Detection of single‐base mutations using 1‐D microfluidic beads array

Abstract

The application of a 1-D microfluidic beads array that is composed of individually addressable functionalized SiO2 beads has been demonstrated for detection of single-base mutations based on "sandwich" hybridization assay without additional sample labeling and PCR amplification. We concentrated on detection of mutations in the human p53 tumor suppressor gene with more than 50% mutation frequency in the known human cancers. Using a microinjection system, functionalized beads could be selectively and linearly arrayed in a single microfluidic channel comprising many periodic chambers. This 1-D microfluidic beads array was sufficiently sensitive to identify single-nucleotide mutations in 40 pM quantities of DNA targets and could discriminate the mutated alleles in an excess of nonmutated alleles at a level of one mutant in 100 wild-type sequences. The surface of beads was regenerated and rehybridized up to six times without obvious loss of signal. The entire reaction process was done at room temperature within minutes, and only 2-10 microL sample solution was needed to complete the whole detection process. The p53 genotypes of A549, CNE2, and SKBr-3 cell lines were also correctly evaluated by using mRNA extracts as target without need for sample labeling and amplification. Thus, this platform enabled rapid and exact discrimination of gene mutations with the advantages of reusability, simple handling of liquid, low cost, and little reagent consumption.

Self-regenerating and hybrid irreversible/reversible PDMS microfluidic devices

“…Other methods of regeneration are related, in reality, to the reusability of chips following channel-cleaning processes by removing clogging from particles or chemically anchored species. Such methods include anti-fouling coatings 51 , rinsing at high flow rates, harsh chemicals 52 , desorption techniques 53 54 55 56 and more recently, thermal treatment 57 . The deployment of reversible bonding methods is another powerful output 40 .…”

Ex situ integration of multifunctional porous polymer monoliths into thermoplastic microfluidic chips

“…A variety of strategies have been reported for the integration of arrays of porous or 3-dimensional detection elements into microfluidic chips including integration of discrete functionalized capillary segments,[15] patterning of hydrogel micropatches,[16] and 1-dimensional microbead arrays. [17] For the case of porous monoliths, the in situ formation of a multifunctional array within a sealed microchannel is possible, but this approach would require a laborious sequential fabrication process,[18] together with the need for complex flow control to deliver separate functionalization reagents to each array element.…”

Droplet microfluidic preparation of au nanoparticles-coated chitosan microbeads for flow-through surface-enhanced Raman scattering detection

“…With miniaturized beads in channels, the detection spotting region is also much narrowed and real time monitoring can be easily performed. In addition, a bead-based system can be implemented in an array format with beads physically separated from each other for multiple detections (Sato et al 2002;Wen et al 2007;Yang et al 2009;Zhang et al 2007;Zhang et al 2008). However, the conventional way to make bead-based detections needs to prepare beads offline followed by loading them into the desired microfluidic channels and/or microwells (Lim and Zhang 2007).…”

“…However, the conventional way to make bead-based detections needs to prepare beads offline followed by loading them into the desired microfluidic channels and/or microwells (Lim and Zhang 2007). Currently, the loading mainly relies on manual loading assisted by specific micromanipulators Yang et al 2009;Zhang et al 2007;Zhang et al 2008), which is tedious, time consuming, and labor-intensive. In addition, there is a concern about contaminations caused by the subsequent sealing/bonding process if the pre-immobilized probe molecules on beads are sensitive to sealing/bonding reagents used subsequently.…”