Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis

Abstract

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.

Microfluidic and Paper-Based Devices for Disease Detection and Diagnostic Research

“…Another microwell array method by Li et al allowed for the rapid generation of uniform emulsion agarose droplets for single molecule analysis using a droplet generator. The sol-gel switching property of agarose was used to form stable beads after digital amplification, thus maintaining the monoclonality of each droplet in downstream processing [ 135 ]. The monoclonal beads were subsequently retrieved and subjected to DNA sequencing and FACS analysis.…”

Nucleic Acids Analysis

“…Moreover, high-throughput detection techniques, including microarray chip [10,11] and next-generation sequencing (NGS) [12,13], have been developed for simultaneous detection of thousands of and even more nucleic acid sequences of interest. The NGS technique can also be expanded for other biological and biomedical applications, including the analysis of non-nucleic acid targets [14] and the rapid screening of small-molecule drugs [15].…”

A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing

“…Microfluidic technology capable of manipulating small volumes of fluid and integrating a variety of reactions holds great promise for viral nucleic acid testing [18][19][20][21][22][23][24][25][26][27][28][29]. For example, microfluidics combined with a commercially available coffee mug or portable heating device allowed for sensitive and rapid detection of Zika virus by integrating RNA extraction, enrichment, and amplification in a microfluidic device [30][31][32].…”