Isothermal Reciprocal Catalytic DNA Circuit for Sensitive Analysis of Kanamycin.

Inappropriate use of veterinary drugs can result in the presence of antibiotic residues in animal-derived foods, which is a threat to human health. A simple yet efficient antibiotic-sensing method is highly desirable. Programmable DNA amplification circuits have supplemented robust toolkits for food contaminants monitoring. However, they currently face limitations in terms of their intricate design and low signal gain. Herein, we have engineered a robust reciprocal catalytic DNA (RCD) circuit for highly efficient bioanalysis. The trigger initiates the cascade hybridization reaction (CHR) to yield plenty of repeated initiators for activating the rolling circle amplification (RCA) circuit. Then the RCA-generated numerous reconstituted triggers can reversely stimulate the CHR circuit. This results in a self-sufficient supply of numerous initiators and triggers for the successive cross-invasion of CHR and RCA amplifiers, thus leading to exponential signal amplification for the highly efficient detection of analytes. With its flexible programmability and modular features, the RCD amplifier can serve as a universal toolbox for the high-performance and accurate sensing of kanamycin in buffer and food samples including milk, honey, and fish, highlighting its enormous promise for low-abundance contaminant analysis in foodstuffs.

Construction of a simple dual-mode ATP-sensing system for reliable fish freshness evaluation.

Adenosine triphosphate (ATP), the main carrier of chemical energy, plays a key role in various biochemical reactions such as cellular metabolism. Currently, ATP levels are considered important indicators of microbial content in food safety, and food freshness can be determined by detecting ATP content. Some ATP sensing strategies have been applied to evaluate food freshness. However, cumbersome nanomaterial preparation, low sensitivity, and low reliability hamper their widespread application. Herein, a simple, high-performance, and reliable dual-mode sensing system based on hemin-G-quadruplex (G4) DNAzyme was established to detect ATP and assess fish freshness. Two nucleic acid probes, including subunits of the hemin-G4 DNAzyme in inactive structures and anti-ATP aptamer, self-assemble upon the input of ATP into the active hemin-G4 DNAzyme unit. The generated DNAzyme acts as a biocatalyst for colorimetric or fluorescent readout of the sensing process. The colorimetric and fluorescent dual-mode sensing system enables highly sensitive and reliable analysis of target ATP with detection limits of 71 nM and 73 nM, respectively. Moreover, the biosensor exhibited good selectivity for differentiating ATP from other interfering analytes. The proposed system was used to detect ATP in perch samples, and a linear correlation between ATP level and microbial content was confirmed. The established ATP-sensing system reliably evaluated fish freshness. Notably, in comparison with microbiological counts, the proposed DNAzyme-based dual-mode strategy for freshness evaluation is facile, highly efficient, and cost-effective, thus providing a promising method for food safety and quality monitoring.

Construction of a self-sufficient DNA circuit for amplified detection of kanamycin.

Improper use of kanamycin can lead to trace kanamycin residues in animal-derived foods, which can pose a potential threat to public health. Isothermal enzyme-free DNA circuits have provided a versatile toolbox for detecting kanamycin residues in complicated food samples, yet they are always limited by low amplification efficiency and intricate design. Herein, we present a simple-yet-robust nonenzymatic self-driven hybridization chain reaction (SHCR) amplifier for kanamycin determination with 5800-fold sensitivity over that of the conventional HCR circuit. The analyte kanamycin-activated SHCR circuitry can generate numerous new initiators to promote the reaction and improve the amplification efficiency, thus achieving an exponential signal gain. With precise target recognition and multilayer amplification capability, our self-sustainable SHCR aptasensor facilitated the highly sensitive and reliable analysis of kanamycin in buffer, milk, and honey samples, thus holding great potential for the amplified detection of trace contaminants in liquid food matrices.