Biomicrochip-based small molecule multi-link detection kit and preparation method and detection method thereof

Abstract

The application discloses a small-molecule multi-connection detection kit based on a biochip, a preparation method and a detection method thereof. The kit comprises nano-gold particle labeled antibodies, a multi-connection detection chip plate and a detection buffer. The nano-gold particle labeled antibodies comprise nano-gold particle labeled florfenicol antibodies and enrofloxacin antibodies. The multi-connection detection chip plate is coated with florfenicol antigens and enrofloxacin antigens in detection holes. The detection buffer is composed of 10-40 mM phosphate buffer and 0.05-0.5 wt% surfactant S17. The application can simultaneously detect florfenicol and enrofloxacin small molecules by using one detection buffer, thereby saving time and labor.

Description

Technical Field

[0001] This invention belongs to the field of sensing and immunoassay technology, and specifically relates to a small molecule multiplex detection kit based on Nano SPR biochip, as well as its preparation and detection methods. Background Technology

[0002] Plasmon resonance nanopore arrays (NanoSPR), due to their unique three-dimensional structure, exhibit a SPR effect distinct from planar models and a LSPR effect from metallic nanoparticles, simultaneously supporting both SPR and LSPR modes. The plasmon resonance effect in nanopore array biosensors can be directly incident on the nanopore metal structure, instantly exciting the surface light field, thus eliminating the need for complex optical paths and large optical instruments. NanoSPR nanopore array biosensors retain many advantages of traditional SPR sensors, such as real-time operation, label-free operation, absence of background interference, and high resolution. NanoSPR sensors also retain the performance advantages of LSPR sensors; by adjusting parameters such as the pore size, depth, shape, period of the nanopore array, and the type and thickness of the surface metal, a high-quality chip capable of capturing the strongest LSPR signal can be selected, achieving a stronger signal without the need for a large spectrometer.

[0003] Based on the above advantages, the NanoSPR biosensor's detection capabilities can meet the practical needs of biomolecular sensing and detection, and it is widely used in fields such as biomedical detection, drug analysis, food safety, environmental monitoring, and cell biology. Antibiotics are a class of secondary metabolites produced by microorganisms (including bacteria, fungi, and actinomycetes) or higher plants and animals during their life processes, possessing antipathogenic or other activities. The widespread use of antibiotics may lead to antibiotic residues in animal-derived foods. Antibiotics are classified in various ways, such as sulfonamides, fluoroquinolones, β-lactam antibiotics, cephalexin, lincomycin, tilmicosin-tylosin, dexamethasone, chloramphenicol, tetracycline, gentamicin, florfenicol, erythromycin, streptomycin, benzoic acid, neomycin, chlortetracycline, oxytetracycline, quinolones, malachite green, or crystal violet. Toxins are toxic substances produced by various organisms (animals, plants, and microorganisms), referring to biologically derived toxic chemical substances that cannot self-replicate, such as aflatoxin M1, aflatoxin B1, or zearalenone.

[0004] Florfenicol belongs to the chloramphenicol class of broad-spectrum antibiotics, primarily used for the prevention and treatment of bacterial infections in fish, pigs, cattle, and poultry. It features broad-spectrum antibacterial activity, good absorption, wide distribution in the body, and high safety and efficacy. Florfenicol has significant advantages over chloramphenicol and thiamphenicol in terms of safety and efficacy, and is already widely used in the aquaculture industry. Enrofloxacin is a rapidly developing class of animal-specific broad-spectrum antibiotics in recent years. Chemically, this class of drugs belongs to the pyruvic acid derivatives, inhibiting bacterial DNA gyrase. It has a broad antibacterial spectrum, high efficacy, low toxicity, and strong tissue penetration. Its antibacterial activity is nearly a thousand times that of sulfonamides, and it can be widely used for the prevention and treatment of bacterial diseases and mycoplasma infections in animals. However, with the development of the livestock and poultry farming industry, the abuse of florfenicol and enrofloxacin has led to huge potential harm in livestock and poultry, endangering public health. my country's GB 31650-2019 "National Food Safety Standard Maximum Residue Limits for Veterinary Drugs in Food" stipulates the residue limits of florfenicol and enrofloxacin in different animal tissues.

[0005] Existing technologies for detecting florfenicol or enrofloxacin include liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), and test strip methods. However, different small molecule detection buffers may not be interchangeable. Therefore, detecting different molecules requires diluting samples with different buffers and performing separate tests, which is time-consuming and labor-intensive. Consequently, there is currently no Nano SPR biochip-based technology that can simultaneously perform multiplex detection of florfenicol and enrofloxacin, or other small molecule antibiotics. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a small molecule multiplex detection kit based on a biochip. The kit contains only one detection buffer, which can be used to simultaneously dilute samples containing florfenicol and enrofloxacin, enabling the simultaneous detection of multiple small molecule antibiotics, saving time and effort.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned biochip-based small molecule multiplex detection kit.

[0008] Another object of the present invention is to provide a detection method using the above-mentioned biochip-based small molecule multiplex detection kit.

[0009] The above-mentioned objective is achieved through the following technical solution.

[0010] A small molecule multi-detection kit based on a biochip includes gold nanoparticle-labeled antibody, a multi-detection chip plate, and a detection buffer.

[0011] The nanoparticle-labeled antibody comprises florfenicol antibody and enrofloxacin antibody labeled with nanoparticles; several detection wells of the multi-detection chip are simultaneously coated with florfenicol antigen and enrofloxacin antigen; the detection buffer consists of 10-40 mM phosphate buffer and 0.05-0.5 wt% surfactant S17.

[0012] The detection buffer of this invention consists only of phosphate buffer and surfactant S17, without the need for additional buffers (such as Tris solution) or reaction-promoting reagents (such as PEG2W, PEG6K, NaCl, EDTA, PVP, etc.). The detection buffer system is simple and can be used to dilute samples containing different antigens, facilitating the simultaneous detection of multiple small molecule antibiotics.

[0013] Preferably, the detection buffer consists of 30 mM phosphate buffer and 0.05 wt% surfactant S17.

[0014] Preferably, the ratio of florfenicol antibody to gold particles in the nano-gold particle-labeled florfenicol antibody is 4 μL: 1.5 mL, and the ratio of enrofloxacin antibody to gold particles in the nano-gold particle-labeled enrofloxacin antibody is 6 μL: 1.5 mL.

