A multi-index integrated detection platform and method for food detection

Abstract

The present application relates to a kind of food detection with multi-index integrated detection platform and detection method, belong to food safety rapid detection field.Platform includes sample pretreatment module, sample solution adjusting module, multi-index detection module, double-mode optical detection module and quality control and result processing module.Sample pretreatment module is according to lipid, pigment and protein interference data, target and MCX's retention relationship and pesticide response compatible relationship, respectively determine including PSA purification and select to carry out MCX fixed bed purification or bypass MCX fixed bed two purification paths.Two homologous purification liquids are adjusted respectively, then time-resolved fluorescence immunochromatography detection and enzyme inhibition colorimetric screening are carried out, and detection result is output after independent quality control.The present application is used for target detection in complex food matrix and organic phosphorus, carbamate pesticide comprehensive screening.

Description

Technical Field

[0001] This invention relates to the field of rapid food safety testing technology, and in particular to a multi-index integrated testing platform and testing method for food testing. Background Technology

[0002] Rapid food testing often requires screening for pesticide residues, veterinary drug residues, mycotoxins, and other hazardous factors simultaneously. Time-resolved fluorescence immunochromatography (TRFI) offers advantages such as high sensitivity, low background interference, and suitability for multi-indicator detection; enzyme inhibition colorimetric assays can comprehensively screen for organophosphates and carbamate pesticides. Integrating these two detection methods onto a single platform helps reduce the number of devices and sample processing steps.

[0003] Existing rapid food testing pretreatment protocols typically have pre-defined fixed procedures based on food type. For example, a defatting step is set for oily samples, a decolorization step for high-pigment samples, and a protein precipitation step for high-protein samples. However, the lipid, pigment, and soluble protein content of the same food can vary significantly depending on the variety, processing technology, storage conditions, and batch. Selecting a fixed pretreatment procedure solely based on the food name can easily lead to insufficient or excessive purification.

[0004] MCX adsorbents combine reverse-phase and cation-exchange effects, making them suitable for the enrichment and purification of certain target analytes in complex matrices. However, different target analytes exhibit varying retention behaviors on MCXs; some analytes are effectively retained, while others may suffer significant loss. If the retention relationship between the target analyte and the MCX is not differentiated and MCX purification is applied uniformly, it may lead to a decrease in analyte recovery.

[0005] MCX may also alter the response of some pesticides in enzyme inhibition detection. If the immunoassay and enzyme inhibition detection are set to the exact same purification process, it may not be possible to simultaneously meet the requirements for target recovery and comprehensive pesticide screening response.

[0006] Time-resolved fluorescence immunochromatography and enzyme inhibition colorimetric reactions have different requirements for the sample solution environment. Immunochromatography usually requires a certain concentration of protein blocking agents and nonionic surfactants to improve chromatographic migration and reduce nonspecific adsorption; enzyme inhibition reactions may be affected by nonionic surfactants, organic solvents, pH, and sample background absorbance. Therefore, the two detection methods should not directly share the same reaction buffer system.

[0007] Furthermore, time-resolved fluorescence immunochromatography and enzyme inhibition colorimetric assays have different quality control requirements. Setting only uniform validity criteria could lead to outputting results even when the quality control of a particular assay channel fails.

[0008] Therefore, there is a need for a multi-index integrated detection platform and detection method that can determine the purification path based on the actual food matrix interference level, the retention relationship between the target and the MCX, and the pesticide response compatibility relationship, and can independently adjust the sample solution and control the quality of the two detection methods. Summary of the Invention

[0009] The purpose of this invention is to provide a multi-index integrated detection platform and detection method for food testing, in order to solve the problems that fixed pretreatment processes are difficult to adapt to different food matrices, MCX purification may cause loss of target substances or changes in pesticide screening response, and inconsistent sample liquid conditions and quality control requirements for different detection channels.

[0010] To achieve the above objectives, the present invention provides a multi-index integrated detection platform for food testing, including a sample pretreatment module, a sample solution conditioning module, a multi-index detection module, a dual-mode optical detection module, and a quality control and result processing module; The sample pretreatment module is used to extract and precipitate the same food sample to obtain matrix interference data including lipid interference data, pigment interference data, and protein interference data. Each matrix interference data is compared with a pre-set corresponding interference threshold to generate sample matrix feature data. When any of the matrix interference data reaches or exceeds the corresponding pre-set interference threshold, the sample matrix feature data includes a corresponding interference tag, which is a lipid interference tag, a pigment interference tag, or a protein interference tag. The sample pretreatment module is also used to split the homologous extraction supernatant of the food sample and determine two purification paths based on the sample matrix characteristic data, the preset retention relationship between the target analyte and the MCX, and the preset response compatibility relationship between the pesticide to be screened and the MCX. Both purification paths include PSA purification, and the module selects to perform MCX fixed bed purification or bypass the MCX fixed bed based on the preset retention relationship and the preset response compatibility relationship, respectively, to form a first purified liquid and a second purified liquid. The sample solution adjustment module is used to dilute or reconstitute the first purified solution with an immunoreaction buffer to form a first sample solution, and to dilute or adjust the volume of the second purified solution with an enzyme reaction buffer to form a second sample solution. The multi-index detection module includes a time-resolved fluorescence immunochromatographic detection unit and an enzyme inhibition colorimetric detection unit. The time-resolved fluorescence immunochromatographic detection unit is used to detect at least one target analyte in the first sample solution, and the enzyme inhibition colorimetric detection unit is used to comprehensively screen organophosphates and carbamates in the second sample solution. The dual-mode optical detection module includes a fluorescence detection optical path for reading time-resolved fluorescence signals and an absorbance detection optical path for reading enzyme inhibition colorimetric absorbance. The quality control and result processing module is used to independently control the detection process of the time-resolved fluorescence immunochromatographic detection unit and the enzyme inhibition colorimetric detection unit, and output the target analyte detection results and pesticide comprehensive screening results when the corresponding quality control is effective.

