Preparation of monoclonal antibody against rhoifolin and its application in enzyme-linked immunosorbent assay of rhoifolin and diosmin.

Both rhoifolin and diosmin belong to flavonoids, which are widely present in citrus. Diosmin is not only used in the medical field in the world, but also used as a dietary supplement in the United States. Rhoifolin has a similar structure to diosmin and also exhibits antioxidant and anti-inflammatory properties. In this study, an anti-rhoifolin monoclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established. The half-maximal inhibitory concentration (IC50) of icELISA was determined to be 4.83 ng/mL, and the detection range was 0.97-33.87 ng/mL. The results of UPLC-MS/MS and icELISA generally demonstrate consistency. Moreover, by exploiting the cross-reactivity of the antibody, diosmin in tablets can be detected by icELISA. The results demonstrate that the developed method has good accuracy, reproducibility, and broad application prospects.

Highly sensitive detection of carbendazim in agricultural products using colorimetric and photothermal lateral flow immunoassay based on plasmonic gold nanostars.

The wide use and high toxicity of carbendazim (CBD) in agriculture pose unprecedented demands for convenient, sensitive, and cost-effective on-site monitoring. Herein, we propose a novel colorimetric and photothermal dual-mode lateral flow immunoassay (LFIA) based on plasmonic gold nanostars (AuNSs) for CBD detection in agricultural products. The AuNSs were synthesized via a rapid seed-mediated growth method (with growth time of ∼5 s). A stable immunoprobe was formed by adsorbing CBD antibodies onto AuNSs. This immunoprobe exhibited high conversion efficiency and sensitivity in photothermal detection with a low limit of detection (LOD) of 0.28 ng mL-1. The LOD of the colorimetric mode was higher (0.48 ng mL-1). The results of CBD detection in various agricultural products aligned well with ultra-performance liquid chromatography tandem mass spectrometry. Overall, our LFIA shows excellent sensitivity, specificity, reproducibility, and rapidness in CBD detection, and thus is a highly potential on-site platform in resource-limited environments.

A CRISPR/Cas12a-based fluorescence method for the amplified detection of total antioxidant capacity.

The CRISPR/Cas12a system is a powerful signal amplification tool that has been widely used in nucleic acid detection. It has also been applied to the assay of non-nucleic acid targets, mainly relying on strategies for converting target determination into nucleic acid detection. Herein, we describe a CRISPR/Cas12a-based fluorescence method for sensitive detection of the total antioxidant capacity (TAC) by utilizing a strategy of converting TAC determination into Mn2+ detection. Specifically, the reduction of MnO2 nanosheets by antioxidants produces plenty of Mn2+, which accelerates the trans-cleavage activity of CRISPR/Cas12a. Thus, a fluorescence enhanced detection method for TAC was established, with a detection limit as low as 0.04 mg L-1 for a typical antioxidant, ascorbic acid. More importantly, this method has been proven to successfully analyze TAC in beverages. The excellent analytical performance of this method demonstrates the great potential of the CRISPR/Cas12a system in simple and sensitive TAC analysis.

Hapten synthesis, monoclonal antibody production and immunoassay development for analyzing spiropidion residues.

Spiropidion developed by Syngenta shows high insecticidal and acaricidal activity against a wide range of sucking pests. In this study, according to the structure of spiropidion, two haptens were synthesized by introducing carboxyl groups from the ester group. After cell fusion, a monoclonal antibody (mAb 8B5) of spiropidion was obtained. The IC50 of the established heterologous indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was 7.36 ng/mL, and its working range was 1.75-34.92 ng/mL. The average recoveries were 76.05-124.78% in the Yangtze River and citrus samples. Moreover, the ic-ELISA results of 15 citrus samples agreed well with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Overall, the established ic-ELISA could be applied for the spiropidion residue monitor in food and agricultural samples.

A photothermal aptasensor based on rolling circle amplification-enriched DNAzyme for portable detection of ochratoxin A in grape juice.

Aptasensors for detection of ochratoxin A (OTA) have been extensively studied, but the majority of them require costly and large-scale equipment as signal readers. Herein, a photothermal aptasensor capable of portable detection of OTA through a thermometer was developed on basis of aptamer structural switching and rolling circle amplification (RCA)-enriched DNAzyme. Oligonucleotides and alkaline phosphatase (ALP) modified magnetic beads were prepared. The binding of aptamers to OTA led to the release of ALP labeled complementary DNA. After magnetic separation, ALP catalyzed the padlock dephosphorylation, inhibiting the subsequent RCA reaction. This process converted the OTA concentration into the amount of the photothermal reagent oxTMB produced from the catalytic reaction induced by RCA-enriched DNAzyme. Under the optimal conditions, the detection limit (LOD) of this aptasensor was 2.28 nM in a clean buffer, while the LOD reached 2.43 nM in 2 % grape juice. The good performance of the photothermal aptasensor makes it possible to measure OTA pollution in low resource environments.

