Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires.

The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.

Enhanced Optical Biosensing by Aerotaxy Ga(As)P Nanowire Platforms Suitable for Scalable Production.

Sensitive detection of low-abundance biomolecules is central for diagnostic applications. Semiconductor nanowires can be designed to enhance the fluorescence signal from surface-bound molecules, prospectively improving the limit of optical detection. However, to achieve the desired control of physical dimensions and material properties, one currently uses relatively expensive substrates and slow epitaxy techniques. An alternative approach is aerotaxy, a high-throughput and substrate-free production technique for high-quality semiconductor nanowires. Here, we compare the optical sensing performance of custom-grown aerotaxy-produced Ga(As)P nanowires vertically aligned on a polymer substrate to GaP nanowires batch-produced by epitaxy on GaP substrates. We find that signal enhancement by individual aerotaxy nanowires is comparable to that from epitaxy nanowires and present evidence of single-molecule detection. Platforms based on both types of nanowires show substantially higher normalized-to-blank signal intensity than planar glass surfaces, with the epitaxy platforms performing somewhat better, owing to a higher density of nanowires. With further optimization, aerotaxy nanowires thus offer a pathway to scalable, low-cost production of highly sensitive nanowire-based platforms for optical biosensing applications.

Output of a valveless Liebau pump with biologically relevant vessel properties and compression frequencies.

Liebau pump is a tubular, non-peristaltic, pulsatile pump capable of creating unidirectional flow in the absence of valves. It requires asymmetrical positioning of the pincher relative to the attachment sites of its elastic segment to the rest of the circuit. Biological feasibility of such valveless pumps remains a hotly debated topic. To test the feasibility of the Liebau-based pumping in vessels with biologically relevant properties we quantified the output of Liebau pumps with their  compliant segments made of a silicone rubber that mimicked the Young modulus of soft tissues. The lengths, the inner diameters, thicknesses of the tested compliant segments ranged from 1 to 5 cm, 3 to 8 mm and 0.3 to 1 mm, respectively. The compliant segment of the setup was compressed at 0.5-2.5 Hz frequencies using a 3.5-mm-wide rectangular piston. A nearest-neighbor tracking algorithm was used to track movements of 0.5-mm carbon particles within the system. The viscosity of the aqueous solution was varied by increased percentage of glycerin. Measurements yielded quantitative relationships between viscosity, frequency of compression and the net flowrate. The use of the Liebau principle of valveless pumping in conjunction with physiologically sized vessel and contraction frequencies yields flowrates comparable to peristaltic pumps of the same dimensions. We conclude that the data confirm physiological feasibility of Liebau-based pumping and warrant further testing of its mechanism using excised biological conduits or tissue engineered components. Such biomimetic pumps can serve as energy-efficient flow generators in microdevices or to study the function of embryonic heart during its normal development or in diseased states.

Single molecule tracking and analysis framework including theory-predicted parameter settings.

Imaging, tracking and analyzing individual biomolecules in living systems is a powerful technology to obtain quantitative kinetic and spatial information such as reaction rates, diffusion coefficients and localization maps. Common tracking tools often operate on single movies and require additional manual steps to analyze whole data sets or to compare different experimental conditions. We report a fast and comprehensive single molecule tracking and analysis framework (TrackIt) to simultaneously process several multi-movie data sets. A user-friendly GUI offers convenient tracking visualization, multiple state-of-the-art analysis procedures, display of results, and data im- and export at different levels to utilize external software tools. We applied our framework to quantify dissociation rates of a transcription factor in the nucleus and found that tracking errors, similar to fluorophore photobleaching, have to be considered for reliable analysis. Accordingly, we developed an algorithm, which accounts for both tracking losses and suggests optimized tracking parameters when evaluating reaction rates. Our versatile and extensible framework facilitates quantitative analysis of single molecule experiments at different experimental conditions.