Open AccessDynamic Aqueous Multiphase Reaction System for One-Pot CRISPR-Cas12a-Based Ultrasensitive and Quantitative Molecular Diagnosis
Author(s) -
Kun Yin,
Xiong Ding,
Ziyue Li,
Hui Zhao,
Kumarasen Cooper,
Changchun Liu
Publication year - 2020
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.0c01459
Subject(s) - crispr , chemistry , nucleic acid , recombinase polymerase amplification , multiplex , microfluidics , molecular diagnostics , biosensor , digital polymerase chain reaction , dna , nanotechnology , computational biology , polymerase chain reaction , loop mediated isothermal amplification , biochemistry , gene , genetics , biology , materials science
Recently, CRISPR-Cas technology has opened a new era of nucleic acid-based molecular diagnostics. However, current CRISPR-Cas-based nucleic acid biosensing has a lack of the quantitative detection ability and typically requires separate manual operations. Herein, we reported a dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis by taking advantage of density difference of sucrose concentration. In the DAMR system, recombinase polymerase amplification (RPA) and CRISPR-Cas12a derived fluorescent detection occurred in spatially separated but connected aqueous phases. Our DAMR system was utilized to quantitatively detect human papillomavirus (HPV) 16 and 18 DNAs with sensitivities of 10 and 100 copies within less than 1 h. Multiplex detection of HPV16/18 in clinical human swab samples were successfully achieved in the DAMR system using 3D-printed microfluidic device. Furthermore, we demonstrated that target DNA in real human plasma samples can be directly amplified and detected in the DAMR system without complicated sample pretreatment. As demonstrated, the DAMR system has shown great potential for development of next-generation point-of-care molecular diagnostics.