Application of NanoSPR-based Biochip in Detecting Small Molecular Compounds in Food

Abstract

The present invention discloses an application of a NanoSPR-based biochip in detecting small molecule compounds in food, belonging to the technical fields of sensing and immunoassay; the application method is to first perform coating antigen modification of small molecule compounds on a chip microplate integrated with a nano-plasmonic resonance biochip; then label colloidal gold particles with antibodies of small molecule compounds and freeze-dry or vacuum-dry them in a common microplate; then add the sample to be tested into the common microplate with the freeze-dried gold particles labeled with antibodies of small molecule compounds obtained in step S2, and mix evenly; then add it into the chip microplate with the small molecule compound antigen modified in step S1; finally, use an enzyme-labeled instrument to record data to complete the detection. This method can achieve high-throughput, rapid, and ultra-sensitive detection of food small molecules. Compared with the 2-hour detection time of the ELISA method, the time-consuming of the entire detection stage of the present invention can be shortened to no more than 30 minutes.

Description

Technical Field

[0001] The present invention belongs to the technical field of sensing and immunoassay, and in particular to the application of a NanoSPR-based biochip in detecting small molecule compounds in food. Background Art

[0002] In the past 20 years of development, a new type of biosensor with two modes of SPR and LSPR, namely plasmonic nanopore array (NanoSPR), has emerged. Due to its unique three-dimensional structure, the NanoSPR is different from the SPR effect of the planar model and the LSPR effect of metal nanoparticles, and can support both SPR and LSPR modes simultaneously.

[0003] The plasmon resonance effect of the nanopore array biosensor can be directly incident on the nanopore metal structure, and the surface optical field is immediately excited, so it does not require the support of complex optical paths and large optical instruments. The NanoSPR nanopore array biosensor retains many advantages of traditional SPR sensors, such as real-time, label-free, background interference-free, and high resolution. The NanoSPR sensor also retains the advantages of LSPR sensors in performance improvement. By adjusting parameters such as the pore diameter, depth, shape, period of the nanopore array, and the type and thickness of the surface metal, a high-quality chip that can capture the strongest LSPR signal can be selected, and a stronger signal can be obtained without a large spectrometer.

[0004] Based on the above advantages, the detection ability of the NanoSPR biosensor can meet the actual needs of biomolecular sensing detection, and is widely used in the fields of biomedical detection, drug analysis, food safety, environmental detection, and cell biology. Sensors based on the SPR principle do not require any labeling reagents; they can be reused after treatment, saving costs; through kinetic studies, the whole process of binding, dissociation, and reactivation between biomolecules on the chip interface can be monitored, and the experimental object can be observed in real time, which can be applied to drug screening and antibody activity testing; it is suitable for miniaturization and high-throughput detection, which is very beneficial for the manufacture of portable sensors. Self-developed small-volume SPR sensors with equivalent functions can be used for in-situ detection of clinical substances and on-site detection of environmental pollutants, etc.; there is no direct contact with the sample to be tested, and the detection is pollution-free and lossless; the sensitivity is high, and the concentration of some analytes directly detected can be lower than 0.01 ng / mL. By using various binding amplification techniques between molecules, the detection limit can be further reduced; the sample pretreatment is simple, and only filtration and dilution are required to meet the requirements.

[0005] In the prior art, for example, Chinese Patent Application CN106442427A provides a surface plasmon resonance immunoassay method for detecting sulfonamides. This method activates a fixed plasmon resonance chip with a mixed solution of EDC and NHS, and then introduces a sulfonamide-OVA solution to complete the modification of the chip with sulfonamide-OVA. It has high sensitivity and accuracy in detecting four sulfonamide drugs (sulfachinoxaline, sulfachloropyridazine, sulfamethoxazole, and sulfamethoxypyridazine), and the detection process is simple and fast. Summary of the Invention

[0006] Aiming at the deficiencies of the above prior art, the present invention provides a nano surface plasmon resonance immunoassay method based on a NanoSPR biochip, which uses a small molecule compound competition method to detect small molecule compounds in food, and can achieve high-throughput, rapid, and ultra-sensitive detection of small molecule compounds in food. The specific implementation is as follows.

