Method for detecting 5-hydroxyfluoroniacin based on metal organic framework enzyme cascade reaction and application

Abstract

The application relates to a method for detecting 5-hydroxyfluoronilicine based on a metal organic framework enzyme cascade reaction. The method utilizes the simulated peroxidase activity of a metal organic framework HKSUT-1 and the regulation of the activity of the metal organic framework HKSUT-1 by PPi / ALP to construct a natural enzyme-nanoenzyme cascade signal amplification path. The method combines the enzyme cascade reaction with a competitive immune reaction, and the detection limit of 5-hydroxyfluoronilicine is 0.12 ng / mL, which is 7.42 times higher than the sensitivity of traditional ELISA for detecting 5-hydroxyfluoronilicine; the quantitative limit of the method for 5-hydroxyfluoronilicine is 0.51 ng / mL, the linear range is 0.51-14.08 ng / mL, and the sensitivity, accuracy and precision all meet the requirements of residual detection; when the method is applied to the detection of milk samples, direct analysis is achieved without extraction, the detection system is simple, and standardization is easy.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and food safety testing technology, and in particular to a method and application for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction. Background Technology

[0002] 5-Hydroxyflunixin (5-Flu), as the main metabolic marker of the nonsteroidal anti-inflammatory drug flunixin, is easily found in animal-derived foods such as milk. Long-term intake can cause health risks such as allergic reactions, gastrointestinal damage, and kidney toxicity in humans. Therefore, establishing a highly sensitive detection method for 5-hydroxyflunixin is of great significance for food safety supervision.

[0003] Currently, the main methods for detecting 5-hydroxyflunixin include instrumental analysis and immunoassay. Instrumental analysis methods, such as high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), offer advantages such as high sensitivity and specificity, but they rely on expensive instruments, require cumbersome sample pretreatment, and demand specialized personnel, making them unsuitable for rapid on-site screening. Immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA), have become the mainstream technology in rapid food safety testing due to their high throughput, ease of operation, and low cost. However, traditional ELISA relies on natural enzymes to catalyze substrate signal generation, which has inherent limitations such as limited sensitivity, poor stability of natural enzymes, and susceptibility to environmental factors. To overcome these bottlenecks, enzyme cascade reactions have attracted widespread attention as a highly efficient signal amplification strategy. This strategy, through the sequential execution of multiple enzymatic reactions, can achieve signal amplification of several orders of magnitude within milliseconds, significantly improving detection sensitivity. Traditional enzyme cascade reactions often employ combinations of two or more natural enzymes, but such systems still fail to completely overcome the inherent instability, easy inactivation, and stringent storage conditions of natural enzymes, limiting their robustness and applicability in practical rapid detection.

[0004] In recent years, the rise of nanozymes has provided a new approach to overcome the shortcomings of natural enzymes. Nanozymes are a class of nanomaterials with enzyme-like catalytic activity, possessing advantages such as low cost, high stability, tunable catalytic activity, and ease of large-scale preparation. Constructing a "natural enzyme-nanozyme" hybrid cascade reaction system by cascading natural enzymes and nanozymes can retain the high substrate specificity and efficient catalytic ability of natural enzymes while leveraging the strong stability and cyclic catalytic properties of nanozymes to achieve multiple signal amplification and long-term stable output.

[0005] Among numerous nanozyme materials, metal-organic frameworks (MOFs) are widely used in immunoassay due to their high specific surface area, tunable pore structure, and excellent enzyme-mimicking activity. HKUST-1, a typical Cu-based MOF, exhibits highly efficient peroxidase-mimicking activity and significantly better environmental stability than natural enzymes, making it a preferred material for replacing natural enzymes or enhancing signals. While there are reports on using MOFs as enzyme mimics in immunoassays, there are still significant shortcomings in the design of compatibility with 5-hydroxyflunixin detection and ELISA systems. Specifically, some methods directly conjugate MOFs (including HKUST-1) as signal markers to antibodies, replacing natural enzymes in antigen-antibody reactions. However, this method suffers from performance degradation after material-antibody binding, making it difficult to further improve detection sensitivity and increasing detection costs, failing to meet the needs of detecting extremely low concentrations of residual antibodies. Furthermore, these methods still rely on specialized equipment such as ELISA readers and electrochemical workstations to read signals, failing to solve the problem of portable signal quantification and overcoming the equipment limitations of on-site detection. More importantly, existing technologies have not established an efficient signal amplification bridge between the immune response and the activity of the HKUST-1 mimic enzyme, specifically targeting the reaction characteristics of the ELISA system. In particular, they have not explored the sequential reaction logic of adding HKUST-1 after alkaline phosphatase (ALP) catalyzes the reaction of pyrophosphate (PPi), thus failing to construct a multi-level signal amplification system through reaction timing regulation.

[0006] In summary, existing detection methods have shortcomings in terms of sensitivity, stability, portability, and cost control. There is an urgent need to develop a rapid, sensitive, accurate, and on-site detection method for 5-hydroxyflunixin to ensure food safety, regulate the rational use of veterinary drugs, and strengthen food supervision. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a method and application for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction.

[0008] The first objective of this invention is to provide a method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction.

[0009] A second objective of this invention is to provide the application of the above method in the qualitative and / or quantitative detection of 5-hydroxyflunixin.