[0015] Preferably, the kit further includes a reconstitution buffer for the gold nanoparticle-labeled antibody, the reconstitution buffer comprising: 25 mM pH 9.0 tris solution, 0.05 wt% polyethylene glycol 20000, 0.4 wt% sucrose, 3 wt% trehalose, and 2 wt% mannitol.

[0016] Preferably, the other detection wells of the multi-detection chip are also coated with other small molecule antigens besides florfenicol and enrofloxacin, and the kit also contains nano-gold particle-labeled antibodies corresponding to the other small molecule antigens.

[0017] The small molecule multiplex detection kit of the present invention utilizes a chip plate (which can be made from a NanoSPR chip and a bottomless 96-well plate) containing different antigens for detection. Small molecules in the test sample compete with the corresponding antigens on the surface of the chip plate for binding to the corresponding gold nanoparticle-labeled antibodies. When there are no small molecules in the test sample, the gold nanoparticle-labeled antibody reacts with the antigens on the surface of the chip plate to produce a large reaction signal. When there are small molecules in the test sample, the gold nanoparticle-labeled antibody binds to the small molecules in the test sample but does not react with the antigens on the surface of the chip plate, thus producing a small or no reaction signal.

[0018] The other small molecules may include one or more of small molecule antibiotics and toxins; the small molecule antibiotics include one or more of sulfonamides, fluoroquinolones, β-lactam antibiotics, cephalexin, lincomycin, tilmicosin-tylosin, dexamethasone, chloramphenicol, tetracycline, gentamicin, florfenicol, erythromycin, streptomycin, benzoic acid, neomycin, chlortetracycline, oxytetracycline, quinolones, malachite green, or crystal violet; the toxins include one or more of aflatoxin M1, B1, or zearalenone. Therefore, this invention can achieve 8-in-1, 16-in-1, 192-in-1, or more combined detection.

[0019] Preferably, the pH of the phosphate buffer in the detection buffer is 7.5.

[0020] The present invention also provides a method for preparing the aforementioned biochip-based small molecule multiplex detection kit, comprising:

[0021] Preparation of the multi-detection chip plate: The NanoSPR chip is assembled with a bottomless microplate to obtain a nano-plasma resonance sensing detection plate; each microwell of the detection plate is sequentially cleaned with ultrapure water and anhydrous ethanol, and dried with nitrogen; florfenicol antigen and enrofloxacin antigen are added to several of the microwells, sealed with a microplate sealing film, and incubated overnight at 4°C to obtain the multi-detection chip plate;

[0022] Preparation of gold nanoparticle-labeled antibodies: Take two groups of gold particle solutions of equal volume, add Tris solution to each group, and mix well; then add florfenicol antibody to the first group and enrofloxacin antibody to the second group, mix well, and let stand; then add bovine serum albumin to each group, mix well, let stand, freeze and centrifuge, remove the supernatant and collect the precipitate to obtain the gold nanoparticle-labeled antibodies; wherein, the volume ratio of florfenicol antibody to gold particle solution in the first group is 4 μL: 1.5 mL, and the volume ratio of enrofloxacin antibody to gold particle solution is 6 μL: 1.5 mL.

[0023] Preferably, in the preparation process of the gold particle-labeled antibody, the tris solution added to the first group and the second group is 4 μL and 6 μL of 0.1M tris solution with pH=9.0, respectively.

[0024] The present invention also provides a detection method for the aforementioned biochip-based small molecule multiplex detection kit, comprising the following steps:

[0025] S1. Preparation of standard curves: Different concentration gradient solutions containing both florfenicol and enrofloxacin were added to the detection wells of the multi-detector chip plate, and the starting point of the full spectrum was detected in the wavelength range of 500-700 nm. Then, florfenicol antibody and enrofloxacin antibody labeled with gold nanoparticles were added to the detection wells of florfenicol antigen + enrofloxacin antigen. After incubation at room temperature for 10 min, the endpoint of the full spectrum was detected in the wavelength range of 500-700 nm. After processing the data by subtracting the starting point reaction value from the endpoint reaction value, the standard curves of florfenicol and enrofloxacin can be obtained simultaneously.

[0026] S2. Sample pretreatment: Take the homogenized egg sample, add acetonitrile, shake to mix, add sodium chloride and anhydrous sodium sulfate, vortex to mix, centrifuge for 5 min, take the supernatant, add anhydrous magnesium sulfate and N-propylethylenediamine, vortex to mix, centrifuge for 5 min, take the supernatant, blow dry, add the detection buffer solution to reconstitute, which is the sample solution to be tested.

[0027] S3. Sample Detection: Add the sample prepared in S2 to the detection wells of the multi-detector chip plate, and detect the full-spectrum start point in the wavelength range of 500-700nm. Then, add florfenicol antibody and enrofloxacin antibody labeled with gold nanoparticles to the detection wells of florfenicol antigen + enrofloxacin antigen. After incubation at room temperature for 10 min, detect the full-spectrum endpoint in the wavelength range of 500-700nm. After processing the data by subtracting the start-point reaction value from the endpoint reaction value, the sample detection signal can be obtained. Substitute the sample detection signal into the standard curve formula in S1 to obtain the contents of florfenicol and enrofloxacin in the sample.

[0028] Preferably, the different concentration gradient solutions in step S1 are: florfenicol 12.8 ng / mL + enrofloxacin 51.2 ng / mL, florfenicol 3.2 ng / mL + enrofloxacin 12.8 ng / mL, florfenicol 0.8 ng / mL + enrofloxacin 3.2 ng / mL, florfenicol 0.2 ng / mL + enrofloxacin 0.8 ng / mL, florfenicol 0.05 ng / mL + enrofloxacin 0.2 ng / mL, and florfenicol 0 ng / mL + enrofloxacin 0 ng / mL.

