Droplet digital recombinase polymerase amplification (ddRPA) reaction unlocking via picoinjection

Johnson Q Cui; Frank X Liu; Hojeong Park; Ka Wai Chan; Tyler Leung; Ben Zhong Tang; Shuhuai Yao

doi:10.1016/j.bios.2022.114019

Droplet digital recombinase polymerase amplification (ddRPA) reaction unlocking via picoinjection

Biosens Bioelectron. 2022 Apr 15:202:114019. doi: 10.1016/j.bios.2022.114019. Epub 2022 Jan 20.

Authors

Johnson Q Cui¹, Frank X Liu¹, Hojeong Park², Ka Wai Chan³, Tyler Leung³, Ben Zhong Tang⁴, Shuhuai Yao⁵

Affiliations

¹ Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
² Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
³ DiagCor Life Science Limited, Kowloon Bay, Kowloon, Hong Kong.
⁴ Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Shenzhen Institute of Molecular Aggregate Science and Engineering, School of Science and Engineering, The Chinese University of Hong Kong, Shenzhen, 2001 Longxiang Boulevard, Longgang District, Shenzhen City, Guangdong, 518172, China. Electronic address: tangbenz@ust.hk.
⁵ Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Electronic address: meshyao@ust.hk.

PMID: 35078139
DOI: 10.1016/j.bios.2022.114019

Abstract

Recombinase polymerase amplification (RPA) has been recognized as a promising isothermal amplification method for nucleic acid detection. However, the digital format of RPA is still challenging to implement due to its MgOAc-initiated reaction feature and the inherent non-specific amplification. Here we develop a Picoinjection Aided Digital reaction unLOCKing (PADLOCK) approach utilizing droplet microfluidics to achieve droplet digital RPA (ddRPA) for absolute nucleic acid quantification. By coupling a microfluidic picoinjector with a droplet generator, the reaction initiator MgOAc is dosed into droplets containing MgOAc-deprived RPA master mix for controlled digital reaction unlocking, which completely circumvents premature amplification. The discretization of the targets to a single-molecule level in confined droplets endows absolute quantification of the copy number. Coupled with CRISPR/Cas13a sensing, the ddRPA demonstrates single molecule detection ability within 30 min with significantly enhanced signal-to-noise ratio (S/N ratio>6) and uniform fluorescence signal reporting, facilitating the precise quantification of nucleic acids. Furthermore, the utility of the PADLOCK-CRISPR assay has been validated with 22 clinical samples, which generated results in 100% concordance with qPCR. We believe the coupling of droplet microfluidic technology with digital RPA will pave the way towards ultrasensitive and precise nucleic acid quantification.

Keywords: Diagnostics; Digital quantification; Droplet microfluidics; Picoinjection; RPA.

MeSH terms

Biosensing Techniques*
Microfluidics
Nucleic Acid Amplification Techniques / methods
Nucleic Acids*
Recombinases

Substances

Nucleic Acids
Recombinases