Digital Nucleic Acid Detection Based on Microfluidic Lab-on-a-Chip Devices

Abstract

Microfluidic lab-on-a-chip (LOC) technologies have been developed as a promising alternative to traditional central laboratory-based analysis approaches over several decades due to the capability of realizing miniaturized multiphase and multistep reactions. In the field of nucleic acid (NA) diagnosis, digital NA detection (dNAD) as a single-molecular-level detection is greatly attributed to the perfect combination of NA amplification and microfluidic LOC techniques. In this chapter, the principle, classification, advances, and application of dNAD will be involved. In particular, the focus will be on chip-based dNAD for giving a deep interpretation of the analysis and evaluation of digital detection. The future prospect of dNAD is also anticipated. It is sure that dNAD by means of microfluidic LOC devices as the promising technique will better serve the ambitious plan of precision medicine through absolute quantitation of NA from individuals.

Target Confinement in Small Reaction Volumes Using Microfluidic Technologies: A Smart Approach for Single-Entity Detection and Analysis

“…Hereby, single NA copies can be isolated in the partitions, enabling the visualization of individually amplified PCR products. This high-throughput approach enabled to analyse 96 samples at once and up to 384 samples per hour 118 . Figure 7.…”

“…This has been used, among others, to perform multigene analysis of bacteria 120 , early diagnosis of cancer 123 and prenatal diagnosis 124 . An even higher throughput was achieved by performing dPCR on more advanced microwell array platforms, generally fabricated in silicon through photolithography 118 . Following this approach, Thermofisher® developed and commercialized its QuantStudio™ 3D Digital PCR system.…”

“…This highthroughput approach enabled analysis of 96 samples at once and up to 384 samples per hour. 118 An increase in throughput of dPCR in microchambers was achieved by introducing a valve-based platform, which resembles the system in Figure 5, and has been commercialized by Fluidigm under the name Biomark. In this system, the sample and PCR premix are loaded into microfluidic channels, which are subsequently separated into microchambers by a NanoFlex valve (i.e., parallel structures, orthogonally placed to the microchannels).…”

“…124 An even higher throughput was achieved by performing dPCR on more advanced microwell array platforms, generally fabricated in silicon through photolithography. 118 Following this approach, Thermofisher developed and commercialized its QuantStudio 3D Digital PCR system. The system consists of a chip, containing an array of 20 000 hexagonal wells of 0.8−1 nL for dPCR over 5 orders of magnitude.…”

Snap-on Adaptor for Microtiter Plates to Enable Continuous-Flow Microfluidic Screening and Harvesting of Crystalline Materials

“…This study aims to develop a snap-on adaptor that can transform any microtiter plate assay into a continuous-flow microfluidic mixer with a minimum requirement for fluid handling systems. The snap-on adapter includes the design of the cyclone mixer, with the merged inlet design where each inlet enters into the cyclone mixer from two channels, providing homogeneous mixing for every well . The inlets of the snap-on adapter can be connected to the flow distributor or concentration gradient generator (CGG). , By offering different combinations of the flow distributor and the CGG, the experimental design becomes more flexible.…”