Dataset Information

A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR.

ABSTRACT: In this work, a double-deck microfluidic chip was presented for digital PCR application. This chip consists of two reverse-placed micro-patterned polydimethylsiloxane (PDMS) layers between the top and bottom glass substrates. Each micropatterned PDMS layer contains more than 20,000 cylindrical micro-chambers to hold the partitioned droplets. The double-deck designs can double the number of chambers and reagent capacity without changing the planar area of the chip. In addition, carbon black was mixed into the pure PDMS gel to obstruct the passage of fluorescence from the positive chambers between the two layers of the chip. Thus, the fluorescence signal of micro-chambers in different layers of the chip after PCR can be collected without mutual interference. The quantitative capability of the proposed chip was evaluated by measuring a 10-fold serial dilution of the DNA template. A high accuracy of the absolute quantification for nucleic acid with a dynamic range of 10⁵ was demonstrated by this chip in this work. Owing to its characteristics of small planar area, large capacity, and sensitivity, the double-deck microfluidic chip is expected to further promote the extensive applications of digital PCR.

SUBMITTER: Xu G

PROVIDER: S-EPMC7759810 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR.

Xu Gangwei G Si Huaqing H Jing Fengxiang F Sun Peng P Zhao Dan D Wu Dongping D

Micromachines 20201124 12

In this work, a double-deck microfluidic chip was presented for digital PCR application. This chip consists of two reverse-placed micro-patterned polydimethylsiloxane (PDMS) layers between the top and bottom glass substrates. Each micropatterned PDMS layer contains more than 20,000 cylindrical micro-chambers to hold the partitioned droplets. The double-deck designs can double the number of chambers and reagent capacity without changing the planar area of the chip. In addition, carbon black was m ...[more]

PMID: 33255151

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Project description:Digital polymerase chain reaction (dPCR) is a powerful tool for genetic analysis, providing superior sensitivity and accuracy. In many applications that demand minuscule reaction volumes, such as single cell analysis, efficient and reproducible sample handling and digitization is pivotal for accurate absolute quantification of targets, but remains a significant technical challenge. In this paper, we described a robust and flexible microfluidic alternating-pull-push active digitization (?APPAD) strategy that confers close to 100% sample digitization efficiency for microwell-based dPCR. Our strategy employs pneumatic valve control to periodically manipulate air pressure inside the chip to greatly facilitate the vacuum-driven partition of solution into microwells, enabling efficient digitization of a small-volume solution with significantly reduced volume variability. The ?APPAD method was evaluated on both tandem-channel and parallel-channel chips, which achieved a digitization efficiency of 99.5 ± 0.3% and 94.6 ± 0.9% within 10.5 min and 2 min, respectively. To assess the analytical performance of the ?APPAD chip, we calibrated it for absolution dPCR quantitation of ?DNA across a range of concentrations. The results obtained with our chip matched well with the theoretical curve computed from Poisson statistics. Compared to the existing methods for highly efficient sample digitization, not only does our technology greatly reduce the constraints on microwell geometries and channel design, but also benefits from the intrinsic amenability of the pneumatic valve technique with device integration and automation. Thus we envision that the ?APPAD technology will provide a scalable and widely adaptable platform to promote the development of advanced lab-on-a-chip systems integrating microscale sample processing with dPCR for a broad scope of applications, such as single cell analysis of tumor heterogeneity and genetic profiling of circulating exosomes directly in clinical samples.

Dataset Information

A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR.

Publications

A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR.

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