Nanochannel Gradient Separations.

Gradient-based electrophoretic separations enable simultaneous separation and concentration of molecules. Compared with conventional injection-based separations, they enable enrichment of low-concentration analytes from larger sample volumes that are not limited by an injection volume. We have demonstrated that a nanochannel, connecting two chemically different reservoirs, can maintain a stationary chemical gradient while trapping biomolecules and effectively averaging out many of the complex physicochemical hydrodynamics that would broaden the bands in a meso- or microscale capillary. Here we describe chemical and physical methods that enable this work.

A mobility shift assay for DNA detection using nanochannel gradient electrophoresis.

Conventional detection of pathogenic or other biological contamination relies on amplification of DNA using sequence-specific primers. Recent work in nanofluidics has shown very high concentration enhancement of biomolecules with some degree of simultaneous separation. This work demonstrates the combination of these two approaches by selectively concentrating a mobility-shifted hybridization product, potentially enabling rapid detection of rare DNA fragments such as highly specific 16S ribosomal DNA. We have performed conductivity gradient electrofocusing within nanofluidic channels and have shown concentration of hybridized peptide nucleic acids and DNA oligomers. We also show selectivity to single base-pair mismatch on 18-mer oligos. This approach may enable sensitive optical detection of small amounts of DNA.

Isoelectric focusing in a silica nanofluidic channel: effects of electromigration and electroosmosis.

Isoelectric focusing of proteins in a silica nanofluidic channel filled with citric acid and disodium phosphate buffers is investigated via numerical simulation. Ions in the channel migrate in response to (i) the electric field acting on their charge and (ii) the bulk electroosmotic flow (which is directed toward the cathode). Proteins are focused near the low pH (anode) end when the electromigration effect is more significant and closer to the high pH (cathode) end when the electroosmotic effect dominates. We simulate the focusing behavior of Dylight labeled streptavidin (Dyl-Strep) proteins in the channel, using a relationship between the protein's charge and pH measured in a previous experiment. Protein focusing results compare well to previous experimental measurements. The effect of some key parameters, such as applied voltage, isoelectric point (pI), bulk pH, and bulk conductivity, on the protein trapping behavior in a nanofluidic channel is examined.

Nanochannel pH gradient electrofocusing of proteins.

We demonstrate matrix-free pH gradient electrofocusing of proteins within an 85 nm deep nanochannel. In contrast to conventional isoelectric focusing where the fluid does not move, this pH gradient method traps protein molecules flowing through a channel by balancing electric forces due to pH-dependent protein charge and viscous drag forces caused by electro-osmosis. The nanoscale depth of the device and the low voltage used limit convection relative to diffusion, thus producing a stable focused band of protein. R-Phycoerythrin (RPE) and Dylight labeled streptavidin (Dyl-Strep) were focused within a nanochannel using applied voltages between 0.4 and 1.6 V. Concentration enhancement factors of over 380 have been achieved within 5 min. Varying the buffer pH (between 2.7 and 7.2) at the boundaries of the nanochannel affected the shape of the focused bands. For RPE, a pH span of 4.5 (pH 2.7 to 7.2) yielded the narrowest peak while a span of 2.4 (pH 2.7 to 5.1) produced a significantly wider peak. Such matrix-free nanofluidic devices with pH gradient electrofocusing may enable on-chip integration of orthogonal separation techniques with mass spectrometry offering labor savings and enhanced performance.