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A microfluidic biosensor based on nucleic acid sequence recognition

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Abstract

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol μL−1 (10 pmol L−1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.

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Acknowledgements

The authors thank former laboratory colleagues Sui Ping Lee and Nicole Schlesinger for their help with the preparation of liposomes. We thank Dr Jun Min, Cornell University for helpful late-night discussions concerning microchannel design. We also thank Dr Laurie Locascio and Dr Susan Barker of the MicroAnalytical Laboratory, NIST, Gaithersburg, Maryland, USA, for microfabrication training. The authors acknowledge financial support for this project from the department of Biological and Environmental Engineering, Cornell University and the Cooperative State Research, Education and Extension Services (NYC-123404). This work was performed in part at the Cornell Nanfabrication Facility (a member of the National Nanofabrication Users Network) which is supported by the National Science Foundation under Grant ECS-9731293, its users, Cornell University and Industrial Affiliates.

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  1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA

    Sylvia Kwakye & Antje Baeumner

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  1. Sylvia Kwakye
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  2. Antje Baeumner
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Correspondence to Antje Baeumner.

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Kwakye, S., Baeumner, A. A microfluidic biosensor based on nucleic acid sequence recognition. Anal Bioanal Chem 376, 1062–1068 (2003). https://doi.org/10.1007/s00216-003-2063-2

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  • DOI: https://doi.org/10.1007/s00216-003-2063-2

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