Preparation of limonin monoclonal antibody and establishment of a sensitive icELISA for analyzing limonin in citrus and herbal samples.

Limonin is an intensely bitter and highly oxidized tetracyclic triterpenoid secondary metabolite, which is abundant in the Rutaceae and Meliaceae, especially in Citrus. In order to detect limonin content in complex substrates such as citrus and traditional Chinese medicine, monoclonal antibodies specifically recognizing limonin were prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The median inhibition concentration (IC50) was 5.40 ng/mL and the linear range was 1.25-23.84 ng/mL. The average recoveries from citrus peel and pulp samples were 95.9%-118.8% and 77.5%-113.1%, respectively. Moreover, the contents of limonin in 6 citrus samples and 4 herbal samples were analyzed by icELISA and UPLC-MS, and the results of the two methods were consistent. This validation is sufficient to demonstrate that the developed immunoassay is applicable for the detection of limonin in citrus and herbal samples and has the advantage of high efficiency, sensitivity, and convenience.

Integrating CRISPR-Cas12a and rolling circle-amplified G-quadruplex for naked-eye fluorescent "off-on" detection of citrus Alternaria.

Alternaria is a plant pathogen that spreads globally and is prone to causing citrus brown spot disease and metabolizing mycotoxins, thus seriously hindering the development of this economic crop industry. Herein, a "label-free" and "turn on" visual fluorescent assay for citrus Alternaria based on CRISPR-Cas12a and rolling circle amplification (RCA) was described. Using ssDNA complementary to RCA primer as a trans-cleavage substrate for CRISPR-Cas12a, the two systems of CRISPR-Cas12a and RCA-amplified G-quadruplex were skillfully integrated. By using a portable light source for excitation, the positive sample produced obvious red fluorescence, while the negative sample remained almost colorless, making them easy to differentiate with the naked eye. In addition, the specificity was demonstrated by distinguishing Alternaria from other citrus disease related pathogens. Moreover, the practicality was verified by analyzing cultured Alternaria and Alternaria in actual citrus leaf and fruit samples. Therefore, this method may contribute to the on-site diagnosis of Alternaria.

Determination, temporal variation and potential health risk assessment of pesticide residues in grapes from South and Southwest China.

Pesticide residues in grapes from South and Southwest China were determined using the QuEChERS procedure and UHPLC-MS/MS and GC-MS/MS methods. The 4-year monitoring and survey showed 94.6% of the 1341 samples of grapes collected from eight main production areas contained one or multiple pesticide residues (above the respective LOQs). Overall, 40 pesticides were detected, including 24 fungicides, 12 insecticides, 2 acaricides and 2 plant growth modulators, of which one pesticide was unauthorised for use in treating grapes. Two or more pesticide residues were discovered in 87.4% of the samples (above the respective LOQs), and pesticide residues in 5.7% of the samples exceeded the MRLs, such as difenoconazole, cyhalothrin, propiconazole, etc. The main risk factors affecting the safety of grape before 2019 were difenoconazole, cyhalothrin and cyazofamid. After 2019, however, the frequency of occurrence of the above pesticides significantly declined, and the banned or restricted pesticides including omethoate were not found, which was credited to the stricter supervision and management policies by local governments. Despite the high detection rates and multi-residue occurrence of pesticides in grapes, about 84% of the samples were compliant with regulatory standards. Moreover, the accumulative chronic diet risk determined from ADI is very low. This study and timely monitoring can ensure that grape growers comply with GAP and minimise the occurrence of residues.

Simultaneous determination of spiropidion and its five major metabolites in sweet orange fruit and various processing by-products using ultra-high performance liquid chromatography-tandem mass spectrometry.

