Preparation method and application of SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of pesticides and veterinary drugs

Abstract

The present invention provides a preparation method and application of a SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of agricultural and veterinary drugs, and relates to the technical field of SERS immunochromatographic test strips. The SERS immunochromatographic test strip described in the present invention includes a test strip and a SERS immunolabel for detection; the test strip includes a bottom plate, a sample pad, a nitrocellulose membrane, a chromatography pad and a water-absorbing pad; a detection line and a quality control line are provided on the nitrocellulose membrane; the detection line and the quality control line are respectively combined with a SERS immunolabel, and the SERS immunolabel is composed of six SERS labels with different Raman scatterings, respectively combined with five agricultural and veterinary drug residues and rabbit antibodies. The SERS immunochromatographic test strip constructed by the present invention can simultaneously complete the detection of five agricultural and veterinary drug residues in a sample, is convenient and fast to operate, and has high sensitivity and strong specificity in the detection result. It is suitable for on-site detection of agricultural products and has important application value for the quality and safety of agricultural products.

Description

Technical Field

[0001] The invention belongs to the technical field of SERS immunochromatographic test strips, and specifically relates to a preparation method and application of a SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of agricultural and veterinary drugs. Background Art

[0002] In order to ensure the safety of agricultural products and eliminate potential hazards, widely developed methods for agricultural product contamination detection and hazard monitoring that combine various instruments (such as chromatography, chromatography-mass spectrometry) have become mainstream. However, instrumental methods face problems such as large instrument size, complex operation, time-consuming pretreatment, and high requirements for operators, making it difficult to meet the needs of rapid analysis and on-site detection. Therefore, the development of portable, cost-effective, and highly sensitive analytical techniques is crucial to achieve rapid detection and timely monitoring of agricultural products.

[0003] Among rapid analytical technologies, immunochromatographic test strips are currently the most advantageous instant testing tools for agricultural product quality and safety due to their cost-effectiveness, portability, and simplicity. However, the test strips use metal nanoparticles as colorimetric indicators, have low sensitivity, and limited quantitative ability, which limits their application in trace target detection. In contrast, immunochromatographic test strips based on surface enhanced Raman scattering (SERS) will have more advantages in trace quantification and high-sensitivity detection. More interestingly, signal molecules containing triple bond groups (e.g., C≡N, C≡C, and C≡O) in the silent zone (1800-2800cm -1 ) exhibits anti-interference scattering signals, which will avoid signal interference from the nitrocellulose membrane of the test strip, agricultural product samples and detection targets. This will further improve the accuracy and practicality of SERS immunochromatographic test strips in agricultural product quality and safety testing. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method and application of a SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of pesticides and veterinary drugs. The SERS immunochromatographic test strip of the present invention can quickly and accurately detect pesticide and veterinary drug residues in agricultural products.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of agricultural and veterinary drugs, wherein the SERS immunochromatographic test strip comprises a chromatography test strip and a SERS immunolabel for detection; the chromatography test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorbent pad;

[0007] The nitrocellulose membrane is provided with detection lines T1, T2, T3, T4, T5 and a quality control line C; the detection lines T1, T2, T3, T4, T5 are attached with five agricultural and veterinary drug antigens, namely neomycin, kanamycin, streptomycin, gentamicin and spectinomycin, and the quality control line C is attached with a goat anti-rabbit secondary antibody; the detection lines and the quality control line are respectively combined with SERS immunolabeling;

[0008] The preparation method of the SERS immunolabeling comprises the following steps:

[0009] The noble metal ions in the Raman beacon molecular suspension are reduced to construct a SERS label; the pH of the SERS label is adjusted, a monoclonal antibody for agricultural and veterinary drugs or a rabbit antibody is added, vortexed, and then a bovine serum albumin solution is added to obtain a SERS immune label.

[0010] Preferably, the Raman beacon molecules include Prussian blue and Prussian blue analogs.

[0011] Preferably, the agricultural and veterinary drug monoclonal antibody includes one or more of neomycin monoclonal antibody, kanamycin monoclonal antibody, streptomycin monoclonal antibody, gentamicin monoclonal antibody or spectinomycin monoclonal antibody.

[0012] Preferably, the final concentration of the pesticide monoclonal antibody is 2-10 μg / mL.

[0013] Preferably, the bottom plate of the chromatography test strip is located below the other parts, and the sample pad and the absorbent pad are overlapped at both ends of the nitrocellulose membrane respectively, and the overlapped parts are located above the nitrocellulose membrane.