[0029] Preferably, the florfenicol antibody and enrofloxacin antibody labeled with gold nanoparticles added in steps S1 and S3 are reconstituted with a reconstitution buffer before addition. The reconstitution buffer comprises: 25 mM pH 9.0 Tris solution, 0.05 wt% polyethylene glycol 20000, 0.4 wt% sucrose, 3 wt% trehalose, and 2 wt% mannitol. The Tris concentration and pH in the reconstitution buffer increase the stability and dispersibility of the gold nanoparticles, resulting in more uniform dispersion. PEG2W facilitates further dispersion of the gold nanoparticles, and trehalose protects the antibody on the surface of the gold nanoparticles in the buffer system, reducing antibody degradation. The reconstitution buffer proposed in this invention makes the gold nanoparticles more stable after reconstitution, less susceptible to aggregation due to external influences.

[0030] Compared with existing technologies, the advantages of this invention are: by optimizing the detection buffer and its combination with other reagents in the detection system, this invention can simultaneously detect florfenicol and enrofloxacin small molecules using only one detection buffer, saving time and effort. This invention can simultaneously detect florfenicol and enrofloxacin small molecules within only 10 minutes, with detection limits as low as 0.05 ng / mL for florfenicol and 0.2 ng / mL for enrofloxacin, respectively. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the formation of a multi-detection chip plate by coating the microwells of the detection plate with florfenicol antigen, enrofloxacin antigen, or other small molecule antigens, as described in a specific embodiment. Figure 1 A is a multi-chip board used to optimize the detection system and for both specific and non-specific detection. Figure 1 B represents the multi-chip board used for linear detection and sample detection.

[0032] Figure 2 The results show the optimization of the gold nanoparticle-labeled florfenicol antibody (Group 1) in Example 1.

[0033] Figure 3 This is the optimization result of the florfenicol basic buffer in Example 2; where PB6.0KB is the blank control sample of phosphate buffer at pH 6.0, and PB6.0 is the sample of phosphate buffer at pH 6.0 with 1 ppb of florfenicol added, and the other sample names are labeled similarly; there is also tris6.36KB, which represents the blank control sample of tris buffer at pH 6.36, and tris6.36, which represents the sample of tris buffer at pH 6.36 with 1 ppb of florfenicol added, and the other sample names are labeled similarly.

[0034] Figure 4This is the optimization result of the florfenicol surfactant species in Example 2; wherein, 0.05% TW20 KB is a blank control sample with 0.05wt% TW20 added to 30mM (pH=7.5) phosphate buffer, and 0.05% TW20 is a small molecule sample of florfenicol with 0.05wt% TW20 added to 30mM (pH=7.5) phosphate buffer and 1ppb florfenicol added. The labeling of other sample names is similar.

[0035] Figure 5 In Example 3, different concentrations of mixed sample 1 were added to the detection wells coated with florfenicol antigen (A) and enrofloxacin antigen (B) on the multi-detection chip plate to test the detection specificity of the method of the present invention.

[0036] Figure 6 In Example 4, mixed sample 2 and mixed sample 3 of different concentrations were added to the chip wells coated with florfenicol antigen (A) and enrofloxacin antigen (B) on the multi-detection chip plate to detect the non-specific results of the detection method of the present invention.

[0037] Figure 7 The results of multi-sample testing in Examples 3 and 4 are obtained by adding mixed samples 1, 2, and 3 of different concentrations to the chip wells coated with florfenicol antigen and enrofloxacin antigen on the multi-sample chip plate, as well as florfenicol small molecule samples and enrofloxacin small molecule samples.

[0038] Figure 8 In Example 5, different concentrations of samples containing both florfenicol and enrofloxacin small molecules were added to one well of the multi-detection chip plate, which was coated with both florfenicol antigen (A) and enrofloxacin antigen (B). This demonstrates that the present invention can achieve multi-detection.

[0039] Figure 9 In Example 6, different concentrations of samples containing both florfenicol and enrofloxacin were added to one well of the multi-detection chip plate, where both florfenicol antigen (A) and enrofloxacin antigen (B) were coated. The resulting gradient reaction indicates that the multi-detection chip plate can perform semi-quantitative or qualitative detection.

[0040] Figure 10 In Example 6, different concentrations of samples containing both florfenicol and enrofloxacin were added to one well of the multi-detection chip plate. The obvious gradient of the results indicates that the multi-detection chip plate can perform semi-quantitative or qualitative detection.

[0041] Figure 11To create a standard curve for the multi-detection chip in Example 6, where different concentrations of florfenicol small molecule samples were added to one well of the chip plate coated with both florfenicol antigen and enrofloxacin antigen, the formula is Y = 0.06268 + 0.30944 / (1 + (x / 0.7275)^0.57985), R 2 A value of 0.998 indicates a good linear relationship, suggesting that this multi-sample test can be used for semi-quantitative or qualitative detection.

[0042] Figure 12 To create a standard curve for the multi-detection chip in Example 6, where different concentrations of enrofloxacin small molecule samples were added to one well of the chip plate coated with both florfenicol antigen and enrofloxacin antigen, the formula is Y = 0.00221 + 0.35911 / (1 + (x / 12.02093)^0.44687), R 2 A value of 0.999 indicates a good linear relationship, suggesting that this multi-sample test can be used for semi-quantitative or qualitative detection. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] In a specific embodiment of the present invention, a small molecule multiplex detection kit based on a biochip is provided, the kit comprising gold nanoparticle-labeled antibody, a multiplex detection chip plate and a detection buffer;

[0045] The gold nanoparticle-labeled antibody comprises one or more of small molecule antibiotics and toxins; the small molecule antibiotics include one or more of sulfonamides, fluoroquinolones, β-lactam antibiotics, cephalexin, lincomycin, tilmicosin-tylosin, dexamethasone, chloramphenicol, tetracycline, gentamicin, erythromycin, streptomycin, benzoic acid, neomycin, chlortetracycline, oxytetracycline, quinolones, malachite green, or crystal violet; the toxins include one or more of aflatoxin M1, B1, or zearalenone.

[0046] The multi-detection chip is coated with antigens, and the types of antigens correspond to the types of antibodies labeled with gold nanoparticles.

[0047] The detectors of the multi-detection chip board include optical detection devices such as spectrometers, enzyme-linked immunosorbent assay (ELISA) readers, and microscopes.

[0048] The gold nanoparticles are prepared using existing methods or obtained commercially; the same gold nanoparticles are used in all the following examples. Specifically, the gold nanoparticles used in this invention have a particle size of 35 nm.