[0011] Furthermore, the matrix interference data includes: Lipid interference data RL, RL = Vo / Vs, where Vo is the total volume of the independent oil phase formed after salting out the same initial extract, and Vs is the total volume of the organic supernatant formed after salting out but excluding the independent oil phase; an emulsification indicator is formed when there is continuous emulsification but no independent oil phase is formed. Pigment interference data generated by the maximum blank-corrected absorbance in the range of 400 nm to 700 nm after the extract supernatant was diluted by a preset factor; and Protein interference data are generated when the soluble protein concentration of parallel measurement solutions of food samples is diluted or removed by organic solvents and reconstituted at a fixed volume, and then measured using the diquinoline carboxylic acid method. The sample solution used for matrix interference measurement is isolated from the sample solution used for detection and does not enter the time-resolved fluorescence immunochromatographic detection unit and the enzyme inhibition colorimetric detection unit.

[0012] Furthermore, when the sample matrix feature data includes lipid interference tags, C18 purification or cryogenic defatting is added to the purification path; When the sample matrix feature data includes pigment interference tags, graphitized carbon black purification is added to the purification path; When the sample matrix feature data includes protein interference tags, protein precipitation, centrifugation, or filtration processes are added to the purification path. When the purification path includes MCX fixed bed purification, and the sample matrix characteristic data includes lipid interference tags or protein interference tags, the amount of MCX used is increased or a tandem MCX fixed bed is used, and the total amount of increased MCX does not exceed twice the original amount of MCX. The materials and dosages used for auxiliary purification are predetermined based on the corresponding interference labels and stored in the sample pretreatment module.

[0013] Furthermore, the extraction and salting-out process uses an aqueous acetonitrile extract and a salting-out agent; The aqueous acetonitrile extract contains acetonitrile to water in a volume ratio of 60:40 to 85:15, and contains formic acid or acetic acid in a volume fraction of 0.1% to 1.0%, and ethylenediaminetetraacetic acid in a mass-volume concentration of 0.01% to 0.10%. The salting-out agent comprises anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate, in a mass ratio of (4-8):(1-2):(0.5-2):(0.25-1). For a purification path that sequentially performs PSA purification and MCX fixed-bed purification, the total dosage of MCX and PSA is 15 mg to 80 mg, and the mass ratio of MCX to PSA is 1:1 to 1:4.

[0014] Furthermore, the immune reaction buffer contains 5 mmol / L to 20 mmol / L of phosphate, 0.5% to 1.5% sodium chloride, 0.1% to 1.0% bovine serum albumin, and 0.01% to 0.10% polysorbate 20, with a pH of 7.0 to 8.0. The enzyme reaction buffer contains 20 mmol / L to 100 mmol / L of phosphate, is free of nonionic surfactants, and has a pH of 7.5 to 8.5. The volume fraction of organic solvent in both the first and second sample solutions is no higher than 10%.

[0015] Furthermore, the time-resolved fluorescence immunochromatographic detection unit includes at least one detection zone and a fluorescence control zone, and outputs the detection result of the target analyte based on the ratio of the time-resolved fluorescence signal of the detection zone to the fluorescence control zone. The enzyme inhibition colorimetric detection unit includes a sample reaction area, a sample blank area, and an enzyme activity control area, and calculates the enzyme inhibition rate based on the absorbance of the sample reaction area, the sample blank area, and the enzyme activity control area. When the signal in the fluorescence control area is within the preset batch allowable range, the time-resolved fluorescence immunochromatographic detection result is valid. When the absorbance of the sample blank area and the absorbance of the enzyme activity control area are both within their respective preset batch allowable ranges, the enzyme inhibition colorimetric detection result is valid. When the test result is invalid, the quality control and result processing module will not output the corresponding test conclusion, but will output a prompt for retesting.

[0016] On the other hand, the present invention also provides a multi-index integrated detection method for food testing, implemented using the above-mentioned multi-index integrated detection platform for food testing, including the following steps: Extraction and salting out were performed on the same food sample to obtain lipid interference data, pigment interference data, and protein interference data, respectively. Each matrix interference data is compared with a pre-set corresponding interference threshold to form sample matrix feature data; when any of the matrix interference data reaches or exceeds the corresponding pre-set interference threshold, a corresponding interference label is generated in the sample matrix feature data; Based on the sample matrix characteristic data, the preset retention relationship between the target analyte and the MCX, and the preset response compatibility relationship between the pesticide to be screened and the MCX, the homologous extraction supernatant of the food sample is divided into at least two portions, and two purification paths are determined for each portion. Both purification paths include PSA purification, and when the interference label is present, auxiliary purification corresponding to the interference label is included. The MCX fixed bed purification or bypassing the MCX fixed bed is selected according to the preset retention relationship and the preset response compatibility relationship to form a first purification liquid and a second purification liquid. The first purified solution was diluted or reconstituted using an immunoreaction buffer to form the first sample solution; The second purified solution was diluted or brought to a final volume using an enzyme reaction buffer to form the second sample solution; The first sample solution was subjected to time-resolved fluorescence immunochromatography, and the second sample solution was subjected to enzyme inhibition colorimetric detection. Determine whether the quality control of the two tests is effective, and output the target analyte test results and pesticide comprehensive screening results only when the corresponding quality control is effective.

[0017] Furthermore, before conducting food sample testing, the corresponding pre-set interference thresholds are determined using the following methods: Select a representative blank food matrix that does not contain the target analyte or the pesticide to be screened, and set no less than five interference levels for lipids, pigments or proteins, with no less than three parallel samples for each interference level; Time-resolved fluorescence immunochromatography and enzyme inhibition colorimetric reactions were performed at each interference level. The lowest level of interference that causes the target analyte spiked response to deviate more than 20% from the unaffected control, or that causes the fluorescence control signal to exceed the batch allowable range, is determined as the first candidate threshold. The lowest level of interference that causes the enzyme inhibition response of a representative pesticide at the same concentration to deviate by more than 20% from the interference-free control, or to cause the signal in the blank area of ​​the sample or the signal in the enzyme activity control area to exceed the batch allowable range, is determined as the second candidate threshold. The lower of the first candidate threshold and the second candidate threshold is determined as the pre-set corresponding interference threshold for the corresponding matrix interference data.

