2 research outputs found
Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis
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
Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis
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