A method for rapidly detecting the content of ractopamine in food

Abstract

The application discloses a kind of analysis methods for rapidly detecting the content of ractopamine in food, belong to food safety detection technical field.The application uses F0F1-ATPase molecular motor biosensor, by determining the chemiluminescence signal of different concentrations RAC standard solution, establishes standard curve;The chemiluminescence signal of the sample solution to be measured is determined after reaction with biosensor, and the content of ractopamine is calculated by substituting into standard curve.The detection limit of the method is as low as 5x10 ‑ 9 mg / kg, and the recovery rate is 87.8%-99.6%, which is highly correlated with the HPLC-MS / MS result.The application has the advantages of rapidness, sensitivity, accuracy, high throughput, etc., and is suitable for quantitative screening detection of ractopamine residues in food.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to an analytical method for rapidly detecting ractopamine content in food. Background Technology

[0002] Ractopamine (RAC) is a β-adrenergic receptor agonist that has been illegally added to animal feed because it can increase lean meat percentage. It easily leaves residues in animal tissues, and long-term intake can lead to poisoning in humans. Most countries have banned its use, but some still permit it; therefore, establishing rapid, sensitive, and accurate detection methods is crucial.

[0003] Current RAC detection technologies are mainly divided into instrumental analysis methods (HPLC-MS / MS, etc.) and immunoassay methods (ELISA, LFIA, etc.). Instrumental methods have high sensitivity but expensive equipment, cumbersome pretreatment, time-consuming, and difficult to achieve high throughput; while immunoassay methods are simple to operate, ELISA / CLIA requires multiple washes and long incubation (more than 1 hour), and LFIA has insufficient sensitivity. Neither of these methods can achieve both speed, ultrasensitivity, and high throughput.

[0004] In recent years, biosensors based on the F0F1-ATPase molecular motor have attracted much attention. This enzyme can be coupled to antibodies via a rotor (ε subunit) or stator (β subunit) to translate antigen recognition into changes in ATP synthesis activity. Stator-loaded modes (such as clenbuterol detection) achieve a sensitivity of up to 10. -12 While the β-subunit loading method results in a high overall load on the F1 cell, significant rotational hindrance, and limited signal transduction efficiency, it also requires continuous recording of fluorescence signal changes during a single detection, making high-throughput rapid screening difficult. In contrast, rotor loading modes (such as the detection of mycotoxins DON) offer advantages in detection time and sensitivity up to 10 g / L. -7 mg / mL, but so far it has not been extended to the detection of β-agonists (such as RAC). Due to the fundamental differences between RAC and DON in terms of molecular structure, physicochemical properties and antibody binding characteristics, directly transferring the rotor regulation mechanism from fungal toxins to RAC detection is not a simple replacement, and its detection performance has significant unpredictability.

[0005] Based on this, the present invention provides an analytical method for rapid detection of ractopamine content in food. A sensor is constructed by connecting ε subunit monoclonal antibody-biotin-neutral avidin-biotin-RAC monoclonal antibody and combined with chemiluminescence detection. The aim is to establish a quantitative relationship model between RAC concentration and chemiluminescence signal, thereby achieving rapid, accurate and high-throughput detection of RAC content in food. Summary of the Invention

[0006] Therefore, this invention provides an analytical method for rapidly detecting ractopamine (RAC) content in food, in order to overcome the shortcomings of existing technologies.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, an analytical method for rapidly detecting ractopamine (RAC) content in food is provided, comprising the following steps: S1. Using the F0F1-ATPase molecular motor biosensor, the chemiluminescence signal of ractopamine (RAC) standard solutions of different concentrations was measured. A linear regression equation was established as a standard curve with the logarithm of ractopamine (RAC) concentration as the abscissa and the chemiluminescence signal value as the ordinate. S2. React the F0F1-ATPase molecular motor biosensor with the test sample solution diluted 10 times, and measure the chemiluminescence signal of the test sample solution as in step S1. S3. Substitute the chemiluminescence signal value measured in step S2 into the standard curve equation to calculate the concentration of ractopamine (RAC) in the sample solution to be tested. Then, based on the mass, volume and dilution factor in the sample pretreatment process, calculate the content of ractopamine (RAC) in the original sample.

[0008] Further, the ractopamine (RAC) standard solution mentioned in step S1 is 0.0001-100 μg / mL.

[0009] Further, the F0F1-ATPase molecular motor biosensor described in step S1 comprises a chromatograph containing F0F1-ATPase, a biotinylated ε-subunit monoclonal antibody, a neutral avidin and a biotinylated ractopamine (RAC) monoclonal antibody in a volume ratio of 15:10:10:1.6.

[0010] Further, the concentration of the chromosome containing F0F1-ATPase is 50 mg / mL, the concentration of the biotinylated ε subunit monoclonal antibody is 0.5 mg / mL, the concentration of the neutral avidin is 0.1 mg / mL, and the concentration of the biotinylated ractopamine (RAC) monoclonal antibody is 0.78 mg / mL.

