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

A novel approach for single-nucleotide detection based on the micropump-integrated microfluidic microbeads array chip and an apyrase-mediated primer extension process was developed. In this method, an integrated chip was constructed by a microfluidic chip, a primers-modified microbeads array and a micro-pump driven by evaporation and capillary effect. The target DNA flowed across the fabricated microfluidic beads array and hybridized with immobilized primer sequences. When the 3′ terminus of primer 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 extended along the template DNA sequence and incorporated the biotin-dCTP into the extended primers and immobilized it onto the surface of microbeads. Then, the streptavidin-labeled quantum dots bond with deposited biotin moities of biotin-dCTP and generated a fluorescence signal. On the contrary, there is no signal when signal-base mismatched duplexes were present in the 3′ terminus of the primer. The limit of detection is 0.2 pM target DNA (S/N > 3) for micro-pump driven chip and 0.5 pM target DNA for liquid pressure driven chip respectively. 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. Since the off-chip assay only detected 0.1 nM target DNA. The fluorescence signals are linear in the target DNA concentration ranging 0.5 pM to 30 pM. This method was also used to detect two multi-drug resistance gene 1 (MDR1)-associated SNP sites (C3435T and G2677T) from a human genomic sample. The fluorescence signals indicated the subject used here possessed both 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.

Graphical abstractAn integrated chip was constructed by a microfluidic chip, a primers-modified microbeads array and a micro-pump driven by evaporation and capillary effect. The apyrase-mediated allele-specific primer extension was applied on the integrated microfluidic chip using quantum dot labels for mutation analysis of gene. This approach displays good specificity, sensitivity and stability for discrimination of gene mutation.Figure optionsDownload full-size imageDownload as PowerPoint slide