capture substrate (1) 10 mL ultrapure water was heated to 40℃, then 200 μL of 10 mM FeCl3solution and 10 mM K4Fe(CN)6·3H2O solution were added to form a clear blue solution, vortex mixed for 5 minutes, then the obtained Prussian blue nanoparticles (PB NPs) were cooled to room temperature, and then stored at 4℃; The above obtained PB NPs were mixed with L-ascorbic acid solution for 1 minute, then 10 mM HAuCl4solution was added, and Au@PB NPs solution was obtained by stirring at 40℃ for 3 minutes; then the solution was centrifuged at 6000 rpm for 10 minutes to remove the supernatant, and the precipitate was washed twice with ultrapure water to obtain Au@PB NPs; TXM ab antibody was combined with Au@PB NPs by electrostatic interaction to construct antibody functionalized SERS probe (Au@PB@TXMab) to realize specific recognition of TXM pesticide; the above obtained Au@PB NPs were adjusted to pH 7.5 with 0.2 mol / L K2CO3solution, and 5.3 mg / mL TXM ab antibody was added, and incubated at room temperature 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 the unbound sites; finally, the SERS probe was centrifuged at 7500 rpm for 5 minutes to remove the unbound TXM ab antibody, and resuspended with PBS solution for storage at 4℃ for standby.

[0043] (2) Preparation of MNPs@TXM-BSA antigen (capture substrate) Take 100 μL of MNPs (carboxylated Fe3O4 magnetic nanoparticles purchased from Sigma-Aldrich) with a concentration of 10 mg / mL in a centrifuge tube, wash twice with PBS buffer under magnetic separation conditions, then add 15 mM MES (2-morpholinoethanesulfonic acid pH 6.0) solution and mix well, repeat washing twice and resuspend with 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), respectively, and shake for 30 minutes to activate the carboxyl groups on the surface of the magnetic nanoparticles; after removing the supernatant under magnetic separation, repeat washing three times with PBS buffer, then resuspend with PBS buffer; after adding TXM antigen, shake for 16 hours at room temperature, add BSA to a final concentration of 1%, and incubate for 1 hour at room temperature to block the remaining non-specific adsorption sites; after coupling is completed, 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.

[0044] II. Establishment of Anti-interference Immunoassay Platform Add SERS probes in a 96-well plate, and add different concentrations of TXM standard solution and capture substrate to each well in turn, mix well, and shake for 30 minutes at room temperature. During this process, the antigen (i.e. TXM) on the capture substrate and the TXM in the TXM standard solution compete with the TXM on the detection probe ab for binding, the antigen on the capture substrate and the antibody on the probe bind and fix, the unbound substances are removed by washing, and after magnetic separation, wash twice with ultrapure water; finally, resuspend the mixture in 330 μL of ultrapure water, place it in the well plate, and the microconvex liquid surface is flattened by the quartz disc on the quartz cover plate. After calibrating the height of the laser probe and performing 96-well plate horizontal correction through the silicon sheet under the quartz disc, perform rapid Raman spectrum detection, determine each sample in triplicate, establish a standard curve, where the concentration of TXM is the independent variable and the Raman intensity is the dependent variable.

[0045] III. Selective Analysis To verify the specific detection of the analysis platform for TXM, acetamiprid, imidacloprid, malathion, and nitenpyram were selected as control samples. After the completion of the competition reaction, the change in Raman intensity of TXM at 2155 cm -1 and other control samples was observed.

[0046] IV. Analysis Process of TXM in Actual Samples Pears, leeks and celery were purchased as test samples, 10 g of the homogenized samples were weighed, and different concentrations of TXM standard solution (10, 30 and 50 ng / mL) were added, and then the samples were placed at room temperature for 2 hours; then 10 mL of acetonitrile solution was added, and the samples were oscillated for 10 minutes; then 1 g of NaCl was added to the samples, and the samples were oscillated vigorously for 1 minute and then centrifuged at 4200 rpm for 5 minutes. The supernatant was filtered through a 0.22 μm filter membrane, and the TXM content was analyzed by using a detection platform and HPLC / MS-MS.

[0047] V. Result analysis 1. Synthesis and characterization of Au@PB NPs After the PB NPs suspension was synthesized by constant temperature and humidification method, L-ascorbic acid and HAuCl4 were added in sequence, and Au@PB NPs were rapidly formed in the process of PB NPs disintegration and gold core formation. The transmission electron microscope (TEM) images (A and B of FIG. 1) clearly showed the Au core (Au@PB NPs) wrapped by a thin layer of PB, and the thickness of the PB shell was about 3 nm, and the total particle size was about 35 nm. Figure 1

[0048] X-ray diffraction (XRD) results (F of FIG. 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 FIG. 1) showed that C, N, O and Fe elements existed in Au@PB NPs and PB NPs, and Au only existed in Au@PB NPs. The synthesized core-shell nanostructure showed two absorption peaks at about 524 nm and 680 nm (E of FIG. 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 exhibited a strong single peak at 2155 cm -1 Figure 1 Figure 1 Figure 1 Figure 1

[0049] 2. Anti-interference Raman response verification and feasibility verification of automated 96-well plate Using Au@PB nanoparticles with unique Raman peaks in the silent zone, the high-throughput operation feasibility of anti-interference Raman analysis in the 96-well plate automation system was verified. ​​​​​​

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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 2 The 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.

