A non-covalently bound redox indicator for electrochemical CRISPR-Cas12a and DNase I biosensors

A rapid and accurate biosensor for detecting disease biomarkers at point-of-care is essential for early disease diagnosis and preventing pandemics. CRISPR-Cas12a is a promising recognition element for DNA biosensors due to its programmability, specificity, and deoxyribonuclease activity initiated in the presence of a biomarker. The current electrochemical CRISPR-Cas12a-based biosensors utilize the single-stranded DNA (ssDNA) self-assembled on an electrode surface and covalently modified with the redox indicator, usually methylene blue (MB). In the presence of a biomarker, the nuclease domain is activated and cleaves ssDNA, decreasing the redox indicator signal. The covalent attachment of the MB to the ssDNA implies complexity and a higher production cost. Alternatively, some redox indicators can noncovalently bind to the ssDNA. Although such indicators have been applied for electrochemical nucleic acid detection, their potential for electrochemical CRISPR-Cas-based biosensors has not been explored. In this work, a ruthenium complex, [Ru(NH₃)₆]³⁺, was investigated as a redox indicator non-covalently binding to the ssDNA. Voltammetric studies and the optimization resulted in a simple and robust electrochemical method that was tested for deoxyribonuclease I (DNase I) activity detection and applied in the CRISPR-Cas12a-based biosensor for viral DNA (HPV-16). The biosensors revealed good analytical properties and represent an alternative to reported biosensors for nuclease activity requiring a covalent attachment of the redox indicator. Moreover, the developed method offers prospects for advancement and can be transformed to operate with other Cas nucleases to detect RNA and other analytes.