[0010] To achieve the above objectives, the present invention is implemented through the following solution: This invention claims protection for a method for detecting 5-hydroxyflunixin using a metal-organic framework enzyme cascade reaction, comprising the following steps: S1. Add the coating agent at a concentration of 0.1-2 μg / mL to the enzyme-labeled wells at a rate of 50-150 μL / well, incubate thoroughly, block, and dry to obtain the coated enzyme-labeled plate; The coating material is 5-hydroxyflunixin-BSA; the preparation method of 5-hydroxyflunixin-BSA is as follows: using the active ester method, 5-hydroxyflunixin with carboxyl active sites is covalently coupled with BSA carrier protein, and then purified by dialysis. S2. Add the sample to be tested and 5-hydroxyflunixin monoclonal antibody at a concentration of 1-5 μg / mL to the coated enzyme-labeled wells obtained in step S1 at a rate of 25-75 μL / well. After incubation, discard the liquid. Then add enzyme-labeled antibody at a rate of 50-150 μL / well. After sufficient reaction, wash to obtain the enzyme-labeled well plate after sample loading. The 5-hydroxyflunixin monoclonal antibody was obtained by immunizing animals with 5-hydroxyflunixin-OVA as an immunogen. The preparation method of the 5-hydroxyflunixin-OVA is as follows: using the active ester method, 5-hydroxyflunixin with a carboxyl active site is covalently coupled to the OVA carrier protein, and then purified by dialysis to obtain the product. S3. Add 50-150 μL of 0.1-0.2 mM PPi solution to each well of the enzyme-labeled plate after sample loading, and incubate at 25-45 °C for 15-40 min. Then add 5-30 μL of 0.5-2 mg / mL metal-organic framework solution to each well and allow it to react completely. Next, add 5-30 μL of mixed substrate solution to each well and allow it to react completely to obtain the enzyme-labeled plate to be tested. The mixed substrate contains o-phenylenediamine (OPD) at a final concentration of 1–5 mM and hydrogen peroxide (H2O2) at a volume concentration of 0.005–0.02%. S4. Under light irradiation at an excitation wavelength of 365 nm, obtain the RGB color values ​​in the enzyme label wells to be tested, and detect 5-hydroxyflunixin in the sample by combining the standard curve shown in Formula I. Formula I: y=0.1+{0.3 / [1+(x / 2.65)] 0.83}; Where y is R / (R+G+B) and x is the concentration of 5-hydroxyflunixin.

[0011] Preferably, in step S1, the coating agent with a concentration of 0.5 μg / mL is added to the enzyme-labeled well at a rate of 50–150 μL / well.

[0012] More preferably, in step S1, the coating agent with a concentration of 0.5 μg / mL is added to the enzyme-labeled well at a rate of 100 μL / well.

[0013] Preferably, the full incubation in step S1 is incubation at 25–45 °C for 6–18 h.

[0014] More preferably, the full incubation is incubation at 37 °C for 12 h.

[0015] Preferably, the sealing is performed using skim milk powder.

[0016] More preferably, skim milk powder with a mass fraction of 6% is sealed at 37 °C for 3 h.

[0017] Preferably, in step S2, the sample to be tested and 5-hydroxyflunixin monoclonal antibody at a concentration of 1-5 μg / mL are added to the coated enzyme-labeled wells obtained in step S1 at a rate of 50 μL / well.

[0018] Preferably, the full incubation in step S2 is incubation at 25–45 °C for 15–50 min.

[0019] More preferably, the full incubation in step S2 is incubation at 37 °C for 30 min.

[0020] Preferably, in step S2, enzyme-labeled antibody is added at a rate of 100 μL / well.

[0021] More preferably, the enzyme-labeled antibody is an ALP-labeled goat anti-mouse secondary antibody.

[0022] Preferably, the complete reaction in step S2 is an incubation at 25–35 °C for 15–50 min.

[0023] More preferably, the complete reaction in step S2 is an incubation at 37 °C for 30 min.

[0024] Preferably, the washing in step S2 is performed using PBST buffer.

[0025] Preferably, in step S3, 0.175 mM PPi solution is added to the enzyme-labeled plate at a rate of 50–150 μL / well.

[0026] More preferably, in step S3, 0.175 mM PPi solution is added to the microplate after sample loading at a rate of 100 μL / well.

[0027] Preferably, in step S3, the reaction is carried out at 37 °C for 30 min.

[0028] Preferably, in step S3, a metal-organic framework solution of 0.5–2 mg / mL is added at a rate of 20 μL / well to allow for complete reaction.

[0029] More preferably, a metal-organic framework solution of 1.5 mg / mL is added at a rate of 20 μL / well and allowed to react completely.

[0030] More preferably, the method for preparing the metal-organic framework is as follows: S31. Copper nitrate trihydrate {Cu(NO3)2·3H2O} and polyvinylpyrrolidone (PVP) were mixed at a mass ratio of 0.5-1:0.2-0.5. After the mixture was fully reacted, pyromellitic acid (H3BTC) was added and the mixture was reacted at 25-40 °C for 12-24 h. The precipitate was collected by solid-liquid separation, washed and dried to obtain the metal-organic framework HKSUT-1. The quality ratio of H3BTC to PVP is 0.1–0.5: 0.2–0.5; S32. Dissolve the metal-organic framework obtained in step S31 in water to obtain a metal-organic framework solution.

[0031] Preferably, in step S31, Cu(NO3)2·3H2O and PVP are mixed at a mass ratio of 8.75:4.

[0032] Preferably, in step S31, the mass ratio of H3BTC to PVP is 2.1:4.

[0033] Preferably, in step S31, the mixture is stirred at 25 °C for 24 h.

[0034] This invention also claims protection for the use of any of the methods described above in the qualitative and / or quantitative detection of 5-hydroxyflunixin.

