Digital PCR using micropatterned superporous absorbent array chips

Wang, Yazhen; Southard, Kristopher M.; Zeng, Yong

doi:10.1039/c6an00164e

Cited by 22 publications

(10 citation statements)

References 56 publications

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“…Single-copy DNA amplification can take place in droplets because of the excellent biocompatibility of agarose . The sol–gel switching property of agarose enables formation of stable beads by rapid cooling after amplification, thus, ensuring effective maintenance of monoclonality of each droplet for downstream processing.…”

Section: Resultsmentioning

confidence: 99%

“…The agarose droplet array method offers a flexible way to retrieve and store the amplified DNA products for further analysis and sequencing. , In the agarose droplet array, the droplets were generated in each microwell and isolated and immobilized by physical barriers (PDMS walls). Thus, the uncertainty problem of droplet floating and moving in the oil phase by other droplet microfluidic methods was easily overcome.…”

Section: Resultsmentioning

confidence: 99%

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

Zhang

et al. 2018

Anal. Chem.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Zhang

et al. 2018

Anal. Chem.

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“…This SPC named chip for dPCR with high-throughput benefited automation and integration without complex pipeline. Wang et al [77] achieved the similar goal that the microchambers in array could be spontaneously filled using a superporous absorbent array chip (Figure 5). The chip packed with microbeads performed superior ability for sample absorption.…”

Section: Mutation Detection Of Cfdnamentioning

confidence: 99%

“…dPCR using a superporous absorbent array chip [77]. ( A ) Schematic of fabrication of the ready-to –use microdevice.…”

Section: Figurementioning

confidence: 99%

Microfluidic Technologies for cfDNA Isolation and Analysis

Qiao

2019

Micromachines

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Cell-free DNA (cfDNA), which promotes precision oncology, has received extensive concern because of its abilities to inform genomic mutations, tumor burden and drug resistance. The absolute quantification of cfDNA concentration has been proved as an independent prognostic biomarker of overall survival. However, the properties of low abundance and high fragmentation hinder the isolation and further analysis of cfDNA. Microfluidic technologies and lab-on-a-chip (LOC) devices provide an opportunity to deal with cfDNA sample at a micrometer scale, which reduces required sample volume and makes rapid isolation possible. Microfluidic platform also allow for high degree of automation and high-throughput screening without liquid transfer, where rapid and precise examination and quantification could be performed at the same time. Microfluidic technologies applied in cfDNA isolation and analysis are limited and remains to be further explored. This paper reviewed the existing and potential applications of microfluidic technologies in collection and enrichment of cfDNA, quantification, mutation detection and sequencing library construction, followed by discussion of future perspectives.

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“…Future work should investigate the potential and limitations for downscaling the encapsulated liquid volumes, the suitability of the method for batch manufacturing, and the use of the method for release of chemical compounds in applications including organson-a-chip [28,29], ingestible non-invasive electronic devices [10], or nanoliter multiplexed dPCR arrays [30,31].…”

Section: Future Outlookmentioning

confidence: 99%