Author: Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude
Title: Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids Document date: 2016_4_13
ID: 0tmd9knh_5
Snippet: In this work, we developed a picoliter well array (PWA) chip to perform real-time dRPA for absolute quantification of nucleic acids. The PWA chip was manufactured from a silicon substrate and etched with 27,000 consistently sized picoliter wells. We demonstrated that sample loading using a scraping liquid blade is suitable for the PWA chip, which was simpler, faster, and resulted in less reaction dropout than droplet-based approaches. Additionall.....
Document: In this work, we developed a picoliter well array (PWA) chip to perform real-time dRPA for absolute quantification of nucleic acids. The PWA chip was manufactured from a silicon substrate and etched with 27,000 consistently sized picoliter wells. We demonstrated that sample loading using a scraping liquid blade is suitable for the PWA chip, which was simpler, faster, and resulted in less reaction dropout than droplet-based approaches. Additionally, unlike other microfluidic chips, it did not require a complex fabrication process or a pneumatic control system. Passivating the PWA chip surface using a methoxy-PEG-silane agent eliminated cross-contamination. For wide-field fluorescence imaging in situ, we creatively placed an optical cube between the chip and object lens to reduce the loss of light intensity in a conventional microscope. Not only did this ensure that the light intensity was able to excite the fluorescence signal, but it also avoided the need to obtain images by scan shooting and stitching serial small images together. Finally, we sealed the PWA chip in a homemade copper chamber filled with oil and successfully performed real-time dRPA on an isothermal incubation setup for the absolute quantification of serial dilutions of a Listeria monocytogenes gDNA stock solution.
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