Aptasensor-based assay for dual-readout determination of aflatoxin B1 in corn and wheat via an electrostatic force-mediated FRET strategy.

A rapid and sensitive aptasensor was established for the dual-readout determination of aflatoxin B1 (AFB1) utilizing an electrostatically mediated fluorescence resonance energy transfer (FRET) signal amplification strategy. In the presence of AFB1, the aptamer preferentially bound to AFB1, resulting in the aggregation of bare gold nanoparticles (AuNPs) induced by NaCl, accompanied by a change of AuNP solution from wine-red to purple. This color change was used for colorimetric channel analysis. Then, the positively charged quantum dots were introduced into reaction system and interacted with negatively charged AuNPs, which successfully converted the color signal into a more sensitive fluorescence signal through FRET. The fluorescence quenching efficiency decreased with increasing concentrations of AFB1, and the fluorescence of aptasensor gradually recovered. The variation of fluorescence intensity was employed for fluorometric channel analysis. Under the optimal conditions, the color and fluorescence signals exhibited excellent response to AFB1 concentration within the ranges 10-320 ng·mL-1 and 3-320 ng·mL-1, respectively, and the limit of detection was as low as 7.32 ng·mL-1 and 1.48 ng·mL-1, respectively. The proposed aptasensor exhibited favorable selectivity, good recovery (85.3-113.4% in spiked corn and wheat samples), stable reproducibility (RSD<13.3%), and satisfactory correlation with commercial kits (R2=0.998). The aptasensor developed integrates advantages of modification-free, dual-readout, self-calibration, easy operation, and cost-effectiveness, while providing a simple and universal strategy for rapid and sensitive detection of mycotoxins in foodstuffs.

MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples.

Recently, urinary tract infection (UTI) triggered by bacteria carrying pan-drug-resistant genes, including carbapenem resistance gene blaNDM and blaKPC, colistin resistance gene mcr-1, and tet(X) for tigecycline resistance, have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of-care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 102 CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug-resistant genes in clinical UTI samples and provides the necessary medication guidance for UTI treatment.

Construction of a Degradation-Free DNA Conjugated Nanoprobe and Its Application in Rapid Field Screening for Sulfur Mustard.

Sulfur mustard (SM) is a notorious blistering chemical warfare agent. Rapid field screening for trace SM is of vital significance for the detection of antiterrorism and timely treatment. Here, a visual assay for SM was constructed on the basis of its inhibition for the G-quadruplexes/hemin DNAzyme. Specifically, multiple guanine (G)-rich single stranded oligonucleotides (ssODN) named S1 (80% of G in the total bases), i.e., the precursor for G-quadruplex, which could oxide tetramethylbenzidine (TMB) to its green product, were conjugated on the nonfouling polymer brush grafted magnetic beads (MB@P(C-H)). SM could specifically alkylate the N7 and O6 sites of G in the S1; thus, it failed to form the DNAzyme based signal reporter. It was demonstrated that the nonfouling P(C-H) interface on the magnetic bead (MB) could protect the conjugated ssODN from nuclease degradation, thus ensuring its well sensing performance in complex samples. Under the optimized conditions, this method achieved good sensitivity and selectivity with a limit of detection (LOD) as low as 0.26 µmol L-1, and the recoveries ranging from 86% to 117% were obtained for different SM spiked real samples. Above all, this method combining low cost and ready operation could be suited for rapid field SM screening in a wide range of environmental matrices.

Mismatched duplexed aptamer-isothermal amplification-based nucleic acid-nanoflower for fluorescent detection of okadaic acid.

We developed a highly sensitive fluorescent assay to detect okadaic acid (OA), a prevalent aquatic toxin posing serious health risks. Our approach uses a mismatched duplexed aptamer (DA) immobilized on streptavidin-conjugated magnetic beads (SMBs) to create a DA@SMB complex. In the presence of OA, the cDNA unwinds, hybridizes with a G-rich segment pre-encoding circular template (CT), and undergoes rolling circle amplification (RCA) to produce G-quadruplexes, which are detected using the fluorescent dye thioflavine T (ThT). The method has a LOD of 3.1 × 10-3 ng/mL, a linear range of 0.1 âˆ¼ 1.0 × 103 ng/mL, and was successfully applied to shellfish samples with spiked recoveries of 85.9% ∼ 102.2% and RSD less than 13%. Furthermore, instrumental analysis confirmed the accuracy and reliability of this rapid detection method. Overall, this work represents a significant advancement in the field of rapid aquatic toxin detection and has important implications for public health and safety.

Magnetic amino-rich hyper-crosslinked polymers for fat-rich foodstuffs pretreatment in nontargeted analysis of chemical hazards.

Nontargeted analysis for chemical hazards is highly desirable in controlling food safety to ensure human health. As the dominating interference in fat-rich foodstuffs, lipids removal is a great challenge in sample pretreatment. Herein, diverse lipids from both animal and vegetable oils are effectively removed and 565 chemical hazards with various physicochemical properties are used for method validation. These benefits are from the designed magnetic amino-rich hyper-crosslinked core-shell polymeric composites (Fe3O4@poly(MAAM-co-EGDMA)) and the application of an auto extraction system. Among them, the amino groups are the key factors for lipid removal. Theoretical calculations, isothermal titration calorimetry (ITC), and functional monomer replacement demonstrated that the mechanisms to universally capture free fatty acids (FFAs) and triglycerides (TGs) are electrostatic interaction and supplemented by hydrogen bonding. Overall, this work highlights the great application potentials of polymeric adsorbents as sample pretreatment materials for nontargeted analysis in food safety.

