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.

A polydimethylsiloxane (PDMS)-based self-priming microfluidic chip with cushion chambers is presented in this study for robust and easy-operation digital polymerase chain reaction (dPCR). The chip has only one inlet and can partition samples autonomously through negative pressure, provided by a de-gassed PDMS layer with a multi-level vertical branching microchannel design. Meanwhile, cushion chambers make the chip capable of very robust use for sample partitioning. Finally, the proposed microfluidic chip showed excellent performance in the absolute quantification of a target gene by performing quantitative detection of a 10-fold serial dilution DNA template. Owing to its characteristics of easy operation, low cost, and high robustness, the proposed dPCR chip is expected to further promote the extensive application of digital PCR, especially in resource-limited settings.

Deoxyribonucleic acid (DNA) sequencing technology provides important data for the disclosure of genetic information and plays an important role in gene diagnosis and gene therapy. Conventional sequencing devices are expensive and require large and bulky optical structures and additional fluorescent labeling steps. Sequencing equipment based on a semiconductor chip has the advantages of fast sequencing speed, low cost and small size. The detection of DNA base pairing is the most important step in gene sequencing. In this study, a large-scale ion-sensitive field-effect transistor (ISFET) array chip with more than 13 million sensitive units is successfully designed for detecting the DNA base pairing. DNA base pairing is successfully detected by the sensor system, which includes the ISFET microarray chip, microfluidic system, and test platform. The chip achieves a high resolution of at least 0.5 mV, thus enabling the recognition of the change of 0.01 pH value. This complementary metal-oxide semiconductor (CMOS) compatible and cost-efficient sensor array chip, together with other specially designed components, can form a complete DNA sequencing system with potential application in the molecular biology fields.

It has been known that the slow response of ion-sensitive field-effect-transistors (ISFETs), with tremendous challenges on meeting the requirements of the accuracy and long-term stability, handicaps extensive application of ISFET sensors. In this paper, dual-mechanism slow site-response (DSSR) model, combining enriched buried sites theory and the mechanism of specific adsorption activity (SAA) of effective sites, is proposed to systematically understand the slow response characteristics of ISFETs during a test of pH variation. The SAA variation occurs during the whole slow response process, while the effects of buried sites dominate only at the short-term slow response stage. Experiments based on extended-gate ion-sensitive field-effect-transistors using both aluminum oxide and silicon nitride as sensing layer were carried out. It is found that the DSSR model can excellently explain the slow response characteristics of the measured ISFETs. Furthermore, the slow response characteristics of ISFETs with silicon nitride are revealed to be superior to that of ISFETs with aluminum oxide in the slow response performance.

Digital PCR, as a nucleic acid absolute quantification method at the single-molecule level, has been widely applied in early cancer screening, single-cell analysis, and other biomedical fields. However, existing digital PCR methods still suffer from high costs, complex operations, and low detection dynamic range, which limit their applications. In the study, we developed a microfluidic chip-based digital PCR with a high-density vertical structure using PDMS (polydimethylsiloxane) flexible material. The chip features a three-layer structure of glass–PDMS–glass, with the PDMS structural layer containing 30,000 reaction chambers, each with a volume of 0.713 nL. This vertical-structured chip can increase the total volume and the total number of chambers by 50% without changing the chip area and chamber volume, thereby significantly enhancing dynamic range and sensitivity of the chip detection. This chip is theoretically capable of achieving a nucleic acid detection dynamic range close to 10⁵. Moreover, the digital PCR quantitative detection results of five different concentrations of serially diluted KRAS plasmid DNA templates using this chip also validated the accuracy and reliability of the nucleic acid quantitative detection results. The vertical-structured digital PCR chip, with its simple manufacturing process, uniform and stable sample partitioning, wide detection dynamic range, and low cost, will promote the widespread application of digital PCR.