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Sample Preparation-Free, Real-Time Detection of microRNA in Human Serum Using Piezoelectric Cantilever Biosensors at Attomole Level

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    Sample Preparation-Free, Real-Time Detection of microRNA in Human Serum Using Piezoelectric Cantilever Biosensors at Attomole Level
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    Department of Chemical and Biological Engineering, Drexel University, Philadelphia, Pennsylvania 19104, United States
    *E-mail: [email protected]. Tel: (215) 895-2236. Fax: (215) 895-5837.
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    Analytical Chemistry

    Cite this: Anal. Chem. 2012, 84, 23, 10426–10436
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    https://doi.org/10.1021/ac303055c
    Published October 28, 2012
    Copyright © 2012 American Chemical Society
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    Abstract

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    A sensitive, selective, sample preparation-free method for near real-time detection of microRNA in buffer and human serum is given using gold (Au)-coated dynamic piezoelectric cantilever sensors. Sensor response to thiolated DNA probe chemisorption, hsa-let-7a hybridization, labeled-DNA hybridization, and Au nanoparticle-functionalized DNA hybridization was monitored continuously in flowing liquid samples using custom flow-cells. The assay showed successful detection of target let-7a with a dynamic range spanning 6 orders of magnitude (10 fM–1 nM) with a limit of detection of less than 10 attomoles (∼4 fM). The serum background had negligible effect on sensitivity relative to the results obtained in the buffer due to reduction in nonspecific binding caused by continuous sensor vibration. Both hybridization and nonspecific binding reduction were confirmed using fluorescence-based assays to support sensor-based results. The sensor-based method demonstrated excellent selectivity for the microRNA target in comparison with similar microRNA differing by only a single nucleotide (hsa-let-7c) and random microRNA sequences. Au nanoparticle-based amplification of sensor response was investigated and led to an order of magnitude improvement in the detection limit and a 128% amplification of sensor response over the entire dynamic range. Au nanoparticle amplification was verified by scanning electron microscopy. The cantilever sensor-based microRNA assay provides competitive sensitivity with current microRNA detection methods and has the advantage of requiring no sample preparation, even when working with biological samples that contain a complex background.

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    A schematic of the experimental apparatus (Figure S.1), sensor response to gold nanoparticle (Au NP) amplification of let-7a (Figure S.2), and scanning electron micrographs of Au NP-amplified samples versus Au control samples are given (Figure S.3). We also present the reproducibility of the laboratory-fabricated cantilever sensors design, whereby we include the fabrication-associated error instead of the assay-associated error which is presented in the main body of the manuscript (Figures S.4 and S.5). This material is available free of charge via the Internet at http://pubs.acs.org.

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    Analytical Chemistry

    Cite this: Anal. Chem. 2012, 84, 23, 10426–10436
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ac303055c
    Published October 28, 2012
    Copyright © 2012 American Chemical Society
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