[0014] The present invention also provides an application of the SERS immunochromatographic test strip in qualitative and quantitative detection of pesticide and veterinary drug residues.

[0015] The present invention also provides a method for multiple detection of pesticide residues in agricultural products, comprising the following steps: mixing the sample to be tested with the SERS immunolabel in the SERS immunochromatographic test strip to obtain a mixed solution, inserting the chromatography test strip into the mixed solution, and performing qualitative and quantitative detection.

[0016] Preferably, the qualitative detection is: if the sample contains the target drug, the target drug in the sample binds to the SERS immunolabeling, and the color of the detection line becomes weaker; if the sample does not contain the target drug, the SERS immunolabeling binds to the antigen on the detection line, and the detection line develops normally.

[0017] Preferably, the quantitative detection is: measuring the Raman intensity on the detection line by a Raman spectrometer to perform quantitative analysis on the target drug.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The invention provides a SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of agricultural and veterinary drugs, comprising a chromatographic test strip for chromatography and immunoadsorption and a SERS immunolabel for detection; the SERS immunolabel is provided by gold and silver nanoparticles wrapped by Prussian blue (Fe4[Fe(CN)6]3) and Prussian blue analogs (Cu2[Fe(CN)6], Pb2[Fe(CN)6]) to provide Raman signals, and its Raman characteristic peak is located at 2042cm -1 、2085cm -1 、2125cm -1 、2140cm -1 、2160cm -1 and 2087cm -1 , are located in the Raman silence region of biomolecules (1800-2800cm -1 ), which can effectively avoid the spectral interference between the sample and the test strip. The SERS immunolabeling of the present invention is mixed with the sample, and after the immunochromatography of the test strip, it can aggregate and develop color on the detection line, and at the same time provide a colorimetric signal for semi-quantitative analysis by the naked eye and a Raman signal for precise quantification that can be obtained by a portable spectrometer; the SERS immunochromatography test strip can simultaneously complete the detection of five kinds of pesticide residues, namely neomycin, kanamycin, streptomycin, gentamicin and spectinomycin, in the sample within 10 minutes, and the operation is convenient and fast, and the detection result has high sensitivity, strong anti-interference and strong specificity, which is suitable for on-site detection of various agricultural products and has important application value for the quality and safety of agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The combination positions of the test lines and quality control lines on the test strip designed by the present invention and the six SERS immunolabelings;

[0021] Figure 2 are the Raman signal positions of the six SERS immunolabelings in the present invention;

[0022] Figure 3 The spectral anti-interference characteristics of the Raman signals of the six SERS immunolabelings in the present invention;

[0023] Figure 4 The SERS immunolabeling transmission electron microscopy morphology analysis diagram and elemental analysis in the present invention;

[0024] Figure 5 The optimization analysis results of the loading amount of neomycin antigen on the test strip of the present invention and the modification amount of neomycin antibody on the SERS labeled surface are as follows;

[0025] Figure 6The optimization analysis results of the kanamycin antigen loading amount on the test strip of the present invention and the kanamycin antibody modification amount on the SERS labeled surface;

[0026] Figure 7 The results of the optimization analysis of the loading amount of streptomycin antigen on the test strip of the present invention and the modification amount of streptomycin antibody on the SERS labeled surface;

[0027] Figure 8 The optimization analysis results of the gentamicin antigen loading amount on the test strip of the present invention and the optimization analysis results of the gentamicin antibody modification amount on the SERS labeled surface;

[0028] Fig. 9 The results of the optimization analysis of the loading amount of spectinomycin antigen on the test strip of the present invention and the modification amount of spectinomycin antibody on the SERS labeled surface;

[0029] Fig.10 The standard curve for detecting neomycin drug residues of the present invention;

[0030] Fig.11 The standard curve for detecting kanamycin drug residues of the present invention;

[0031] Fig.12 It is the detection standard curve of streptomycin drug residues of the present invention;

[0032] Fig.13 The standard curve for detecting gentamicin drug residues of the present invention;

[0033] Fig.14 The standard curve for detecting the residual spectinomycin drug of the present invention;

[0034] Fig.15 The specific detection result of the test strip of the present invention on the neomycin drug residue;

[0035] Fig.16 The specific detection result of the test strip of the present invention on the kanamycin drug residue;

[0036] Fig.17 The specific detection result of the test strip of the present invention on streptomycin drug residues;

