Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.

We present a technique which enables the separation of small RNAs-such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs)-from >or=66 nucleotide RNAs and other biomolecules contained in a cell lysate. In particular, the method achieves separation of small RNAs from precursor miRNAs (pre-miRNAs) in less than 3 min. We use on-chip isotachophoresis (ITP) for the simultaneous extraction, isolation, preconcentration and quantitation of small RNAs (approximately 22 nucleotides) and employ the high-efficiency sieving matrix Pluronic F-127; a thermo-responsive triblock copolymer which allows convenient microchannel loading at low temperature.

A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins.

Microfabricated regular sieving structures hold great promise as an alternative to gels to improve the speed and resolution of biomolecule separation. In contrast to disordered porous gel networks, these regular structures also provide well defined environments ideal for the study of molecular dynamics in confining spaces. However, the use of regular sieving structures has, to date, been limited to the separation of long DNA molecules, however separation of smaller, physiologically relevant macromolecules, such as proteins, still remains a challenge.

Molecular sieving using nanofilters: past, present and future.

Filtration of molecules by nanometer-sized structures is ubiquitous in our everyday life, but our understanding of such molecular filtration processes is far less than desired. Until recently, one of the main reasons was the lack of experimental methods that can help provide detailed, microscopic pictures of molecule-nanostructure interactions. Several innovations in experimental methods, such as nuclear track-etched membranes developed in the 70s, and more recent development of nanofluidic molecular filters, played pivotal roles in advancing our understanding.

Electrical detection of fast reaction kinetics in nanochannels with an induced flow.

Nanofluidic channels can be used to enhance surface binding reactions, since the target molecules are closely confined to the surfaces that are coated with specific binding partners. Moreover, diffusion-limited binding can be significantly enhanced if the molecules are steered into the nanochannels via either pressure-driven or electrokinetic flow. By monitoring the nanochannel impedance, which is sensitive to surface binding, low analyte concentrations have been detected electrically in nanofluidic channels within response times of 1-2 h.

pH-controlled diffusion of proteins with different pI values across a nanochannel on a chip.

Electrostatic interactions dominate the diffusion of proteins in a nanochannel, which was measured and modeled for pH values above and below the isoelectric points of three lectin proteins. Maximal diffusion coefficients were obtained when the proteins were neutral at their pI. This pH-controlled transport led to a separation of biomolecules across the nanochannel.

Ionic transport phenomena in nanofluidics: experimental and theoretical study of the exclusion-enrichment effect on a chip.

In nanometer-sized apertures with charged surfaces, the extension of the electrical double layer results in the electrostatic exclusion of co-ions and enrichment in counterions, which affects the permselectivity of such structures. A modeling of this phenomenon is proposed and is compared with quantitative measurements of the ionic permeability change of a Pyrex nanoslit at low ionic strength. The comparison of experimental results with theoretical predictions justifies that electrostatic forces are the governing forces in nanofluidics.