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.

Chronic hepatitis B poses a serious threat to human health and life, and early diagnosis is essential to improving patient cure rates. Hepatitis B virus (HBV) and Alpha-fetoprotein (AFP) are two key biomarkers for diagnosing chronic hepatitis B. In this study, we propose a silicon nanowire array field effect transistor (SiNW-array FET) biosensor that enables highly sensitive, real-time, and low-cost joint detection of both HBV and AFP. The SiNW-array FET is fabricated using traditional micro-nano fabrication techniques such as self-limiting oxidation and anisotropic etching, and its morphology and electrical properties were tested. The results show that the diameters of the fabricated silicon nanowires (SiNWs) are uniform and the SiNW-array FET exhibits a strong output signal and high signal-to-noise ratio. Through specific chemical modification on the surface of SiNWs, the SiNW-array FET is highly sensitive and specific to HBV-DNA fragments and AFP, with ultralow detection limits of 0.1 fM (HBV-DNA) and 0.1 fg/mL (AFP). The detection curve of the SiNW-array FET exhibits good linearity within the HBV-DNA concentration range of 0.1 fM to 100 pM and AFP concentration range of 0.1 fg/mL to 1000 pg/mL. More importantly, the device could also detect HBV-DNA successfully in serum samples, laying a solid foundation for the highly sensitive clinical detection of chronic hepatitis B.

The detection and monitoring of ions are essential for a broad range of applications, including industrial process control and biomedical diagnostics. Traditional ion-sensitive field-effect transistors require bulky and expensive reference electrodes, which face several limitations, including device miniaturization, high fabrication costs, and incompatibility with semiconductor manufacturing processes. Here, we introduce a reference-less semiconductor ion sensor (RELESIS) that utilizes anodic aluminum oxide film as both the sensitive and dielectric layer. The RELESIS is composed of a metal-oxide-semiconductor field-effect transistor and an interdigital electrode, which fundamentally eliminates the need for a reference electrode, thereby enabling device miniaturization. During fabrication, the anodic oxidation process is employed in place of the expensive atomic layer deposition method, significantly reducing manufacturing costs while maintaining high surface quality. In practical measurements, the RELESIS device demonstrated an excellent pH sensitivity of 57.8 mV/pH with a low hysteresis of 7 mV. As a proof-of-concept application, the RELESIS device was employed for real-time, non-destructive monitoring of milk freshness, accurately detecting pH changes from fresh to spoiled in milk samples. The combination of reference-less structure, low-cost fabrication, and superior sensing performance positions this technology as a promising platform for next-generation portable ion sensing systems in food safety, environmental monitoring, and point-of-care diagnostics.