[0018] Furthermore, by passing the standard solution of the target analyte through an MCX fixed bed and an inert control fixed bed respectively, and comparing the recovery rates of the two, a pre-defined retention relationship between the target analyte and the MCX was established. When the recovery rate of MCX fixed bed decreases by more than 20% relative to the recovery rate of inert control fixed bed, the corresponding target analyte is classified as an MCX-retaining target analyte. When the decrease in the recovery rate of the MCX fixed bed relative to the recovery rate of the inert control fixed bed does not exceed 20%, the corresponding target analyte is classified as an MCX non-retention target analyte. When the target analyte is an MCX-retaining target analyte, the purification path of the first purification solution includes MCX fixed-bed purification. When the target analyte is a non-retention type target analyte of MCX, the purification path of the first purification solution bypasses the MCX fixed bed; The purification path of the second purification solution is determined independently based on the preset response compatibility relationship between the pesticide to be screened and the MCX. When the enzyme inhibition response deviation of the representative pesticide after treatment with the MCX fixed bed does not exceed 20%, the purification path of the second purification solution includes purification with the MCX fixed bed. When the enzyme inhibition response deviation exceeds 20%, the purification path of the second purification solution bypasses the MCX fixed bed.

[0019] Furthermore, the food sample is extracted at a ratio of 4 to 10 ml of aqueous acetonitrile extract per gram of food sample, wherein the volume ratio of acetonitrile to water in the aqueous acetonitrile extract is 60:40 to 85:15, and contains 0.1% to 1.0% formic acid or acetic acid by volume and 0.01% to 0.10% ethylenediaminetetraacetate by mass and volume. A salting-out agent comprising anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate is added in a mass ratio of (4–8):(1–2):(0.5–2):(0.25–1). The supernatant after salting out was purified by PSA, and a first purified solution and a second purified solution were formed based on the retention relationship between the target analyte and the MCX and the response compatibility relationship between the pesticide to be screened and the MCX. The first purification solution was diluted or reconstituted using an immunoreaction buffer containing 5 mmol / L to 20 mmol / L phosphate, 0.5% to 1.5% sodium chloride (w / v), 0.1% to 1.0% bovine serum albumin, and 0.01% to 0.10% polysorbate 20 with a pH of 7.0 to 8.0 to form the first sample solution. The second purification solution was diluted or brought to volume using an enzyme reaction buffer containing 20 mmol / L to 100 mmol / L of phosphate, free of nonionic surfactants, and with a pH of 7.5 to 8.5 to form a second sample solution. The volume fraction of organic solvent in both the first and second sample solutions was no higher than 10%. The time-resolved fluorescence signal was read 5 to 15 minutes after the first sample solution reacted. The second sample solution was pre-incubated with acetylcholinesterase at 30°C to 40°C for 5 to 15 minutes. After adding acetylthiocholine and 5,5′-dithiobis(2-nitrobenzoic acid) and reacting for 2 to 10 minutes, the absorbance was read in the range of 405 nm to 420 nm. Beneficial effects

[0020] This invention obtains matrix interference data characterizing the degree of interference from lipids, pigments, and proteins through extraction and salting out. It also combines the retention relationship between the target analyte and the MCX adsorbent with pesticide response compatibility, breaking the limitations of traditional fixed purification schemes based on food names. This invention enables the determination of the purification path as needed based on the actual degree of matrix interference and the properties of the target analyte, taking into account both matrix purification effect and detection response retention rate.

[0021] This invention determines a first purification path and a second purification path for homologous extraction supernatant of the same food sample, so that time-resolved fluorescence immunochromatographic detection and enzyme inhibition colorimetric detection can adopt appropriate purification methods according to their respective detection principles, reducing the adverse effects of a uniform purification process on different detection channels.

[0022] This invention adjusts the two purification solutions using immunoreaction buffer and enzyme reaction buffer respectively, and limits the volume fraction of organic solvents in the sample solution, which helps to ensure the stability of immunochromatographic migration, antigen-antibody reaction and enzyme inhibition reaction.

[0023] This invention sets up a fluorescence quality control area, a sample blank area, and an enzyme activity control area, which can identify situations such as chromatography failure, abnormal sample background absorbance, abnormal enzyme activity, abnormal reaction temperature, or abnormal reaction time, and stop outputting the corresponding detection conclusion when quality control is ineffective, thereby improving the reliability of the detection results. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the connection structure of the multi-index integrated detection platform for food testing according to the present invention. Detailed Implementation

[0025] To facilitate understanding of the present invention by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0026] In this invention, the target analyte to be detected refers to the target analyte that can be specifically detected by time-resolved fluorescence immunochromatography, and can be veterinary drugs, mycotoxins, food additives, allergens, or other food hazard factors for which an immunochromatographic detection method can be established.

[0027] The comprehensive pesticide screening results described in this invention refer to the category screening results of organophosphate and carbamate pesticides through acetylcholinesterase inhibition reaction, and are not used to distinguish individual pesticide types.

[0028] Homologous extraction supernatant refers to the supernatant obtained from the same food sample through the same extraction and salting-out process, and then subjected to separate processing in subsequent steps.

[0029] MCX is a hybrid cation exchange adsorbent that combines reverse phase interaction and strong cation exchange interaction.

[0030] PSA is a primary and secondary amine adsorbent used to remove some organic acids, fatty acids, sugars, and polar matrix interferences.

[0031] C18 is an octadecyl-bonded silica gel adsorbent used to remove some lipids and hydrophobic matrix interferences.