In Situ Growth Large Area Silver Nanostructure on Metal Phenolic Network Coated NAAO Film and Its SERS Sensing Application for Monofluoroacetic Acid.

Rapid screening monofluoroacetic acid (FAcOH) is responsible for preventing chemical poisoning and food safety events. Whereas surface enhanced Raman scattering (SERS) spectra is an effective tool for detecting forbidden chemicals, it is difficult to directly detect FAcOH due to its small Raman scattering cross section as well as weak adsorption on SERS substrates. In this work, the metal phenolic supramolecular networks (MPNs, i.e., the tannic acid and Fe3+ complex) were fabricated on the commercial nanoanodic aluminum oxide film (NAAO) for assisting in situ chemical deposition highly uniform Ag nanostructure over large areas (the NAAO@AgNS). The low cost and simple fabrication process made the NAAO@AgNS a single-use consumable. For FAcOH detection, a specific derivative reaction between FAcOH and thiosalicylic acid (TSA) was introduced. By taking TSA as the Raman probe, its SERS signal attenuated constantly with the increasing amount of FAcOH. For improving quantitative accuracy, thiocyanate (SCN-) was introduced on the NAAO@AgNS as an internal standard; thus, the characteristic peak intensity ratios associated with TSA and SCN- (I1035/I2125) were fitted to the concentration of FAcOH. It was demonstrated that the SERS assay achieved good sensitivity and selection toward FAcOH with the limit of quantitation (LOD) as low as 50 nmol L-1. The NAAO@AgNS featured with highly sensitive, uniform, and consistent SERS performances could easily extend to wide SERS applications.

An Immunoaffinity Purification Method for the Simultaneous Analysis of Triclocarban and Triclosan in Foodstuffs by Liquid Chromatography Tandem Mass Spectrometry.

Triclocarban (TCC) and triclosan (TCS) have been simultaneously detected in five kinds of foodstuffs using an immunoaffinity purification method coupled with ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the first time. Two highly specific monoclonal antibodies against TCC and TCS were produced and coupled to N-hydroxysuccinimide-activated Sepharose 6B gel to prepare the immunosorbent. Under the optimal conditions, mean recoveries from spiked samples by the IAC-UHPLC-MS/MS method were 70.1-92.8% for TCC and 76.6-102.5% for TCS. Intraday relative standard deviations were below 14.5%. The limits of quantification (LOQs) of TCC were 1 ng/L for beverage samples and 0.01-0.02 µg/kg for food samples. The LOQs of TCS were 0.03 µg/L for beverage samples and 0.2-0.3 µg/kg for food samples. The applicability of the method has been proven by analyzing TCC and TCS in different samples from supermarkets in Beijing. The proposed method is sufficiently sensitive and reliable for monitoring trace concentrations of TCC and TCS in food samples.

Development of low matrix effects method for the analysis of bisphenol A and bisphenol S in aquatic products by immunoaffinity purification.

In this study, a high sensitive and reproducible method based on immunoaffinity cleanup followed by ultra-performance liquid chromatography-tandem mass spectrometry has been developed for the simultaneous determination bisphenol A (BPA) and bisphenol S (BPS) in aquatic products. The immunoaffinity column was prepared by coupling N-Hydroxysuccinimide-activated Sepharose 4B gel with monoclonal antibodies of BPA and BPS, which can selectively recognize the target analytes to achieve a low matrix interference. The matrix effects of different aquatic products were all less than ±10%. The limits of detection in four kinds of aquatic products was 0.060 µg/kg for BPA and 0.012 µg/kg for BPS. The recoveries of BPA and BPS in matrices varied from 74% to 106% with relative standard deviations <12%. The proposed method was successfully applied to real aquatic products.

Development of an immunoaffinity column for the highly sensitive analysis of bisphenol A in 14 kinds of foodstuffs using ultra-high-performance liquid chromatography tandem mass spectrometry.

An immunoaffinity clean-up material based on a monoclonal antibody (mAb) has been prepared for concentrating and purifying bisphenol A (BPA) in 14 kinds of foodstuffs at trace level. Haptens and immunogen of bisphenol A have been synthesized and comprehensively characterized. An mAb towards BPA was prepared and cross-reactivities with 14 BPA analogues were below 5%. The prepared antibody was coupled to N-hydroxysuccinimide-activated Sepharose 4B to manufacture an immunoaffinity column (IAC), which was applied to purify BPA in 14 kinds of foodstuffs. The analyte was then detected by means of ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Under the optimized conditions, compared with two traditional SPE clean-up methods, the IAC showed better selectivity (matrix effect <16.8%) and higher sensitivity. The limits of detection for BPA in 14 kinds of foodstuffs ranged from 0.001 µg L-1 to 0.01 µg kg-1, and the limits of quantification were in the range from 0.003 µg L-1 to 0.04 µg kg-1. The recoveries of BPA from spiked samples ranged from 82.0% to 104.9%, with RSDs below 13.8%. Besides, the IAC exhibited good reusability, with 40% column capacity remaining and no significant loss of recovery after 25 application cycles in real sample detection. These results demonstrated that the developed IAC-UPLC-MS/MS approach has wide applicability for purifying and detecting BPA in various foodstuffs.