[0037] Fig.18 The specific detection result of the test strip of the present invention on gentamicin drug residue;

[0038] Fig.19 The results are specific detection results of the test strip of the present invention on the spectinomycin drug residue. DETAILED DESCRIPTION

[0039] The present invention provides a SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of agricultural and veterinary drugs. The SERS immunochromatographic test strip preferably comprises a chromatography test strip and a SERS immunolabel for detection; the chromatography test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorbent pad;

[0040] The nitrocellulose membrane is provided with detection lines T1, T2, T3, T4, T5 and a quality control line C; the detection lines T1, T2, T3, T4, T5 are attached with five agricultural and veterinary drug antigens, namely neomycin, kanamycin, streptomycin, gentamicin and spectinomycin, and the quality control line C is attached with a goat anti-rabbit secondary antibody; the detection lines and the quality control line are respectively combined with SERS immunolabeling;

[0041] The preparation method of the SERS immunolabeling preferably comprises the following steps:

[0042] The noble metal ions in the Raman beacon molecular suspension are reduced to construct a SERS label; the pH of the SERS label is adjusted, a monoclonal antibody for agricultural and veterinary drugs or a rabbit antibody is added, vortexed, and then a bovine serum albumin solution is added to obtain a SERS immune label.

[0043] In the present invention, the SERS immune marker is preferably prepared into a SERS immune marker liquid, and the preparation method of the SERS immune marker liquid preferably includes the following steps: reducing the noble metal ions in the Raman beacon molecule suspension, vortexing to obtain the SERS marker; adjusting the pH of the SERS marker, adding a monoclonal antibody for agricultural and veterinary drugs or a rabbit antibody, vortexing, and then adding a bovine serum albumin solution to obtain the SERS immune marker; centrifuging the prepared SERS immune marker, removing the supernatant, and re-dispersing it with a re-solution to obtain a SERS immune marker concentrated solution; mixing the prepared SERS immune marker concentrated solution with the re-solution at a volume ratio of 8-15:1 to obtain the SERS immune marker liquid; the Raman beacon molecules include Prussian blue and Prussian blue analogs.

[0044] In the present invention, the Raman beacon molecule suspension is preferably obtained by diluting the Raman beacon molecule with ultrapure water, and the volume ratio of the ultrapure water to the Raman beacon molecule is preferably 2-8:1, more preferably 5:1. In the present invention, the Raman beacon molecule is preferably vortexed at 600-700rpm after being mixed with ultrapure water; 0.1g / mL 100-200μL ascorbic acid is added during vortexing, and HAuCl3 or AgNO3 is added after continuous vortexing for 1-3min to construct precious metal nanoparticles encapsulating the Raman beacon molecule as a SERS marker.

[0045] In the present invention, the pH of the SERS immunolabeling is preferably adjusted to 7-7.5. The reagent for adjusting the pH can be conventionally selected according to actual needs. In the present invention, phosphate buffer (pH=8.6) is preferably used for pH adjustment.

[0046] In the present invention, the Prussian blue is an excellent blue dye, and its chemical name is ferric ferrocyanide or hexacyanoferrate (II) iron (III), and its chemical formula is Fe4[Fe(CN)6]3. The Prussian blue analog is a class of compounds containing cyano and transition metal ions, and its molecular formula can be expressed as M[Fe(CN)6], wherein M represents a metal ion, such as K, Na, Co, Ni, etc. In the present invention, the Prussian blue analog is preferably Cu2[Fe(CN)6] (copper ferrocyanide) or Pb2[Fe(CN)6] (lead ferrocyanide). In the present invention, the Prussian blue and Prussian blue analogs can be purchased from conventional commercial products or synthesized by conventional chemical reactions. In the present invention, the Fe4[Fe(CN)6]3 is preferably obtained by successively adding 800 μL FeCl3 (100 mmol / L) and 1000 μL K4[Fe(CN)6] (100 mmol / L) into 20 mL ultrapure water, and vortexing continuously for 30 minutes at 700 rpm; the Cu2[Fe(CN)6] is preferably obtained by successively adding 800 μL CuCl2 (100 mmol / L) and 800 μL K4[Fe(CN)6] (100 mmol / L) into 20 mL ultrapure water, and vortexing continuously for 30 minutes at 700 rpm; the Pb2[Fe(CN)6] is preferably obtained by successively adding 800 μL PbCl2 (100 mmol / L) and 1000 μL K4[Fe(CN)6] (100 mmol / L) into 10 mL ultrapure water, and vortexing continuously for 15 minutes at 700 rpm.