[0011] Furthermore, in step S2, the volume ratio of the F0F1-ATPase molecular motor biosensor to the 10-fold diluted test sample solution is 1:1; the reaction conditions of the F0F1-ATPase molecular motor biosensor and the 10-fold diluted test sample solution are: temperature 37℃, time 10min.

[0012] Furthermore, the method for detecting changes in ATP synthesis activity in step S2 is as follows: The F0F1-ATPase molecular motor biosensor, the sample solution to be tested, and the ATP synthesis buffer were mixed evenly at a volume ratio of 1:1:3 and incubated at 37°C for 10 min to obtain the incubation product. The incubation product was then mixed evenly with the buffer solution to terminate the reaction and obtain the reaction product. The reaction product was then immediately mixed rapidly with the bioluminescent ATP detection reagent at a ratio of 5:3, and the chemiluminescent signal was measured.

[0013] Furthermore, the ATP synthesis buffer contains N-tris(hydroxymethyl)methylglycine-sodium hydroxide (Tricine-NaOH), magnesium chloride (MgCl2), disodium hydrogen phosphate (Na2HPO4), and adenosine diphosphate (ADP).

[0014] Furthermore, the volume ratio of the incubation product to the buffer solution is 1:9, and the buffer solution is PBS buffer.

[0015] Furthermore, the concentration of the PBS buffer is 0.01 mol / L and the pH is 7.4.

[0016] Furthermore, the bioluminescent ATP detection reagent includes, but is not limited to, luciferase / luciferin.

[0017] Furthermore, the preparation method of the sample solution to be tested in step S2 is as follows: Fresh pork leg meat was taken, and visible fat and fascia were removed. The meat was then minced and mixed thoroughly to obtain a minced sample. The minced sample was mixed with a methanol-ammonia solution (methanol:ammonia = 100:5, v / v) at a ratio of 4 g:20 mL and sonicated for 20 min. After treatment, the mixture was centrifuged and the supernatant was collected. The supernatant was then evaporated at 40 °C to near dryness to obtain residue 1. Ethyl acetate was mixed with residue 1 and sonicated for 10 min. The supernatant was then centrifuged and the supernatant was collected. The supernatant was then evaporated at 40 °C to near dryness to obtain residue 2. Residue 2 was reconstituted with PBS buffer to obtain a mixture. This mixture was mixed with n-hexane at a volume ratio of 1:1. After standing and separating the layers, the upper n-hexane layer was discarded, and the lower aqueous phase was collected to obtain the sample solution to be tested.

[0018] Furthermore, the centrifugation parameters are as follows: rotation speed 4,300 r / min, time 10 min.

[0019] Furthermore, the linear regression equation described in step S1 is: y =0.0361 x +0.3493, correlation coefficient R 2 =0.9544.

[0020] Furthermore, the formula for calculating the ractopamine (RAC) content in the original sample in step S3 is as follows: ; Note: C 1: The RAC concentration (μg / mL) of the sample solution to be tested; V This indicates the final volume (mL) of the sample solution to be tested. m This indicates the mass (g) of the sample taken. ; Note: D This indicates the additional dilution factor of the sample before measurement.

[0021] Furthermore, based on the ractopamine (RAC) content in the original sample calculated in step S3, it is further determined whether the sample solution to be tested contains ractopamine (RAC). The criteria for determination are as follows: like C 样品 If the RAC residue in the sample is ≥0.01 mg / kg, it is determined that the RAC residue exceeds the maximum residue limit recommended by the Codex Alimentarius Commission. like C 样品 If the concentration is <0.01 mg / kg and higher than the detection limit, it is considered qualified but caution is required; like C 样品 If the value is below the detection limit, it is considered undetectable.

[0022] Furthermore, the detection limit is 5 × 10⁻⁶. -9 mg / kg.

[0023] According to a second aspect of the invention, the analytical method is provided for the application of the method in detecting ractopamine (RAC) content in food.

[0024] The present invention has the following advantages: This invention, for the first time, extends the ε-subunit-regulated F0F1-ATPase molecular motor sensor from fungal toxin detection to the detection of β-agonists (ractopamine), overcoming the technical obstacles caused by differences in antibody recognition characteristics among different target molecules. Experiments show that the detection limit of this method is as low as 5 × 10⁻⁶. -9 The spiked recoveries were 87.8%–99.6% at mg / kg, and were highly correlated with the HPLC-MS / MS results (R0.05). 2 =0.986), with intra-batch coefficient of variation of 9.5%-11.3% and inter-batch coefficient of variation of 10.1%-12.9%, both below 15.0%, meeting the requirements of the guidelines for biological sample analysis methods and exhibiting good sensitivity, accuracy, and precision. Furthermore, this invention provides a complete conversion formula from chemiluminescence signal to RAC content in the original sample, which can be directly applied to the quantitative analysis of actual samples and is suitable for high-throughput screening and detection of ractopamine residues in food. Attached Figure Description

