A high-specificity anti-triazole cross-interference ophiopogon polysperma paclobutrazol exclusive colloidal gold rapid detection method

Abstract

The application discloses a kind of high specificity anti-triazole cross interference's ophiopogon japonicus paclobutrazol exclusive colloidal gold rapid detection method, belong to traditional Chinese medicinal materials pesticide residue rapid detection technical field.For existing paclobutrazol colloidal gold detection technology identifies triazole public parent nucleus, cause uniconazole, tebuconazole, fenbuconazole, propiconazole Serious cross reaction;Ophiopogon japonicus high polysaccharide high saponin matrix interference is big, false positive rate is high;Prior art is only adapted to fruit and vegetable, not suitable for the defects of traditional Chinese medicinal materials detection.The application is modified by the unique t-butyl site of paclobutrazol directional hapten, accesses C2 Short carbon chain carboxyl linker, ophiopogon japonicus matrix-four interferants mixed antibody screening, ophiopogon japonicus special flocculation extraction buffer, no GCB specific d-SPE purification system, chromatography membrane whole system synergistic modification five innovations, constructs ophiopogon japonicus paclobutrazol single target exclusive detection system.The application is less than 5% to uniconazole, tebuconazole, fenbuconazole, propiconazole cross reaction rate, solves the problem of false positive of ophiopogon japonicus matrix non-specific adsorption;Sensitivity matches "Chinese Pharmacopoeia 2025 edition" ophiopogon japonicus paclobutrazol 0.1mg / kg screening threshold, can realize large quantities of ophiopogon japonicus sample on-site preliminary screening, positive sample can use LC-MS / MS pharmacopoeia method to confirm, has very high industrial application value.

Description

Technical Field

[0001] This invention relates to the field of rapid detection technology for plant growth regulator residues in Chinese medicinal materials, and in particular to a highly specific colloidal gold rapid detection method for paclobutrazol-specific compound that resists cross-interference from triazole pesticides. Background Technology

[0002] Ophiopogon japonicus is a major medicinal and edible herb in my country. The General Chapter 0212 of the 2025 edition of the Chinese Pharmacopoeia clearly stipulates that the residue limit of paclobutrazol in Ophiopogon japonicus shall not exceed 0.1 mg / kg. Confirmatory testing uses LC-MS / MS, corresponding to General Chapter 2342. While LC-MS provides accurate results, it involves high equipment investment, a long testing cycle, and cumbersome sample pretreatment, making it difficult to meet the actual needs of the industry for Ophiopogon japonicus procurement, warehousing acceptance, and large-scale on-site screening.

[0003] Colloidal gold immunochromatographic assay technology boasts advantages such as rapid detection speed, ease of operation, low cost, and on-site detection capability, making it a mainstream technique for rapid on-site testing of agricultural products and traditional Chinese medicinal materials. Existing patents CN119350247A and CN114133357A disclose the preparation technology of paclobutrazol colloidal gold test strips; however, this existing technology has inherent technical defects and cannot be directly applied to the detection of Ophiopogon japonicus samples. Specific problems are as follows: First, the design of hapten molecules has inherent flaws. Existing patents all select the common backbone site of triazole core and chlorobenzene ring to derive artificial antigens from the linker arm. The antibodies induced mainly recognize the common core structure of triazole pesticides, thus causing serious cross-reactivity with uniconazole, tebuconazole, cyproconazole, and propiconazole, resulting in a high false positive rate.

[0004] Secondly, there is a lack of a dedicated matrix processing system for traditional Chinese medicine. Existing extraction and purification systems are all developed for fruit and vegetable samples, and there are no dedicated flocculation and anti-interference reagents developed for the complex matrix of Ophiopogon japonicus, which is high in viscosity, polysaccharides, and saponins. During the detection of Ophiopogon japonicus samples, the non-specific adsorption phenomenon caused by the matrix is ​​prominent, which seriously interferes with the interpretation of test strips.

[0005] Third, antibody screening conditions are overly idealized. Current technology only uses buffer solution with a single paclobutrazol standard for antibody screening, without introducing a real matrix or coexisting interfering pesticides. While antibodies show good specificity under laboratory conditions, they exhibit significant deviations when detecting actual samples.

[0006] Fourth, the purification system has defects. Commercially available rapid testing d-SPE purification packing materials generally contain GCB graphitized carbon black. GCB has a strong adsorption effect on triazole pesticides, and paclobutrazol in the sample will be adsorbed by the packing material, directly causing false negatives.