The present work reported the application of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous analysis of spiropidion and its five major metabolites in sweet orange fruit and by-products throughout the whole industrial juicing process of the orange fruit. The reversed-dispersive solid phase extraction (r-DSPE) with multi-walled carbon nanotubes (MWCNTs) was employed for the extraction and purification. The established method was validated and satisfactory parameters (linearity, trueness, precision, sensitivity, matrix effect and stability) were obtained. And then, the field trial of spiropidion on sweet oranges has been conducted and the effect of commercial juicing processing on the residue of spiropidion and its metabolites was further investigated. The various processing factors (PFs) for washing, juicing, sterilization, concentrating and essential oil collecting were also determined. The final results indicated that washing processing reduced residues by 18.4%; the juicing step allowed a significant decrease of the spiropidion residue by 34.2-70.8%, with PFs value in the range of 0.290-0.658. However, high level of residual spiropidion (ranging from 4.016 to 4.205 mg/kg) was detected in orange essential oil, with PFs value of 17.157. All the above results demonstrated the efficiency of the established method in the routine control analysis of spiropidion residues in sweet orange fruits and their by-products, and will facilitate the further intensive research on its spatial distribution, transfer and degradation during the different processing procedures of the sweet orange fruits.

Determination, Quality, and Health Assessment of Pesticide Residues in Kumquat in China.

Pesticide residues in kumquat fruits from China, and the quality and chronic/acute intake risks in Chinese consumers, were assessed using the QuEChERS procedure and UHPLC-MS/MS and GC-MS/MS methods. Our 5-year monitoring and survey showed 90% of the 573 samples of kumquat fruits collected from two main production areas contained one or multiple residual pesticides. Overall, 30 pesticides were detected, including 16 insecticides, 7 fungicides, 5 acaricides, and 2 plant growth modulators, of which 2 pesticides were already banned. Two or more residual pesticides were discovered in 81% of the samples, and pesticide residues in 9.4% of the samples surpassed the MRLs, such as profenofos, bifenthrin, triazophos, avermectin, spirodiclofen, difenoconazole, and methidathion. The major risk factors on the safety of kumquat fruits before 2019 were profenofos, bifenthrin, and triazophos, but their over-standard frequencies significantly declined after 2019, which was credited to the stricter supervision and management policies by local governments. Despite the high detection rates and multi-residue occurrence of pesticides in kumquat fruits, about 81% of the samples were assessed as qualified. Moreover, the accumulative chronic diet risk determined from ADI is very low. To better protect the health of customers, we shall formulate stricter organic phosphorus pesticide control measures and stricter use guidelines, especially for methidathion, triazophos, chlorpyrifos, and profenofos. This study provides potential data for the design of kumquat fruit quality and safety control guidelines and for the reduction in health risks to humans.

Ascorbic acid-mediated in situ growth of gold nanostars for photothermal immunoassay of ochratoxin A.

Currently, the development of efficient mycotoxins detection methods, particularly using portable devices as readout devices, remains a great challenge. Herein, a photothermal enzyme-linked immunosorbent assay (ELISA) based on gold nanostars (AuNSs) for the detection of ochratoxin A (OTA) using a "thermometer" was proposed for the first time. AuNSs with photothermal conversion capacity were parepared using an ascorbic acid (AA)-mediated in situ growth methd. Quantification was based on the alkaline phosphatase catalyzing the dephosphorylation of ascorbic acid 2-phosphoate to AA, thereby converting OTA concentration to the amount of in situ synthesized AuNSs, thus achieving straightforward readout by temperature. Benefiting from the classical tyramine signal amplification strategy, a detection limit of 0.39 ng mL-1 was obtained. The recoveries of grape juice and maize samples spiked with 10 ng mL-1 and 30 ng mL-1 OTA ranged from 86.53% to 116.9%. Our method has great potential in on-site OTA detection for food safety.

RPA-CRISPR/Cas12a Combined with Rolling Circle Amplification-Enriched DNAzyme: A Homogeneous Photothermal Sensing Strategy for Plant Pathogens.

Alternaria is an endemic fungus associated with brown spot disease, which is one of the most serious citrus diseases. In addition, the mycotoxins metabolized by Alternaria threaten human health seriously. Herein, a novel homogeneous and portable qualitative photothermal method based on recombinase polymerase amplification (RPA), CRISPR/Cas12a, and rolling circle amplification (RCA) for the detection of Alternaria is described. Using RCA primers as substrates for CRISPR/Cas12a trans-cleavage, the two systems, RPA-CRISPR/Cas12a and RCA-enriched G-quadruplex/hemin DNAzyme, are intelligently combined. Target DNA at fg/µL levels can be detected with high specificity. Additionally, the practicability of the proposed method is demonstrated by analyzing cultured Alternaria from different fruit and vegetable samples, as well as citrus fruit samples collected in the field. Furthermore, the implementation of this method does not require any sophisticated equipment and complicated washing steps. Therefore, it has great potential to screen Alternaria in poor laboratories.

Preparation of spirodiclofen monoclonal antibody and establishment of indirect competitive enzyme-linked immunosorbent assay.