[0054] 3. Optimization of probe synthesis 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 3Based 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.

[0055] 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.

[0056] 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. -1 The 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.

[0057] 4. Analytical performance of the SERS analysis platform 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.

[0058] 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.

[0059] 5. Specificity and stability of the SERS analysis platform Specificity is a key parameter for the reliability of automated 96-well plate SERS analysis platforms. To evaluate specificity, the system was used to test the responses of TXM and control samples (including acetamiprid, imidacloprid, acetamiprid, malathion, and nitenpyram) at the same concentrations (0, 30, 50, 100, 300, and 500 ng / mL) under identical experimental conditions. Figure 5 As shown, 2155cm -1 The SERS intensity at the site decreased significantly with increasing TXM concentration. In contrast, the SERS response of other pesticides remained constant throughout the tested concentration range, confirming the platform's high specificity for TXM detection.

[0060] To evaluate the stability of the platform, the performance of three batches of surface-bound TXMab SERS probes was verified over 60 days. Figure 5 The display shows 2155cm. -1 The SERS intensity at the site remained stable over time, with a relative standard deviation (RSD) of less than 4.2%. These results demonstrate the platform's excellent long-term reproducibility and confirm its reliability for TXM detection.

[0061] 6. Analysis of real samples To evaluate the application of the SERS analysis platform in the detection of TXM in real samples, TXM solutions with different concentrations (10, 30 and 50 ng / mL) were added into 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%, and the relative standard deviation (RSD) was 1.1-4.6%. In addition, the results obtained by the method were similar to those determined by HPLC-MS / MS, indicating that the SERS analysis platform can be used for high-accuracy quantitative determination of TXM in real samples.

[0062] Table 1. Detection results of TXM content in real samples

[0063] 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 belong to the protection scope of the present application.

Claims

1. A detection platform for SERS detection, characterized in that, The quartz cover plate is covered on the hole plate, and the lower surface of the quartz cover plate is provided with a plurality of quartz discs corresponding to the hole positions of the hole plate one by one, and the diameter of the quartz disc is not greater than the diameter of the hole position. The lower surface of the quartz disc at the four corners is provided with a silicon wafer, and the area of the silicon wafer is not greater than the area of the quartz disc. The thickness of the quartz cover plate is 0.45-0.55 mm. The thickness of the quartz disc is 1.4-1.5 mm. The thickness of the silicon wafer is 0.5-1 mm.

2. The detection platform for SERS detection according to claim 1, wherein, The hole plate includes a 96-hole plate, a 24-hole plate, a 48-hole plate or a 384-hole plate.

3. The detection platform for SERS detection of claim 1, wherein, The diameter of the quartz disc is 3.9-4.1 mm.

4. The method for constructing a detection platform for SERS detection according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: (1) placing the quartz cover plate in a positioning clamp, coating ultraviolet curing glue on the center point of each predetermined hole position, then adhering the quartz disc to the surface of the ultraviolet curing glue, and performing ultraviolet curing to obtain a cover plate with the quartz disc adhered thereto; (2) coating ultraviolet curing glue on the surface of the quartz disc at the four corners, then adhering the silicon wafer thereto, and performing ultraviolet curing to obtain a cover plate with the silicon wafer adhered thereto; (3) covering the cover plate with the silicon wafer adhered thereto on the hole plate, so that the side with the quartz disc adhered thereto is in contact with the hole plate, to obtain the detection platform for SERS detection.

5. The construction method according to claim 4, characterized in that, In step (1), the coating amount of ultraviolet curing glue on each predetermined hole position is 0.5-1 μL, and the coating diameter of the ultraviolet curing glue is 1 mm; In the adhering process, the quartz disc is also subjected to pressure.

6. The construction method of claim 4, wherein, After the adhering, another quartz cover plate is arranged on the surface of all the quartz discs, and the quartz discs are flattened, the flattening pressure is 1 kg, and the pressure holding time is 10 min. The condition of the UV curing in the step (1) includes: the wavelength of the UV light source is 365 nm, the light intensity is 20 mW / cm 2 , and the time is 60 s.

7. The construction method of claim 4, wherein, The condition of the UV curing in the step (2) includes: the wavelength of the UV light source is 365 nm, the light intensity is 20 mW / cm 2 , and the time is 90 s.

8. A SERS detection method, characterized by, The method comprises the following steps: Mixing a detection probe and a solution of a to-be-detected substance, placing them in the detection platform, and performing SERS detection; The detection platform is the detection platform for SERS detection according to any one of claims 1-3 or the detection platform for SERS detection constructed by the construction method according to any one of claims 4-7; The mixed solution obtained by mixing is in contact with the quartz discs in the detection platform; The detection probe is an antibody-functionalized modified Prussian blue nanoparticle; The antibody is obtained by immunization of the to-be-detected substance; The modified Prussian blue nanoparticle comprises Prussian blue and nanogold wrapped inside the Prussian blue; The SERS detection includes qualitative detection or quantitative detection.

9. The SERS detection method of claim 8, wherein, The to-be-detected substance in the solution of the to-be-detected substance includes thiamethoxam, pyridaben, acetamiprid, imidacloprid, malathion or nitenpyram.

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