[0047] In the present invention, the agricultural veterinary drug monoclonal antibody includes one or more of neomycin monoclonal antibody, kanamycin monoclonal antibody, streptomycin monoclonal antibody, gentamicin monoclonal antibody or spectinomycin monoclonal antibody. In the present invention, the neomycin monoclonal antibody (ZD5070), kanamycin monoclonal antibody (ZD5064), streptomycin monoclonal antibody (ZD5069), gentamicin monoclonal antibody (ZD5063), spectinomycin monoclonal antibody (ZD5066) and neomycin-BSA (ZD6070), kanamycin-BSA (ZD6064), streptomycin-BSA (ZD6069), gentamicin-BSA (ZD6063), and spectinomycin-BSA artificial antigen (ZD6066) are all purchased from Bio-Long Company.

[0048] In the present invention, the final concentration of the monoclonal antibody against pesticides and veterinary drugs is preferably 2-10 μg / mL; more preferably 4-8 μg / mL. Specifically, the final concentration of the monoclonal antibody against pesticides and veterinary drugs may be 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, or 10 μg / mL.

[0049] In the present invention, the reconstitution solution preferably uses phosphate buffer as the reconstitution solution solvent, and sucrose and bovine serum albumin are added; based on the total volume of phosphate buffer, the volume fraction of sucrose is preferably 2-6%, and the volume fraction of bovine serum albumin is preferably 1-5%; the volume fraction of sucrose is more preferably 5%, and the volume fraction of bovine serum albumin is more preferably 4%.

[0050] The present invention also provides a SERS immunolabeling liquid obtained by the preparation method. In the present invention, the SERS immunolabeling comprises six SERS labels, Au@Fe4[Fe(CN)6]3, Au@Cu2[Fe(CN)6], Ag@Fe4[Fe(CN)6]3, Au@Pb2[Fe(CN)6], Ag@Cu2[Fe(CN)6], and Ag@Pb2[Fe(CN)6], with a substrate wrapped with Prussian blue and its analogs and enhanced; the morphology of the SERS label Au@Fe4[Fe(CN)6]3 under an electron microscope is a typical core-shell morphology and has obvious signals of elements such as Au, Fe, and N; the morphology of the Au@Cu2[Fe(CN)6] is relatively thin and has obvious characteristic signals of elements such as Au, Fe, and Cu; and the Au@Pb2[Fe(CN)6] has an obvious Pb2[Fe(CN)6] shell and has obvious characteristic signals of elements such as Au, Fe, and Pb. At the same time, the microstructures of Ag@Fe4[Fe(CN)6]3, Ag@Cu2[Fe(CN)6], and Ag@Pb2[Fe(CN)6] with silver as the reinforcement substrate all show nanoaggregates wrapped in transparent shells, and show the elemental signals of Ag, Fe, N, Ag, Fe, Cu, and Ag, Fe, Pb, respectively ( Figure 4 In the present invention, the six SERS markers are combined with monoclonal antibodies and rabbit antibodies against five target drugs to obtain SERS immunolabeling liquid. The SERS immunolabeling liquid provided by the present invention can effectively avoid spectral interference from agricultural products and the test strip itself, and its Raman characteristic peak is located at 2042cm -1 、2085cm -1 、2125cm -1 、2140cm -1 、2160cm -1 and 2087cm -1 , are located in the Raman silence region of biomolecules (1800-2800cm-1 ). At the same time, the SERS immunolabeling Raman signal is only provided by the cyano ligands of Prussian blue and its analogs affected by the enhanced substrate and metal ions, and the Raman signal exists only at the characteristic peak position, so the signal overlap between the labels is very small. In addition, Prussian blue and its analogs use abundant cyano groups as their bridging ligands, and the relative content of Raman signal active groups (cyano groups) far exceeds that of other Raman signal molecules, which can provide super strong Raman signals. The SERS immunolabeling provided by the present invention can provide high-intensity, pure, and anti-interference Raman signals, and can give the test strips sensitive and accurate detection results when used for the detection of pesticide and veterinary drug residues in agricultural products.

[0051] The present invention also provides an application of the SERS immunochromatographic test strip in qualitative and quantitative detection of food pesticide and veterinary drug residues.