[0049] Specifically, the preparation method of the small molecule multiplex detection kit includes:

[0050] (1) Fabrication of the multi-detector chip board:

[0051] A tapered nanopillar quartz substrate mold was fabricated using photolithography. Then, a UV-curable polymer (e.g., NOA61, NOA68T) was uniformly coated onto the mold. After UV curing, the cured material was peeled off to obtain the chip substrate. Peptides, silver, and gold were then sequentially deposited onto the chip substrate via electron beam evaporation to obtain the Nano SPR chip.

[0052] The same Nano SPR chip is used in all the following embodiments;

[0053] Next, attach the NanoSPR chip to the bottom of the bottomless 96-well plate to complete the assembly of the nano-plasma resonance sensing detection plate.

[0054] Each detection well of the detection plate is sequentially cleaned with ultrapure water and anhydrous ethanol, and then dried with nitrogen. One or more of florfenicol antigen, enrofloxacin antigen, or other small molecule antigens are added to each detection well, as shown in the schematic diagram. Figure 1 As shown, after sealing with a detection plate sealing film, it is incubated at 4°C overnight to obtain a multi-chip detection board;

[0055] like Figure 1 As shown, florfenicol, enrofloxacin antigen, or other small molecule antigens can be immobilized on a detection plate integrated with a nanoplasma resonance biochip, enabling 8-in-1, 16-in-1, 192-in-1, or more combined detection.

[0056] (2) Preparation of gold nanoparticle-labeled antibody: Eight centrifuge tubes were divided into two groups. 1.5 mL of gold particle solution was added to each group. 4 μL and 6 μL of 0.1 M tris (pH = 9.0) solution were added to the first group and the second group, respectively, and mixed well. 1 μL, 2 μL, 4 μL, and 6 μL of 0.89 μg / mL florfenicol antibody were added to the four tubes of the first group, respectively. 1 μL, 2 μL, 4 μL, and 6 μL of 0.89 μg / mL enrofloxacin antibody were added to the four tubes of the second group, respectively. Each tube was mixed well and allowed to stand. 150 μL of 10 wt% bovine serum albumin was added to each group, mixed well, blocked, allowed to stand, centrifuged at freezing, and the supernatant was removed before collecting the precipitate to obtain the gold nanoparticle-labeled antibody.

[0057] (3) The detection buffer solution includes 10-40 mM phosphate buffer and 0.05-0.5 wt% surfactant S17.

[0058] The detection method for florfenicol antigen and enrofloxacin antigen using the above-mentioned small molecule multiplex detection kit includes the following steps:

[0059] S1. Preparation of the standard curve: A gradient solution containing both florfenicol and enrofloxacin small molecules was added to each of the multi-detector chip plates, specifically to wells coated with both florfenicol and enrofloxacin antigen. (The corresponding concentrations of florfenicol + enrofloxacin in the gradient solutions were florfenicol 12.8 ng / mL + enrofloxacin 51.2 ng / mL, florfenicol 3.2 ng / mL + enrofloxacin 12.8 ng / mL, florfenicol 0.8 ng / mL + enrofloxacin 3.2 ng / mL, and florfenicol...) The reagents were prepared as follows: 0.2 ng / mL florfenicol + 0.8 ng / mL enrofloxacin, 0.05 ng / mL florfenicol + 0.2 ng / mL enrofloxacin, and 0 ng / mL florfenicol + 0 ng / mL enrofloxacin. The starting point of the full spectrum was detected in the wavelength range of 500-700 nm. Then, in the same detection well coated with both florfenicol antigen and enrofloxacin antigen, florfenicol antibody and enrofloxacin antibody coated with gold nanoparticles (prepared in step (2) above) were added simultaneously. After incubation at room temperature for 10 min, the endpoint of the full spectrum was detected in the wavelength range of 500-700 nm. After processing the data by subtracting the starting point reaction value from the endpoint reaction value, the standard curves of florfenicol and enrofloxacin could be obtained simultaneously. The formulas are as follows:

[0060] Florfenicol Y = 0.06268 + 0.30944 / (1 + (x / 0.7275)^0.57985), R 2 It is 0.998;

[0061] Enrofloxacin Y = 0.00221 + 0.35911 / (1 + (x / 12.02093)^0.44687), R 2 It is 0.999;

[0062] The detection limits were as low as 0.05 ng / mL for florfenicol and 0.2 ng / mL for enrofloxacin.

[0063] S2. Sample pretreatment: Take the homogenized egg sample and add acetonitrile, shake to mix, add sodium chloride and anhydrous sodium sulfate, vortex to mix, centrifuge for 5 min, take the supernatant and add anhydrous magnesium sulfate and N-propylethylenediamine, vortex to mix, centrifuge for 5 min, take the supernatant, blow dry, add the detection buffer solution to reconstitute, which is the sample solution to be tested.

[0064] S3. Sample detection: Add the sample prepared in S2 to the multi-detection chip plate, i.e., to the detection wells coated with both florfenicol antigen and enrofloxacin antigen, and detect the full spectrum start point in the wavelength range of 500-700nm. Then, add the florfenicol antibody and enrofloxacin antibody (prepared in step (2) above) labeled with gold nanoparticles from the kit to the detection wells coated with both florfenicol antigen and enrofloxacin antigen. After incubation at room temperature for 10 min, detect the full spectrum endpoint in the wavelength range of 500-700nm. After processing the data by subtracting the start point reaction value from the endpoint reaction value, the sample detection signal can be obtained. Substituting the sample detection signal into the standard curve formula, the content of florfenicol and enrofloxacin in the sample can be obtained simultaneously, thereby achieving semi-quantitative detection.

[0065] The florfenicol antibody and enrofloxacin antibody labeled with gold nanoparticles added in steps S1 and S3 were both reconstituted in a buffer solution containing 25 mM tris (pH = 9.0), 0.05 wt% PEG2W (polyethylene glycol 20000), 0.4 wt% sucrose, 3 wt% trehalose, and 2 wt% mannitol before use.