[0025] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0026] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0027] Figure 1 This is a standard curve of ractopamine (RAC) in Example 1 of the present invention; where the horizontal axis represents the logarithm of the RAC concentration. C RAC The vertical axis represents relative fluorescence intensity, and the linear regression equation is: y =0.0361 x +0.3493, correlation coefficient R 2 =0.9544; Figure 2 This is a comparative analysis result of the analytical method for rapid detection of ractopamine (RAC) content in food according to Example 4 of the present invention and the standard HPLC-MS / MS method. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0029] According to a first aspect of the present invention, an analytical method for rapidly detecting ractopamine (RAC) content in food is provided, comprising the following steps: S1. Using the F0F1-ATPase molecular motor biosensor, the chemiluminescence signal of ractopamine (RAC) standard solutions of different concentrations was measured. A linear regression equation was established as a standard curve with the logarithm of ractopamine (RAC) concentration as the abscissa and the chemiluminescence signal value as the ordinate. S2. React the F0F1-ATPase molecular motor biosensor with the test sample solution diluted 10 times, and measure the chemiluminescence signal of the test sample solution as in step S1. S3. Substitute the chemiluminescence signal value measured in step S2 into the standard curve equation to calculate the concentration of ractopamine (RAC) in the sample solution to be tested. Then, based on the mass, volume and dilution factor in the sample pretreatment process, calculate the content of ractopamine (RAC) in the original sample.

[0030] Furthermore, the ractopamine (RAC) standard solution in step S1 is 0.0001-100 μg / mL.

[0031] Further, in step S1, the F0F1-ATPase molecular motor biosensor comprises a chromatograph containing F0F1-ATPase, a biotinylated ε-subunit monoclonal antibody, a neutral avidin and a biotinylated ractopamine (RAC) monoclonal antibody in a volume ratio of 15:10:10:1.6.

[0032] Furthermore, the concentration of the chromosome containing F0F1-ATPase was 50 mg / mL, the concentration of the biotinylated ε subunit monoclonal antibody was 0.5 mg / mL, the concentration of neutral avidin was 0.1 mg / mL, and the concentration of the biotinylated ractopamine (RAC) monoclonal antibody was 0.78 mg / mL.

[0033] Furthermore, in step S2, the volume ratio of the F0F1-ATPase molecular motor biosensor to the 10-fold diluted test sample solution is 1:1; the reaction conditions between the F0F1-ATPase molecular motor biosensor and the 10-fold diluted test sample solution are: temperature 37℃, time 10min.

[0034] Furthermore, the method for detecting changes in ATP synthesis activity in step S2 is as follows: The F0F1-ATPase molecular motor biosensor, the sample solution to be tested, and the ATP synthesis buffer were mixed evenly at a volume ratio of 1:1:3 and incubated at 37°C for 10 min to obtain the incubation product. The incubation product was then mixed evenly with the buffer solution to terminate the reaction and obtain the reaction product. The reaction product was then immediately mixed rapidly with the bioluminescent ATP detection reagent at a ratio of 5:3, and the chemiluminescent signal was measured.

[0035] Furthermore, the ATP synthesis buffer contains N-tris(hydroxymethyl)methylglycine-sodium hydroxide (Tricine-NaOH), magnesium chloride (MgCl2), disodium hydrogen phosphate (Na2HPO4), and adenosine diphosphate (ADP).

[0036] Furthermore, the volume ratio of the incubation product to the buffer solution was 1:9, and the buffer solution was PBS buffer.

[0037] Furthermore, the concentration of the PBS buffer was 0.01 mol / L and the pH was 7.4.

[0038] Furthermore, bioluminescent ATP detection reagents include, but are not limited to, luciferase / luciferin.

[0039] Furthermore, the preparation method of the sample solution to be tested in step S2 is as follows: Fresh pork leg meat was taken, and visible fat and fascia were removed. The meat was then minced and mixed thoroughly to obtain a minced sample. The minced sample was mixed with a methanol-ammonia solution (methanol:ammonia = 100:5, v / v) at a ratio of 4 g:20 mL and sonicated for 20 min. After treatment, the mixture was centrifuged and the supernatant was collected. The supernatant was then evaporated at 40 °C to near dryness to obtain residue 1. Ethyl acetate was mixed with residue 1 and sonicated for 10 min. The supernatant was then centrifuged and the supernatant was collected. The supernatant was then evaporated at 40 °C to near dryness to obtain residue 2. Residue 2 was reconstituted with PBS buffer to obtain a mixture. This mixture was mixed with n-hexane at a volume ratio of 1:1. After standing and separating the layers, the upper n-hexane layer was discarded, and the lower aqueous phase was collected to obtain the sample solution to be tested.

[0040] Furthermore, the centrifugation parameters were as follows: rotation speed 4,300 r / min, time 10 min.

[0041] Furthermore, the linear regression equation in step S1 is: y =0.0361 x +0.3493, correlation coefficient R 2 =0.9544.