[0052] The present invention also provides a method for multiple detection of pesticide residues in agricultural products, comprising the following steps: mixing the sample to be tested with the SERS immunolabel in the SERS immunochromatographic test strip to obtain a mixed solution, inserting the chromatography test strip into the mixed solution, and performing qualitative and quantitative detection.

[0053] In the present invention, the SERS immunolabeling is preferably prepared into a SERS immunolabeling liquid; the SERS immunolabeling liquid can be directly mixed with a liquid sample. If the sample is a solid sample, it is mixed in an extracting liquid at a concentration of 1g / mL, and then mixed with the SERS immunolabeling liquid. In the present invention, the extracting liquid is preferably a phosphate buffer (pH=8.6) containing 5wt% sucrose, 4wt% BSA and 2wt% polyvinyl pyrrolidone.

[0054] In the present invention, the bottom plate of the chromatography test strip is located below other parts, the sample pad and the absorbent pad are overlapped at both ends of the nitrocellulose membrane respectively, and the overlapped part is located above the nitrocellulose membrane.

[0055] In the present invention, the optimization of the concentration of the antigen fixed on the nitrocellulose membrane and the amount of the antibody fixed on the SERS immunolabeling surface has an important influence on the detection performance of the test strip. The present invention completes the optimization by controlling the concentration of the antigen solution used to draw the detection line on the nitrocellulose and the final concentration of the antibody when preparing the SERS immunolabeling. In the optimization of antigen and antibody, the controlled concentrations of various antigens during antigen optimization are 20, 40, 60, 80, and 100 μg / mL, and the final concentrations of various antibodies are controlled to be 1, 2, 3, 4, and 5 μg / mL; the inhibition rate is used to evaluate the optimization results, and the calculation method is the Raman intensity (I0) of the test strip when detecting the blank minus the Raman intensity (I i ) strength difference (I0-I i ) to I0 [(I0-Ii ) / I0]. After inhibition rate evaluation, the optimization results are as follows: the optimal amount of antigen fixed on T1 is 60μg / mL, and the optimal concentration of antibody corresponding to SERS immunolabeling construction is 4μg / mL; the optimal amount of antigen fixed on T2 is 80μg / mL, and the optimal concentration of antibody corresponding to SERS immunolabeling construction is 4μg / mL; the optimal amount of antigen fixed on T3 is 80μg / mL, and the optimal concentration of antibody corresponding to SERS immunolabeling construction is 3μg / mL; the optimal amount of antigen fixed on T4 is 40μg / mL, and the optimal concentration of antibody corresponding to SERS immunolabeling construction is 2μg / mL; the optimal amount of antigen fixed on T5 is 80μg / mL, and the optimal concentration of antibody corresponding to SERS immunolabeling construction is 4μg / mL ( Figure 5-Figure 9 ).

[0056] After various conditions are optimized, the test strips are used to detect target drugs in samples. First, a calibration curve for detecting five drugs is constructed to measure the concentration of target drugs in samples. After the test strips are tested, the test results are used to construct a calibration curve for detecting five pesticide residues. Figure 10-14 As shown, the calibration curve of the test strip for drug 1 is y=10692.3-3030.24×lgx(R 2 =0.996), the calibration curve for drug 2 is y=5211.2-1487.9×lgx(R 2 =0.994), the calibration curve for drug 3 is y=11938.7-3278.0×lgx(R 2 =0.995), the calibration curve for drug 4 is y=10171.7-2438.4×lgx(R 2 =0.995), the calibration curve for drug 5 is y=17892.5-4911.6×lgx(R 2 =0.995).

[0057] In the present invention, during sample detection, the SERS immunolabeling mixed in the sample is fixed on the detection lines T1-T5 and the quality control line C, respectively, in the following specific manner: the C line carries the goat anti-rabbit secondary antibody, and the Au@Fe4[Fe(CN)6]3 bound to the rabbit antibody is fixed; the T1 line carries the drug 1 antigen, and the Au@Cu2[Fe(CN)6] bound to the drug 1 antibody is fixed; the T2 line carries the drug 2 antigen, and the Ag@Fe4[Fe(CN)6]3 bound to the drug 2 antibody is fixed; the T3 line carries the drug 3 antigen, and the Au@Pb2[Fe(CN)6] bound to the drug 3 antibody is fixed; the T4 line carries the drug 4 antigen, and the Ag@Cu2[Fe(CN)6] bound to the drug 4 antibody is fixed; the T5 line carries the drug 5 antigen, and the Ag@Pb2[Fe(CN)6] bound to the drug 5 antibody is fixed (see Figure 1). The qualitative detection is preferably: if the sample contains the target drug, the target drug in the sample binds to the SERS immunolabeling, and the color of the detection line becomes weaker; if the sample does not contain the target drug, the SERS immunolabeling binds to the antigen on the detection line, and the detection line develops color normally.

