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.