A Microfluidic Microbeads Array Chip Integrated with Micro-fluid Driven Micro-pump for Discrimination of Gene Mutation

Abstract

A new approach was developed for the single-nucleotide detection based on the micropump-integrated microfluidic microbeads array chip and an apyrase-mediated primer extension process.The microfluidic chip,primers-modified microbeads array and the micro-pump driven by evaporation and capillary effect were integrated to construct the detection chip.The target DNA flowed across fabricated microfluidic beads array and hybridized with immobilized primer sequences.When the 3′ terminus of primer was matched with the target DNA in the single-base mutation site of interest,under synergistic effect of apyrase and exonuclease-deficient Klenow DNA polymerase,the matched primer could be extended along the template DNA sequence and the biotin-dCTP could be incorporated into extended primers immobilized on the surface of microbeads.However,single-base mismatched duplexes in the 3′ terminus of primer could not be extended.Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal.The results indicated the on-chip single-nucleotide discrimination driven by micro-pump and liquid pressure could detect 0.2 pmol/L target DNA(S/N3) and 0.5 pmol/L target DNA respectively,but off-chip assay only discriminated 0.1 nmol/L of target DNA.The chip-based signal enhancement for single-nucleotide discrimination using micro-pump integrated microfluidic chip resulted in 500 times higher sensitivity than that of an off-chip test.The fluorescence signals were correlated to the concentration of the target DNA in the range of 0.5-30 pmol/L.Meanwhile,two multi-drug resistance gene 1(MDR1)-associated SNP sites(C3435T and G2677T) from a human genomic sample were determined using the proposed method.The fluorescence signals indicated the subject used here possessed the MDR1 3435CT and MDR1 2677TT genotypes,which were consistent with the results by DNA sequencing.This approach displays good specificity,sensitivity and stability for discrimination of gene mutation.