[0007] Fifth, there is a lack of a joint verification system for multiple interfering agents. Existing patents have not conducted joint interference verification under conditions of coexistence of multiple triazole pesticides, and therefore cannot confirm the anti-interference ability of the technical solution in real complex samples of Ophiopogon japonicus.

[0008] In summary, existing publicly available technologies cannot meet the on-site screening requirements for the 0.1 mg / kg limit of paclobutrazol in the 2025 edition of the Chinese Pharmacopoeia. The industry urgently needs a rapid single-target paclobutrazol detection technology that is compatible with the Ophiopogon japonicus matrix across the entire chain, from antigen molecule modification, antibody screening, extraction buffer, purification process, chromatography material modification, and validation standards. Summary of the Invention

[0009] (1) Purpose of the invention The purpose of this invention is to overcome the shortcomings of existing paclobutrazol colloidal gold detection technologies, such as severe cross-interference from triazole pesticides, poor adaptability to Chinese medicinal material matrices, and frequent false positives and false negatives. This invention provides a highly specific, triazole-resistant, rapid colloidal gold detection method specifically for paclobutrazol in Ophiopogon japonicus. This invention reduces the broad-spectrum cross-recognition of triazole pesticides at the molecular level, adapts to the complex matrix of Ophiopogon japonicus (high in polysaccharides and saponins), and exhibits cross-reactivity rates of less than 5% for uniconazole, tebuconazole, cyproconazole, and propiconazole. The detection sensitivity matches the 0.1 mg / kg limit threshold for paclobutrazol in the 2025 edition of the Chinese Pharmacopoeia, enabling large-scale on-site initial screening of Ophiopogon japonicus samples. Positive samples can be confirmed using LC-MS / MS methods, filling the technological gap in compliant on-site screening of paclobutrazol in Chinese medicinal materials.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A highly specific colloidal gold rapid detection method for Ophiopogon japonicus paclobutrazol-specific paclobutrazol with anti-triazole cross-interference includes the following steps: 1) Targeted preparation of paclobutrazol-specific hapten and artificial antigen; 2) Preparation and forced screening of highly specific monoclonal antibodies based on Ophiopogon japonicus matrix and four triazole mixed interfering agents; 3) A dedicated extraction buffer system adapted to the high polysaccharide and high saponin matrix of Ophiopogon japonicus; 4) A one-step purification system with low adsorption specificity of d-SPE and no GCB; 5) Synergistic anti-interference modification process for the entire colloidal gold chromatography material system; 6) Specificity verification process for Ophiopogon japonicus with multiple interfering substances.

[0011] Furthermore, the paclobutrazol hapten is derived and modified by selecting the tert-butyl side chain hydroxyl site unique to the paclobutrazol molecule and attaching it to the C2 short carbon chain carboxyl linker arm; the derivatization process avoids the triazole core and chlorobenzene ring structural sites common to triazole pesticides, so that the immunogenic epitope is concentrated in the tert-butyl characteristic group unique to paclobutrazol; the obtained hapten is coupled to the carrier proteins BSA and OVA respectively to obtain the immunogen and the coating antigen, with the antigen-to-conjugate ratio controlled at 8-12:1.

[0012] Furthermore, in the monoclonal antibody screening stage, a mixed interference system was constructed using Ophiopogon japonicus negative matrix solution, with the addition of four interfering agents: paclobutrazol, tebuconazole, cyproconazole, and propiconazole, to conduct ELISA forced negative screening; hybridoma cell lines meeting the following criteria were obtained: paclobutrazol IC50 50 The concentration was 0.08-0.12 mg / kg, and the cross-reactivity rate (CR) of the four triazole analogs (uniconazole, tebuconazole, cyproconazole, and propiconazole) was <5%.

[0013] Furthermore, the specific extraction buffer for Ophiopogon japonicus is a phosphate buffer system with a pH of 7.2-7.6. The buffer components include 0.03%-0.08% Tween 20, 0.15%-0.3% sodium caseinate, and 0.2%-0.4% plant polysaccharide flocculation components by mass fraction. This extraction buffer can quickly precipitate Ophiopogon japonicus mucus, colloids, and macromolecular saponin impurities, and eliminate non-specific adsorption interference caused by the matrix.

[0014] Furthermore, the one-step d-SPE purification system does not contain any GCB graphitized carbon black; the filler ratio is: anhydrous magnesium sulfate 110-130mg, PSA 35-45mg, C18 10-15mg; the filler can remove pigments and organic acid impurities from the sample, while avoiding false negatives caused by the adsorption of paclobutrazol by graphitized carbon black.