[0035] Compared with the prior art, the present invention has the following beneficial effects: This application relates to a method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction. This method utilizes the peroxidase-mimicking activity of the metal-organic framework HKSUT-1 and the regulatory effect of PPi / ALP on its activity to construct a natural enzyme-nanozyme cascade signal amplification pathway. Specifically, PPi inhibits the enzymatic catalytic activity of HKSUT-1 by complexing it with copper ions, and this inhibition is concentration-dependent; ALP catalyzes the decomposition of PPi, reducing the concentration of PPi in the system, thereby weakening its inhibitory effect on the catalytic activity of HKSUT-1 and achieving precise signal regulation. The method combines the enzyme cascade reaction with a competitive immunoassay, achieving a detection limit of 0.12 ng / mL for 5-hydroxyflunixin, which is 7.42 times more sensitive than the traditional ELISA method. The method also has a quantification limit of 0.51 ng / mL and a linear range of 0.51–14.08 ng / mL, with sensitivity, accuracy, and precision meeting the requirements for residue detection. When applied to milk sample testing, the method allows for direct analysis without extraction, and the detection system is simple and easy to standardize. Attached Figure Description

[0036] Figure 1 This is a scanning electron microscope image of the metal-organic framework HKUST-1 in Example 1; Figure 2 This is an X-ray diffraction diagram of the metal-organic framework HKUST-1 in Example 1; Figure 3 The following are the X-ray photoelectron analysis results of the metal-organic framework HKUST-1 in Example 1: a is the overall XPS spectrum; b is the Cu 2p high-resolution narrow spectrum; c is the O 1s high-resolution narrow spectrum; d is the C 1s high-resolution narrow spectrum; e is the N 1s high-resolution narrow spectrum. Figure 4 This is a graph showing the stability measurement results of the fluorescence signal parameters in Example 3; Figure 5 The graph shows the sensitivity test results in Example 3; Figure 6 Figure a shows the sample adaptability results in Example 3; a is the result of simultaneous detection of 5-hydroxyflunixin in 10 milk samples based on metal-organic framework enzyme cascade reaction and LC-MS / MS; b is the correlation analysis of the detection results of the two methods. Figure 7 The graph shows the sensitivity results of HRP-ELISA for detecting 5-hydroxyflunixin in Comparative Example 1. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0038] Example 1: Preparation and Characterization of the Metal-Organic Framework HKUST-1 I. Experimental Methods 0.875 g of Cu(NO3)2·3H2O and 0.4 g of PVP were dispersed in 50 mL of methanol solution and stirred for 30 min to mix evenly. Then, 50 mL of methanol solution containing 0.42 g of H3BTC was added and stirred continuously at 25 °C for 24 h. The blue precipitate was then collected by centrifugation and washed three times with methanol. After washing, the precipitate was dried at 60 °C to obtain the metal-organic framework HKUST-1.

[0039] The prepared metal-organic framework HKUST-1 was observed using scanning electron microscopy, and the scanning electron microscopy results were recorded. The metal-organic framework HKUST-1 was analyzed using X-ray photoelectron spectroscopy (XPS). The specific procedure was as follows: The prepared metal-organic framework HKUST-1 was placed in the sample chamber of a Thermo Scientific Nexsa XPS instrument, with the pressure in the sample chamber less than 3.0 × 10⁻⁶. -7 At mbar, the prepared metal-organic framework HKUST-1 was sent into the analysis chamber with a spot size of 400 μm; the full spectrum scan pass energy was 200 eV with a step size of 1 eV; the narrow spectrum scan pass energy was 50 eV with a step size of 0.1 eV, and the X-ray photoelectron spectroscopy characterization results were recorded.

[0040] The prepared metal-organic framework HKUST-1 was analyzed using X-ray diffraction (XRD) technology. The specific operation was as follows: the prepared metal-organic framework HKUST-1 was placed in a Bruker D8 ADVANCE instrument, the scanning angle range was 5° to 60°, the scanning speed was 4° / min, and the testing time was 10 to 15 min.

[0041] II. Experimental Results Scanning electron microscopy results of the metal-organic framework HKUST-1 are as follows: Figure 1 As shown in the figure, the results show that HKUST-1 exhibits a typical octahedral microcrystalline structure with a size of about 500 nm, a smooth surface with sharp edges and corners, and good crystallinity.

[0042] XRD results of the metal-organic framework HKUST-1 are as follows Figure 2 As shown. The results show that the diffraction peaks of HKUST-1 appear at 6.90°, 9.70°, 11.83°, 13.60°, 17.68° and 19.2°. These sharp and clear peaks correspond to the (200), (220), (331), (222), (400) and (600) crystal planes of the product, respectively. The results indicate that the prepared HKUST-1 is a pure phase, and the sharp and clear peaks show the high crystallinity of HKUST-1.

[0043] XPS analysis results of the metal-organic framework HKUST-1 are as follows: Figure 3 As shown, Figure 3 In this context, 'a' represents the XPS total spectrum. Figure 3 In the image, b represents a high-resolution narrow spectrum of Cu 2p. Figure 3 c in the image represents the high-resolution narrow spectrum of the O 1s spectral region. Figure 3 In the image, d represents the high-resolution narrow spectrum of C 1s. Figure 3 In the image, 'e' represents the N 1s high-resolution narrow spectrum.

[0044] The results showed that the XPS full spectrum of HKUST-1 (a) confirmed the presence of four elements, Cu, O, N, and C, consistent with the chemical composition of HKUST-1; the Cu 2p spectrum (b) showed two main peaks at 934.6 eV and 954.4 eV, corresponding to Cu 2p... 3 / 2 and Cu 2p 1 / 2 Meanwhile, a distinct satellite peak appears at 942-944 eV, confirming that Cu... 2+ Successful coordination with the pyromellitic acid ligand formed the HKUST-1 framework structure; the O 1s spectrum (c) was fitted with two peaks, the main peak at 531.8 eV corresponding to the O–C=O bond in the ligand H3BTC, and the shoulder peak at 533.2 eV belonging to the –OH or C–O bond adsorbed on the material surface; the C 1s spectrum (d) could be fitted with three peaks, of which the peak at 288.6 eV was assigned to the C=O bond, the peak at 286.3 eV to the C–O bond, and the peak at 284.8 eV to the C–C bond, consistent with the chemical structure of the ligand H3BTC; the single peak in the N 1s spectrum (e) at 400.1 eV corresponds to the residual PVP or the N–H bond in the ligand.

