Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

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• 作者：Jason E. Kreutz ; Todd Munson ; Toan Huynh ; Feng Shen ; Wenbin Du ; Rustem F. Ismagilov
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：November 1, 2011
• 年：2011
• 卷：83
• 期：21
• 页码：8158-8168
• 全文大小：1064K
• 年卷期：v.83,no.21(November 1, 2011)
• ISSN：1520-6882

文摘

This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for 鈥渄igital鈥?(single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12鈥?00 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, <500 molecules/mL to >1鈥?00鈥?00 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011, DOI: 10.1021/ja2060116), this approach is used to design and test digital RT-PCR devices for quantifying RNA.