Rapid and low-cost droplet pcr using a novel microfluidic setup with a numerical study on micromixer effects

• A thermal cycler setup, an optical DNA detection system, and a droplet-based. • microfluidic chip was developed. • 3D-printing for the fabrication of the chip was implemented. • The effect of the micro-mixers on the PCR test efficiency was investigated. • Experiments showed relatively monodispersed droplet diameters. • Fluorescent images and gel electrophoresis tests showed comparable results with a commercial PCR device. Droplet microfluidics, recognized for its precise dispensing and sample handling capabilities at microliter and lower volumes, has emerged as a popular method for miniaturizing polymerase chain reaction (PCR) platforms. This technology enables the accurate detection of low-abundance biological variations through droplet PCR (dPCR). In this study, we introduce a low-cost PCR thermal cycler setup, an optical system for DNA detection, and a 3D-printed droplet-based microfluidic device engineered to produce hundreds of nanoliter water-in-oil droplets. The microfluidic chip incorporates 21 cycles, each equipped with a micromixer. For the first time, we investigate the impact of integrating micromixers into the PCR chip cycles and demonstrate that this integration enhances the efficiency and specificity of the PCR process. Following the completion of the PCR cycles, the droplets are gathered in a chamber located at the end of the chip for amplification detection. A simple fluorescent optical setup is developed for fluorescence detection and accurate measurement of DNA concentration in the sample. This setup comprises a cost-effective high-resolution camera, a dichroic mirror, and an LED light source. Subsequently, the fluorescent images captured are processed using Python (OpenCV library) and analyzed for fluorescent emission intensity and droplet size distribution. The outcomes of the dPCR conducted with the microfluidic chip are subsequently assessed through a gel electrophoresis test and compared to the identical results from a commercial quantitative PCR (qPCR) device. Our study demonstrates the ability to perform rapid dPCR analysis via endpoint imaging utilizing a cost-effective and easy-to-fabricate setup.