[0045] The results above demonstrate that the metal-organic framework HKUST-1 material was successfully synthesized.

[0046] Example 2: Establishment of a method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction. I. Establishment of the Standard Curve 1. Experimental Methods Solid-phase carrier coating: The coating precursor (5-hydroxyflunixin-BSA) was dissolved in carbonate (CB) buffer (pH 9.6, 50 mM, prepared by dissolving 1.69 g of Na2CO3 and 2.95 g of NaHCO3 in 1000 mL of ultrapure water) to prepare a coating solution of 0.5 μg / mL. Then, 100 μL / well was added to the microplate and incubated in a 37 ℃ water bath for 12 h. After incubation, the liquid in the wells of the microplate was discarded, and the plates were washed twice with phosphate (PBST) buffer {pH 7.4, 10 mM, containing 0.05% (v / v) Tween-20}. After washing, blocking buffer (6 wt% skim milk powder) was added to 120 μL / well and the plates were blocked at 37 ℃ for 3 h. After blocking, the liquid in the wells was discarded, the plates were patted dry, and then dried at 37 ℃ for 30 min to obtain the coated microplates. The preparation method of 5-hydroxyflunixin-BSA is as follows: using the active ester method, 5-hydroxyflunixin with a carboxyl active site is covalently coupled to BSA carrier protein, and 5-hydroxyflunixin-BSA is obtained after dialysis purification.

[0047] Establishment of the immune competitive reaction: Milk samples containing a series of concentrations (0.03, 0.06, 0.12, 0.24, 0.48, 0.97, 1.95, 3.90, 7.81, 15.62, 31.25, 62.5, 125, 250, 350 ng / mL) of 5-hydroxyflunixin standard (VETRANAL brand, catalog number 32463) were added to each well of a coated ELISA plate. Then, 50 μL of 5-hydroxyflunixin monoclonal antibody (3 μg / mL, obtained by immunizing experimental animals with 5-hydroxyflunixin-OVA, prepared by covalently coupling 5-hydroxyflunixin with a carboxyl active site to an OVA carrier protein using the active ester method, followed by dialysis purification) was added to each well. The plate was then incubated at 37°C. After incubation at ℃ for 30 min, discard the liquid in the wells, wash 5 times with PBST buffer, pat dry, and add 100 μL / well of ALP-labeled goat anti-mouse secondary antibody (1:1000 dilution, Beyotime, catalog number A0258). Incubate in a 37 ℃ water bath for 30 min. After incubation, wash 5 times with PBST buffer and pat dry to obtain microplates containing different concentrations of 5-hydroxyflunixin standard.

[0048] Generation of cascade signals: 100 μL of 0.175 mM PPi solution was added to each well of an ELISA plate containing different concentrations of 5-hydroxyflunixin standard. The plate was reacted in a 37 °C water bath for 30 min. Then, 1.5 mg / mL HKUST-1 solution (prepared by dissolving the metal-organic framework HKUST-1 obtained in Example 1 in ultrapure water) was added to each well. After mixing thoroughly, the plate was incubated at 25 °C for 10 min. Then, 20 μL of mixed substrate solution was added to each well, and the plate was reacted for 15 min to obtain the ELISA plate to be tested. The mixed substrate solution was a mixed solution containing 3 mM OPD and 0.01 v / v% H2O2; Signal reading and standard curve establishment: The enzyme-labeled plate to be tested was transferred to a dark box and irradiated with light at an excitation wavelength of 365 nm. The plate was photographed under light irradiation, and ImageJ was used to analyze the RGB (red, green, blue) color values ​​of the enzyme-labeled wells corresponding to each concentration of 5-hydroxyflunixin standard. A standard curve was constructed with the concentration of 5-hydroxyflunixin standard as the x-axis and the R / (R+G+B) value of 5-hydroxyflunixin standard in the enzyme-labeled wells at that concentration as the y-axis, and the formula for the standard curve was given.

[0049] 2. Experimental Results Following the steps above, after reacting with a series of concentrations of 5-hydroxyflunixin standards, the standard curve for detecting 5-hydroxyflunixin is shown in Formula I. Formula I: y=0.1+{0.3 / [1+(x / 2.65)] 0.83}; Where y is the R / (R+G+B) value and x is the concentration of 5-hydroxyflunixin.