[0032] Bypassing the MCX fixed bed means that the sample solution does not pass through the MCX fixed bed, but can pass through an inert empty column or directly enter the subsequent processing unit through a bypass pipeline.

[0033] Continuous emulsification refers to the presence of a stable emulsion layer in the extract after treatment under specified centrifugation conditions, and the inability to obtain a clear organic supernatant that meets the preset volume requirements.

[0034] The detection platform of this embodiment includes a sample pretreatment module, a sample liquid conditioning module, a multi-index detection module, a dual-mode optical detection module, and a quality control and result processing module.

[0035] The modules work together via sample tubing and data connections. The sample pretreatment module includes extraction containers, salting-out containers, a centrifugation unit, a splitting unit, an adsorbent purification unit, and a fixed-bed purification unit. The sample conditioning module includes a quantitative pipetting unit, a buffer storage unit, and a mixing unit. The multi-index detection module includes a time-resolved fluorescence immunochromatography detection unit and an enzyme inhibition colorimetric detection unit. The dual-mode optical detection module includes fluorescence detection and absorbance detection optical paths. The quality control and result processing module is connected to each optical detection unit, temperature sensor, and time control unit.

[0036] The sample pretreatment module first extracts and precipitates the same food sample, obtaining a supernatant for detection and a sample solution for matrix interference measurement. The sample solution for matrix interference measurement is only used to generate lipid interference data, pigment interference data, and protein interference data, and does not enter the subsequent detection channels.

[0037] The supernatant used for detection is divided into at least two portions based on sample matrix characteristic data, the preset retention relationship between the target analyte and the MCX, and the preset response compatibility relationship between the pesticide to be screened and the MCX. Both purification paths include PSA purification, and corresponding auxiliary purification is included when interfering labels are present. The first portion is selected to pass through or bypass the MCX fixed bed according to the preset retention relationship between the target analyte and the MCX, forming the first purified solution. The second portion is selected to pass through or bypass the MCX fixed bed according to the preset response compatibility relationship between the pesticide to be screened and the MCX, forming the second purified solution.

[0038] The sample pretreatment module pre-stores matrix interference thresholds, retention relationships between target analytes and MCXs, response compatibility relationships between pesticides to be screened and MCXs, as well as auxiliary purification materials and dosages corresponding to different interference labels.

[0039] Lipid interference data (RL) is obtained by the ratio of the total volume Vo of the independent oil phase after salting out to the total volume Vs of the organic supernatant excluding the independent oil phase, i.e., RL = Vo / Vs. If no independent oil phase is formed, Vo is recorded as zero; if emulsification persists after centrifugation, an emulsification marker is generated, and this marker is used as one of the triggering conditions for lipid or protein-related auxiliary purification.

[0040] Pigment interference data were obtained by diluting the extraction supernatant by a preset factor and scanning the absorbance in the range of 400 nm to 700 nm. After subtracting the absorbance of the blank obtained from the same dilution, the maximum absorbance was taken as the pigment interference data.

[0041] Protein interference data are generated using parallel measurement solutions of food samples. These parallel solutions can be directly diluted and measured using the quinoline carboxylic acid method, or the organic solvent can be removed first, and then the solutions can be reconstituted with water or buffer before measurement. A calibration curve is established using standard protein solutions, and the absorbance of the sample solutions is converted into soluble protein concentration.

[0042] The protein measurement solution and the detection supernatant are isolated from each other to prevent copper ions and colorimetric reagents in the diquinoline carboxylic acid method from entering the immunoassay channel or the enzyme inhibition detection channel.

[0043] The sample preprocessing module compares each matrix interference data with a pre-set corresponding interference threshold. When any matrix interference data reaches or exceeds the corresponding threshold, the sample matrix feature data includes the corresponding lipid interference tag, pigment interference tag, or protein interference tag.

[0044] The matrix interference threshold was determined through validation experiments before formal testing. During validation, a representative blank food matrix containing neither the target analyte nor the pesticide to be screened was selected, and no fewer than five interference levels for lipids, pigments, or proteins were set, with no fewer than three parallel samples for each interference level.

[0045] For time-resolved fluorescence immunochromatography, the lowest interference level that causes the target analyte spiked response to deviate more than 20% from the non-interference control, or causes the fluorescence control signal to exceed the batch allowable range, is determined as the first candidate threshold.

[0046] For colorimetric detection of enzyme inhibition, the lowest interference level that causes the enzyme inhibition response of a representative pesticide of the same concentration to deviate by more than 20% from the interference-free control, or to cause the signal in the blank area of ​​the sample or the signal in the enzyme activity control area to exceed the batch allowable range, is determined as the second candidate threshold.

[0047] The lower of the first and second candidate thresholds is determined as the official interference threshold for the corresponding matrix interference data. When changing the main reagent batch, adsorbent material batch, or test card batch, the corresponding threshold can be re-verified and calibrated.

[0048] When the sample matrix characteristic data includes lipid interference tags, add C18 purification or cryogenic defatting to the purification path; when the sample matrix characteristic data includes pigment interference tags, add graphitized carbon black purification to the purification path; when the sample matrix characteristic data includes protein interference tags, add protein precipitation, centrifugation, or filtration to the purification path. When the corresponding purification path includes MCX fixed-bed purification, if the sample matrix characteristic data includes lipid interference tags or protein interference tags, increase the amount of MCX or use a tandem MCX fixed-bed, but the total amount of increased MCX should not exceed twice the original amount.

[0049] The auxiliary purification materials and their dosages are predetermined based on the validation results and stored in the sample pretreatment module. For example, the dosages of C18, graphitized carbon black, and PSA can be set in stages according to the volume of the supernatant and the corresponding interference level.

[0050] The pre-defined retention relationship between the target analyte and the MCX was established through a fixed-bed comparative experiment. Standard solutions of the target analyte at the same concentration were passed through the MCX fixed bed and the inert control fixed bed, respectively, and the recovery rate was determined under the same elution and collection conditions.

