Microfluidic bead-based assay for microRNAs using quantum dots as labels and enzymatic amplification

作者：Zhang, He^*; Liu, Yuan; Fu, Xin; Yuan, Lihua; Zhu, Zhenjun

来源：Microchimica Acta, 2015, 182(3-4): 661-669.

DOI：10.1007/s00604-014-1372-9

• Microfluidic beads array
• miRNA-29
• Quantum dots
• Primer extension
• Enzymatic elongation

We report on a microfluidic assay for microRNA using quantum dots as labels and capture probes immobilized in a bead array. Target microRNA flows along the microfluidic channel to hit the beads array where it hybridizes with the immobilized capture probes. Next, the hybrid is labeled by using the bound microRNAs as a primer for enzymatic elongation with biotin-labeled nucleotides. Due to the specificity of (a) the hybridization assay and (b) the enzymatic elongation step, this assay is quite selective and only the completely matched duplex can be labeled, in a final step, with streptavidin-labeled quantum dots. The method was applied to the specific detection of microRNAs that occur in the miRNA-29 family and display minute differences only in their nucleotide sequence. It does not require (a) a labeling step before hybridization and (b) no amplification. This on-chip assay for microRNA can detect concentrations as low as 0.1 pmol center dot L-1 (at an SNR of > 3) when using synthetic microRNA. The 200-fold better sensitivity than that of an off-chip test is ascribed to the microfluidic-based signal enhancement. Other features include rapid binding kinetics, the advantages of a homogeneous assay in a suspended microbead array, the detection sensitivity resulting from the use of quantum dots, small reagent consumption, short assay time, and parallel detection.

• 出版日期2015-2
• 单位湖南工程学院