[0050] II. A method for detecting 5-hydroxyflunixin based on metal-organic framework enzyme cascade reaction A method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction includes the following steps: S1. The coating agent (5-hydroxyflunixin-BSA) was dissolved in CB buffer (pH 9.6, 50 mM, prepared by dissolving 1.69 g of Na2CO3 and 2.95 g of NaHCO3 in 1000 mL of ultrapure water) to prepare a coating solution of 0.5 μg / mL. Then, 100 μL / well was added to the microplate and incubated in a 37 ℃ water bath for 12 h. After incubation, the liquid in the wells was discarded, and the wells were washed twice with PBST buffer {pH 7.4, 10 mM, containing 0.05% (v / v) Tween-20}. After washing, 120 μL / well was added with blocking buffer (6 wt% skim milk powder), and the wells were blocked at 37 ℃ for 3 h. After blocking, the liquid in the wells was discarded, the wells were patted dry, and the wells were dried at 37 ℃ for 30 min to obtain the coated microplate. S2. In the coated microplate obtained in step S1, add the sample to be tested at 50 μL / well, followed by 50 μL / well of 5-hydroxyflunixin monoclonal antibody (3 μg / mL, obtained by immunizing experimental animals with 5-hydroxyflunixin-OVA, wherein the preparation method of 5-hydroxyflunixin-OVA is: using the active ester method, 5-hydroxyflunixin with carboxyl active site is covalently coupled with OVA carrier protein, and 5-hydroxyflunixin-OVA is obtained after dialysis purification). After incubation at 37 ℃ for 30 min, discard the liquid in the well, wash 5 times with PBST buffer, pat dry, and add 100 μL / well of ALP-labeled goat anti-mouse secondary antibody (1:1000 dilution). Incubate in a water bath at 37 ℃ for 30 min. After incubation, wash 5 times with PBST buffer and pat dry to obtain the microplate containing the sample to be tested. S3. In the enzyme-labeled plate containing the sample to be tested obtained in step S2, add 100 μL of 0.175 mM PPi solution to each well and react in a 37 ℃ water bath for 30 min. Then, add 20 μL of 1.5 mg / mL HKUST-1 solution (prepared by dissolving the metal-organic framework HKUST-1 obtained in Example 1 in ultrapure water), mix well, and incubate at 25 ℃ for 10 min. Then, add 20 μL of mixed substrate solution to each well and react for 15 min to obtain the enzyme-labeled plate to be tested. The mixed substrate solution was a mixed solution containing 3 mM OPD and 0.01 v / v% H2O2; S4. Transfer the enzyme-labeled plate to be tested to a dark box, irradiate it with light at an excitation wavelength of 365 nm, take a picture of the enzyme-labeled plate under light irradiation, and use ImageJ software to analyze the RGB (red, green, blue) color values ​​of the enzyme-labeled wells of the sample to be tested; Based on the RGB color values ​​of the enzyme-labeled wells of the sample to be tested, the R / (R+G+B) value is calculated and combined with the standard curve shown in Formula I to calculate the concentration of 5-hydroxyflunixin in the sample to be tested.

[0051] Example 3 Performance evaluation of a method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction I. Experimental Methods 1. Stability of fluorescence signal parameters The stability of the fluorescence intensity parameters was verified through batch-to-batch repeatability testing. HKUST-1, OPD, and H2O2 were diluted to working concentrations (0.5 mg / mL HKUST-1, 3 mM OPD, and 0.01 v / v% H2O2), respectively. Detection was performed according to the method described in Example 2, "A Method for Detecting 5-Hydroxyflunixin Based on a Metal-Organic Framework Enzyme Cascade Reaction." Fluorescence intensity was recorded using a smartphone, and the RGB values ​​of the images were extracted using ImageJ software. Eight parallel reactions were set up.

[0052] The fluorescence intensity of 8 parallel reaction systems was recorded, and the R / (R+G+B) value and R+G value of the 8 parallel reaction systems were calculated and compared. The standard relative deviation (RSD) value between the data was used as an indicator to evaluate the signal stability. The lower the RSD value, the more stable the signal.

[0053] 2. Sensitivity Test Different concentrations (0.03, 0.06, 0.12, 0.24, 0.48, 0.97, 1.95, 3.90, 7.81, 15.62, 31.25, 62.5, 125, 250, 350 ng / mL) of 5-hydroxyflunixin were added to different milk samples, and the assays were performed in parallel three times according to the method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction as shown in Example 2. The limit of detection (LOD), limit of quantitation (LOQ), and linear detection range were determined. All milk samples were purchased from the local market in Guangzhou and were confirmed to be free of 5-hydroxyflunixin residue by LC-MS / MS. The standard curve was plotted with 5-hydroxyflunixin concentration on the x-axis and R / (R+G+B) value on the y-axis. LOD and LOQ were defined as the mean of 20 blank sample measurements plus 3 or 10 times the standard deviation, respectively; the inhibition rate on the standard curve was 20% to 80% (IC50). 20 -IC 80 The concentration range corresponding to ) is used as the linear detection range of this method.

[0054] 3. Accuracy and precision testing For milk samples, three different concentrations (0.51, 20, and 40 ng / mL) of 5-hydroxyflunixin standard were prepared and added to construct a spiked sample system. All milk samples were purchased from the local market in Guangzhou and were confirmed to have no 5-hydroxyflunixin residue by LC-MS / MS. According to the method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction as shown in Example 2, three parallel determinations were performed for each concentration gradient. The recovery rate was calculated according to Formula II, and the coefficient of variation (CV) was calculated according to Formula III to evaluate the accuracy and precision. Formula II: Recovery rate (%) = Actual detected concentration / Spiked concentration × 100%; Formula III: CV (%) = standard deviation / mean × 100%.

[0055] 4. Stability test (1) Accelerated stability test The coated ELISA plates from Example 2 were placed in sealed bags containing desiccant and stored at 37 °C. They were retrieved on days 0, 7, 14, 21, and 28, and tested with milk samples spiked at concentrations of 0 and 2 ng / mL, according to the method described in Example 2, "A method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction." The inhibition rate was calculated, and the stability under accelerated conditions was evaluated. Milk samples spiked at 0 ng / mL were used as negative samples during the testing process. All milk samples were purchased from the local market in Guangzhou and were confirmed to be free of 5-hydroxyflunixin residue by LC-MS / MS.

[0056] (2) Room temperature stability test The coated ELISA plates from Example 2 were placed in sealed bags containing desiccant and stored at 25 °C. They were retrieved on days 0, 7, 14, 21, and 28, and milk samples with spiked concentrations of 0 and 2 ng / mL were tested according to the method described in Example 2, "A Method for Detecting 5-Hydroxyflunixin Based on a Metal-Organic Framework Enzyme Cascade Reaction." The inhibition rate was calculated, and the stability at room temperature (25 °C) was evaluated. Milk samples with a spiked concentration of 0 ng / mL were used as negative samples during the testing process.

[0057] (3) Long-term stability test The coated ELISA plates from Example 2 were placed in sealed bags containing desiccant and stored at 4 °C. They were retrieved on days 0, 30, 60, 90, 120, 150, 180, 210, and 240, respectively. Milk samples with spiked concentrations of 0 and 2 ng / mL were tested according to the method described in Example 2, "A Method for Detecting 5-Hydroxyflunixin Based on a Metal-Organic Framework Enzyme Cascade Reaction." The inhibition rate was calculated according to Formula IV to assess its stability during long-term storage. Milk samples with a spiked concentration of 0 ng / mL were used as negative samples during the testing process. Formula IV: IR (%) = (negative - positive) / negative × 100%.

