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

Abstract

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.

Nitric Oxide and a Conditioned Medium Affect the Hematopoietic Development in a Microfluidic Mouse Embryonic Stem Cell/OP9 Co-Cultivation System

“…Polydimethylsiloxane (PDMS) devices ( Figure 1a) were fabricated by molding as previously described [25][26][27]. The degassed PDMS prepolymer was poured onto a master with an I-shaped channel structure (depth × width × length, 0.2 mm, 0.5 mm, or 1 mm × 1 mm × 10 mm) to a thickness of 4 mm and baked at 65 • C for 1 h. The PDMS replica was peeled off from the master, mounted on a glass slide (26 mm × 76 mm), and baked again at 100 • C for 1 h. Through-holes were made at either end of the channel with a 1.0 mm biopsy punch.…”

Influence of Culture Conditions on Cell Proliferation in a Microfluidic Channel

“…Microchannels were fabricated by a molding process as described previously. 2,24 In brief, the degassed PDMS mixture was poured onto a master to a thickness of 4 mm, which has channel structure of 0.065 × 0.3 × 20, 0.25 × 1 × 10, or 0.5 × 1 × 10 mm (H × W × L), and then baked at 65 C for 1 h. The PDMS replica was peeled off from the master and adhered to a glass slide (26 × 76 mm), and then baked further at 100 C for 1 h. Through-holes were made at both ends of the microchannel with a 1.0-mm biopsy punch. Both bonding surfaces of the PDMS sheet and a cover slip were exposed to oxygen plasma at 100 W for 35 s.…”

A Microfluidic Cell Stretch Device to Investigate the Effects of Stretching Stress on Artery Smooth Muscle Cell Proliferation in Pulmonary Arterial Hypertension

“…Microchannels were fabricated by molding as described previously [23,24]. The degassed PDMS mixture was poured to a thickness of 4 mm (upper sheet) or 1 mm (lower sheet), with upper (0.5 × 1 × 10 mm) or lower (0.5 × 2 × 15 mm) channel structures, respectively, and baked at 65 • C for 1 h. The PDMS replica was adhered to a glass slide (26 × 76 mm) and baked at 100 • C for 1 h. Through-holes were made at both ends of the upper microchannel with a 1.0 mm biopsy punch.…”