Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions

The fluorescence in situ hybridization (FISH)-based padlock probe and rolling circle amplification (RCA) method allows for the detection of point mutations. However, it requires multiple reaction steps and solution exchanges, making it costly, labor-intensive, and time-consuming. In this study, we aimed to improve the efficiency of padlock/RCA by determining the effects of microchannel shape and ultrasonic solution mixing. Using a circular-shaped microchamber and ultrasonic mixing, the efficiency of microfluidic padlock/RCA was improved, and the consumption of the expensive probe solution was reduced from 10 µL to approximately 3.5 µL. Moreover, the fluorescent probe hybridization time was reduced to 5 min, which is four times faster than that of the standard protocol. We used this method to successfully detect mitochondrial DNA and transcripts of β-actin and K-ras proto-oncogene codon 12 in cells. Our method offers improvements over current padlock/RCA methods and will be helpful in optimizing other microfluidics-based FISH-related analyses.