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

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., Mg(2+) 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.

Nonviral gene vector formation in monodispersed picolitre incubator for consistent gene delivery.

A novel picolitre incubator based microfluidic system for consistent nonviral gene carrier formulation is presented. A cationic lipid-based carrier is the most attractive nonviral solution for delivering plasmid DNA, shRNA, or drugs for pharmaceutical research and RNAi applications. The size of the cationic lipid and DNA complex (CL-DNA), or the lipoplex, is one of the important variations for consistency of gene transfection. CL-DNA size, in turn, may be controlled by factors such as the cationic lipid and DNA mixing order, mixing rate, and mixture incubation time. The Picolitre Microfluidic Reactor and Incubator (PMRI) system described here is able to control these parameters in order to create homogeneous CL-DNA. Compared with conventional CL-DNA preparation techniques involving hand-shaking or vortexing, the PMRI system demonstrates a greater ability to constantly and uniformly mix cationic lipids and DNA simultaneously. After mixing in the picolitre droplet reactors, the cationic lipid and DNA is incubated within the picolitre incubator to form CL-DNA. The PMRI generates a narrower size distribution band, while also turning the sample loading, mixing and incubation steps into an integrated process enabling the consistent formation of CL-DNA. The coefficient of variation (CV) of transfection efficiency is 0.05 and 0.30 for PMRI-based and conventional methods, respectively. In addition, this paper demonstrates that the gene transfection efficiency of lipoplex created in the PMRI is more reproducible.