[0015] Furthermore, the improved process of the colloidal gold chromatography system includes: selecting colloidal gold with a particle size of 25-30 nm; precisely controlling the pH of the colloidal gold labeling at 8.0-8.3; using a low-adsorption NC membrane; modifying the sample pad for protein blocking; reducing the original coating concentration of the T-line coating to compress the broad-spectrum antibody recognition window and achieve precise recognition of paclobutrazol single target.

[0016] Furthermore, the specificity verification process for multiple interfering agents specific to Ophiopogon japonicus was set up with 7 verification systems: blank Ophiopogon japonicus matrix group, paclobutrazol 0.1 mg / kg spiked group, uniconazole 0.5 mg / kg spiked group, tebuconazole 0.5 mg / kg spiked group, cyproconazole 0.5 mg / kg spiked group, propiconazole 0.5 mg / kg spiked group, and a mixed interference group of four triazole pesticides at 0.5 mg / kg each; the judgment criteria were: only the paclobutrazol 0.1 mg / kg group showed a positive result, and all other groups were negative, with no false positive inhibition.

[0017] This invention also provides a rapid detection kit for Ophiopogon japonicus using colloidal gold and paclobutrazol, prepared by the aforementioned detection method. Under the real matrix conditions of Ophiopogon japonicus, the kit shows no cross-false positives at spiking concentrations of 0.5 mg / kg for uniconazole, tebuconazole, cyproconazole, and propiconazole, and its specificity meets the screening limit requirement of 0.1 mg / kg for Ophiopogon japonicus in the 2025 edition of the Chinese Pharmacopoeia.

[0018] Compared with existing published patents CN119350247A and CN114133357A, the present invention has the following beneficial effects: 1. Innovative Molecular Antigen Design: This invention avoids the common core structure of triazoles and selects the unique tert-butyl hydroxyl site of paclobutrazol for hapten derivatization and modification, inserting a C2 short carbon chain carboxyl linker. The immunodominant epitope is concentrated on the paclobutrazol-specific group, reducing cross-recognition with uniconazole, tebuconazole, nifedipine, and propiconazole at the molecular structure level, which is different from all existing triazole rapid detection antigen design ideas.

[0019] 2. Innovative antibody screening process: The first-ever forced negative screening mode with the coexistence of Ophiopogon japonicus negative matrix and four triazole interfering agents was developed to screen for highly specific cell lines, solving the technical pain point of high specificity in laboratory conditions but high false positive rate in real sample detection that is common in the industry.

[0020] 3. Reagent formulation innovation: We developed a special flocculation extraction buffer for Ophiopogon japonicus, which is designed to flocculate and settle large molecular impurities in the high viscosity, polysaccharide and saponin matrix of Ophiopogon japonicus, reduce non-specific adsorption of the matrix, and is specially adapted to Chinese medicinal material samples, which is different from the general extraction system for fruits and vegetables.

[0021] 4. Innovative purification process: The GCB-free d-SPE purification formula avoids the strong adsorption effect of GCB on paclobutrazol, eliminates the risk of false negatives, and effectively removes pigments and organic acid impurities, ensuring stable sample recovery rate.

[0022] 5. Full-chain closed-loop technology innovation: This invention is not a single process parameter improvement, but a six-dimensional closed-loop technology system that realizes hapten modification - antibody forced screening - dedicated extraction solution - GCB-free purification - chromatography material modification - multi-interference verification. The technical barrier is significantly higher than that of existing single-point improvement patents.

[0023] 6. High industrial application value: The detection sensitivity matches the 0.1 mg / kg limit of paclobutrazol in the 2025 edition of the Chinese Pharmacopoeia, and can be used for large-scale on-site screening in Ophiopogon japonicus producing areas, purchasing and processing enterprises, and wholesale markets; positive samples can be sent to the laboratory for confirmation by LC-MS / MS, filling the technical gap in the rapid on-site detection of paclobutrazol in Ophiopogon japonicus; the raw materials for the whole process are readily available and the conditions are mild, making it suitable for the industrial mass production of the reagent kit. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the overall six-dimensional closed-loop innovative process of this invention. The diagram fully illustrates the closed-loop process from the targeted preparation of paclobutrazol-specific hapten, the forced screening of Ophiopogon japonicus matrix-specific antibodies, the exclusive flocculation extraction, the GCB-free purification treatment, the modification and assembly of the chromatography system, to the joint verification of multiple interfering substances, demonstrating the systematic innovative advantages of this invention compared to traditional single-point improvement technologies.