[0051] When the recovery rate of MCX fixed bed is reduced by more than 20% relative to the recovery rate of inert control fixed bed, the corresponding target analyte is classified as an MCX-retained target analyte; when the recovery rate of MCX fixed bed is reduced by no more than 20% relative to the recovery rate of inert control fixed bed, the corresponding target analyte is classified as an MCX-non-retained target analyte.

[0052] For MCX-retaining target analytes, the first purification pathway includes MCX fixed-bed purification. After the sample solution flows through the MCX fixed-bed, it can be eluted with a suitable elution buffer, and the organic solvent can be removed by nitrogen blowing or vacuum concentration, followed by reconstitution with immunoreaction buffer.

[0053] For non-retention target substances of MCX, the purification path of the first purification solution bypasses the MCX fixed bed. After PSA purification and corresponding auxiliary purification, it is directly diluted or concentrated and reconstituted.

[0054] The pre-defined response compatibility between the pesticides to be screened and the MCX was established through a comparative experiment on the enzyme inhibition response of representative pesticides. Representative organophosphates and carbamates were treated in an MCX fixed bed and an inert control fixed bed, respectively, and the enzyme inhibition response was measured under the same concentration and reaction conditions.

[0055] When the enzyme inhibition response deviation of a representative pesticide after treatment with an MCX fixed bed does not exceed 20%, the pesticide is deemed to be compatible with the MCX response, and the purification path of the second purification solution includes MCX fixed bed purification.

[0056] When the enzyme inhibition response deviation exceeds 20%, it is determined that the pesticide is incompatible with the MCX response, and the purification path of the second purification solution bypasses the MCX fixed bed.

[0057] After the first purification solution enters the sample conditioning module, it is diluted or reconstituted using an immunoreaction buffer. The immunoreaction buffer contains 5 mmol / L to 20 mmol / L phosphate, 0.5% to 1.5% sodium chloride (w / v), 0.1% to 1.0% bovine serum albumin (BSA) and 0.01% to 0.10% polysorbate 20 (v / v), with a pH of 7.0 to 8.0.

[0058] After the second purification solution enters the sample conditioning module, it is diluted or brought to volume using enzyme reaction buffer. The enzyme reaction buffer contains 20 mmol / L to 100 mmol / L of phosphate, does not contain nonionic surfactants, and has a pH of 7.5 to 8.5.

[0059] The organic solvent volume fraction in both the first and second solutions is not higher than 10% by dilution, nitrogen blowing concentration followed by resolution, or vacuum concentration followed by resolution.

[0060] The time-resolved fluorescence immunochromatographic assay unit can employ a competitive detection structure. The assay card includes a sample pad, a conjugation pad, a nitrocellulose membrane, an absorbent pad, at least one detection zone, and a fluorescence control zone. The time-resolved fluorescent label can be europium chelate microspheres or other labels with delayed fluorescence properties.

[0061] After the first sample is added to the detection card, it migrates along the membrane under capillary action. After a reaction of 5 to 15 minutes, the fluorescence detection optical path in the dual-mode optical detection module reads the time-resolved fluorescence signals of the detection area and the fluorescence control area. The fluorescence detection optical path performs acquisition after a preset delay following the excitation pulse to reduce the influence of sample autofluorescence and short-lived background signals.

[0062] The detection result can be calculated based on the ratio of the signal in the detection zone to the signal in the fluorescence control zone. For competitive detection structures, the signal in the detection zone typically decreases as the target analyte concentration increases. The quality control and result processing module outputs negative, positive, or quantitative results based on a pre-established standard curve or judgment threshold.

[0063] The enzyme inhibition colorimetric detection unit includes a sample reaction area, a sample blank area, and an enzyme activity control area. The sample reaction area contains a second sample solution, acetylcholinesterase, acetylthiocholine, and 5,5′-dithiobis(2-nitrobenzoic acid). The sample blank area does not contain acetylcholinesterase and is used to correct for sample background absorbance. The enzyme activity control area uses enzyme reaction buffer without the sample instead of the second sample solution to evaluate acetylcholinesterase activity.

[0064] The second sample solution was pre-incubated with acetylcholinesterase at 30°C to 40°C for 5 to 15 minutes, and then acetylthiocholine and 5,5′-dithiobis(2-nitrobenzoic acid) were added. After reacting for 2 to 10 minutes, the absorbance was read in the range of 405 nm to 420 nm.

[0065] The enzyme inhibition rate I can be calculated using the following formula: I=[1-(As-Ab) / Ac]×100% Where As is the absorbance of the sample reaction area, Ab is the absorbance of the sample blank area, and Ac is the absorbance of the enzyme activity control area.

[0066] When the absorbance of the sample blank area and the absorbance of the enzyme activity control area are both within the preset batch allowable range, the enzyme inhibition colorimetric detection result is valid. The quality control and result processing module compares the calculated enzyme inhibition rate with the preset screening threshold and outputs a negative or suspected positive result. A suspected positive result may suggest further confirmation using chromatographic or mass spectrometric methods.

[0067] The quality control and result processing module independently determines the results of the two detection channels. When the signal in the fluorescence control zone is within the preset batch allowable range, the time-resolved fluorescence immunochromatography detection result is valid; otherwise, no target analyte detection conclusion is output, and a retest is prompted.

[0068] The enzyme inhibition colorimetric detection result is valid when both the absorbance of the blank area and the absorbance of the enzyme activity control area are within the preset batch allowable range; otherwise, the pesticide comprehensive screening conclusion will not be output, and a retest will be prompted.

[0069] If the reaction temperature exceeds the preset range, the reaction time exceeds the preset range, the sample volume is insufficient, or the optical reading is abnormal during the detection process, the corresponding detection channel will be deemed invalid.

[0070] The following is a specific testing process.

