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.

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.

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.

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.

Construction of a portable immunosensor for the sensitive detection of carbendazim in agricultural products using a personal glucose meter.

Portable and sensitive detection of carbendazim (CBD) is highly desirable for food safety and environmental protection. Herein, a portable immunosensor for the sensitive detection of CBD is proposed based on alkaline phosphatase (ALP)-labeled and secondary antibody-modified gold nanoparticles (AuNPs). The quantification is based on ALP catalyzing the dephosphorylation of glucose-1-phosphate disodium salt to generate glucose, thus converting the concentration of CBD into glucose, thereby realizing the portable detection of CBD by personal glucose meter. Benefiting from signal amplification strategy that integrates the large specific surface area of AuNPs, the enzymatic reactions of terminal deoxynucleotidyl transferase and ALP, a low detection limit of 0.37 ng/mL for CBD is achieved. When this portable method is used to analyze citrus fruit, canned citrus, and cabbage, good-consistency results are obtained with the UPLC-MS/MS method. The good performance demonstrates the great potential of this portable method for CBD monitoring in resource-poor settings.