Enzyme-based digital bioassay technology - key strategies and future perspectives

Digital bioassays based on single-molecule enzyme reactions represent a new class of bioanalytical methods that enable the highly sensitive detection of biomolecules in a quantitative manner. Since the first reports of these methods in the 2000s, there has been significant growth in this new bioanalytical strategy. The principal strategy of this method is to compartmentalize target molecules in micron-sized reactors at the single-molecule level and count the number of microreactors showing positive signals originating from the target molecule. A representative application of digital bioassay is the digital enzyme-linked immunosorbent assay (ELISA). Owing to their versatility, various types of digital ELISAs have been actively developed. In addition, some disease markers and viruses possess catalytic activity, and digital bioassays for such enzymes and viruses have, thus, been developed. Currently, with the emergence of new microreactor technologies, the targets of this methodology are expanding from simple enzymes to more complex systems, such as membrane transporters and cell-free gene expression. In addition, multiplex or multiparametric digital bioassays have been developed to assess precisely the heterogeneities in sample molecules/systems that are obscured by ensemble measurements. In this review, we first introduce the basic concepts of digital bioassays and introduce a range of digital bioassays. Finally, we discuss the perspectives of new classes of digital bioassays and emerging fields based on digital bioassay technology.