The exploration of droplet digital branched rolling circle amplification based ultrasensitive biosensor for gastric cancer cell-derived extracellular vesicles detection

Abstract

Techniques for the ultrasensitive detection of cancer cell derived extracellular vesicles (EVs) surface protein are limited. Detection of cancer EVs directly from clinical samples is even more challengeable. Here we developed a droplet digital branched rolling circle amplification (ddBRCA) platform for the rapid and sensitive detection of cancer EVs. The design includes a hairpin structure DNA containing a cancer EVs specific aptamer sequence and BRCA primer sequence. The presence of target EVs could cause the conformational switching of the hairpin DNA, which subsequently triggers the BRCA. The reaction mixture was encapsulated in nanoliter droplets and allowed to incubate at 30 °C for 45 min. The fluorescent signal of each droplet was then measured using a microchip scanner. The results showed that our platform could detect EVs effectively with a limit of detection down to 12 particles/μL. Furthermore, the platform was tested using serum samples to validate its good prospect for clinical applicability. To sum it up, our platform was the first droplet digital RCA method so far, with advancements such as favorable rapidity (∼2 h), high sensitivity, and no washing step requirement, the proposed biosensor is expected to become a promising tool to be applied in clinical routines to assist in cancer diagnosis.

Keywords

• Extracellular vesicles
• Droplet digital
• Branched rolling circle amplification
• Washing-free