[0058] 5. Sample adaptability To assess the sample suitability of this method, 10 milk samples randomly purchased from the Guangzhou market were used in the experiment, and LC-MS / MS was used to confirm that the milk samples did not contain 5-hydroxyflunixin.

[0059] Ten milk samples were randomly added with 5-hydroxyflunixin (0, 5, or 10 ng / mL of 5-hydroxyflunixin were added randomly). The samples were then detected in parallel by LC-MS / MS according to the method described in Example 2, “A method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction”. Each sample was measured three times, and the consistency of the detection results of the two methods was compared.

[0060] The LC-MS / MS method is operated as follows: Milk samples are tested according to the "SN / T 2190-2008 Method for the Determination of Non-steroidal Anti-inflammatory Drug Residues in Imported and Exported Animal-Derived Foods - Liquid Chromatography-Mass Spectrometry / Mass Spectrometry"; The ultra-high performance liquid chromatography (UPLC) conditions are as follows: Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid. A gradient elution program was used, with a total run time of 5.5 min and a constant flow rate of 0.3 mL / min. The gradient settings were as follows: 0–0.5 min, 10% B; 0.5–2.5 min, linearly up to 100% B; 2.5–4.5 min, maintain 100% B; 4.5–5.0 min, return to 10% B, and then equilibrate to 5.0 min. The injection volume was 10 μL, the column temperature was 40 °C, and the column was a Waters BEHC18 column (50 mM × 2.1 mM, 1.7 μm).

[0061] Mass spectrometry detection was performed using an electrospray ionization source in positive ion mode with multiple reaction monitoring. The quantitative ion pair was m / z 313.11 > 279.95, the qualitative ion pair was m / z 313.11 > 295.05, the declustering voltage was 40 V, and the collision energies were 33 eV and 22 eV, respectively.

[0062] II. Experimental Results 1. Stability of fluorescence signal parameters The results of the fluorescence signal stability measurement are as follows: Figure 4 As shown, the results indicate that for the 8 parallel reactions, the RSD of R+G is 5.1%, while the RSD of R / (R+G+B) is 0.1%. The R / (R+G+B) value is more stable than the R+G value, indicating that the normalized ratio parameter has better repeatability and stability, and can ensure the stability of the output results of the detection method shown.

[0063] 2. Sensitivity Test Sensitivity test results as follows Figure 5 As shown, the results indicate that the method shown in Example 2 has a low LOD of 0.12 ng / mL and a LOQ of 0.51 ng / mL for 5-hydroxyflunixin, and the quantitative linear range is 0.51–14.08 ng / mL, indicating that the method shown in Example 2 has excellent detection sensitivity for 5-hydroxyflunixin.

[0064] 3. Accuracy and precision testing The accuracy and precision test results of the detection of 5-hydroxyflunixin based on metal-organic framework enzyme cascade reaction are shown in Table 1.

[0065] Table 1. Accuracy and precision test results of 5-hydroxyflunixin detection based on metal-organic framework enzyme cascade reaction.

[0066] The results showed that when 5-hydroxyflunixin was detected in milk samples using the method shown in Example 2, the spiked recovery rate reached 94.1%–111.2%, and the variation rate was 4.6%–7.2%, indicating that the method shown in Example 2 has excellent accuracy and precision in detecting 5-hydroxyflunixin.

[0067] 4. Stability Results The stability results of 5-hydroxyflunixin based on metal-organic framework enzyme cascade reaction are shown in Table 2.

[0068] Table 2. Results of stability assay for 5-hydroxyflunixin based on metal-organic framework enzyme cascade reaction.

[0069] The results showed that after 28 days of storage at 37 ℃ and 25 ℃, the fluorescence intensity of both negative and positive samples did not change significantly, and the inhibition rate remained within 3%, indicating that the inhibition rate remained stable. Under storage conditions at 4 ℃, although the colorimetric response decreased to some extent with increasing storage time, the fluctuation in the inhibition rate was still controlled within 3%. In conclusion, the established method has good stability and can maintain excellent detection performance for 90 days under storage conditions at 4 ℃.

[0070] 5. Sample adaptability Sample fitness results as follows Figure 6 As shown, Figure 6 Figure 'a' in the graph shows the results of simultaneous detection of 5-hydroxyflunixin in 10 milk samples based on metal-organic framework enzyme cascade reaction and LC-MS / MS. Figure 6 Figure b in the figure represents the correlation analysis of the detection results of the two methods. The results show that: the method for detecting 5-hydroxyflunixin based on metal-organic framework enzyme cascade reaction as shown in Example 2 and the simultaneous analysis of 10 milk samples by LC-MS / MS showed good correlation between the data obtained by the two detection methods, with a correlation coefficient of 0.991. This indicates that the method has excellent reliability and can be applied to the rapid detection of 5-hydroxyflunixin in milk samples.

[0071] Comparative Example 1: A method for detecting 5-hydroxyflunixin based on HRP-ELISA I. Establishment of Standard Curve 1. Experimental Methods Solid-phase carrier coating: The coating precursor (5-hydroxyflunixin-BSA) was dissolved in CB buffer (pH 9.6, 50 mM, prepared by dissolving 1.69 g of Na2CO3 and 2.95 g of NaHCO3 in 1000 mL of ultrapure water) to prepare a coating solution of 0.1 μg / mL. Then, 100 μL / well was added to the microplate and incubated in a 37 ℃ water bath for 12 h. After incubation, the liquid in the wells of the microplate was discarded, and the plate was washed twice with PBST buffer {pH 7.4, 10 mM, containing 0.05% (v / v) Tween-20}. After washing, blocking buffer (6 wt% skim milk powder) was added to 120 μL / well and the plate was blocked at 37 ℃ for 3 h. After blocking, the liquid in the wells was discarded, the plate was patted dry, and then dried at 37 ℃ for 30 min to obtain the coated microplate 1. The preparation method of 5-hydroxyflunixin-BSA is as follows: using the active ester method, 5-hydroxyflunixin with a carboxyl active site is covalently coupled to BSA carrier protein, and 5-hydroxyflunixin-BSA is obtained after dialysis purification.

