TriD-LAMP: A pump-free microfluidic chip for duplex droplet digital loop-mediated isothermal amplification analysis

• Published in: Analytica chimica acta Vol. 1233; p. 340513
• Main Authors: Wu, Cui, Liu, Linbo, Ye, Zunzhong, Gong, Jingjing, Hao, Pei, Ping, Jianfeng, Ying, Yibin
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.11.2022
• Subjects: Digital analysis; Droplet; Dual detection; Microfluidic chip; Nucleic acids; Microfluidic chip; Digital analysis; Nucleic acids; Droplet; Dual detection
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2022.340513

Digital nucleic acid amplification techniques are powerful and attractive approaches for providing sensitive and absolute quantification in biology. Among these, digital loop-mediated isothermal amplification (dLAMP) shows the potential for field detection, since its robustness and independence from thermal cycling. The key of dLAMP is to generate a large number of individual droplets or microwells. However, an auxiliary precision pump is always required for sample digitalization. In addition, current systems for droplet dLAMP usually need to transfer the droplets after digitalization or amplification. Herein, we developed and evaluated a pump-free microfluidic chip for duplex droplet dLAMP (TriD-LAMP) detection. This chip was designed based on step emulsification and contains a droplet generation zone and a droplet storage zone. Droplets are formed through the step due to the difference in Laplace pressure. There are 64 parallel nozzles that could generate tens of thousands of uniform droplets manually (variation <5%). The storage zone for droplets collection was previously filled with oil containing fluorosurfactant that keeps the droplets from fusing and evaporation during the amplification. Therefore, this custom chip is able to perform all stages of the dLAMP process without transferring droplets. Combined with the optimized fluorescent probe method, the chip achieves accurate quantification of the E. coli DNA down to 19.8 copies/μL. As a proof of concept, the simultaneous quantification of two targets was successfully realized on this custom chip. Conclusively, this integrated, pump-free TriD-LAMP chip can provide a promising tool for multiple targets detection in clinical diagnostics and point-of-care applications. [Display omitted] •A pump-free microfluidic chip for dual detection is developed.•The chip is easy to operate and can manually generate uniform droplets (CV<5%).•Absolute quantification of different targets is successfully achieved on the chip.•The entire process of droplet dLAMP can be completed in one chip within 40 min.