[0042] Furthermore, the formula for calculating the ractopamine (RAC) content in the original sample in step S3 is as follows: ; Note: C 1: The RAC concentration (μg / mL) of the sample solution to be tested; V This indicates the final volume (mL) of the sample solution to be tested. m This indicates the mass (g) of the sample taken. ; Note: D This indicates the additional dilution factor of the sample before measurement.

[0043] Furthermore, based on the ractopamine (RAC) content in the original sample calculated in step S3, it is further determined whether the test sample solution contains ractopamine (RAC). The criteria for determination are as follows: likeC 样品 If the RAC residue in the sample is ≥0.01 mg / kg, it is determined that the RAC residue exceeds the maximum residue limit recommended by the Codex Alimentarius Commission. like C 样品 If the concentration is <0.01 mg / kg and higher than the detection limit, it is considered qualified but caution is required; like C 样品 If the value is below the detection limit, it is considered undetectable.

[0044] Furthermore, the detection limit is 5×10⁻⁶. -9 mg / kg.

[0045] According to a second aspect of the invention, an analytical method is provided for the detection of ractopamine (RAC) content in food.

[0046] The materials and chemical reagents used in this invention are as follows: (+)-Biotin N-hydroxysuccinimide ester was purchased from Sigma-Aldrich (Bux, Switzerland). The anti-ractopamine (RAC) monoclonal antibody was purchased from Thermo Fisher Scientific (USA).

[0047] Neutral affinity was purchased from Sigma-Aldrich (Bux, Switzerland). Roseobacterium thermophilum ( Rhodothermus obamensis strain wa0073 (ATCC27502) was purchased from the American Type Culture Collection (ATCC, USA). ENLITEN @ Luciferase / luciferin reagent was purchased from Promega (USA); Standard RAC and ADP were purchased from Sigma-Aldrich (Bux, Switzerland).

[0048] The instruments and equipment used in this invention are as follows: Centro XS3 LB 960 microplate chemiluminescence analyzer (Germany); The Fluoroskan Ascent fluorescence chemiluminescence reader was purchased from Thermolabsystems (Finland).

[0049] The statistical analysis methods used in this invention are as follows: Statistical analysis was performed using GraphPad Prism 5.0. The comparison between the two paired datasets used the Student pairing method. tTests were performed; one-way ANOVA was used for comparisons among multiple groups, followed by Tukey's multiple comparison test. p <0.05 is considered statistically significant.

[0050] To better illustrate the technical effects of this invention, the following embodiments are provided.

[0051] Preparation Example 1 Preparation of ATCC Medium 655 liquid culture medium Take 1.8g of MnCl2·4H2O, 4.4g of Na2B4O7·10H2O, 0.22g of ZnSO4·7H2O, 0.05g of CuCl2·H2O, 0.03g of Na2MoO4·2H2O, and 0.03g of VOSO4·2H2O, dissolve them in 1L of deionized water, and adjust the pH to (2.0±0.2) with dilute sulfuric acid (H2SO4) to obtain a salt solution. Store at 4°C for later use.

[0052] Take 1g of yeast extract, 1g of tryptone, 1.3g of ammonium sulfate, 0.247g of magnesium sulfate heptahydrate, 0.28g of potassium dihydrogen phosphate, 0.074g of calcium chloride dihydrate, and 0.019g of ferric chloride hexahydrate, add 1mL of the above salt solution, and make up to 1L with deionized water. Mix well. Adjust the pH to (8.5±0.2) with 1M NaOH. Dispense into Erlenmeyer flasks and autoclave at 121℃ for 15min to obtain ATCC Medium 655 liquid culture medium. After cooling, store at 4℃ for later use.

[0053] Preparation Example 2 Preparation of chromatographs containing F0F1-ATPase Rose-colored heat bacteria ( Thermomicrobium roseumThe wa0073 strain was inoculated into ATCC Medium 655 liquid medium as in Preparation Example 1 and cultured at 70°C with shaking at 150 rpm until the logarithmic growth phase (OD600 = 0.8–1.0). The culture was centrifuged at 4°C and 8,500 rpm for 20 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were resuspended in pre-cooled disruption buffer (composed of 50 mM Tris-HCl, 5 mM MgCl2, 1 mM DTT, and 0.25 M sucrose, pH 8.0), centrifuged at 4°C and 8,500 rpm for 20 min, the supernatant was discarded, and the process was repeated once, for a total of two washes. The bacterial resuspended in lysis buffer (composed of 50 mM Tris-HCl, 5 mM MgCl2, 1 mM DTT, and 0.25 M sucrose, pH 8.0) at a 1:10 (w / v) ratio and sonicated under ice bath conditions (200 W, 5 s operation time, 10 s interval, total sonication time 30 min). The lysate was centrifuged at 33,400 rpm for 30 min at 4°C, the precipitate was discarded, and the supernatant was retained. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 36,600 rpm for 90 min at 4°C, the supernatant was discarded, and the precipitate was the crude chromatophore. The precipitate was resuspended in lysis buffer and centrifuged again at 33,400 rpm for 60 min, and the supernatant was discarded. The final precipitate was resuspended in a small amount of storage buffer (composed of 50 mM Tris-HCl, 5 mM MgCl2, 1 mM DTT, and 50% glycerol, pH 8.0) to obtain a chromatograph containing F0F1-ATPase, and its concentration was adjusted to approximately 50 mg / mL (as determined by the Bradford method, using BSA as the standard).