[0058] In the present invention, the quantitative detection is preferably: the target drug is quantitatively analyzed by measuring the Raman intensity on the detection line by a Raman spectrometer. The intensity of the Raman spectrum curve on the T1-T5 line is collected by a Raman spectrometer to construct a standard curve of signal intensity and detection target concentration. Thus, the subsequent quantitative detection of the target drug content in the sample is achieved by measuring the Raman intensity against the constructed detection standard curve. The present invention mixes the above-mentioned SERS immunolabeling liquid with the sample, and obtains the detection result through a portable Raman spectrometer after detection by a chromatographic test strip. Since the target drug contained in the test sample is combined with the SERS immunolabeling, the color of the detection line T line becomes weak, so there is a competitive relationship between the target drug in the sample and the antigen on the detection line, and the Raman intensity at the characteristic peak measured on the T line is negatively correlated with the target drug, thereby achieving quantitative detection of the detection target.

[0059] In the present invention, the overlap between the sample pad, the absorbent pad and the nitrocellulose membrane is preferably 1-1.5 mm, and the length of the SERS immunochromatography test strip is preferably 6-8 cm, and the width is preferably 4-5 mm.

[0060] In the present invention, the length of the nitrocellulose membrane is preferably 2-2.5 cm; the distance between the detection lines is preferably 3-4 mm; the distance between the detection line and the quality control line is preferably 3-4 mm; the line width of the detection line or the quality control line is preferably 300-600 μm.

[0061] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0063] Example 1

[0064] A SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of agricultural and veterinary drugs, the SERS immunochromatographic test strip comprising a chromatography test strip and a SERS immunolabel for detection; the chromatography test strip comprising a bottom plate, a sample pad, a nitrocellulose membrane and a water absorbent pad;

[0065] The bottom plate of the chromatography test strip is located below the other parts, the sample pad and the absorbent pad are overlapped with the two ends of the nitrocellulose membrane respectively, and the overlapped part is located above the nitrocellulose membrane; the nitrocellulose membrane is provided with detection lines T1 (neomycin-BSA artificial antigen), T2 (kanamycin-BSA artificial antigen), T3 (streptomycin-BSA artificial antigen), T4 (gentamicin-BSA artificial antigen), T5 (spectinomycin-BSA artificial antigen) and quality control line C (sheep anti-rabbit anti-secondary antibody); the detection lines and the quality control lines are fixed with six SERS immunolabels combined with antibiotic antibodies or rabbit antibodies respectively;

[0066] The preparation method of the SERS immunolabeling comprises the following steps:

[0067] (1) To three portions of 10 mL ultrapure water vortexed at 700 rpm were added 800 μL FeCl3 (100 mmol / L) + 1000 μL K4[Fe(CN)6] (100 mmol / L), 800 μL CuCl2 (100 mmol / L) + 800 μL K4[Fe(CN)6] (100 mmol / L), and 800 μL PbCl2 (100 mmol / L) + 1000 μL K4[Fe(CN)6] (100 mmol / L), respectively. After continuous vortexing for 15 min, 15 min, and 3 min, three Prussian blue-like substances, Fe4[Fe(CN)6]3, Cu2[Fe(CN)6], and Pb2[Fe(CN)6], were prepared.

[0068] (2) Add 2 mL of freshly prepared Prussian blue / Prussian blue analogs (Fe4[Fe(CN)6]3, Cu2[Fe(CN)6], Pb2[Fe(CN)6]) to each of six portions of 10 mL of ultrapure water vortexed at 700 rpm. Vortex for 30 s and then add 200 μL of ascorbic acid (0.1 g / mL). Continue vortexing for 1 min and then add 200 μL of AuCl3·HCl (100 mmol / L) or AgNO3 (100 mmol / L) solution to each group of Prussian blue + ascorbic acid solutions. After all reaction groups were vortexed for 5 min, six SERS tags, Au@Fe4[Fe(CN)6]3, Au@Cu2[Fe(CN)6], Ag@Fe4[Fe(CN)6]3, Au@Pb2[Fe(CN)6], Ag@Cu2[Fe(CN)6], and Ag@Pb2[Fe(CN)6], were constructed.

