Construction of an Enzyme Cascade Based on the Accurate Adjacent Arrangement of Coupled Enzymes Using a Triblock PolyA DNA Probe.

Intracellular enzyme cascades are essential for various biological processes, and mimicking their functions in artificial systems has attracted significant research attention. However, achieving convenient and efficient spatial organization of enzymes on interfaces remains a critical challenge. In this work, we designed a simple single-DNA scaffold using triblock polyA single-stranded DNA for the arrangement of coupled enzymes. The scaffold was assembled onto a gold electrode through the affinity of polyA-Au, and two enzymes (glucose oxidase and horseradish peroxidase) were captured through hybridization. The molecular distance between the enzymes was regulated by changing the length of the polyA fragment. As a proof of concept, a glucose biosensor was constructed based on the enzyme cascade amplification. The biosensor exhibited excellent detection capability for glucose in human serum samples with a limit of detection of 1.6 µM. Additionally, a trienzyme cascade reaction was successfully activated, demonstrating the potential scalability of our approach for multienzyme reactions. This study provides a promising platform for the development of easy-to-operate, highly efficient, and versatile enzyme cascade systems using DNA scaffolds.

Development of a Single-Molecule Nanobalance Ratiometric Electrochemical DNA Biosensor Using a Triblock Poly-Adenine Probe.

The development of electrochemical DNA biosensors has been limited by their reliability and reproducibility due to many interfering factors such as electrode properties, DNA surface densities, and complex biological samples. In this work, we developed a nanobalance polyA hairpin probe (polyA-HP), which was effectively assembled onto the gold electrode surface through the affinity between the central polyA fragment and the Au surface. One flanking probe of the polyA-HP captured the target sequence together with a MB-labeled signal probe, and the other flanking probe captured a reference probe simultaneously. The MB signal related to the amount of target was normalized by the reference Fc signal; thus, the signal-to-noise (S/N) was as high as 2000, and the reproducibility was remarkably improved to 2.77%, even facing deliberately changed experiment conditions. By designing a hairpin structure at the terminal of the polyA-HP, the selectivity and specificity were dramatically improved for the analysis of mismatched sequences. The analysis performance of biological samples was dramatically improved after normalization, which is critical for its practicability. Our novel biosensor is a universal single-molecule platform for ratiometric biosensors with excellent performance in real samples, indicating great potential for next-generation high-precision electrochemical sensors.

Triblock probe-polyA-probe electrochemical interfacial engineering for the sensitive analysis of RNAi plants.

RNA interference (RNAi) is currently under fast development, which brings improved crop quality and new activity against pests in agriculture, by producing RNAs to specifically inhibit gene expression. This technology, in turn, creates a pressing need for sensitive and specific analytical methods of exogenous RNA molecules in genetically modified (GM) crops for safety assessment and regulation of RNAi plants and their products. In this work, we developed a novel RNA electrochemical biosensor for the analysis of GM maize samples based on a polyA-DNA capturing probe containing three DNA segments: the central polyA segment combined onto a gold electrode surface with adjustable configuration and density, and two flanking DNA probes simultaneously captured the RNA targets through hybridization. Both the assembling and hybridization capability of our probe were demonstrated, and we systematically optimized the analytical conditions. Finally, the ultrasensitive detection of 10 fM RNA was realized without any amplification processes, and the specificity was verified by analyzing non-target maize samples. Our electrochemical biosensor provided a reliable and convenient measurement strategy for RNAi safety and quality assessment, and more importantly, our PAP (probe-polyA-probe) capturing probe exhibited an innovative design for the detection of large RNA molecules with complex secondary structures.