Exploring fluoropolymers for fabrication of femtoliter chamber arrays used in digital bioanalysis.

The global supply of fluoropolymers and fluorinated solvents is decreasing due to environmental concerns regarding polyfluoroalkyl substances. CYTOP has been used for decades primarily as a component of a femtoliter chamber array for digital bioanalysis; however, its supply has recently become scarce, increasing the urgency of fabricating a femtoliter chamber array using alternative materials. In this study, we investigated the feasibility of fabricating a femtoliter chamber array using four types of fluoropolymers in stable supply as candidate substitutes and verified their applicability for digital bioanalysis. Among these candidates, Fluorine Sealant emerged as a viable option for fabricating femtoliter chamber arrays using a conventional photolithography process. To validate its efficacy, we performed various digital bioanalysis using FP-A-based chamber arrays with model enzymes such as CRISPR-Cas, horseradish peroxidase, and ß-galactosidase. The results demonstrated the similar performance to that of CYTOP, highlighting the broader utility of FP-A in digital bioanalysis. Our findings underscore the potential of FP-A to enhance the versatility of digital bioanalysis and foster the ongoing advancement of innovative diagnostic technologies.

Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.

Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level. The peptidase/protease probes were prepared on the basis of a 7-amino-4-methylcoumarin fluorophore. The introduction of a phosphonic acid to the core scaffold made the probe suitable for use in a microdevice-based assay, while phosphonic acid served as the handle for the affinity separation of the probe using Phos-tag. Using this semi-automated scheme, 48 fluorogenic probes for the single-molecule peptidase/protease activity analysis were prepared. Activity-based screening using blood samples revealed altered single-molecule activity profiles of CD13 and DPP4 in blood samples of patients with early-stage pancreatic tumors. The study shows the power of single-molecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.

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.

Automated amplification-free digital RNA detection platform for rapid and sensitive SARS-CoV-2 diagnosis.

In the ongoing COVID-19 pandemic, rapid and sensitive diagnosis of viral infection is a critical deterrent to the spread of SARS-CoV-2. To this end, we developed an automated amplification-free digital RNA detection platform using CRISPR-Cas13a and microchamber device (opn-SATORI), which automatically completes a detection process from sample mixing to RNA quantification in clinical specimens within ~9 min. Using the optimal Cas13a enzyme and magnetic beads technology, opn-SATORI detected SARS-CoV-2 genomic RNA with a LoD of < 6.5 aM (3.9 copies µL-1), comparable to RT-qPCR. Additionally, opn-SATORI discriminated between SARS-CoV-2 variants of concern, including alpha, delta, and omicron, with 98% accuracy. Thus, opn-SATORI can serve as a rapid and convenient diagnostic platform for identifying several types of viral infections.