[0069] (3) The pH of the six constructed SERS labels was adjusted to 7.5 while slowly vortexing at 150 rpm, and 6 μg / mL rabbit antibody, 4 μg / mL neomycin monoclonal antibody, 4 μg / mL kanamycin monoclonal antibody, 3 μg / mL streptomycin monoclonal antibody, 2 μg / mL gentamicin monoclonal antibody and 4 μg / mL spectinomycin monoclonal antibody were added respectively. After continuing to vortex for 20 minutes, 0.5 mL of bovine serum albumin solution (0.2 g / mL) was added and vortexed for 20 minutes to complete the SERS immunolabeling preparation.

[0070] The six SERS immunolabels prepared by the present invention have spectral anti-interference performance, respectively at 2042cm -1 、2085cm -1 、2125cm -1 、2140cm -1 、2160cm -1 and 2087cm -1 There is a Raman scattering peak composed only of near ; Figure 2 The six SERS immunolabelings shown have only Raman scattering in the biomolecule Raman silent region (1800-2800 cm -1 ) appears, this feature completely avoids the spectral signal only in the fingerprint region (600-1800cm -1 ) of various agricultural products and spectral interferences of nitrocellulose membranes ( Figure 3 ).

[0071] Example 2

[0072] A method for multiple detection of pesticide and veterinary drug residues, comprising the following steps:

[0073] (1) The chromatography test strip responsible for chromatography and immunoadsorption in this embodiment includes a bottom plate, a sample pad, a nitrocellulose membrane and a water absorbent pad. The bottom plate is located below the other parts, and the sample pad and the water absorbent pad are overlapped with the two ends of the nitrocellulose membrane respectively, and the overlapping parts are located above the nitrocellulose membrane. The nitrocellulose membrane is provided with detection lines T1 (neomycin-BSA artificial antigen), T2 (kanamycin-BSA artificial antigen), T3 (streptomycin-BSA artificial antigen), T4 (gentamicin-BSA artificial antigen), T5 (spectinomycin-BSA artificial antigen) and quality control line C (sheep anti-rabbit anti-secondary antibody). The detection line and the quality control line are respectively fixed with six SERS immunolabels bound to antibiotic antibodies or rabbit antibodies;

[0074] (2) The six SERS immunolabeling solutions prepared in Example 1 were centrifuged at 7000 rpm for 15 minutes, the supernatant was removed, and the solution was redispersed with 200 μL of the reconstitution solution to prepare six SERS immunolabeling concentrates;

[0075] (3) mixing the prepared six SERS immunolabeling concentrated solutions with the resolvent in a ratio of 10:1 to obtain a mixed labeling solution;

[0076] (4) The prepared mixed marker solution is mixed with the sample and then inserted into a chromatographic test strip to perform accurate quantitative analysis of five types of pesticide residues.

[0077] Example 3

[0078] A method for multiple detection of pesticide and veterinary drug residues in agricultural products, the specific steps are as follows: under a constant temperature environment of 40°C, according to the detection steps in Example 2, the SERS immunolabeling prepared in Example 1 is further prepared into a mixed labeling liquid and a raw milk sample in a ratio of 1:1 to form a 200 μL mixed liquid, mixed for 3 minutes, inserted into a chromatography test strip for detection for 5 minutes, and then the sample pad is scraped off, the colorimetric signal is observed for semi-quantitative analysis, and accurate quantitative reading is performed through a portable Raman spectrometer.

[0079] Example 4

[0080] Different from Example 3, the test sample is fish meat. The fish meat sample should be pulverized by a pulverizer and mixed with an extract (PBS with pH = 8.5, containing 5wt% sucrose, 4wt% BSA and 2wt% polyvinyl pyrrolidone) at 1g / mL for 2 minutes. The extract should be filtered through a 0.2μm organic filter membrane and mixed with the SERS immunolabeling for detection.

[0081] Example 5

[0082] Different from Example 3, the detection sample is celery. The celery sample is crushed in a grinder and mixed with the extract at 1 g / mL for 2 minutes. The extract is filtered through a 0.2 μm organic filter membrane and then mixed with the SERS immunolabeling for detection.

[0083] result Figure 5 As shown, the SERS chromatography test strips have a good linear relationship in the detection of five pesticide residues in agricultural products in the range of 10-2000μg / mL, and the detection sensitivity is 10μg / mL.

