Fluorescence-coded logarithmic-dilution digital droplet PCR for ultrawide-dynamic-range nucleic acid quantification

Qingyuan Shi; Jie Li; Chunchen Liu; Xuanpei Zhai; Long Chen; Ye Zhang; Dezhi Feng; Rong Zhang; Jian Li; Shengjie Ling; Lei Zheng; Yuan Luo; Yifan Liu

doi:10.1016/j.bios.2023.115702

Fluorescence-coded logarithmic-dilution digital droplet PCR for ultrawide-dynamic-range nucleic acid quantification

Biosens Bioelectron. 2023 Dec 1:241:115702. doi: 10.1016/j.bios.2023.115702. Epub 2023 Sep 22.

Authors

Qingyuan Shi¹, Jie Li², Chunchen Liu³, Xuanpei Zhai⁴, Long Chen⁴, Ye Zhang³, Dezhi Feng⁴, Rong Zhang⁴, Jian Li⁴, Shengjie Ling⁴, Lei Zheng⁵, Yuan Luo⁶, Yifan Liu⁷

Affiliations

¹ State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China; School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
² School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address: lijie4@shanghaitech.edu.cn.
³ Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
⁴ School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
⁵ Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China. Electronic address: nfyyzhenglei@smu.edu.cn.
⁶ State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China. Electronic address: yuanluo@mail.sim.ac.cn.
⁷ School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Shanghai Clinical Research and Trial Center, Shanghai, 201210, China. Electronic address: liuyf6@shanghaitech.edu.cn.

PMID: 37751652
DOI: 10.1016/j.bios.2023.115702

Abstract

Digital PCR (dPCR) is considered the next generation of nucleic acid detection for its ability of absolute quantification and high sensitivity. However, when compared to the current gold standard, quantitative PCR (qPCR), dPCR is falling behind by several orders of magnitude in dynamic range, which limits its clinical applicability. Here we present fluorescence-coded logarithmic-dilution digital droplet PCR (Flodd-PCR) that features a dynamic range across 7 orders of magnitude, over 2 orders higher than conventional dPCR (4-5 log range) and approaching that of qPCR (7-8 log range). Flodd-PCR realizes such a wide dynamic range by dividing ∼20,000 droplets into 4 groups, each featuring a unique dilution factor of the loaded DNA template and thus a shifted dynamic range. This is achieved by a microfluidic chip that performs multi-step serial dilution (20-925 folds) and droplet generation. The post-PCR droplets can be clustered in silico based on their dilution indicator fluorescence and analyzed independently. Experimentally, Flodd-PCR can detect 4-20,000,000 copies/μL (cp./μL) of the synthetic human papillomavirus (HPV) DNA and outperforms standard dPCR when analyzing clinical HPV samples. Furthermore, Flodd-PCR can be implemented with existing dPCR system set-up with minimal adjustment, and therefore will also have wide practicality in different applications which conventional dPCR has already demonstrated.

Keywords: Biosensing; Digital PCR; Droplet; Dynamic range; HPV; Nucleic acid.