[0066] Example 1: Preparation and Optimization of Antibodies Labeled with Gold Nanoparticles

[0067] Eight identical centrifuge tubes were divided into two groups (four tubes per group). 1.5 mL of gold particle solution was added to each group. Then, 4 μL of 0.1 M tris (pH = 9.0) solution was added to the first group of tubes, and the mixture was stirred. Next, 1 μL, 2 μL, 4 μL, and 6 μL of 0.89 μg / mL florfenicol antibody were added to the four tubes in the first group, respectively. 6 μL of 0.1 M tris (pH = 9.0) solution was added to the second group of tubes, and the mixture was stirred. Then, 1 μL, 2 μL, 4 μL, and 6 μL of 0.89 μg / mL enrofloxacin antibody were added to the four tubes in the second group, respectively, and the mixture was stirred and allowed to stand. 150 μL of 10 wt% bovine serum albumin was added to each tube, stirred, and the mixture was blocked. The tubes were then allowed to stand. After centrifugation at room temperature, the supernatant was discarded, and the precipitate was collected for later use.

[0068] Detection process: Add 600 μL of reconstitution buffer (25 mM pH = 9.0 Tris solution, 0.05 wt% polyethylene glycol 20000, 0.4 wt% sucrose, 3 wt% trehalose, and 2 wt% mannitol) to the above-prepared nanoparticle-labeled florfenicol antibody and nanoparticle-labeled enrofloxacin antibody precipitates, respectively, and mix well before use.

[0069] After rinsing the prepared multi-detection chip plate twice with PBS phosphate buffer, add 1 ppb of florfenicol small molecules dissolved in PBS phosphate buffer and blank control samples without florfenicol small molecules to the chip wells coated with florfenicol antigen, respectively. Measure the starting point of the full spectrum (500nm-700nm) using a conventional microplate reader. Then, add 7 μL of different volumes of florfenicol antibody labeled with the above-mentioned gold nanoparticles (1 μL, 2 μL, 4 μL, 6 μL), respectively. After mixing, incubate at 37℃ and 700 rpm for 10 min, and then detect the endpoint of the full spectrum (500nm-700nm). The optimized detection process for gold-particle-labeled enrofloxacin antibody is the same as that for the gold-particle-labeled florfenicol antibody: After rinsing the prepared multi-detection chip plate twice with PBS phosphate buffer, add 1 ppb of enrofloxacin small molecules dissolved in PBS buffer and blank control samples without enrofloxacin small molecules to the wells of the chip coated with enrofloxacin antigen, respectively. Measure the starting point of the full spectrum (500nm-700nm) using a conventional microplate reader. Then, add 7 μL of different volumes of enrofloxacin antibody labeled with the above-mentioned gold nanoparticles (1 μL, 2 μL, 4 μL, 6 μL), mix well, and incubate at 37℃ and 700 rpm for 10 min. Then, detect the endpoint of the full spectrum (500nm-700nm).

[0070] The optimized grouping experiments of the above gold nanoparticle-labeled florfenicol antibody (Group 1) are shown in the table below.

[0071]

[0072] The above experimental results are as follows Figure 2 As shown, in the first group, when gold particles bound to 4 μL of florfenicol antibody, the blank sample and the sample with 1 ppb of florfenicol added could be clearly distinguished. Therefore, adding 4 μL of florfenicol antibody to 1.5 mL of gold particles was chosen as the subsequent florfenicol detection condition. Similarly, the inventors found that when 1.5 mL of gold particles bound to 6 μL of enrofloxacin antibody, the blank sample and the sample with 1 ppb of enrofloxacin added could be clearly distinguished. Therefore, adding 6 μL of enrofloxacin antibody to 1.5 mL of gold particles was chosen as the subsequent enrofloxacin detection condition (figure omitted).

[0073] This embodiment also investigated the amount of Tris solution added to the gold nanoparticle solution in two groups. It was found that adding 4 μL of 0.1M Tris solution (pH=9.0) to the first group containing florfenicol antibody and adding 6 μL of 0.1M Tris solution (pH=9.0) to the second group containing enrofloxacin antibody brought the pH value to the optimal condition, which was conducive to the binding of gold nanoparticles with florfenicol antibody or enrofloxacin antibody. The final prepared gold nanoparticle-labeled florfenicol antibody and gold nanoparticle-labeled enrofloxacin antibody were stable and did not aggregate.

[0074] Example 2: Optimization of Multi-Detection Buffer

[0075] After rinsing the prepared multi-detection chip plate twice with PB phosphate buffer, 1 ppb of florfenicol small molecules (denoted as PB6.0, PB6.5, etc., five groups) and control samples without florfenicol small molecules (denoted as PB6.0KB, PB6.5, etc., five groups) were dissolved in 30 mM PB buffer (pH 6.0, 6.5, 7.0, 7.5, 8.0) or Tris buffer (pH 6.36, 7.33, 8.33) respectively into the chip wells coated with florfenicol antigen. The starting point of the full spectrum (500nm-700nm) was measured using a conventional microplate reader. Then, 7 μL of florfenicol antibody labeled with gold nanoparticles was added, mixed well, and incubated at 37℃ and 700 rpm for 10 min. The endpoint of the full spectrum (500nm-700nm) was then measured. Different types of surfactants (Tween 20, Triton, S9, S17) with the same mass concentration were added to the optimized basal buffer. Then, different concentrations of surfactants (0.01wt%, 0.05wt%, 0.1wt%, 0.2wt%, 0.5wt%) were optimized in the basal buffer. Different reaction-promoting reagents (such as PEG2W, PEG6K, NaCl, EDTA, PVP, etc.) were added to this basal buffer to optimize the florfenicol buffer. The optimization method for enrofloxacin buffer is the same.

