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MICROFLUIDIC DEVICES FOR LABEL-FREE AND NON-INSTRUMENTED QUANTITATION OF UNAMPLIFIED NUCLEIC ACIDS BY FLOW DISTANCE MEASUREMENT.

ABSTRACT: Timely biomarker quantitation has potential to improve human health but current methods have disadvantages either in terms of cost and complexity for benchtop instruments, or reduced performance in quantitation and/or multiplexing for point-of-care systems. We previously developed microfluidic devices wherein visually observed flow distances correlated with a model analyte's concentration.¹ Here, we significantly expand over this prior result to demonstrate the measurement of unamplified DNA analogues of microRNAs (miRNAs), biomarkers whose levels can be altered in disease states. We have developed a method for covalently attaching nucleic acid receptors on poly(dimethylsiloxane) microchannel surfaces by silane and cross-linker treatments. We found a flow distance dependence on target concentrations from 10 ?g/mL to 10 pg/mL for DNA in both buffer and synthetic urine. Moreover, flow time in addition to flow distance is correlated with target concentration. We also observed longer flow distances for single-base mismatches compared to the target sequence at the same concentration, indicating that our approach can be used to detect point mutations. Finally, experiments with DNA analogues of miRNA biomarkers for kidney disease (mir-200c-3p) and prostate cancer (mir-107) in synthetic urine showed the ability to detect these analytes near clinically relevant levels. Our results demonstrate that these novel microfluidic assays offer a simple route to sensitive, amplification-free nucleic acid quantitation, with strong potential for point-of-care application.

SUBMITTER: Chatterjee D

PROVIDER: S-EPMC4269297 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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MICROFLUIDIC DEVICES FOR LABEL-FREE AND NON-INSTRUMENTED QUANTITATION OF UNAMPLIFIED NUCLEIC ACIDS BY FLOW DISTANCE MEASUREMENT.

Chatterjee Debolina D Mansfield Danielle S DS Woolley Adam T AT

Analytical methods : advancing methods and applications 20141001 20

Timely biomarker quantitation has potential to improve human health but current methods have disadvantages either in terms of cost and complexity for benchtop instruments, or reduced performance in quantitation and/or multiplexing for point-of-care systems. We previously developed microfluidic devices wherein visually observed flow distances correlated with a model analyte's concentration.<sup>1</sup> Here, we significantly expand over this prior result to demonstrate the measurement of unamplif ...[more]

PMID: 25530814

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Project description:Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental understanding of single-molecule reactions and providing advancements in practical applications such as medical diagnostics, forensics and environmental sampling.

Dataset Information

MICROFLUIDIC DEVICES FOR LABEL-FREE AND NON-INSTRUMENTED QUANTITATION OF UNAMPLIFIED NUCLEIC ACIDS BY FLOW DISTANCE MEASUREMENT.

Publications

MICROFLUIDIC DEVICES FOR LABEL-FREE AND NON-INSTRUMENTED QUANTITATION OF UNAMPLIFIED NUCLEIC ACIDS BY FLOW DISTANCE MEASUREMENT.

Similar Datasets

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