Electrochemical Enzyme-Linked Sandwich Assay with a Cellulase Label for Ultrasensitive Analysis of Synthetic DNA and Cell-Isolated RNA.

Electrochemical enzyme-linked sandwich assays on magnetic beads (MBs) refer to one of the most sensitive approaches for analysis of non-amplified nucleic acid samples, with redox enzymes being routinely used as labels. Here, we report a sensitive and inexpensive electrochemical sandwich assay on MBs that exploits a hydrolytic enzyme cellulase as a label, while MBs are used for pre-concentration and bio-separation of analyzed samples. Binding of target DNA or RNA to capture DNA-modified MB triggers sandwich assembly and its labeling with cellulase. Application of the assembled sandwich to the electrodes covered with insulating nitrocellulose films induces film digestion by the cellulase label and pronounced changes in the electrical properties of the electrodes, the extent of the changes being proportional to the concentration of the analyzed nucleic acids. Down to 1 amole of L. brevis specific synthetic DNA and ribosomal RNA isolated from L. brevis cells could be detected in 1 mL samples in an overall from 2 to 3 h assay. The assay is universal and can be adapted for point-of-care-testing and for in-field environmental and microbiomic analysis of unamplified samples of any DNA/RNA sequences.