Enhancing the Sensitivity of Aptameric Detection of Lysozyme with a “Feed-Forward” Network of DNA-Related Reaction Cycles

Abstract

In this study, a network of DNA-related reaction cycles was established to enhance the sensitivity of lysozyme detection with dual signal amplification, and aptamer-based reactions were integrated into this system to provide high specificity. The network was organized in a feed-forward manner: the "upstream cycles" recognized the lysozyme (the target) and released the "messenger strands" from probe A (a DNA construct); the "downstream cycles" received them and then released the "signal strands" from another DNA construct, probe B, in multiplied quantities to that of the original inputted lysozyme. The upstream cycles centered on "target-displacement polymerization", which circulates the lysozyme to provide primary amplification; the downstream cycles centered on "strand-displacement polymerization", which circulates the messenger strand to provide further amplification. There were also several "nicking-polymerization" cycles in both reaction groups that provide extra signal amplification. In total, the network enclosed eight interconnected and autonomic reaction cycles, with only two probes, two primers, and two enzymes needed as raw feeds, and the network can be operated simply in one-pot mode. With this network, lysozyme could be quantified at lysozyme concentrations as low as 2.0×10(-14) M, with a detection limit of 3.6×10(-15) M (3σ rule), which was seven orders of magnitude lower than that obtained without any amplification(1.8×10(-8) M). Detection of lysozyme in real serum samples confirmed the reliability and practicality of the assay based on this reported reaction network.