Analytical and Bioanalytical Chemistry

, Volume 406, Issue 13, pp 3059–3067

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

Research Paper

DOI: 10.1007/s00216-014-7721-z

Cite this article as:
Hsieh, A.TH., Pan, P.J. & Lee, A.P. Anal Bioanal Chem (2014) 406: 3059. doi:10.1007/s00216-014-7721-z

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.

Figure

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.

Keywords

Molecular beacons FRET Titration Lab-on-a-chip Microfluidics 

Supplementary material

216_2014_7721_MOESM1_ESM.pdf (191 kb)
ESM 1(PDF 190 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringUniversity of California, IrvineIrvineUSA

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