[0084] Example 6

[0085] Raw milk samples were used as the detection object. 2 g of sample was weighed and one of five agricultural and veterinary drugs, namely neomycin, kanamycin, streptomycin, gentamicin, and spectinomycin, was added at 50, 100, 200, and 400 μg / g, respectively, and mixed with the SERS immunolabeling for detection.

[0086] The results are as follows Figure 15-19 As shown, each detection line only responds to its detection target, and the corresponding Raman intensity only decreases with the increase of the target drug concentration.

[0087] Example 7

[0088] Raw milk, fish and celery samples were used as test objects. 2g of samples were weighed and added with 50, 100 and 200ng / g of neomycin, kanamycin, streptomycin, gentamicin and spectinomycin. The results were used for addition recovery analysis after test strip detection. The results are shown in Table 1.

[0089] Table 1 Addition recovery analysis results of the samples to be tested

[0090]

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SERS immunochromatographic test strip for qualitative and quantitative detection of multiple residues of pesticides and veterinary drugs, characterized in that: The SERS immunochromatographic test strip comprises a chromatographic test strip and a SERS immunolabel for detection; the chromatographic test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorbent pad; The nitrocellulose membrane is provided with detection lines T1, T2, T3, T4, T5 and a quality control line C; the detection lines T1, T2, T3, T4, T5 are attached with five agricultural and veterinary drug antigens, namely neomycin, kanamycin, streptomycin, gentamicin and spectinomycin, and the quality control line C is attached with a goat anti-rabbit secondary antibody; the detection lines and the quality control line are respectively combined with SERS immunolabeling; The preparation method of the SERS immunolabeling comprises the following steps: The noble metal ions in the Raman beacon molecular suspension are reduced to construct a SERS marker; the pH of the SERS marker is adjusted, a monoclonal antibody for agricultural and veterinary drugs or a rabbit antibody is added, vortexed, and then a bovine serum albumin solution is added to obtain a SERS immunolabeling; The Raman beacon molecules include Prussian blue and Prussian blue analogs; The SERS immunolabeling is composed of six SERS labels, namely Au@Fe4[Fe(CN)6]3, Au@Cu2[Fe(CN)6], Ag@Fe4[Fe(CN)6]3, Au@Pb2[Fe(CN)6], Ag@Cu2[Fe(CN)6], and Ag@Pb2[Fe(CN)6], with a Prussian blue and its analogues wrapped enhanced substrate; the six SERS labels are respectively combined with monoclonal antibodies and rabbit antibodies against five target drugs to obtain SERS immunolabeling liquid.

2. The SERS immunochromatography test strip according to claim 1, characterized in that: The agricultural and veterinary drug monoclonal antibody includes one or more of neomycin monoclonal antibody, kanamycin monoclonal antibody, streptomycin monoclonal antibody, gentamicin monoclonal antibody or spectinomycin monoclonal antibody.

3. The SERS immunochromatography test strip according to claim 1, characterized in that: The final concentration of the pesticide monoclonal antibody is 2-10 μg / mL.

4. The SERS immunochromatographic test strip according to claim 1, characterized in that: The bottom plate of the chromatography test strip is located below the other parts, and the sample pad and the water absorbent pad are overlapped at both ends of the nitrocellulose membrane respectively, and the overlapped part is located above the nitrocellulose membrane.

5. Use of the SERS immunochromatographic test strip according to any one of claims 1 to 4 in qualitative and quantitative detection of pesticide and veterinary drug residues.

6. A method for multiple detection of pesticide and veterinary drug residues in agricultural products, characterized in that: The method comprises the following steps: mixing a sample to be tested with the SERS immunolabel in the SERS immunochromatographic test strip according to any one of claims 1 to 4 to obtain a mixed solution, inserting a chromatography test strip into the mixed solution, and performing qualitative and quantitative detection.

7. The method according to claim 6, characterized in that The qualitative detection is as follows: if the sample contains the target drug, the target drug in the sample combines with the SERS immunolabeling, and the color of the detection line becomes weaker; If the sample does not contain the target drug, the SERS immunolabeling will bind to the antigen on the detection line and the detection line will develop color normally.

8. The method according to claim 6, characterized in that The quantitative detection is: measuring the Raman intensity on the detection line by a Raman spectrometer to perform quantitative analysis on the target drug.

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