[0076] The optimization results of the buffer solution are as follows: Figure 3 As shown, under 30mM PB (pH=7.5) buffer conditions, the reaction values ​​of the blank control and the small molecule sample containing 1ppb florfenicol showed a significant difference. The blank reaction value was the largest at this point, and the blank reaction values ​​of the Tris buffer at different pH values ​​were not as large as those of the PB buffer. Therefore, 30mM PB (pH=7.5) buffer was chosen as the base buffer for further optimization. The optimization results for surfactant types are shown below. Figure 4As shown, under basal buffer conditions, different types of surfactants were added at the same mass concentration ratio (0.05 wt%). The blank control with 0.05 wt% S17 showed the largest reaction value, and the reaction value of the blank control was significantly different from that of the sample containing 1 ppb florfenicol. Therefore, 30 mM PB (pH = 7.5) and 0.05 wt% S17 buffer (denoted as PBS17 phosphate buffer) were selected as the basal buffer for further optimization. Then, different concentrations of surfactant S17 (0.01 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, and 0.5 wt%) were optimized in the basal buffer. The results showed that the blank control with 0.05 wt% S17 showed the largest reaction value, and the reaction value of the blank control was significantly different from that of the sample containing 1 ppb florfenicol (figure omitted). Adding different reaction-promoting reagents (such as PEG2W, PEG6K, NaCl, EDTA, PVP, etc.) to the basic buffer did not promote the reaction results (figure omitted). Furthermore, the blank control reaction value was sufficient to meet the experimental requirements. Therefore, the optimized 30mM PB (pH=7.5), 0.05wt% S17 buffer (PBS 17 phosphate buffer) was selected for subsequent detection experiments of florfenicol. The buffer optimization method for enrofloxacin was the same as that for florfenicol. The results showed that in 30mM PB (pH=7.5) 0.05wt% S17 buffer, the enrofloxacin blank control reaction value was the highest and close to that of the florfenicol blank control. The reaction value of the blank control was significantly different from that of the small molecule sample containing 1ppb enrofloxacin. Therefore, the optimized 30mM PB (pH=7.5), 0.05wt% S17 buffer (PBS 17 phosphate buffer) was selected for subsequent detection experiments of enrofloxacin.

[0077] Example 3: Multiplex detection for small molecule specificity

[0078] After rinsing the prepared multi-detection chip plate twice with PBS-17 phosphate buffer, different concentrations of mixed solution 1 (0.05–10 ng / mL) were added to the wells coated with florfenicol and enrofloxacin antigens. This solution was prepared by diluting the stock solution (1.8 ng / mL florfenicol, 7.2 ng / mL enrofloxacin, 10 ng / mL sulfonamide, 10 ng / mL malachite green, and 10 ng / mL chloramphenicol) six-fold, resulting in concentrations of 1.8 ng / mL florfenicol + 7.2 ng / mL enrofloxacin + 10 ng / mL sulfonamide + 10 ng / mL malachite green + 10 ng / mL chloramphenicol) and 0.3 ng / mL florfenicol + 1.2 ng / mL enrofloxacin + 1.7 ng / mL chloramphenicol. Sulfonamide + 1.7 ng / mL malachite green + 1.7 ng / mL chloramphenicol, 0.05 ng / mL florfenicol + 0.2 ng / mL enrofloxacin + 0.3 ng / mL sulfonamide + 0.3 ng / mL malachite green + 0.3 ng / mL chloramphenicol, 0 ng / mL florfenicol + 0 ng / mL enrofloxacin + 0 ng / mL sulfonamide + 0 ng / mL malachite green + 0 ng / mL chloramphenicol were used. The starting point (500 nm-700 nm) of the full spectrum was measured using a conventional microplate reader. 7 μL of gold particles labeled with florfenicol and enrofloxacin antibodies were added respectively, mixed well, and incubated at 37℃ and 700 rpm for 10 min. Then the endpoint (500 nm-700 nm) of the full spectrum was measured.

[0079] Data analysis was performed by subtracting the start point from the end point of the full spectrum. For example... Figure 5 A, B and Figure 7 The presence of a gradient reaction indicates that this multiplex detection method has good specificity.

[0080] Example 4: Multi-detection small molecule nonspecific detection

[0081] After rinsing the multi-detection chip plate twice with PBS-17 phosphate buffer, add mixed solutions of different concentrations (0.2–10 ng / mL) to the wells coated with florfenicol and enrofloxacin antigens. These solutions are prepared by 6-fold dilution of stock solutions containing 7.2 ng / mL enrofloxacin, 10 ng / mL sulfonamide, 10 ng / mL malachite green, and 10 ng / mL chloramphenicol, resulting in concentrations of 7.2 ng / mL enrofloxacin + 10 ng / mL sulfonamide + 10 ng / mL malachite green + 10 ng / mL chloramphenicol. 1.2 ng / mL chloramphenicol, 1.2 ng / mL enrofloxacin + 1.7 ng / mL sulfonamide + 1.7 ng / mL malachite green + 1.7 ng / mL chloramphenicol, 0.2 ng / mL enrofloxacin + 0.3 ng / mL sulfonamide + 0.3 ng / mL malachite green + 0.3 ng / mL chloramphenicol, 0 ng / mL enrofloxacin + 0 ng / mL sulfonamide + 0 ng / mL malachite green + 0 ng / mL chloramphenicol) and 0.05–10 ng / mL mixed solutions 3 (concentration 1.8n) The stock solutions of florfenicol (1.8 ng / mL), sulfonamide (10 ng / mL), malachite green (10 ng / mL), and chloramphenicol (10 ng / mL) were diluted 6-fold to obtain the following concentrations: 1.8 ng / mL florfenicol + 10 ng / mL sulfonamide + 10 ng / mL malachite green + 10 ng / mL chloramphenicol; 0.3 ng / mL florfenicol + 1.7 ng / mL sulfonamide + 1.7 ng / mL malachite green + 1.7 ng / mL chloramphenicol; and 0.05 ng / mL florfenicol + 0.3 ng / mL chloramphenicol. The starting point (500nm-700nm) of the full spectrum was measured using a standard microplate reader. 7μl of gold particles labeled with florfenicol and enrofloxacin antibodies were added to each sample, mixed, and incubated at 37℃ and 700rpm for 10 min. The endpoint (500nm-700nm) was then measured.

[0082] like Figure 6 A, B and Figure 7 As shown, when mixed samples without florfenicol and enrofloxacin were added to the surface of the antigen chip coated with florfenicol and enrofloxacin, respectively, there was no reaction, indicating that the multiplex test did not have nonspecific binding.

