A compact centrifugal multi-volume digital PCR disk (MV-dPCR disk) with integrated pneumatic stabilization.

Abstract

Digital polymerase chain reaction (dPCR) enables absolute nucleic acid quantification with high sensitivity and has strong potential for decentralized and integrated analytical applications. However, most current dPCR platforms rely on bulky pressure-driven instrumentation and large-footprint cartridges, limiting their suitability for compact and fully integrated microfluidic systems that incorporate upstream sample processing. Here, we introduce a centrifugal multi-volume digital PCR (MV-dPCR) disk designed to perform dPCR assays. The disk incorporates two primary innovations: (i) a radially arranged, multi-scale partitioning array with four distinct microcavity volumes (60 nl, 12 nl, 2.4 nl, and 0.48 nl), achieving compact layout while preserving a broad dynamic range; and (ii) an integrated vapor-based pneumatic pressurization module (∼70 kPa) that maintains stable partition integrity during thermal cycling without requiring external pressure hardware. These features are implemented in a monolithic cyclic olefin copolymer (COC) cartridge fabricated <i>via</i> thermoforming and sealed through thermo-diffusive bonding, occupying only one-third of a standard 140 mm disk. This compact design preserves plentiful space for future integration of upstream sample preparation modules. The MV-dPCR disk consistently achieves uniform partition filling with volume coefficients of variation (CVs) below 12% and low dead volume (<9%), and covers a 4.6 log₁₀ dynamic range using only 720 partitions. Validation experiments using synthetic DNA over four ten-fold dilution series demonstrate excellent linearity (<i>R</i>² > 0.99) and quantification accuracy comparable to commercial dPCR systems. Collectively, these advancements render the MV-dPCR disk a versatile, affordable, and easily scalable platform with potential for integration into more complex automated microfluidic workflows.