[0007] The application of a NanoSPR-based biochip in detecting small molecule compounds in food includes the following steps:

[0008] S1. Perform coating antigen modification of small molecule compounds on a chip microplate integrated with a nano plasmon resonance biochip;

[0009] S2. Label colloidal gold particles with antibodies of small molecule compounds, and freeze-dry or vacuum-dry them in a common microplate;

[0010] S3. Add the sample to be tested into the common microplate with freeze-dried gold particles labeled with antibodies of small molecule compounds obtained in step S2, and mix evenly; then add it to the chip microplate with modified small molecule compound antigens obtained in step S1;

[0011] S4. Use an enzyme-labeled instrument to record data to complete the detection.

[0012] Preferably, in the detection method of the above application, the small molecule compounds in food are antibiotics, toxins, and additional additives; the antibiotics are sulfonamides, fluoroquinolones, β-lactam antibiotics, cephalexin, lincomycin, tilmicosin-tylosin, dexamethasone, chloramphenicol, tetracycline, gentamicin, florfenicol, erythromycin, streptomycin, benzoic acid, neomycin, chlortetracycline, oxytetracycline, flumequine, malachite green, and crystal violet; the toxins are aflatoxin M1, B1, zearalenone, and the additional additives are melamine, diethylstilbestrol, bisphenol A, and β-lactamase.

[0013] The present invention effectively combines the high specificity of immunoassay with the high sensitivity, rapidity, etc. of the nano surface plasmon resonance technology. The detection process is simple. First, the corresponding products are prepared in the chip microplate and the ordinary microplate respectively, and then the product in the ordinary microplate is added to the chip microplate. Such a two-step high-throughput method enables rapid detection of a large number of samples. The constructed nano surface plasmon resonance biochip immunosensing detection microplate has the advantages of high detection accuracy and sensitivity, reusability, and good result reproducibility, and can be used for the analysis and detection of small molecule drug compounds in samples such as milk, eggs, chicken, beef, fish, etc., such as sulfonamides, quinolones, flouxins, fipronil, etc.

[0014] More preferably, when the food small molecule is sulfonamide, the detection method for sulfonamide is as follows:

[0015] P1. Each micro-well of the chip microplate integrated with the nano plasmon resonance biochip is successively washed with ultrapure water and absolute ethanol, and dried with nitrogen; 2 - 100 μg / ml of sulfonamide antigen is added to each micro-well for the first incubation, washed with PBST buffer, and dried with nitrogen; then 0.5 - 8% bovine serum albumin blocking solution is added for the second incubation, washed with PBST buffer, and dried with nitrogen for standby;

[0016] P2. Take the colloidal gold solution, add potassium carbonate solution, and mix well; then add sulfonamide antibody and mix well, let stand; then add PEG 20000 and mix well, let stand; freeze-centrifuge, discard the supernatant, take the precipitate and add the reconstitution solution, mix well; then add it to the ordinary microplate, dry it in vacuum and seal for standby;

[0017] P3. Take the sample to be tested (generally 30 - 100 μl) and add it to the ordinary microplate with the dried gold particles labeled with sulfonamide antibody obtained in step P2, mix well and react for 5 - 10 min; then add the mixture in each micro-well of the ordinary microplate to each micro-well of the chip microplate modified with sulfonamide antigen obtained in step P1; use an enzyme-linked immunosorbent assay (ELISA) reader to record data to complete the detection.

[0018] Further preferably, in step P1, the conditions for the first incubation are incubation at 4 - 37 °C for 2 - 24 h, and the conditions for the second incubation are incubation at 4 - 37 °C for 0.5 - 2 h.

[0019] Further preferably, step P2 is specifically: take 1 ml of colloidal gold solution, add 6 - 20 μl of 0.1 M potassium carbonate solution, and mix well; then add 0.5 - 5 μg of sulfonamide antibody and mix well, let stand; then add 10 μl of 10% PEG 20000 and mix well, let stand; freeze-centrifuge, discard the supernatant, take the precipitate and add 100 μl of reconstitution solution, mix well; then take this colloidal gold reconstitution solution and add it to the ordinary microplate, dry it in vacuum or by vacuum pumping and seal for standby.

[0020] More preferably, in step P2, the conditions for cryo-centrifugation are centrifugation at 4°C, 7000 - 9000 rpm for 20 - 30 min.

[0021] More preferably, in step P2, the reconstitution solution is a PBST buffer containing 1 - 5% sucrose, 1 - 5% glucose, 0.5 - 4% mannitol, and 0.1 - 5% BSA.