[0025] Figure 2 This is a schematic diagram of the molecular structure of the paclobutrazol hapten-specific modification site of the present invention; The diagram clearly distinguishes the common inert structure of triazole core and chlorobenzene ring shared by triazole pesticides, accurately marks the unique tert-butyl hydroxyl modification site of paclobutrazol, and the attached C2 short carbon chain carboxyl linker arm, intuitively demonstrating the molecular design features of this invention that target modification, avoid common triazole structures, and eliminate cross-interference from the source.

[0026] Figure 3 This is a flowchart of the process for extracting and purifying Ophiopogon japonicus samples according to the present invention. The diagram illustrates the complete process of Ophiopogon japonicus powder sampling, oscillation extraction with a dedicated flocculation buffer, flocculation and sedimentation of macromolecular impurities, one-step purification without GCB d-SPE, and centrifugation to obtain the test solution. It clearly demonstrates the core improved process of Ophiopogon japonicus-specific matrix anti-interference treatment and false negative purification.

[0027] Figure 4 This is a schematic diagram comparing the hapten modification sites of the present invention with those of existing patents; The left side shows the defects of existing technologies in modifying the common core and chlorobenzene ring structure of triazoles, which easily leads to cross-reactions with broad-spectrum triazole pesticides and a high false positive rate. The right side shows the exclusive design of this invention, which modifies only the unique tert-butyl site of paclobutrazol and combines it with the C2 short carbon chain carboxyl linker, which can form a specific immune epitope and completely avoid cross-recognition of triazoles.

[0028] Figure 5 This is a comparison chart showing the specificity verification effect of the combined polytriazole interferon of the present invention; Seven parallel validation groups were set up, including a blank matrix group, a pharmacopoeia-limited paclobutrazol group, single interference groups of four triazoles, and a mixed interference group. The blank Ophiopogon japonicus matrix group and all triazole interference groups were negative, with the negative criterion being the development of the C-line and T-line. Only the 0.1 mg / kg paclobutrazol-limited group showed a positive reaction, with the T-line significantly inhibited, demonstrating that the present invention possesses excellent anti-triazole cross-interference performance and exhibits no false positives.

[0029] Figure 6 This is a schematic diagram comparing the detection effects of the present invention and existing technologies on Ophiopogon japonicus samples; Existing technologies suffer from severe matrix interference from Ophiopogon japonicus, cloudy test strip background, band tailing, and a tendency to produce false positives and false negatives. The test strip of this invention has a clean background, clear and sharp bands, no tailing, and no discoloration, enabling accurate interpretation of paclobutrazol results under the complex matrix of Ophiopogon japonicus, and significantly improving the resistance to matrix interference. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0031] By selecting the unique tert-butyl side chain hydroxyl site of the paclobutrazol molecule and introducing a C2 short carbon chain carboxyl linker, a paclobutrazol-specific hapten was synthesized. The hapten was conjugated to BSA and OVA carrier proteins respectively using the carbodiimide method to prepare immunogens and coating antigens. The conjugation products were characterized and verified by ultraviolet spectroscopy, and the antigen-to-conjugation ratio was controlled at 8-12:1.

[0032] Balb / c mice were immunized with the immunogen obtained in Example 1 to complete cell fusion and prepare hybridoma cells. During the screening process, Ophiopogon japonicus negative matrix was used, and 0.5 mg / kg each of tebuconazole, tebuconazole, cyproconazole and propiconazole were added to construct a mixed interference system for forced negative screening by ELISA. Stable hybridoma cell lines were obtained through screening.

[0033] Cell line detection performance: Paclobutrazol IC 50 =0.092 mg / kg; cross-reactivity rate (CR) of uniconazole = 2.6%; CR of tebuconazole = 3.1%; CR of fenbendazole = 3.5%; CR of propiconazole = 2.7%; the cross-reactivity rates of all four interfering substances were less than 5%.

[0034] Weigh 2.0g of Ophiopogon japonicus powder sample, add 10mL of the Ophiopogon japonicus-specific extraction buffer of this invention, and shake for 3min; transfer the supernatant of the upper sample into a d-SPE purification tube without GCB; vortex for 1min, centrifuge at 3000r / min for 2min, take the supernatant, and use it for colloidal gold test paper detection; the processed sample solution is clear and there is no obvious matrix interference.

[0035] Purification pipe packing: 120mg anhydrous magnesium sulfate, 40mg PSA, 12mg C18.

[0036] Extraction buffer: pH 7.4 phosphate buffer containing 0.05% Tween 20, 0.22% sodium caseinate, and 0.3% plant polysaccharide flocculant.

