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Microfluidic chips with reversed-phase monoliths for solid phase extraction and on-chip labeling.

ABSTRACT: The integration of sample preparation methods into microfluidic devices provides automation necessary for achieving complete micro total analysis systems. We have developed a technique that combines on-chip sample enrichment with fluorescence labeling and purification. Polymer monoliths made from butyl methacrylate were fabricated in cyclic olefin copolymer microdevices and used for solid phase extraction. We studied the retention of fluorophores, amino acids and proteins on these columns. The retained samples were subsequently labeled with both Alexa Fluor 488 and Chromeo P503, and unreacted dye was rinsed off the column before sample elution. Additional purification was obtained from the differential retention of proteins and fluorescent labels. A linear relation between the eluted peak areas and concentrations of on-chip labeled heat shock protein 90 samples demonstrated the utility of this method for on-chip quantitation. Our fast and simple method of simultaneously concentrating and labeling samples on-chip is compatible with miniaturization and desirable for automated analysis.

SUBMITTER: Nge PN

PROVIDER: S-EPMC3463737 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Microfluidic chips with reversed-phase monoliths for solid phase extraction and on-chip labeling.

Nge Pamela N PN Pagaduan Jayson V JV Yu Ming M Woolley Adam T AT

Journal of chromatography. A 20120901

The integration of sample preparation methods into microfluidic devices provides automation necessary for achieving complete micro total analysis systems. We have developed a technique that combines on-chip sample enrichment with fluorescence labeling and purification. Polymer monoliths made from butyl methacrylate were fabricated in cyclic olefin copolymer microdevices and used for solid phase extraction. We studied the retention of fluorophores, amino acids and proteins on these columns. The r ...[more]

PMID: 22995197

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Project description:Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 ? 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (?SPE) consisting of a plastic chip that is activated with UV/O3 to generate surface-confined carboxylic acid functionalities for the ?SPE of cfDNA. The ?SPE uses an immobilization buffer (IB) consisting of polyethylene glycol and salts that induce cfDNA condensation onto the activated plastic microfluidic surface. The ?SPE consists of an array of micropillars to increase extraction bed load (scalable to loads >700 ng of cfDNA) and can be produced at low-cost using replication-based techniques. The entire ?SPE can be fabricated in a single molding step negating the need for adding additional extraction supports to the device simplifying production and keeping device and assay cost low. The ?SPE allowed for recoveries >90% of model cfDNA fragments across a range of sizes (100-700 bp) and even the ability to extract efficiently short cfDNA fragments (50 bp, >70%). In addition, the composition of the IB allowed for reducing the interference of co-extracted genomic DNA. We demonstrated the clinical utility of the ?SPE by quantifying the levels of cfDNA in healthy donors and patients with non-small-cell lung and colorectal cancers. ?SPE extracted cfDNA from plasma samples was also subjected to a ligase detection reaction (LDR) for determining the presence of mutations in the KRAS gene for colorectal and non-small cell lung cancer patients.

Dataset Information

Microfluidic chips with reversed-phase monoliths for solid phase extraction and on-chip labeling.

Publications

Microfluidic chips with reversed-phase monoliths for solid phase extraction and on-chip labeling.

Similar Datasets

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