[0022] Based on the above application method of the NanoSPR biochip for detecting small molecule compounds in food, the present invention also provides a detection kit for detecting small molecule compounds in food, including a chip microplate integrated with a nano - surface plasmon resonance biochip, a common microplate, a small molecule compound antibody, a small molecule compound coated antigen, and a reconstitution solution. The above detection kit uses the indirect competition method. The sulfonamide drugs in the sample specifically compete with the antigen immobilized on the chip microplate for binding to the antibody, thereby generating a surface plasmon resonance effect with the nanopore chip, causing a change in absorbance at a specific wavelength. A large reading indicates a small content; a small reading indicates a large content.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a nano - surface plasmon resonance immunoassay method for food small molecule detection based on a NanoSPR biochip, using the small molecule competition method, which can achieve high - throughput, rapid, and ultra - sensitive detection of food small molecules. Compared with the 2 - hour detection time of the ELISA method, the total detection time of the present invention can be shortened to no more than 30 min. Description of the Drawings

[0024] Figure 1 It is a photo of a silicon wafer chip obtained by photolithography; among them, (a) is a silicon wafer after nano - scale photolithography and etching processing; (b), (c) are photos of the NanoSPR chip; (d) is an electron microscope photo of the microscopic structure of the chip surface;

[0025] Figure 2 It is a 96 - well microplate integrated with a nano - surface plasmon resonance biochip;

[0026] Figure 3 It is a schematic diagram of the principle of the NanoSPR biochip for detecting small molecule compounds;

[0027] Figure 4 It is the full - spectrum diagram of the NanoSPR biochip in Example 1 for detecting sulfonamide small molecules at different concentrations;

[0028] Figure 5 It is a bar chart of the NanoSPR biochip in Example 1 for detecting sulfonamide small molecules at different concentrations in 5 min;

[0029] Figure 6Standard curve graph of the 5 - minute NanoSPR biochip in Example 1 for detecting sulfonamide small molecules with different concentrations;

[0030] Figure 7 Bar graph of the 10 - minute NanoSPR biochip in Example 1 for detecting sulfonamide small molecules with different concentrations;

[0031] Figure 8 Standard curve graph of the 10 - minute NanoSPR biochip in Example 1 for detecting sulfonamide small molecules with different concentrations;

[0032] Figure 9 Bar graph of the 15 - minute NanoSPR biochip in Example 1 for detecting sulfonamide small molecules with different concentrations;

[0033] Figure 10 Standard curve graph of the 15 - minute NanoSPR biochip in Example 1 for detecting sulfonamide small molecules with different concentrations. Detailed implementation manners

[0034] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0035] Example 1

[0036] Taking sulfonamide small molecule compounds as an example, for the collected samples to be tested, the detection of sulfonamide small molecule compounds is carried out.

[0037] The specific materials, reagents and instruments used in the following detection methods are as follows:

[0038] The preparation method of the chip microplate of the nano - plasmonic resonance biochip is as follows: (1) A nano - hole mold can be fabricated by photolithography on a silicon oxide wafer; (2) The nano - structure on the 12 - inch silicon wafer nano - mold is transferred to a polymer flexible material substrate by nano - imprinting method to form an inverse wafer - level nano - device structure; (3) After plating a noble metal film layer on its surface (for example, 9 nm of titanium and 70 nm of gold), it becomes a plasmonic sensing chip;

[0039] Sulfonamide antigen and sulfonamide antibody: Purchased from Guangzhou Youkangduo Biotechnology Co., Ltd.;

[0040] Bovine serum albumin: Purchased from Wuhan Qianzesui Biotechnology Co., Ltd.;

[0041] The preparation method of the colloidal gold solution is as follows:

[0042] (1) Add 400 ml of ultrapure water to a clean three-necked round-bottom flask. Place the three-necked round-bottom flask in an oil bath, adjust the temperature of the oil bath to 120 °C, install a condenser, and turn on the water flow switch of the condenser.

[0043] (2) After heating to boiling, add 6 ml of 1% sodium citrate tribasic, and stopper the bottle mouth.

[0044] (3) Continue heating until it boils again, add 4 ml of 1% chloroauric acid, observe the color change, and start counting down for 10 min after the color remains red and does not change.

[0045] (4) After the timing ends, stop heating, turn off the oil bath, take out the three-necked round-bottom flask, pour out the prepared colloidal gold solution into a 500 ml clean glass bottle, let it cool naturally to room temperature, then tighten the bottle cap and seal it for standby;

[0046] Microplate reader: A microplate reader with specific wavelengths (such as 580 nm, 590 nm, 600 nm, 610 nm) for nano-plasmon resonance biochips, such as Xelement SPR100, or use a full-spectrum microplate reader (such as Biotek Epoch) or an optical detection device with these wavelength coverage ranges. The Xelement SPR100 is used in the following examples.