[0037] The colloidal gold particles were 27 nm in diameter and labeled with a pH controlled at 8.1. The sample pad was double-sealed with BSA and casein. A low-adsorption NC membrane was selected. The original concentration of the T-line coating was optimized by reducing its volume, and the colloidal gold test strip was assembled.

[0038] Spiking validation was conducted using Ophiopogon japonicus matrix: Paclobutrazol 0.1 mg / kg showed a clear positive result at the pharmacopoeia limit point; under conditions of single spikes of 0.5 mg / kg for uniconazole, tebuconazole, cyproconazole, and propiconazole, as well as a mixed spike of 0.5 mg / kg for the four pesticides, the test strips showed no T-line inhibition and no false positives.

[0039] Referring to the technical solutions of patents CN119350247A and CN114133357A: a triazole nucleus site-derived hapten, common fruit and vegetable extracts, GCB-containing graphitized carbon black purification tubes, and a single standard buffer were used to screen antibodies. This system was applied to the detection of Ophiopogon japonicus matrix samples; the results showed that 0.5 mg / kg of uniconazole, tebuconazole, cyproconazole, and propiconazole in the Ophiopogon japonicus matrix all showed significant T-line inhibition, resulting in serious false positives, and therefore could not be used for the detection of Ophiopogon japonicus samples.

Claims

1. A rapid colloidal gold detection method for Ophiopogon japonicus paclobutrazol with high specificity and resistance to triazole cross-interference, characterized in that, Includes the following steps: 1) Targeted preparation of paclobutrazol-specific hapten and artificial antigen; 2) Preparation and forced screening of highly specific monoclonal antibodies based on Ophiopogon japonicus matrix and a mixture of four triazole interfering agents; 3) A dedicated extraction buffer system adapted to the high polysaccharide and high saponin matrix of Ophiopogon japonicus; 4) A one-step purification system with low adsorption specificity of d-SPE and no GCB; 5) Synergistic anti-interference modification process for the entire colloidal gold chromatography material system; 6) Specificity verification process for Ophiopogon japonicus with multiple interfering substances.

2. The method according to claim 1, characterized in that: The paclobutrazol hapten is derived and modified by selecting the tert-butyl side chain hydroxyl site unique to the paclobutrazol molecule and attaching it to the C2 short carbon chain carboxyl linker; avoiding the triazole core and chlorobenzene ring structural sites common to triazoles; so that the immunodominant epitope is completely concentrated in the tert-butyl characteristic group unique to paclobutrazol.

3. The method according to claim 1, characterized in that: In the monoclonal antibody screening stage, Ophiopogon japonicus negative matrix solution was directly used, and four interfering agents—paclobutrazol, tebuconazole, cyproconazole, and propiconazole—were simultaneously added to perform ELISA forced negative screening; the screening retained antibodies that met the following criteria: paclobutrazol IC50. 50 A specific cell line with a cross-reactivity rate (CR) of 0.08–0.12 mg / kg for four triazole analogs < 5%.

4. The method according to claim 1, characterized in that: The specific extraction buffer solution for Ophiopogon japonicus is a phosphate system with a pH of 7.2–7.6, containing: 0.03%–0.08% Tween 20, 0.15%–0.3% sodium caseinate, and 0.2%–0.4% plant polysaccharide flocculation components; it can quickly precipitate Ophiopogon japonicus mucus, colloids, and macromolecular saponin impurities, and eliminate non-specific matrix interference.

5. The method according to claim 1, characterized in that: The one-step d-SPE purification system contains no GCB graphitized carbon black. The filler ratio is: 110–130 mg anhydrous magnesium sulfate, 35–45 mg PSA, and 10–15 mg C18. This system removes pigments and organic acid impurities while completely avoiding false negatives caused by the adsorption of paclobutrazol by GCB.

6. The method according to claim 1, characterized in that: The improved chromatography system includes: 25–30 nm colloidal gold particles, pH 8.0–8.3 precise labeling, low adsorption NC membrane, protein blocking modification of the sample pad, T-line coating concentration reduction optimization, and compression of the antibody broad-spectrum recognition window to achieve precise single-target recognition.

7. A rapid detection kit for Ophiopogon japonicus paclobutrazol colloidal gold prepared by the method according to any one of claims 1-6, characterized in that: In the real matrix of Ophiopogon japonicus, there were no cross-false positive reactions to tebuconazole, fenbendazole, cyproconazole and propiconazole at a concentration of 0.5 mg / kg, and the specificity met the pharmacopoeia limit screening requirement of 0.1 mg / kg.

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