Abstract
The generation of droplets is one of the most critical steps in the droplet digital polymerase chain reaction (ddPCR) procedure. In this study, the mechanism of droplet formation in microchannel structure and factors affecting droplet formation were studied. The physical field of laminar two-phase flow level was used to simulate the process of droplet generation through microfluidic technology. The effect of the parameters including flow rate, surface tension, and viscosity on the generated droplet size were evaluated by the simulation. After that, the microfluidic chip that has the same dimension as the simulation was then, fabricated and evaluated. The chip was made by conventional SU-8 photolithography and injection molding. The accuracy of the simulation was validated by comparing the generated droplets in the real scenario with the simulation result. The relative error (RE) between experimentally measured droplet diameter and simulation results under different flow rate, viscosity, surface tension and contact angle was found less than 3.5%, 1.8%, 1.4%, and 1.2%, respectively. Besides, the coefficient of variation (CV) of the droplet diameter was less than 1%, which indicates the experimental droplet generation was of high stability and reliability. This study provides not only fundamental information for the design and experiment of droplet generation by microfluidic technology but also a reliable and efficient investigation method in the ddPCR field.
Highlights
Droplet digital polymerase chain reaction based on the microfluidic chip has been widely used in a variety of applications as the replacement to the conventional
Photoresist SU-8 (Micro Chem 2050), copolymers of cycloolefin (COC)(TOPAS 5031), trichlorosilane (United Chemical Technologies, Inc., Bristol, PA), an inverted microscope (IX73, Olympus Corp.) was used to observe and image the experiments, Manta G-1236 CMOS camera, UV light, silicon wafer, tabletop homogenizer, incubator, plasma cleaning machine, air pumps, proportional valve, solenoid valve, gas cylinder, silicon oils (5 cst, 20 cst, 60 cst, Dow Corning, USA) with different viscosity were used as the continuous phase, with 0.05% Triton X-100 (T9284, Sigma Aldrich) and 2% ABIL EM90 supplemented as the surfactant, adjusting the concentration of the surfactant to obtain different surface tensions
The process of droplet generation in the droplet generation chip was simulated by level set interface of laminar two-phase flow using COMSOL
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The definition of digital polymerase chain reaction (dPCR) was proposed by B.Vogelstein in 1990, and a mutated oncogene was detected in the feces of patients with colorectal cancer through this method [1]. In digital PCR, the DNA or RNA is separated into numerous similar volumes of droplets, in which molecules are randomly distributed. The fluorescence could digitally read and count after the amplification procedure [2,3]. Droplet digital polymerase chain reaction (ddPCR) based on the microfluidic chip has been widely used in a variety of applications as the replacement to the conventional
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