[0054] Preparation Example 3 Preparation of biotinylated ε subunit monoclonal antibody (1) Preparation of ε subunit antigen Synthetic nucleotide sequences of *Thermophyton floccosum* as shown in SEQ ID NO. 1 ( Thermomicrobium roseum The ε subunit encoding gene was cloned into a pET-22b(+) expression vector (purchased from Shanghai Zaikang Biotechnology Co., Ltd.) with a His-tag, resulting in a recombinant plasmid. After sequencing verification, the recombinant plasmid with the correct gene sequence was transformed into... E. coliBL21(DE3) was induced to express the target protein (1 mM) at 37°C for 3 h using IPTG. Bacterial cells were collected and sonicated on ice (200 W, 5 s operation time, 10 s interval, total sonication time 30 min). The supernatant was loaded onto a Ni-NTA affinity column and eluted sequentially with buffers containing 20 mM, 50 mM, 100 mM, 200 mM, and 300 mM imidazole. The eluent fractions at each imidazole concentration were collected. After SDS-PAGE identification, the eluent fractions containing the target protein (16 kDa) were combined. The combined eluent was purified using a Sartorius ultrafiltration tube with a molecular weight cutoff of 10 kDa to obtain the purified ε subunit protein. The purified ε subunit protein was identified as a single band by SDS-PAGE.

[0055] (2) Preparation of ε subunit monoclonal antibody Endotoxin was removed from the purified ε subunit in step (1) using the Triton X-114 phase separation method to obtain purified endotoxin-free ε subunits. The purified endotoxin-free ε subunits were used as the immunogen to immunize 6-8 week old BALB / c mice. For the primary immunization, the antigen was thoroughly emulsified with an equal volume of Freund's complete adjuvant (FCA) and then injected subcutaneously at multiple points in the abdomen (80 μg antigen per mouse). Booster immunizations were then performed every 2 weeks using Freund's incomplete adjuvant (FIA), for a total of 4 immunizations. Two weeks after the fourth immunization, blood was collected via the tail vein, and serum antibody titers were detected using an indirect ELISA method, with the purified endotoxin-free ε subunits as the target antigen. Mice with titers greater than 1:100,000 were selected and given a booster immunization three days before cell fusion via intravenous injection of an adjuvant-free antigen solution.

[0056] Spleen cells from mice three days after booster immunization were mixed with logarithmic growth phase SP2 / 0 myeloma cells at an 8:1 ratio and fused in 50% PEG 1500 (pH 8.0). After fusion, the cells were seeded in 96-well plates, and positive hybridoma cells were selected using RPMI-1640 medium containing 20% ​​fetal bovine serum and HAT. On day 12 post-fusion, the supernatant from positive wells was used for ELISA identification. Positive wells were cloned three times using the limiting dilution method to obtain hybridoma cell lines stably secreting ε-subunit monoclonal antibodies.

[0057] Hybridoma cells were injected into the peritoneal cavity of mice pretreated with liquid paraffin (1 × 10⁶ cells per mouse), and ascites fluid was collected 8 days later. The ascites fluid was initially purified by fractional precipitation with 33% ammonium sulfate, and the IgG fraction was then separated by Sephadex G-200 size exclusion chromatography to obtain the ε subunit monoclonal antibody. The concentration of the ε subunit monoclonal antibody was determined by the Bradford method, adjusted to 0.5 mg / mL, and stored at -20°C for later use.

[0058] (3) Preparation of biotinylated ε subunit monoclonal antibody Take 500 μL of ε-subunit monoclonal antibody (0.5 mg / mL) and place it in a centrifuge tube. Add 2 μL of (+)-biotin-N-hydroxysuccinimide ester (10 mM, freshly prepared with anhydrous DMSO) and incubate at room temperature in the dark for 1 h. After the reaction is complete, dialyze three times with PBS (0.01 mol / L, pH 7.4) to remove excess (+)-biotin-N-hydroxysuccinimide ester, obtaining biotinylated ε-subunit monoclonal antibody. Aliquot and store at -20℃ in the dark, avoiding repeated freeze-thaw cycles.

[0059] Preparation Example 4 Preparation of biotinylated RAC monoclonal antibodies The concentration of anti-ractopamine (RAC) monoclonal antibody was determined using the Bradford method, adjusted to 0.78 mg / mL, and stored at -20°C for later use.