[0083] Example 5: Linear Detection of Small Molecules Using Multiple Detection Systems

[0084] After rinsing the multi-detection chip plate (i.e., the wells containing both florfenicol and enrofloxacin antigens) twice with PBS 17 phosphate buffer, add different concentrations (0.05–7.2 ng / mL) of a small molecule solution containing both florfenicol and enrofloxacin (florfenicol 1.8 ng / mL, enrofloxacin 7.2 ng / mL) to the wells containing both antigens, and then dilute 6-fold, resulting in concentrations of 1.8 ng / mL florfenicol + 7.2 ng / mL enrofloxacin. The following formulations were used: Florfenicol (0.3 ng / mL + 1.2 ng / mL + Enrofloxacin), 0.05 ng / mL Florfenicol (0.2 ng / mL + Enrofloxacin), and 0 ng / mL Florfenicol (0 ng / mL + Enrofloxacin). The starting point (500 nm - 700 nm) of the full spectrum was measured using a spectrometer. Simultaneously, 7 μL of florfenicol antibody-labeled gold particles and 7 μL of enrofloxacin antibody-labeled gold particles were added, mixed, and incubated at 37°C and 700 rpm for 10 min. The endpoint (500 nm - 700 nm) of the full spectrum was then measured. The data were analyzed by subtracting the starting point from the endpoint.

[0085] The results are as follows Figure 8 Figures A and B show the linear detection of florfenicol (A) and enrofloxacin (B) small molecules, respectively. The detection curves for florfenicol are compared with the specificity test results for florfenicol. Figure 5 The similarity between A and B indicates that florfenicol has good specificity and reproducibility; the detection curve of enrofloxacin is similar to that of the enrofloxacin specificity test (A). Figure 5 B) The similarity indicates that the specificity of enrofloxacin is good, as is its repeatability. It also indicates that the linearity of florfenicol and enrofloxacin is good, which can achieve linear detection of multiplex detection chips.

[0086] Example 6: Multi-detection semi-quantitative or qualitative detection of small molecules

[0087] After rinsing the multi-detection chip plate (i.e., wells coated with both florfenicol and enrofloxacin antigen) twice with PBS 17 phosphate buffer, different concentrations of solutions containing both florfenicol and enrofloxacin (12.8 ng / mL florfenicol and 51.2 ng / mL enrofloxacin) were added to the wells coated with both antigens. These solutions were then diluted four-fold to obtain concentrations of florfenicol 12.8 ng / mL + enrofloxacin 51.2 ng / mL and florfenicol 3.2 ng / mL + enrofloxacin 12.8 ng / mL, respectively. The following solutions were prepared: florfenicol 0.8 ng / mL + enrofloxacin 3.2 ng / mL, florfenicol 0.2 ng / mL + enrofloxacin 0.8 ng / mL, florfenicol 0.05 ng / mL + enrofloxacin 0.2 ng / mL, and florfenicol 0 ng / mL + enrofloxacin 0 ng / mL. The starting point of the full spectrum (500 nm - 700 nm) was measured using a spectrometer. Simultaneously, 7 μL of florfenicol antibody-labeled gold particles and 7 μL of enrofloxacin antibody-labeled gold particles were added, mixed well, and incubated at 37 °C and 700 rpm for 10 min. Then, the endpoint of the full spectrum (500 nm - 700 nm) was measured.

[0088] The results are as follows Figure 9 Figures A and B show the standard curve detection graphs for florfenicol (A) and enrofloxacin (B), respectively. These are the full-wavelength spectra obtained by subtracting the starting point from the endpoint. The graphs show a uniform curve distribution. Figure 10 The figure shows a bar chart of small molecule detection of florfenicol and enrofloxacin obtained by subtracting the wavelengths from 600-575 nm. Curve fitting was performed to obtain the standard curves for florfenicol and enrofloxacin, with formulas Y = 0.06268 + 0.30944 / (1 + (x / 0.7275)^0.57985), R0 and R0 respectively. 2 0.998 ( Figure 11 ); Y=0.00221+0.35911 / (1+(x / 12.02093)^0.44687), R 2 0.999 ( Figure 12 Thus, the multi-detection chip plate of the present invention can be obtained, that is, multiple antigens are coated in one chip well, which can simultaneously detect multiple small molecule compounds in a semi-quantitative or qualitative manner.

[0089] Application Example 1: Spiking Detection of Actual Samples

[0090] Five negative egg samples were crushed and mixed thoroughly, then randomly divided into three parallel groups (A, B, and C, 10g per group). Each group contained both florfenicol and enrofloxacin standard stock solutions, ensuring the samples contained both. Specifically, group A contained 20 μg / kg florfenicol and 50 μg / kg enrofloxacin; group B contained 10 μg / kg florfenicol and 20 μg / kg enrofloxacin; and group C contained 5 μg / kg florfenicol and 10 μg / kg enrofloxacin.

[0091] Sample pretreatment:

[0092] Weigh 10g of homogenized egg sample and add 20mL of acetonitrile. Vortex to mix. Add 4g of sodium chloride and 6g of anhydrous sodium sulfate, vortex, and centrifuge at 5000rpm / min for 5min. Transfer 4mL of the supernatant to a new centrifuge tube, add 600mg of anhydrous magnesium sulfate and 200mg of N-propylethylenediamine, vortex for 0.5min, and centrifuge at 5000rpm / min for 5min. Take 2mL of the supernatant, dry it under nitrogen, and reconstitute it with 400μL of detection buffer (30mM PB buffer, 0.05wt% surfactant S17). This is the sample solution to be tested.

[0093] Testing of actual samples:

[0094] After rinsing the detection wells of the multi-detection chip plate coated with both florfenicol and enrofloxacin antigen twice with PBS17 phosphate buffer, blank samples were added to each well coated with both florfenicol and enrofloxacin antigen. Group A eggs contained 20 μg / kg florfenicol and 50 μg / kg enrofloxacin; Group B eggs contained 10 μg / kg florfenicol and 20 μg / kg enrofloxacin; and Group C eggs contained 5 μg / kg florfenicol and 10 μg / kg enrofloxacin. The starting point of the full spectrum was measured using a spectrometer. At the same time, 7 μL of florfenicol antibody labeled with gold nanoparticles and 7 μL of enrofloxacin antibody labeled with gold nanoparticles (prepared in step (2) above) were added from the kit. After mixing, the mixture was incubated at 37°C and 700 rpm for 10 min, and then the endpoint of the full spectrum was measured. The data were analyzed by subtracting the starting point from the endpoint of the full spectrum.

