A Real-Time Centrifugal Microfluidic Chip with Dual-Valving Strategy for Multiplexed PCR Detection at Point-of-Care Testing

Different from isothermal amplification, for polymerase chain reaction (PCR), highly reliable valving for PCR chamber, significantly shortened thermal cycling time, and concise multiplexed detection are always challenges for microfluidic-based devices. Here, we present a real-time, centrifugal, plastic microfluidic chip for multiplexed PCR detection specifically based on the mechanism of cooperating valving. To achieve consistent amplification, a concise dual-valving strategy was developed. Instantly melted wax is centrifuged and completely filled into the narrow channel and hole to act as the compact wax valve. Meanwhile, an elastic and sticky membrane is depressed to seal the hole to act as the membrane valve. The wax valve is protected by the membrane valve from being damaged by both mechanical deformation and thermal corroding caused by the hot vapor with high pressure from the PCR chamber. A double-sided heating strategy is adopted to reduce the thermal cycling time; meanwhile, a balanced mechanism is used to achieve real-time amplification by rotating the centrifugal chip between the heating and detection positions in turn. As a proof-of-concept, the performance of the centrifugal chip with four parallel units is demonstrated by successfully detecting purified DNA templates or the extracted DNA templates from cells as well within 20 min.