[0060] 500 μL of RAC monoclonal antibody (0.78 mg / mL) was placed in a centrifuge tube, and 2 μL of (+)-biotin N-hydroxysuccinimide ester (10 mM, freshly prepared with anhydrous DMSO) was added. The mixture was incubated at room temperature in the dark for 1 h. After the reaction, the mixture was dialyzed three times with PBS (0.01 mol / L, pH 7.4) to remove excess (+)-biotin N-hydroxysuccinimide ester, yielding the biotinylated RAC monoclonal antibody. After aliquoting, the antibody was stored at -20°C in the dark, avoiding repeated freeze-thaw cycles.

[0061] Example 1 Establishment of standard curve 1. Preparation of RAC standard solution Take standard RAC samples and prepare a series of RAC standard solutions with concentrations ranging from 0.0001 μg / mL, 0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, to 100 μg / mL using PBS buffer (0.01 mol / L, pH 7.4). Prepare three replicates for each concentration.

[0062] Construction of 2 F0F1-ATPase molecular motor biosensor Take 15 μL of chromosomes containing F0F1-ATPase and 10 μL of biotinylated ε subunit monoclonal antibody (0.5 mg / mL), dilute to 1 mL with PBS buffer (0.01 mol / L, pH 7.4), and incubate at 37 °C for 60 min. Centrifuge at 19,000 rpm for 20 min at 4 °C, discard the supernatant, and resuspend the precipitate in 500 μL of PBS buffer (0.01 mol / L, pH 7.4) to obtain suspension 1; Add 10 μL of neutral avidin (0.1 mg / mL) to suspension 1, dilute to 1 mL with PBS (0.01 mol / L, pH 7.4), and incubate at 37 °C for 10 min. Centrifuge at 19,000 rpm for 20 min at 4 °C, discard the supernatant, and resuspend the precipitate in 500 μL of PBS (0.01 mol / L, pH 7.4) to obtain suspension 2. Add 1.6 μL of biotinylated RAC monoclonal antibody (0.78 mg / mL) to suspension 2, dilute to 1 mL with PBS, and incubate at 37 °C for 10 min. Centrifuge at 19,000 rpm for 20 min at 4 °C, discard the supernatant, and resuspend the precipitate in 500 μL PBS (0.01 mol / L, pH 7.4) to obtain the F0F1-ATPase molecular motor biosensor, and store at 4 °C.

[0063] 3. Determination of chemiluminescence signal 10 μL of the F0F1-ATPase molecular motor biosensor constructed in step 2 above, 10 μL of RAC standard solutions of different concentrations prepared in step 1 above, and 30 μL of ATP synthesis buffer (prepared by mixing 20 mM Tricine-NaOH (pH 8.0), 2 mM MgCl2, 2 mM Na2HPO4, and 0.5 mM ADP) were mixed thoroughly and incubated at 37 °C for 10 min to obtain the incubation product. The reaction was terminated by adding 450 μL of PBS (0.01 mol / L, pH 7.4). 50 μL of the reaction solution was immediately mixed with 30 μL of ENLITEN. ® Luciferase / luciferin reagent was rapidly mixed and the chemiluminescence signal (expressed as relative fluorescence intensity) was measured using a Fluoroskan Ascent chemiluminescence analyzer. Each concentration was measured three times, and the results are expressed as mean ± standard deviation.

[0064] 4. Establishment of the standard curve equation The logarithm of RAC concentration (Log C RAC The unit of C is μg / mL) is the x-axis. x The measured relative fluorescence intensity value is used as the ordinate. y ), plot the standard curve of RAC (e.g. Figure 1 (As shown).

[0065] The obtained linear regression equation is: y =0.0361 x +0.3493, correlation coefficient R 2 =0.9544. The limit of detection (LOD) was calculated as 10 times the standard deviation of the mean signal of the blank control (0.01 mol / L, pH 7.4 PBS buffer), which was approximately 10.-8 μg / mL. The results show that the analytical method for rapid detection of ractopamine content in food in this invention has extremely high sensitivity, and the entire detection process (from sample addition to reading) only takes about 10 minutes, without the need for sealing, labeling or washing steps.

[0066] Example 2 Determination of RAC content in the sample to be tested 1 Sample pretreatment Take (4.0±0.2) g of minced pork into a 15 mL centrifuge tube, add 20 mL of methanol-ammonia water mixed solution (20∶1, v / v), sonicate for 20 min, centrifuge and take 10 mL of supernatant, evaporate to near dryness at 40℃ to obtain residue 1; take 10 mL of ethyl acetate and mix with residue 1, sonicate for 10 min for extraction; after extraction, centrifuge and take 8 mL of supernatant, evaporate to near dryness at 40℃ to obtain residue 2; redissolve residue 2 with 2 mL of PBS (0.01 mol / L, pH 7.4) to obtain a mixture; add 2 mL of n-hexane to the mixture and mix, let stand for layering and discard the upper n-hexane (containing lipids), collect the lower aqueous phase (about 1 mL), which is the sample solution to be tested.