Spirodiclofen, a spirocyclic tetronic acid derivative, has excellent acaricidal effect and is used worldwide to control the majority of important mite species. For monitoring its residue in food and environmental samples, two haptens containing different spacer arms were synthesized, a monoclonal antibody (mAb 5A4) against spirodiclofen was prepared, and a heterologous indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established. The 50% inhibition concentration (IC50) of ic-ELISA was 25.46 ng/mL, and the working range was 5.59-133.85 ng/mL. The ic-ELISA showed no cross-reactivity with structural analogs of spirodiclofen and other commonly-used acaricides. The average recoveries from Shiranui citrus samples and Yangtze River water were 85.62%-97.74% and 85.95%-99.30%, respectively. In the analysis of 12 citrus samples, the results of the ic-ELISA were quite similar to those of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Hence, the new immunosorbent assay provides a substitute method for the qualitative and quantitative of spirodiclofen in food and environmental samples.

Seed-mediated in situ growth of photothermal reagent gold nanostars: Mechanism study and preliminary assay application.

Photothermal reagent-mediated portable detection platforms using thermometers as signal readers have received extensive attention due to their simplicity, low cost, and practicality. However, exploitation photothermal reagent with excellent photothermal conversion effect, convenient to synthesize, preferably without any modification for biosensing application, is still challenging. Herein, a simple and rapid seed-mediated in situ synthesis strategy has been developed for the preparation of gold nanostars (AuNSs) with remarkable photothermal conversion effect. By simply changing the seed size and component concentrations involved in the in situ synthesis process, AuNSs have adjustable geometries, allowing the photothermal conversion to be tuned to a high level optimal for biosensing. Meanwhile, an accurate understanding of the photothermal conversion mechanism is obtained by studying the relationship between the morphology of AuNSs and the photothermal effect. Subsequently, using ascorbic acid (AA) as a model target, the preliminary application of AuNSs in constructing a portable photothermal detection platform has been demonstrated. This in situ preparation strategy of AuNSs not only exhibits remarkable photothermal conversion effect, but also avoids complicated and time-consuming synthesis and modification. Therefore, it has great potential to be extended to portable detection of other targets by simply converting the concentration of the target to that of AA.

A CRISPR/Cas12a-based photothermal platform for the portable detection of citrus-associated Alternaria genes using a thermometer.

The outbreak of citrus brown spot because of Alternaria is one of the most destructive citrus diseases. Additionally, Alternaria species produce highly toxic mycotoxins. Mass screening is a valid method to control the spread of Alternaria. Morphological analysis and polymerase chain reaction combined with gene-sequencing technique are the most commonly used techniques for detecting Alternaria. However, they are limited by either low convenience and accuracy or low instrument accessibility and high cost. To balance the convenience, accuracy, test availability, and low cost, we develop a CRISPR/Cas12a-based photothermal platform for the portable detection of Alternaria genes using a thermometer. Using this platform, the Alternaria genes from the synthetic sequences and cultured fungus of citrus, tomato, and apple can be detected using a thermometer with a detection limit of 1.5 pM. With the aid of the CRISPR/Cas12a system, citrus-associated Alternaria can be specifically differentiated from other citrus disease-associated microorganisms. When the photothermal platform is applied to analyze the citrus fruit samples collected in the field, good-consistency results are obtained with the gene-sequencing technology. The excellent performance of this portable method shows that it can be applied to screen for Alternaria in resource-poor settings.

Simultaneous enantioselective determination of two succinate- dehydrogenase-inhibitor fungicides in plant-origin foods by ultra-high performance liquid chromatography-tandem mass spectrometry.

Fluindapyr and penthiopyrad are two new succinate-dehydrogenase-inhibitor fungicides both employed as racemic mixtures of enantiomers to control various fungal pathogens. In the present work, a robust and highly-sensitive method for simultaneous determination of fluindapyr and penthiopyrad enantiomers in plant-origin foods (cereals, fruits and vegetables) was developed using UPLC-MS/MS combined with a chiral stationary phase. Rapid baseline chiral separation of four stereoisomers of fluindapyr and penthiopyrad was obtained within 4.2 min on chiral MX(2)-RH column under reversed-phase conditions (with the eluent of acetonitrile/0.1% formic acid in water =70/30 (V:V) and column temperature maintained at 30 °C). The plant-origin samples were extracted quickly with acetonitrile and purified with multi-walled carbon nanotubes. Excellent linearity for the target analytes was observed in the concentration ranging from 1 to 250 µg/L with regression coefficient no less than 0.9967. The mean recoveries of fluindapyr and penthiopyrad enantiomers from six matrices were 77.1-107.2%, with all relative standard deviations values lower than 9.1%. The limit of quantification of four stereoisomers of two target chiral fungicides was 5 µg/kg. The analysis of real samples reveal that the developed method is suitable for the simultaneous chiral determination of fluindapyr and penthiopyrad residues in cereals, fruits and vegetables samples at enantiomeric level and can support their further investigation on enantioselective environmental behaviors and residue surveillance.

CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products.

Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn2+ ions to activate the Mn2+-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.

Dissipation of imidacloprid and its metabolites in Chinese prickly ash (Zanthoxylum) and their dietary risk assessment.

Dissipation of imidacloprid (IMI) and its metabolites (urea, olefin, 5-hydroxy, guanidine, 6-chloronicotinic acid) in Chinese prickly ash (CPA) was investigated using QuEChERS combined with UPLC-MS/MS. Good linearity (r2 ≥0.9963), accuracy (recoveries of 71.8-104.3%), precision (relative standard deviations of 0.9-9.4%), and sensitivity (limit of quantification ≤0.05 mg kg-1) were obtained. After application of IMI at dosage of 467 mg a.i. L-1 for three times with interval of 7 d, the dissipation dynamics of IMI in CPA followed first-order kinetics, with half-life of 6.48-7.29 d. IMI was the main compound in CPA, followed by urea and guanidine with small amounts of olefin, 5-hydroxy, and 6-chloronicotinic acid. The terminal residues of total IMI and its metabolites at PHI of 14-21 d were 0.16-7.80 mg kg-1 in fresh CPA and 0.41-10.44 mg kg-1 in dried CPA, with the median processing factor of 3.62. Risk assessment showed the acute (RQa) and chronic dietary risk quotients (RQc) of IMI in CPA were 0.020-0.083% and 0.052-0.334%, respectively. Based on the dietary structures of different genders and ages of Chinese people, the whole dietary risk assessment indicated that RQc was less than 100% for the general population except for 2- to 7-year-old children (RQc of 109.9%), implying the long-term risks of IMI were acceptable to common consumers except for children.

Ultrasound-assisted dispersive liquid-phase microextraction by solidifying L-menthol-decanoic acid hydrophobic deep eutectic solvents for detection of five fungicides in fruit juices and tea drinks.

An ecofriendly and efficient ultrasound-assisted deep eutectic solvents dispersive liquid-phase microextraction by solidifying the deep eutectic solvents-rich phase was developed to determine azoxystrobin, fludioxonil, epoxiconazole, cyprodinil, and prochloraz in fruit juices and tea drinks by high-performance liquid chromatography. A varieties of environmental hydrophobic deep eutectic solvents serving as extraction agents were prepared using L-menthol and decanoic acid as hydrogen-bond acceptor and hydrogen-bond donor, respectively. The deep eutectic solvents were ultrasonically dispersed in sample solutions, solidified in a freezer and easily harvested. The main variables were optimized by one-factor-at-a-time and response surface test. The new method performs well with relative recovery of 71.75-109.40%, linear range of 2.5-5000 µg/L (r ≥ 0.9968), detection limit of 0.75-8.45 µg/L, quantification limit of 2.5-25 µg/L,, and inter- and intraday relative standard deviations below 13.53 and 14.84%, respectively. As for the extraction mechanism, deep eutectic solvents were disposed into many fine particles in the solution and captured the analytes based on the changes of particle size and quantity in deep eutectic solvents droplets after extraction. The environmental method can successfully detect fungicide residues in real fruit juices and tea drinks.

A sensitive and practical ELISA for analyzing naringenin in pummelo and herb samples.

Naringenin, a flavonoid compound found in pummelo, is a key biological active compound in some traditional Chinese medicines, including Citri reticulatae pericarpium, Citri reticulatae pericarpium viride, Aurantii fructus immaturus, and Aurantii fructus. These Chinese medicinal preparations are the peels or immature fruits of certain citrus species. Aiming at detecting naringenin in complex matrices such as pummelo and traditional Chinese medicines, we put forward a sensitive and practical indirect competitive enzyme-linked immunosorbent assay (icELISA) based on anti-naringenin monoclonal antibodies (anti-Nar-mAbs). The median inhibitory concentration (IC50) was 4.43 ng/mL, and the working range was 1.15-15.81 ng/mL. The findings of the icELISA for the analysis of naringenin in pummelo and herb samples had a good correlation with the ultra performance liquid chromatography (UPLC) methodology and showed good accuracy and reproducibility. These data demonstrated that the developed icELISA is reliable, accurate, and suitable for detecting naringenin in pummelo and traditional Chinese medicines.