[0071] Weigh the food sample and add the extract at a ratio of 4 to 10 ml of aqueous acetonitrile extract per gram of food sample. The volume ratio of acetonitrile to water in the extract is 60:40 to 85:15, and it contains 0.1% to 1.0% formic acid or acetic acid by volume, and 0.01% to 0.10% ethylenediaminetetraacetate by mass and volume.

[0072] After shaking extraction, a salting-out agent consisting of anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate was added in a mass ratio of (4–8):(1–2):(0.5–2):(0.25–1). After thorough mixing, the mixture was centrifuged to separate the layers.

[0073] Record the total volume Vo of the independent oil phase and the total volume Vs of the organic supernatant excluding the independent oil phase, and calculate RL. If persistent emulsification occurs, generate an emulsification indicator.

[0074] A portion of the supernatant was diluted using a preset ratio, and the maximum blank-corrected absorbance in the range of 400 nm to 700 nm was measured to generate pigment interference data.

[0075] A parallel measurement solution of another food sample was taken, diluted or had its organic solvent removed and reconstituted to a fixed volume, and the concentration of soluble protein was determined using the diquinoline carboxylic acid method to generate protein interference data.

[0076] Sample matrix feature data is generated based on three types of matrix interference data and pre-set corresponding interference thresholds. When any matrix interference data reaches or exceeds the corresponding threshold, a corresponding interference tag is generated in the sample matrix feature data. Based on the interference tag, it is determined whether to add C18, graphitized carbon black, protein precipitation, centrifugation, or filtration.

[0077] The supernatant for testing was divided into a first fraction and a second fraction. Both fractions were purified with PSA. For pathways requiring sequential PSA and MCX fixed-bed purification, the total amount of MCX and PSA was 15 mg to 80 mg, with a mass ratio of MCX to PSA of 1:1 to 1:4.

[0078] For MCX-retaining target analytes, the first fraction is purified by the MCX fixed bed, and if necessary, eluted and concentrated to form the first purified solution. For MCX-non-retaining target analytes, the first fraction bypasses the MCX fixed bed and is purified by PSA and corresponding auxiliary purification to form the first purified solution.

[0079] The second portion is selected based on the preset response compatibility relationship between the pesticide to be screened and the MCX, either passing through the MCX fixed bed or bypassing the MCX fixed bed, to form the second purification solution.

[0080] The first purified solution was diluted or reconstituted using an immunoreaction buffer to form the first sample solution, and the second purified solution was diluted or brought to a final volume using an enzyme reaction buffer to form the second sample solution, ensuring that the volume fraction of organic solvent in both sample solutions did not exceed 10%.

[0081] The first sample solution was added to the time-resolved fluorescence immunochromatographic detection unit, and the second sample solution was added to the enzyme inhibition colorimetric detection unit. The dual-mode optical detection module reads the time-resolved fluorescence signal and absorbance signal, respectively.

[0082] The quality control and result processing module first determines whether the quality control of each detection channel is effective, and then outputs the corresponding detection results.

[0083] The detection method of the present invention can also be implemented according to the following steps: Step 1: Extract and salt out the food sample to obtain the supernatant for detection and the sample solution for measuring matrix interference, which are isolated from each other; Step 2: Based on the ratio of oil phase volume to organic supernatant volume, the maximum blank-corrected absorbance of the supernatant, and the soluble protein concentration of the parallel measurement solutions, lipid interference data, pigment interference data, and protein interference data are generated. Step 3: Compare the three types of matrix interference data with the corresponding preset interference thresholds to form sample matrix feature data. When any matrix interference data reaches or exceeds the corresponding threshold, generate the corresponding interference label in the sample matrix feature data. Step 4: Based on the sample matrix characteristic data, the preset retention relationship between the target analyte and the MCX, and the preset response compatibility relationship between the pesticide to be screened and the MCX, the homologous extraction supernatant is split; both purification paths undergo PSA purification, and auxiliary purification corresponding to the interference label is performed when interference labels are present; the first fraction selects to pass through or bypass the MCX fixed bed based on the preset retention relationship between the target analyte and the MCX, and the second fraction independently selects to pass through or bypass the MCX fixed bed based on the preset response compatibility relationship between the pesticide to be screened and the MCX. Step 5: Adjust the first purification solution with an immunoreaction buffer to form the first sample solution; adjust the second purification solution with an enzyme reaction buffer to form the second sample solution. Step 6: Perform time-resolved fluorescence immunochromatography on the first sample and enzyme inhibition colorimetric detection on the second sample; Step 7: Determine the validity of the detection results based on the fluorescence control area signal, the absorbance of the sample blank area, and the absorbance of the enzyme activity control area, respectively. Step 8: Output the target analyte detection results and pesticide comprehensive screening results only when the corresponding quality control is effective.

[0084] The types of target analytes, the number of immunochromatographic detection zones, and the types of antibodies or antigens in this invention can be adjusted according to actual testing needs. A single test card can be configured with one or more detection zones to detect one or more target analytes.

[0085] The sample pretreatment module in this invention can employ manual pipetting, semi-automatic pipetting, or fully automatic flow path control. The MCX fixed bed can be a solid-phase extraction column, a micro fixed bed, or an integrated purification chip. PSA, C18, and graphitized carbon black can be extracted using dispersed solid-phase extraction or a fixed bed method.

[0086] The dual-mode optical detection module can use a shared sample chamber and separate fluorescence detection and absorption detection optical paths, or it can use two independent sample chambers. The fluorescence detection and absorption detection optical paths are uniformly controlled by the quality control and result processing module, but each reads data according to the time and optical parameters of its corresponding detection channel.

[0087] This invention enables different detection principles to operate in a coordinated manner on the same platform by employing homologous extraction, split purification, independent sample liquid conditioning, and independent quality control, while reducing repeated sampling and extraction.

[0088] 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. Those skilled in the art can adjust, substitute, or combine the above embodiments without departing from the concept of the present invention, and the resulting technical solutions should still fall within the scope of protection of the present invention.