[0072] Establishment of the competitive immune response: In a coated ELISA plate (well 1), 50 μL of milk sample containing a series of concentrations (0.05, 0.10, 0.20, 0.40, 0.80, 1.60, 3.13, 6.25, 12.5, 25, 50, 75, 100, 200 ng / mL) of 5-hydroxyflunixin standard (VETRANAL brand, catalog number 32463) was added to each well. Then, 50 μL of 5-hydroxyflunixin monoclonal antibody (0.05 μg / mL, obtained by immunizing experimental animals with 5-hydroxyflunixin-OVA, prepared by covalently coupling 5-hydroxyflunixin with a carboxyl active site to an OVA carrier protein using the active ester method, followed by dialysis purification) was added to each well. The plate was incubated at 37 ℃ for 30 minutes. After 1 minute, discard the liquid in the wells, wash 5 times with PBST buffer, pat dry, and add horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (1:5000 dilution, TransGold, catalog number HS201-01) at 100 μL / well. Incubate in a 37 ℃ water bath for 30 minutes. After incubation, wash 5 times with PBST buffer and pat dry to obtain ELISA plates 1 containing different concentrations of 5-hydroxyflunixin standard.

[0073] Catalytic signal generation: TMB chromogenic solution (TransGold, catalog number HE101-01) was added to ELISA plate 1 containing different concentrations of 5-hydroxyflunixin standard at a rate of 100 μL / well, and the plate was incubated in a 37 ℃ water bath for 10 min.

[0074] Signal reading and standard curve establishment: After adding 50 μL of stop solution (10% sulfuric acid solution) to each well, the plate to be tested was immediately transferred to a microplate reader (Thermo Fisher Scientific, model MultiskanFC). The absorbance of the solution in the well at 450 nm was read and recorded as B. Milk samples that did not contain 5-hydroxyflunixin were recorded as negative samples, and the absorbance of the negative samples was recorded as B0.

[0075] A standard curve was constructed using the concentration of 5-hydroxyflunixin standard in milk samples as the x-axis and the B / B0 value as the y-axis, and the formula for the standard curve was given.

[0076] 2. Experimental Results Following the steps above, when using milk samples containing a series of concentrations of 5-hydroxyflunixin standards in combination with HRP-ELISA for reaction, the standard curve for detecting 5-hydroxyflunixin is shown in Equation V. Formula V: y=0.09+{0.86 / [1+(x / 8.02)]}; Where y is the B / B0 value and x is the concentration of 5-hydroxyflunixin.

[0077] II. HRP-ELISA-based method for detecting 5-hydroxyflunixin The method for detecting 5-hydroxyflunixin based on HRP-ELISA includes the following steps: S1. The coating precursor (5-hydroxyflunixin-BSA) was dissolved in CB buffer (pH 9.6, 50 mM, prepared by dissolving 1.69 g of Na2CO3 and 2.95 g of NaHCO3 in 1000 mL of ultrapure water) to prepare a coating solution of 0.1 μg / mL. Then, 100 μL / well was added to the microplate and incubated in a 37 ℃ water bath for 12 h. After incubation, the liquid in the wells was discarded, and the wells were washed twice with PBST buffer {pH 7.4, 10 mM, containing 0.05% (v / v) Tween-20}. After washing, 120 μL / well was added with blocking buffer (6 wt% skim milk powder), and the wells were blocked at 37 ℃ for 3 h. After blocking, the liquid in the wells was discarded, the wells were patted dry, and the wells were dried at 37 ℃ for 30 min to obtain coated microplate 1. S2. In the coated microplate 1 obtained in step S1, add 50 μL of the sample to be tested per well, followed by 50 μL of 5-hydroxyflunixin monoclonal antibody (0.05 μg / mL, obtained by immunizing experimental animals with 5-hydroxyflunixin-OVA, wherein the preparation method of 5-hydroxyflunixin-OVA is: using the active ester method, 5-hydroxyflunixin with carboxyl active site is covalently coupled with OVA carrier protein, and 5-hydroxyflunixin-OVA is obtained after dialysis purification). After incubating at 37℃ for 30 min, discard the liquid in the well, wash 5 times with PBST buffer, pat dry, and add 100 μL of HRP-labeled goat anti-mouse secondary antibody (1:1000 dilution) per well. Incubate in a 37℃ water bath for 30 min. After incubation, wash 5 times with PBST buffer and pat dry to obtain microplate 1 with the sample to be tested. S3. In the enzyme-labeled plate 1 containing the sample to be tested obtained in step S2, add TMB chromogenic solution (Full Gold, catalog number HE101-01) at 100 μL / well and incubate in a 37 ℃ water bath for 10 min; S4. After adding 50 μL of stop solution (10% sulfuric acid solution) to each well, immediately transfer the plate to be tested to a microplate reader (Thermo Fisher Scientific, Multiskan FC). Use the microplate reader to read the absorbance of the solution in the well at 450 nm and record it as B; at the same time, use a milk sample without 5-hydroxyflunixin as a negative sample and record the absorbance of the negative sample at 450 nm after processing as shown above (B0). The concentration of 5-hydroxyflunixin in the sample was calculated by combining B / B0 with the standard curve shown in Equation V.