[0067] Note: All centrifugation parameters above are: rotation speed 4,300 r / min, time 10 min.

[0068] 2. Measurement of chemiluminescence signal of sample The above-mentioned sample solution was diluted 10-fold with PBS (0.01 mol / L, pH 7.4), and the chemiluminescence signal (expressed as relative fluorescence intensity) was measured according to the steps in Example 1. Each sample was tested in triplicate, and the average value was taken. An unspecified blank control (i.e., 0 mg / kg) and a negative control (0.01 mol / L, pH 7.4 PBS buffer) were also included.

[0069] 3. Calculation of RAC content in the sample (1) Calculate the RAC concentration in the sample solution to be tested. The relative fluorescence intensity value of the sample solution to be tested ( y Substituting into the standard curve equation, the concentration of RAC in the sample solution can be calculated by reverse calculation. C 1 (μg / mL), the specific calculation formula is as follows: ; Note: D This indicates the additional dilution factor of the sample before measurement.

[0070] (2) Calculate the RAC content in the original sample Calculate the RAC content in the original pork sample based on the mass, volume, and dilution factor during pretreatment.C 样品 (mg / kg), and the specific calculation formula is as follows: ; Note: C 1: RAC concentration measured in the test sample solution (μg / mL); V represents the final constant volume of the test sample solution (mL); m represents the mass of the weighed sample (g).

[0071] (3) Result Judgment If C 样品 ≥ 0.01 mg / kg, it is determined that the RAC residue in the sample exceeds the maximum residue limit recommended by the Codex Alimentarius Commission; If C 样品 < 0.01 mg / kg and higher than 5×10 -9 mg / kg, the result is determined as qualified but requires attention; If C 样品 lower than 5×10 -9 mg / kg, it is determined as not detected.

[0072] Experimental results show that the average relative fluorescence intensity of the blank control and negative control y is 0.00166. Substituting into the standard curve equation to calculate the concentration of the diluted test solution C 1 is 2.3×10 -9 μg / mL, calculated according to the formula C 样品 is 1.17×10 - 9 mg / kg (lower than 5×10 -9 mg / kg). The judgment result is not detected, indicating that the detection system has no matrix interference. The average relative fluorescence intensity of the test sample y is 0.24968. Substituting into the standard curve equation to calculate the concentration of the diluted test solution C 1 is 0.01739 μg / mL, calculated according to the formula C 样品 is 0.087 mg / kg, which exceeds the maximum residue limit recommended by the Codex Alimentarius Commission (≥0.01 mg / kg).

[0073] Example 3 To verify the accuracy and precision of the rapid analytical method for detecting ractopamine content in food of the present invention, spiked recovery and precision tests were carried out.

[0074] 1 Determination of spiked recovery Blank pork samples, verified to be RAC-free by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), were spiked with RAC standards to achieve final RAC concentrations (fresh weight) of 0.01 mg / kg, 0.1 mg / kg, and 0.5 mg / kg, respectively. Three replicates were set up for each concentration, and results are expressed as mean ± standard deviation. Pretreatment and determination were performed according to the method in Example 2. The measured concentrations of each spiked sample were calculated, and the recovery rate was calculated using the following formula: ; Note: C 1 indicates the concentration measured by RAC; C 2 indicates the concentration of RAC added.

[0075] The results are shown in Table 1 below: Table 1. Results of RAC spike recovery in pork samples (n=3)

[0076] As shown in Table 1 above, the recovery rate determined using the standard calibration curve ranged from 87.8% to 99.6%.

[0077] 2. Precision determination Intra-batch precision: Within the same batch, samples with the same spiked concentration are measured three times in parallel, and the coefficient of variation is calculated. CV The specific calculation formula is as follows: ; Inter-batch precision: For three consecutive days, samples with the same spiked concentration were measured each day (n=3), and the results were calculated. CV The results are shown in Table 2 below: Table 2. Precision determination results of RAC spiked recovery rate in pork samples (n=3)

[0078] As shown in Table 2 above, the intra-batch concentrations of all spiked concentrations (0.01 mg / kg, 0.1 mg / kg, 0.5 mg / kg) were... CV Between 9.5% and 11.3%, batch-to-batch CV The percentages were between 10.1% and 12.9%, all below 15.0%, which meets the requirements of the guidelines for biological sample analysis methods.

[0079] Example 4 To verify the accuracy of the analytical method for rapid detection of ractopamine content in food according to the present invention, a comparative analysis was conducted with the standard HPLC-MS / MS method.

[0080] Spiked pork samples (0.01 mg / kg, 0.1 mg / kg, and 0.5 mg / kg) identical to those in Example 3 were taken and analyzed using the rapid detection method for ractopamine in food according to this invention and the standard HPLC-MS / MS method. HPLC-MS / MS conditions: Chromatographic conditions: C18 column (4.6 mm × 150 mm, 3.5 μm); mobile phase A: water (containing 0.1% formic acid); mobile phase B: acetonitrile (containing 0.1% formic acid); flow rate: 0.2 mL / min; injection volume: 20 μL. Gradient elution program: 0 min, 95% A; 2 min, 95% A; 8 min, 20% A; 21 min, 77% A; 22 min, 5% A; 25 min, 5% A; 25.5 min, 95% A.