Claims

1. A multi-index integrated detection platform for food testing, characterized in that, It includes a sample pretreatment module, a sample solution conditioning module, a multi-index detection module, a dual-mode optical detection module, and a quality control and result processing module; The sample pretreatment module is used to extract and precipitate the same food sample to obtain matrix interference data including lipid interference data, pigment interference data, and protein interference data. Each matrix interference data is compared with a pre-set corresponding interference threshold to generate sample matrix feature data. When any of the matrix interference data reaches or exceeds the corresponding pre-set interference threshold, the sample matrix feature data includes a corresponding interference tag, which is a lipid interference tag, a pigment interference tag, or a protein interference tag. The sample pretreatment module is also used to split the homologous extraction supernatant of the food sample and determine two purification paths based on the sample matrix characteristic data, the preset retention relationship between the target analyte and the MCX, and the preset response compatibility relationship between the pesticide to be screened and the MCX. Both purification paths include PSA purification, and the module selects to perform MCX fixed bed purification or bypass the MCX fixed bed based on the preset retention relationship and the preset response compatibility relationship, respectively, to form a first purified liquid and a second purified liquid. The sample solution adjustment module is used to dilute or reconstitute the first purified solution with an immunoreaction buffer to form a first sample solution, and to dilute or adjust the volume of the second purified solution with an enzyme reaction buffer to form a second sample solution. The multi-index detection module includes a time-resolved fluorescence immunochromatographic detection unit and an enzyme inhibition colorimetric detection unit. The time-resolved fluorescence immunochromatographic detection unit is used to detect at least one target analyte in the first sample solution, and the enzyme inhibition colorimetric detection unit is used to comprehensively screen organophosphates and carbamates in the second sample solution. The dual-mode optical detection module includes a fluorescence detection optical path for reading time-resolved fluorescence signals and an absorbance detection optical path for reading enzyme inhibition colorimetric absorbance. The quality control and result processing module is used to independently control the detection process of the time-resolved fluorescence immunochromatographic detection unit and the enzyme inhibition colorimetric detection unit, and output the target analyte detection results and pesticide comprehensive screening results when the corresponding quality control is effective.

2. The multi-index integrated detection platform for food testing according to claim 1, characterized in that, The matrix interference data includes: Lipid interference data RL, RL = Vo / Vs, where Vo is the total volume of the independent oil phase formed after salting out the same initial extract, and Vs is the total volume of the organic supernatant formed after salting out but excluding the independent oil phase; an emulsification indicator is formed when there is continuous emulsification but no independent oil phase is formed. Pigment interference data generated by the maximum blank-corrected absorbance in the range of 400 nm to 700 nm after the extract supernatant was diluted by a preset factor; and Protein interference data are generated when the soluble protein concentration of parallel measurement solutions of food samples is diluted or removed by organic solvents and reconstituted at a fixed volume, and then measured using the diquinoline carboxylic acid method. The sample solution used for matrix interference measurement is isolated from the sample solution used for detection and does not enter the time-resolved fluorescence immunochromatographic detection unit and the enzyme inhibition colorimetric detection unit.

3. The multi-index integrated detection platform for food testing according to claim 1, characterized in that: When the sample matrix feature data includes lipid interference tags, C18 purification or cryogenic defatting is added to the purification path; When the sample matrix feature data includes pigment interference tags, graphitized carbon black purification is added to the purification path; When the sample matrix feature data includes protein interference tags, protein precipitation, centrifugation, or filtration processes are added to the purification path. When the purification path includes MCX fixed bed purification, and the sample matrix characteristic data includes lipid interference tags or protein interference tags, the amount of MCX used is increased or a tandem MCX fixed bed is used, and the total amount of increased MCX does not exceed twice the original amount of MCX. The materials and dosages used for auxiliary purification are predetermined based on the corresponding interference labels and stored in the sample pretreatment module.

4. The multi-index integrated detection platform for food testing according to claim 1, characterized in that, The extraction and salting-out process uses an aqueous acetonitrile extract and a salting-out agent. The aqueous acetonitrile extract contains acetonitrile to water in a volume ratio of 60:40 to 85:15, and contains formic acid or acetic acid in a volume fraction of 0.1% to 1.0%, and ethylenediaminetetraacetic acid in a mass-volume concentration of 0.01% to 0.10%. The salting-out agent comprises anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate, in a mass ratio of (4-8):(1-2):(0.5-2):(0.25-1). For a purification path that sequentially performs PSA purification and MCX fixed-bed purification, the total dosage of MCX and PSA is 15 mg to 80 mg, and the mass ratio of MCX to PSA is 1:1 to 1:

4.

5. The multi-index integrated detection platform for food testing according to claim 1, characterized in that: The immune reaction buffer contains 5 mmol / L to 20 mmol / L phosphate, 0.5% to 1.5% sodium chloride, 0.1% to 1.0% bovine serum albumin, and 0.01% to 0.10% polysorbate 20, with a pH of 7.0 to 8.

0. The enzyme reaction buffer contains 20 mmol / L to 100 mmol / L of phosphate, is free of nonionic surfactants, and has a pH of 7.5 to 8.

5. The volume fraction of organic solvent in both the first and second sample solutions is no higher than 10%.

6. The multi-index integrated detection platform for food testing according to claim 1, characterized in that: The time-resolved fluorescence immunochromatographic detection unit includes at least one detection zone and one fluorescence control zone, and outputs the detection result of the target analyte based on the ratio of the time-resolved fluorescence signal of the detection zone to the fluorescence control zone. The enzyme inhibition colorimetric detection unit includes a sample reaction area, a sample blank area, and an enzyme activity control area, and calculates the enzyme inhibition rate based on the absorbance of the sample reaction area, the sample blank area, and the enzyme activity control area. When the signal in the fluorescence control area is within the preset batch allowable range, the time-resolved fluorescence immunochromatographic detection result is valid. When the absorbance of the sample blank area and the absorbance of the enzyme activity control area are both within their respective preset batch allowable ranges, the enzyme inhibition colorimetric detection result is valid. When the test result is invalid, the quality control and result processing module will not output the corresponding test conclusion, but will output a prompt for retesting.

