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A real-time characterization method to rapidly optimize molecular beacon signal for sensitive nucleic acids analysis

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Abstract

This research demonstrates an integrated microfluidic titration assay to characterize the cation concentrations in working buffer to rapidly optimize the signal-to-noise ratio (SNR) of molecular beacons (MBs). The “Microfluidic Droplet Array Titration Assay" (MiDATA) integrated the functions of sample dilution, sample loading, sample mixing, fluorescence analysis, and re-confirmation functions all together in a one-step process. It allows experimentalists to arbitrarily change sample concentration and acquire SNR measurements instantaneously. MiDATA greatly reduces sample dilution time, number of samples needed, sample consumption, and the total titration time. The maximum SNR of molecular beacons is achieved by optimizing the concentrations of the monovalent and divalent cation (i.e., Mg2+ and K+) of the working buffer. MiDATA platform is able to reduce the total consumed reagents to less than 50 μL, and decrease the assay time to less than 30 min. The SNR of the designated MB is increased from 20 to 126 (i.e., enhanced the signal 630 %) using the optimal concentration of MgCl2 and KCl determined by MiDATA. This novel microfluidics-based titration method is not only useful for SNR optimization of molecular beacons but it also can be a general method for a wide range of fluorescence resonance energy transfer (FRET)-based molecular probes.

The concentration of monovalent (K+) and divalent (Mg2+) cation in working buffer influences the signal-to-noise ratio (SNR) of molecular beacon (MB). Thus, optimizing the cationic concentrations in working buffer is necessary to achieve optimal SNR of MB assays for sensitive nucleic acids analysis.

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Acknowledgments

This work was supported in part by the Defense Advanced Research Projects Agency (DARPA) N/MEMS S&T Fundamentals program under grant number HR001-06-1-0500 issued to the Micro/Nano Fluidics Fundamentals Focus (MF3) Center; and The National Aeronautics and Space Administration (NASA) through the University Affiliated Research Center under grant number NAS2-03144.

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  1. Department of Biomedical Engineering, University of California, Irvine, 3120 Natural Sciences II, Irvine, CA, 92697-2715, USA

    Albert Tsung-Hsi Hsieh, Patrick J. Pan & Abraham P. Lee

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  1. Albert Tsung-Hsi Hsieh
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  2. Patrick J. Pan
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  3. Abraham P. Lee
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Correspondence to Albert Tsung-Hsi Hsieh or Abraham P. Lee.

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Hsieh, A.TH., Pan, P.J. & Lee, A.P. A real-time characterization method to rapidly optimize molecular beacon signal for sensitive nucleic acids analysis. Anal Bioanal Chem 406, 3059–3067 (2014). https://doi.org/10.1007/s00216-014-7721-z

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  • DOI: https://doi.org/10.1007/s00216-014-7721-z

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