[0095] The test results are shown in the table below. The sample recovery rate ranged from 101% to 107%, indicating that the multi-detection chip plate of the present invention, i.e., multiple antigens are coated in one chip well, is suitable for multi-detection of samples.

[0096]

[0097] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A small molecule multiplex detection kit based on a biochip, characterized in that, The kit contains gold nanoparticle-labeled antibodies, a multi-detection chip, and a detection buffer. The nanoparticle-labeled antibody comprises florfenicol antibody and enrofloxacin antibody labeled with nanoparticles; several detection wells of the multi-detection chip are simultaneously coated with florfenicol antigen and enrofloxacin antigen; the detection buffer consists of 30mM phosphate buffer at pH 7.5 and 0.05wt% surfactant S17. The multi-detection chip board is a nano-plasma resonance sensing detection board obtained by assembling a NanoSPR chip with a bottomless microporous plate. In the case of florfenicol antibody labeled with gold nanoparticles, the ratio of florfenicol antibody to gold nanoparticles was 4 μL: 1.5 mL. In the case of enrofloxacin antibody labeled with gold nanoparticles, the ratio of enrofloxacin antibody to gold nanoparticles was 6 μL: 1.5 mL.

2. The small molecule multiplex detection kit based on a biochip according to claim 1, characterized in that, It also includes a reconstitution buffer for the antibody labeled with the gold nanoparticles, the reconstitution buffer comprising: 25 mM pH9.0 tris solution, 0.05 wt% polyethylene glycol 20000, 0.4 wt% sucrose, 3 wt% trehalose, and 2 wt% mannitol.

3. The small molecule multiplex detection kit based on a biochip according to claim 1, characterized in that, The detection wells of the multi-detection chip are also coated with other small molecule antigens, and the kit also contains nano-gold particle-labeled antibodies corresponding to the types of the other small molecule antigens.

4. The method for preparing the small molecule multiplex detection kit based on a biochip according to any one of claims 1 to 3, characterized in that, include: Fabrication of the multi-detection chip board: The NanoSPR chip is assembled with a bottomless microporous plate to obtain a nano-plasma resonance sensing detection board; each detection well of the detection board is cleaned sequentially with ultrapure water and anhydrous ethanol, and then dried with nitrogen gas; Florfenicol antigen and enrofloxacin antigen are added to several of the detection wells, sealed with microplate sealing film, and incubated overnight at 4°C to obtain the multi-detection chip plate. Preparation of gold nanoparticle-labeled antibodies: Take two groups of gold particle solutions of equal volume, add Tris solution to each group, and mix well; then add florfenicol antibody to the first group and enrofloxacin antibody to the second group, mix well, and let stand; then add bovine serum albumin to each group, mix well, let stand, freeze and centrifuge, remove the supernatant and collect the precipitate to obtain the gold nanoparticle-labeled antibodies; wherein, the volume ratio of florfenicol antibody to gold particle solution in the first group is 4 μL: 1.5 mL, and the volume ratio of enrofloxacin antibody to gold particle solution is 6 μL: 1.5 mL.

5. The method for preparing the small molecule multiplex detection kit based on a biochip according to claim 4, characterized in that, In the preparation of the gold particle-labeled antibody, the tris solution added to the first group and the second group is 4 μL and 6 μL of 0.1M tris solution with pH = 9.0, respectively.

6. A detection method using the biochip-based small molecule multiplex assay kit according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Preparation of standard curves: Different concentration gradient solutions containing both florfenicol and enrofloxacin were added to the detection wells of the multi-detector chip plate, and the starting point of the full spectrum was detected in the wavelength range of 500-700 nm. Then, florfenicol antibody and enrofloxacin antibody labeled with gold nanoparticles were added to the detection wells of florfenicol antigen + enrofloxacin antigen. After incubation at room temperature for 10 min, the endpoint of the full spectrum was detected in the wavelength range of 500-700 nm. After processing the data by subtracting the starting point reaction value from the endpoint reaction value, the standard curves of florfenicol and enrofloxacin can be obtained simultaneously. S2. Sample pretreatment: Take the homogenized egg sample, add acetonitrile, shake to mix, add sodium chloride and anhydrous sodium sulfate, vortex to mix, centrifuge for 5 min, take the supernatant and add anhydrous magnesium sulfate and N-propylethylenediamine. Vortex mix, centrifuge for 5 minutes, take the supernatant, blow dry, and reconstitute with the detection buffer solution to obtain the sample solution to be tested; S3. Sample Detection: Add the sample prepared in S2 to the detection wells of the multi-detector chip plate, and detect the full-spectrum start point in the wavelength range of 500-700nm. Then, add florfenicol antibody and enrofloxacin antibody labeled with gold nanoparticles to the detection wells of florfenicol antigen + enrofloxacin antigen. After incubation at room temperature for 10 min, detect the full-spectrum endpoint in the wavelength range of 500-700nm. After processing the data by subtracting the start-point reaction value from the endpoint reaction value, the sample detection signal can be obtained. Substitute the sample detection signal into the standard curve formula in S1 to obtain the contents of florfenicol and enrofloxacin in the sample.

7. The detection method of the small molecule multiplex assay kit based on biochips according to claim 6, characterized in that, The different concentration gradient solutions mentioned in step S1 are: florfenicol 12.8 ng / mL + enrofloxacin 51.2 ng / mL, florfenicol 3.2 ng / mL + enrofloxacin 12.8 ng / mL, florfenicol 0.8 ng / mL + enrofloxacin 3.2 ng / mL, florfenicol 0.2 ng / mL + enrofloxacin 0.8 ng / mL, florfenicol 0.05 ng / mL + enrofloxacin 0.2 ng / mL, and florfenicol 0 ng / mL + enrofloxacin 0 ng / mL.

8. The detection method of the small molecule multiplex assay kit based on a biochip according to claim 6, characterized in that, The florfenicol antibody and enrofloxacin antibody labeled with gold nanoparticles added in steps S1 and S3 were both reconstituted with a reconstitution buffer before being added. The reconstitution buffer consisted of: 25 mM pH = 9.0 tris solution, 0.05 wt% polyethylene glycol 20000, 0.4 wt% sucrose, 3 wt% trehalose, and 2 wt% mannitol.

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