Abstract:

More accurate, sensitive, and convenient methods are eagerly expected for quantifying nucleic acid in precision medicine. Therefore, in this study, a high-density picoliter-level microfluidic chip is developed for the precise detection of nucleic acid, which is combined with a dual fluorescence system based on isothermal multiple self-matching-initiated amplification (IMSA). Multilayer soft lithography technique is applied to the fabrication of the mold of chip. Then, the chip is produced by molding with the material of polydimethylsiloxane (PDMS). In addition, a simple nano-waterproof layer lying up on the microcell array is able to efficiently prevent the water from evaporation during the IMSA. On the chip, a total of 120000 reactors with picoliter volume for each are integrated and the density reaches up to 7000 reactors per cm². This device allows three samples counted through partitioning each into 40000 reactors at the same time. The sample can be sucked into each microcell of the chip by negative pressure equally without external power. Then, thermo-coagulation oil fills the channels to separate microcells automatically, avoiding the need of any valve. The microcells which contain the target are distinguished by a dual fluorescence system based on IMSA. Then, the accurate concentration of target can be calculated by the formula of Poisson distribution. As a proof-of-concept assay, the IMSA-based quantitative detection of hepatitis B virus (HBV) artificial plasmid DNAs by using this chip device was conducted. As indicated by the results, this device achieves a dynamic range of 10⁶ and the upper limit of quantitation reaches 1.13×10⁶ copies nucleic acid in 36 μL sample. Accordingly, this portable device with accurate quantitation ability and fast sample processing is very beneficial for precision detection.

Key words: accurate quantification, driven by negative pressure, high-density, isothermal multiple self-matching-initiated amplification (IMSA), microfluidic chip