[0047] Ultrapure water: Prepared by a Merck Millipore water purifier;

[0048] PBST buffer: 29 g of disodium hydrogen phosphate dodecahydrate, 2.72 g of sodium dihydrogen phosphate, 2 g of potassium chloride, 20 g of sodium chloride, 5 mL of Tween-20, 5 mL of preservative Proclin300, 10 L of double-distilled water;

[0049] The specific detection method is as follows:

[0050] P1. Add 200 μl of sterilized ultrapure water to each well of the chip microplate integrated with nano-plasmon resonance biochips using a multi-channel pipette, wash twice, then wash twice with 200 μl of absolute ethanol, and dry with nitrogen; add 10 μg / ml sulfonamide antigen to each well for the first incubation, and the incubation conditions are incubation at 4 - 37 °C for 2 h; wash twice with PBS buffer and dry with nitrogen; then add 1% bovine serum albumin blocking solution for the second incubation, incubate at 37 °C for 0.5 h, wash with PBS buffer, and dry with nitrogen for standby;

[0051] P2. Take 1 ml of colloidal gold solution and add it to a 1.5 ml centrifuge tube. Add 20 μl of 0.1 M potassium carbonate solution and mix well by inverting the tube up and down. Then add 4 μg of sulfonamide antibody to the centrifuge tube and mix well by inverting the tube up and down. Let it stand at room temperature for 10 min. Then add 10 μl of 10% PEG 20000 to the centrifuge tube and mix well by inverting the tube up and down. Let it stand for 10 min. Centrifuge at 7000 rpm at 4 °C for 22 min. Take out the centrifuge tube, gently remove the supernatant, and take the precipitate (the precipitate of gold particles labeled with sulfonamide antibody) and add it to 100 μl of the reconstitution solution and mix well. Take 2 μl each and add it to each well of a common microplate. After freeze-drying, seal it and store it in a dry environment for later use. The reconstitution solution is a PBS buffer containing 1-5% sucrose, 1-5% glucose, 0.5-4% mannitol, and 0.1-5% BSA;

[0052] P3. Dilute and pretreat the test sample (the diluted concentrations are 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 ng / ml respectively, and set a blank control of 0 ng / ml at the same time); Take 50 μl each and add it to each well of the common microplate with freeze-dried gold particles labeled with sulfonamide antibody obtained in step P2 and mix well. Then add it to the chip microplate modified with sulfonamide antigen obtained in step P1. Use an enzyme-linked immunosorbent assay (ELISA) reader to record the initial value, then shake the plate at a speed of 300 rpm for 5 min, and use an ELISA reader to record the end value respectively to complete the detection.

[0053] P4. Dilute and pretreat the test sample (the diluted concentrations are 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 ng / ml respectively, and set a blank control of 0 ng / ml at the same time); Take 50 μl each and add it to each well of the common microplate with freeze-dried gold particles labeled with sulfonamide antibody obtained in step P2 and mix well. Then add it to the chip microplate modified with sulfonamide antigen obtained in step P1. Use an ELISA reader to record the initial value, then shake the plate at a speed of 300 rpm for 10 min, and use an ELISA reader to record the end value respectively to complete the detection.

[0054] P5. Dilute and pretreat the test sample (the diluted concentrations are 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 ng / ml respectively, and set a blank control of 0 ng / ml at the same time); Take 50 μl each and add it to each well of the common microplate with freeze-dried gold particles labeled with sulfonamide antibody obtained in step P2 and mix well. Then add it to the chip microplate modified with sulfonamide antigen obtained in step P1. Use an ELISA reader to record the initial value, then shake the plate at a speed of 300 rpm for 15 min, and use an ELISA reader to record the end value respectively to complete the detection.

[0055] The detection process and specific detection results of P3 - P5 are as follows Figures 1 - 10 shown

[0056] Figure 1 In Figures 1 - 10 , (a) is a silicon wafer after nano - scale lithography and etching processes; (b) and (c) are photos of the NanoSPR chip; (d) is an electron microscope photo of the microscopic structure of the chip surface; Figure 2 is a 96 - well microplate for an integrated nano - plasmon resonance biochip;

[0057] From Figures 4 - 10 it can be seen that the detection method of this embodiment can detect sulfonamide content in the range of 0.25 - 16 ng / ml, with very high detection sensitivity, short detection time, and high detection throughput.

