Miniaturized Continuous-Flow Digital PCR for Clinical-Level Serum Sample Based on the 3D Microfluidics and CMOS Imaging Device

Abstract

In recent years, the development of polymerase chain reaction (PCR) technology has focused on digital PCR, which depends on the microfluidics. Based on continuous-flow microfluidic technology, this paper designed a miniaturized digital PCR amplification system, and greatly reduced the area required for microdroplet generation and reaction. The core rod. made of polydimethylsiloxane (PDMS), was combined with the Teflon tube to form 3D microfluidics, which requires only one heating source to form the temperature difference required for gene amplification. Only two 34 g needles can form and transmit micro-droplets in a 4-fold tapered Teflon tube, which is the simplest method to generate digital PCR droplets as far as we know, which allows the microdroplet generation device to be free from dependence on expensive chips. A complementary metal oxide semiconductor (CMOS) camera was used as a detection tool to obtain fluorescence video for the entire loop area or a specified loop area. In addition, we developed a homebrew for automatic image acquisition and processing to realize the function of digital PCR. This technique realizes the analysis of clinical serum samples of hepatitis B virus (HBV) and obtained the same results as real-time quantitative PCR. This system has greatly reduced the size and cost of the entire system, while maintaining a stable response.

A sample-to-answer DNA detection microfluidic system integrating sample pretreatment and smartphone-readable gradient plasmonic photothermal continuous-flow PCR

“…The PDMS microchannel depth is only 0.1 mm, much smaller than the inner diameter (0.3–1 mm) of most microtubules. 47 This extremely shallow channel design means less thermal inertia and faster heat transfer, enabling thermal synchronization of PCR reagents with an external thermal field and shortening the 2–4 m pipeline in the tubular CF-PCR to only 0.94 m. 48 In addition, the flow promotes full contact of the reagent components, which can also accelerate the PCR reaction. 20 Although this CF-PCR has a speed gap compared to some ultra-fast PCR devices that complete amplification within 1 minute, it still has significant practical value because its time consumption is at least 50% shorter than that of most benchtop PCR instruments, which greatly simplifies thermal control.…”

Single-shot multi-channel plasmonic real-time polymerase chain reaction for multi-target point-of-care testing

“…29–31 Conventional fluorescence microscopes face practical problems at the point-of-care level due to their bulky and complicated optical components and long total track length for high magnification. 32,33 Recent near-patient molecular diagnostic assays utilize smartphone-based microscopic fluorescence imaging to provide simple and portable integration into mobile healthcare devices. 34,35 However, they have some technical limitations such as additional lens attachment, different optical paths, and mechanical replacement of fluorescence detection elements for multi-target diagnostics.…”

Microfluidic viscometers for biochemical and biomedical applications: A review

“…In the recent past, microfluidics has become an influential technology transforming biological [1], biochemical [2], biomedical [3] studies, and analysis by replicating the existing bench-top laboratory procedure on a chipsize miniaturized device [4]. Microfluidics has a vast range of applications in the area of drug delivery, tissue culture, nucleic acid amplification, electrocatalytic domains [5,6]. Microfluidics offers benefits like ideal delivery of sample, precise dosage, controlled and sustainable release, multiple dosing, with negligible side effects [7].…”