[0078] III. Sensitivity Test for Detection of 5-Hydroxyflunixin Based on HRP-ELISA 1. Experimental Methods Different concentrations (0.05, 0.1, 0.20, 0.40, 0.80, 1.6, 3.13, 6.25, 12.5, 25, 50, 75, 100, 200 ng / mL) of 5-hydroxyflunixin were added to different milk samples. Milk samples without 5-hydroxyflunixin were used as negative samples. The above-mentioned "Detection of 5-hydroxyflunixin based on HRP-ELISA" was performed three times in parallel to determine the LOD, LOQ and linear detection range. All milk samples were purchased from the local market in Guangzhou and were confirmed to be free of 5-hydroxyflunixin residue by LC-MS / MS. The standard curve was plotted with the concentration of 5-hydroxyflunixin on the x-axis and the B / B0 value on the y-axis; where B is the absorbance of milk samples with different concentrations of 5-hydroxyflunixin added at a wavelength of 450 nm, and B0 is the absorbance of the negative sample at a wavelength of 450 nm.

[0079] LOD and LOQ are defined as the average of 20 blank sample measurements plus 3 or 10 times the standard deviation, respectively; with an inhibition rate of 20% to 80% on the standard curve (IC50). 20 -IC 80 The concentration range corresponding to ) is used as the linear detection range of this method.

[0080] 2. Experimental Results Sensitivity test results for HRP-ELISA detection of 5-hydroxyflunixin are as follows: Figure 7 As shown, the results indicate that, based on HRP-ELISA detection of 5-hydroxyflunixin, the LOD for 5-hydroxyflunixin in milk samples was 0.89 ng / mL, the LOQ was 2.01 ng / mL, and the quantitative linear range was 2.01–31.88 ng / mL.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting 5-hydroxyflunixin based on a metal-organic framework enzyme cascade reaction, characterized in that, Includes the following steps: S1. Add the coating agent with a concentration of 0.1-2 μg / mL to the microplate at a rate of 50-150 μL / well, incubate thoroughly, block, and dry to obtain the coated microplate. The coating material is 5-hydroxyflunixin-BSA; the preparation method of 5-hydroxyflunixin-BSA is as follows: using the active ester method, 5-hydroxyflunixin with carboxyl active sites is covalently coupled with BSA carrier protein, and then purified by dialysis. S2. Add the test sample and 5-hydroxyflunixin monoclonal antibody at a concentration of 1-5 μg / mL to the coated ELISA plate obtained in step S1 at a rate of 25-75 μL / well. After incubation, discard the liquid. Then add the ELISA antibody at a rate of 50-150 μL / well. After the reaction is complete, wash to obtain the ELISA plate after sample loading. The 5-hydroxyflunixin monoclonal antibody was obtained by immunizing animals with 5-hydroxyflunixin-OVA as an immunogen. The preparation method of the 5-hydroxyflunixin-OVA is as follows: using the active ester method, 5-hydroxyflunixin-OVA with carboxyl active sites is prepared. Hydroxyflunixin is obtained by covalently coupling it with OVA carrier protein and then purifying it by dialysis. S3. Add 0.1–0.2 mM pyrophosphate solution at 50–150 μL / well to the microplate after sample loading, incubate at 25–45 °C for 15–40 min, then add 0.5–2 mg / mL metal-organic framework solution at 5–30 μL / well and react fully. Next, add mixed substrate solution at 5–30 μL / well and react fully to obtain the microplate to be tested. The mixed substrate contains o-phenylenediamine at a final concentration of 1–5 mM and hydrogen peroxide at a volume concentration of 0.005–0.02%. S4. Under light irradiation at an excitation wavelength of 365 nm, obtain the RGB color values ​​in the enzyme label wells to be tested, and detect 5-hydroxyflunixin in the sample by combining the standard curve shown in Formula I. Formula I: y = 0.1 + {0.3 / [1 + (x / 2.65)] 0.83}; Where y is R / (R+G+B) and x is the concentration of 5-hydroxyflunixin.

2. The method according to claim 1, characterized in that, In step S1, the coating agent with a concentration of 0.5 μg / mL is added to the enzyme-labeled well at a rate of 50–150 μL / well.

3. The method according to claim 2, characterized in that, In step S1, the coating agent with a concentration of 0.5 μg / mL is added to the microplate at a rate of 100 μL / well.

4. The method according to claim 1, characterized in that, In step S2, add enzyme-labeled antibody at a rate of 100 μL / well.

5. The method according to claim 1, characterized in that, In step S3, 0.175 mM pyrophosphate solution is added to the microplate after sample loading at a rate of 50–150 μL / well.

6. The method according to claim 5, characterized in that, In step S3, 0.175 mM pyrophosphate solution is added to the microplate at a rate of 100 μL / well.

7. The method according to claim 1, characterized in that, In step S3, a metal-organic framework solution of 0.5–2 mg / mL is added at a rate of 20 μL / well and allowed to react completely.

8. The method according to claim 7, characterized in that, The preparation method of the metal-organic framework solution in step S3 is as follows: S31. Copper nitrate trihydrate and polyvinylpyrrolidone are mixed in a mass ratio of 0.5–1:0.2–0.

5. After the mixture has reacted completely, pyromellitic acid is added and the mixture is reacted at 25–40 °C for 12–24 h. The precipitate is collected by solid-liquid separation, washed, and dried to obtain a metal-organic framework. The mass ratio of pyromellitic acid to polyvinylpyrrolidone is 0.1–0.5:0.2–0.

5. S32. Dissolve the metal-organic framework obtained in step S31 in water to obtain a metal-organic framework solution.

9. The method according to claim 8, characterized in that, In step S31, copper nitrate trihydrate and polyvinylpyrrolidone are mixed at a mass ratio of 8.75:

4.

10. The use of the method according to any one of claims 1 to 9 in the qualitative and / or quantitative detection of 5-hydroxyflunixin.

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