Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids

Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

The substrate for the PWA chip was an n-type (100) single crystalline silicon wafer, with a thickness of 500 μm and a diameter of 100 mm. The silicon wafer was chemically cleaned by sequential immersion in acetone, ethanol, and deionized (DI) water for 15 min, with sonication. Then, it was immersed in a Piranha solution (H 2 SO 4 :H 2 O 2 = 3:1) at 180°C for 30 min, rinsed thoroughly with DI water, and dried with nitrogen. Positive photoresist AZ 6130 (thickness, 5 μm) was spin-coated on the silicon wafer for subsequent lithography (MA6; SUSS MicroTec, Garching, Germany) with a photolithography mask. Under the protection of the photoresist, a picoliter well array was formed using deep reactive-ion etching (PlasmaLab System 100; Oxford Instruments, Concord, MA, USA) and the remaining photoresist was removed with acetone. After cleaning, a 200-nm-thick silicon dioxide (SiO 2 ) layer was added to the wafer in a thermal oxidation furnace at 1050°C.