Compact wide-field femtoliter-chamber imaging system for high-speed and accurate digital bioanalysis.

The femtoliter-chamber array is a bioanalytical platform that enables highly sensitive and quantitative analysis of biological reactions at the single-molecule level. This feature has been considered a key technology for "digital bioanalysis" in the biomedical field; however, its versatility is limited by the need for a large and expensive setup such as a fluorescence microscope, which requires a long time to acquire the entire image of a femtoliter-chamber array. To address these issues, we developed a compact and inexpensive wide-field imaging system (COWFISH) that can acquire fluorescence images with a large field of view (11.8 mm × 7.9 mm) and a high spatial resolution of ∼ 3 μm, enabling high-speed analysis of sub-million femtoliter chambers in 20 s. Using COWFISH, we demonstrated a CRISPR-Cas13a-based digital detection of viral RNA of SARS-CoV-2 with an equivalent detection sensitivity (limit of detection: 480 aM) and a 10-fold reduction in total imaging time, as compared to confocal fluorescence microscopy. In addition, we demonstrated the feasibility of COWFISH to discriminate between SARS-CoV-2-positive and -negative clinical specimens with 95% accuracy, showing its application in COVID-19 diagnosis. Therefore, COWFISH can serve as a compact and inexpensive imaging system for high-speed and accurate digital bioanalysis, paving a way for various biomedical applications, such as diagnosis of viral infections.