[0058] Example 2

[0059] The sample to be tested is diluted and pretreated (the diluted concentrations are 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 ng / ml respectively, and a blank control of 0 ng / ml is set. At the same time, sulfonamide standards with concentrations of 0.5, 2.0, 8.0 ng / mL are added to the samples respectively, and three parallel samples are prepared for each concentration to detect the sample recovery rate); 50 μl is taken and added to each well of the ordinary microplate with freeze - dried gold particles labeled with sulfonamide antibody obtained in step P2, and mixed evenly; then it is added to the chip microplate modified with sulfonamide antigen obtained in step P1; an enzyme - labeled instrument is used to record the initial value, and then the plate is shaken at a speed of 300 rpm for 15 min, and the enzyme - labeled instrument is used to record the end - point value respectively to complete the detection. The detection results are shown in Table 1 below.

[0060] Table 1 Recovery rate of detecting sulfonamide small molecules in samples by the 15 - min NanoSPR biochip in Example 2

[0061]

[0062] As can be seen from Table 1 above, by using the method of the present invention, the detection recovery rate of sulfonamide small molecules in the sample meets the requirements (80 - 120%), and it is a fast, sensitive, accurate, and reliable detection method.

Claims

1. Application of the NanoSPR-based biochip in detecting small molecule compounds in food, characterized in that, When the food small molecule is sulfonamide, the following steps are included: P1. Each micro-well of the chip micro-well plate integrated with the nano-plasmon resonance biochip is successively washed with ultrapure water and absolute ethanol, and dried with nitrogen; 2-100 μg / ml sulfonamide antigen is added to each micro-well for the first incubation, washed with PBST buffer, and dried with nitrogen; then 0.5-8% bovine serum albumin blocking solution is added for the second incubation, washed with PBST buffer, and dried with nitrogen for standby; The preparation method of the chip micro-well plate of the nano-plasmon resonance biochip is as follows: (1) A nano-hole mold can be made by photolithography on a silicon oxide wafer; (2) The nano-structure on the 12-inch silicon wafer nano-mold is transferred to the polymer flexible material substrate by nano-imprinting to form an inverted wafer-level nano-device structure; (3) After a noble metal film layer is plated on its surface, the chip micro-well plate of the nano-plasmon resonance biochip is obtained; P2. Take a colloidal gold solution, add a potassium carbonate solution, and mix well; then add a sulfonamide antibody and mix well and let stand; then add PEG20000 and mix well and let stand; freeze-centrifuge, take the precipitate after removing the supernatant and add a reconstitution solution, and mix well; then add it to a common micro-well plate, freeze-dry and seal for standby; P3. Take a test sample and add it to the common micro-well plate with freeze-dried gold particles labeled with sulfonamide antibody obtained in step P2, mix well and react for 5-10 min; then add the mixed solution in each micro-well of the common micro-well plate to each micro-well of the chip micro-well plate modified with sulfonamide antigen obtained in step P1; Use an enzyme-linked immunosorbent assay (ELISA) reader to record data to complete the detection.

2. The application of the NanoSPR-based biochip according to claim 1 in detecting small molecule compounds in food, characterized in that, In step P1, the conditions for the first incubation are incubation at 4-37 °C for 2-24 h, and the conditions for the second incubation are incubation at 4-37 °C for 0.5-2 h.

3. Use of the NanoSPR-based biochip according to claim 1 in detecting small molecule compounds in food, characterized in that, Step P2 is specifically: Take 1 ml of colloidal gold solution, add 6-20 μl of 0.1 M potassium carbonate solution, and mix well; then add 0.5-5 μg of sulfonamide antibody and mix well and let stand; then add 10 μl of 10% PEG 20000 and mix well and let stand; Freeze-centrifuge, take the precipitate after removing the supernatant and add 100 μl of reconstitution solution, and mix well; then take the colloidal gold reconstitution solution and add it to a common micro-well plate, freeze-dry or vacuum-dry and seal for standby.

4. Use of the NanoSPR-based biochip according to claim 3 in detecting small molecule compounds in food, characterized in that, In step P2, the conditions for freeze-centrifugation are centrifugation at 4 °C, 7000-9000 rpm for 20-30 min.

5. Use of the NanoSPR-based biochip according to claim 3 in the detection of small molecule compounds in food, characterized in that, In step P2, the reconstitution solution is a PBST buffer containing 1-5% sucrose, 1-5% glucose, 0.5-4% mannitol, and 0.1-5% BSA.

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