7. A multi-index integrated detection method for food testing, characterized in that, The food testing multi-index integrated detection platform according to any one of claims 1 to 6 is implemented by including the following steps: Extraction and salting out were performed on the same food sample to obtain lipid interference data, pigment interference data, and protein interference data, respectively. Each matrix interference data is compared with a pre-set corresponding interference threshold to form sample matrix feature data; when any of the matrix interference data reaches or exceeds the corresponding pre-set interference threshold, a corresponding interference label is generated in the sample matrix feature data; Based on the sample matrix characteristic data, the preset retention relationship between the target analyte and the MCX, and the preset response compatibility relationship between the pesticide to be screened and the MCX, the homologous extraction supernatant of the food sample is divided into at least two portions, and two purification paths are determined for each portion. Both purification paths include PSA purification, and when the interference label is present, auxiliary purification corresponding to the interference label is included. The MCX fixed bed purification or bypassing the MCX fixed bed is selected according to the preset retention relationship and the preset response compatibility relationship to form a first purification liquid and a second purification liquid. The first purified solution was diluted or reconstituted using an immunoreaction buffer to form the first sample solution; The second purified solution was diluted or brought to a final volume using an enzyme reaction buffer to form the second sample solution; The first sample solution was subjected to time-resolved fluorescence immunochromatography, and the second sample solution was subjected to enzyme inhibition colorimetric detection. Determine whether the quality control of the two tests is effective, and output the target analyte test results and pesticide comprehensive screening results only when the corresponding quality control is effective.

8. The multi-index integrated detection method for food testing according to claim 7, characterized in that, Before conducting food sample testing, the corresponding pre-set interference thresholds are determined using the following methods: Select a representative blank food matrix that does not contain the target analyte or the pesticide to be screened, and set no less than five interference levels for lipids, pigments or proteins, with no less than three parallel samples for each interference level; Time-resolved fluorescence immunochromatography and enzyme inhibition colorimetric reactions were performed at each interference level. The lowest level of interference that causes the target analyte spiked response to deviate more than 20% from the unaffected control, or that causes the fluorescence control signal to exceed the batch allowable range, is determined as the first candidate threshold. The lowest level of interference that causes the enzyme inhibition response of a representative pesticide at the same concentration to deviate by more than 20% from the interference-free control, or to cause the signal in the blank area of ​​the sample or the signal in the enzyme activity control area to exceed the batch allowable range, is determined as the second candidate threshold. The lower of the first candidate threshold and the second candidate threshold is determined as the pre-set corresponding interference threshold for the corresponding matrix interference data.

9. The multi-index integrated detection method for food testing according to claim 7, characterized in that: By passing the standard solution of the target analyte through an MCX fixed bed and an inert control fixed bed respectively, and comparing the recovery rates of the two, a pre-defined retention relationship between the target analyte and the MCX was established. When the recovery rate of MCX fixed bed decreases by more than 20% relative to the recovery rate of inert control fixed bed, the corresponding target analyte is classified as an MCX-retaining target analyte. When the decrease in the recovery rate of the MCX fixed bed relative to the recovery rate of the inert control fixed bed does not exceed 20%, the corresponding target analyte is classified as an MCX non-retention target analyte. When the target analyte is an MCX-retaining target analyte, the purification path of the first purification solution includes MCX fixed-bed purification. When the target analyte is a non-retention type target analyte of MCX, the purification path of the first purification solution bypasses the MCX fixed bed; The purification path of the second purification solution is determined independently based on the preset response compatibility relationship between the pesticide to be screened and the MCX. When the enzyme inhibition response deviation of the representative pesticide after treatment with the MCX fixed bed does not exceed 20%, the purification path of the second purification solution includes purification with the MCX fixed bed. When the enzyme inhibition response deviation exceeds 20%, the purification path of the second purification solution bypasses the MCX fixed bed.

10. The multi-index integrated detection method for food testing according to claim 9, characterized in that: The food sample was extracted by adding 4 to 10 ml of aqueous acetonitrile extract per gram of food sample. The aqueous acetonitrile extract contained acetonitrile to water in a volume ratio of 60:40 to 85:15 and contained 0.1% to 1.0% formic acid or acetic acid by volume and 0.01% to 0.10% ethylenediaminetetraacetate by mass and volume. A salting-out agent comprising anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate is added in a mass ratio of (4–8):(1–2):(0.5–2):(0.25–1). The supernatant after salting out was purified by PSA, and a first purified solution and a second purified solution were formed based on the retention relationship between the target analyte and the MCX and the response compatibility relationship between the pesticide to be screened and the MCX. The first purification solution was diluted or reconstituted using an immunoreaction buffer containing 5 mmol / L to 20 mmol / L phosphate, 0.5% to 1.5% sodium chloride (w / v), 0.1% to 1.0% bovine serum albumin, and 0.01% to 0.10% polysorbate 20 with a pH of 7.0 to 8.0 to form the first sample solution. The second purification solution was diluted or brought to volume using an enzyme reaction buffer containing 20 mmol / L to 100 mmol / L of phosphate, free of nonionic surfactants, and with a pH of 7.5 to 8.5 to form a second sample solution. The volume fraction of organic solvent in both the first and second sample solutions was no higher than 10%. The time-resolved fluorescence signal was read 5 to 15 minutes after the first sample solution reacted. The second sample solution was pre-incubated with acetylcholinesterase at 30°C to 40°C for 5 to 15 minutes. After adding acetylthiocholine and 5,5′-dithiobis(2-nitrobenzoic acid) and reacting for 2 to 10 minutes, the absorbance was read in the range of 405 nm to 420 nm.

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