[0081] Mass spectrometry conditions: Electrospray ionization positive ion mode (ESI+); multiple reaction monitoring (MRM) scan. Drying gas temperature 330℃; gas flow rate 8 L / min; nebulizer pressure 35 psi; capillary voltage 3500 V. The precursor ion of the RAC was m / z 302, and the daughter ions were m / z 284 and 164, with m / z 164 used for quantification.

[0082] The x-axis represents the values ​​determined by HPLC-MS / MS. x The value measured by this method is the ordinate ( y Linear regression analysis was performed, and the results are as follows: Figure 2 As shown.

[0083] The obtained linear regression equation is: y =0.6764 x +0.0253, R 2 =0.986. This result indicates that the method provided by this invention is highly correlated with the HPLC-MS / MS determination results, verifying the accuracy of the analytical method for rapid detection of ractopamine content in food.

[0084] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A rapid analytical method for detecting ractopamine content in food, characterized in that, Includes the following steps: S1. Using the F0F1-ATPase molecular motor biosensor, the chemiluminescence signal of ractopamine standard solutions of different concentrations was measured. A linear regression equation was established as a standard curve with the logarithm of ractopamine concentration as the abscissa and the chemiluminescence signal value as the ordinate. S2. React the F0F1-ATPase molecular motor biosensor with the test sample solution diluted 10 times, and measure the chemiluminescence signal of the test sample solution as in step S1. S3. Substitute the chemiluminescence signal value measured in step S2 into the standard curve equation to calculate the concentration of ractopamine in the sample solution to be tested. Then, based on the mass, volume and dilution factor in the sample pretreatment process, calculate the content of ractopamine in the original sample.

2. The analytical method as described in claim 1, characterized in that, The ractopamine standard solution mentioned in step S1 is 0.0001-100 μg / mL.

3. The analytical method as described in claim 1, characterized in that, The F0F1-ATPase molecular motor biosensor described in step S1 comprises a chromatograph containing F0F1-ATPase in a volume ratio of 15:10:10:1.6, a biotinylated ε-subunit monoclonal antibody, a neutral avidin, and a biotinylated ractopamine monoclonal antibody.

4. The analytical method as described in claim 3, characterized in that, The concentration of the chromosome containing F0F1-ATPase is 50 mg / mL, the concentration of the biotinylated ε subunit monoclonal antibody is 0.5 mg / mL, the concentration of the neutral avidin is 0.1 mg / mL, and the concentration of the biotinylated ractopamine monoclonal antibody is 0.78 mg / mL.

5. The analytical method as described in claim 1, characterized in that, In step S2, the volume ratio of the F0F1-ATPase molecular motor biosensor to the 10-fold diluted sample solution is 1:1; the reaction conditions of the F0F1-ATPase molecular motor biosensor and the 10-fold diluted sample solution are: temperature 37℃, time 10min.

6. The analytical method as described in claim 1, characterized in that, The method for detecting changes in ATP synthesis activity in step S2 is as follows: The F0F1-ATPase molecular motor biosensor, the sample solution to be tested, and the ATP synthesis buffer were mixed evenly at a volume ratio of 1:1:3 and incubated at 37°C for 10 min to obtain the incubation product. The incubation product was then mixed evenly with the buffer solution to terminate the reaction and obtain the reaction product. The reaction product was then immediately mixed rapidly with the bioluminescent ATP detection reagent at a ratio of 5:3, and the chemiluminescent signal was measured.

7. The analytical method as described in claim 1, characterized in that, The linear regression equation mentioned in step S1 is: y =0.0361 x +0.3493, correlation coefficient R 2 =0.9544.

8. The analytical method as described in claim 1, characterized in that, The formula for calculating the ractopamine content in the original sample in step S3 is as follows: ； Note: C 1: The RAC concentration (μg / mL) of the sample solution to be tested; V This indicates the final volume (mL) of the sample solution to be tested. m This indicates the mass (g) of the sample taken. ； Note: D Indicates the additional dilution factor of the sample before measurement.

9. The analytical method as described in claim 1, characterized in that, Based on the ractopamine content in the original sample calculated in step S3, it is further determined whether the sample solution to be tested contains ractopamine. The criteria for determination are as follows: If C 样品 If the RAC residue in the sample is ≥0.01 mg / kg, it is determined that the RAC residue exceeds the maximum residue limit recommended by the Codex Alimentarius Commission. If C 样品 If the concentration is <0.01 mg / kg and higher than the detection limit, it is considered qualified but caution is required; If C 样品 If the value is below the detection limit, it is considered undetectable.

10. The application of the analytical method according to any one of claims 1-9 in the detection of ractopamine content in food.

Images