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We developed a reliable but simple integration method of polymeric nanostructure in a poly(dimethylsiloxane) (PDMS)-based microfluidic channel, for nanofluidic applications. The Nation polymer junction was creased by infiltrating polymer solution between the gaps created by mechanical cutting, without any photolithography or etching processes. The PDMS can seal itself with the heterogeneous polymeric nanoporous material between the PDMS/PDMS gap due to its flexibility without any (covalent) bonding between PDMS and the polymer materials. Thus, one can easily integrate the nanoporous-junction into a PDMS microchip in a leak-free manner with excellent repeatability. We demonstrated nanofluidic preconcentration of proteins ([beta]-phycoerythrin) using the device. Because the polymeric junction spans across the entire microchannel height, the preconcentration was achieved with high-pressure field or even in large channels, with the dimensions of 1000 [micro]m width x 100 [micro]m depth.

We report a novel method of increasing both the reaction rate and the sensitivity of low-abundance enzyme assay using a micro/nanofluidic preconcentration chip. The disposable preconcentration device made out of PDMS with a surface-patterned ion-selective membrane increases local enzyme/substrate concentrations for rapid monitoring of enzyme activity. As a model system, we used trypsin as the enzyme and BODIPY FL casein as the fluorogenic substrate. We demonstrated that the reaction rate of trypsin--BODIPY FL was significantly enhanced by increasing the local concentrations of both trypsin and BODIPY FL casein in the preconcentration chip. The reaction time required to turn over substrates at 1 ng/ mL was only ~10 min compared to ~1 h without preconcentration, which demonstrates a significantly higher reaction rate through the increase of the concentrations of both the enzyme and substrate. Furthermore, trypsin activity can be measured down to a concentration level of 10 pg/mL, which is a ~100 fold enhancement in sensitivity compared to the result without the preconcentration step. This micro/nanofluidic preconcentrator chip could be used as a generic micro reaction platform to study any enzyme--substrate systems, or other biochemical reaction systems in low concentration ranges.

Simple and efficient sample concentration tools are the key to the application of proteomics in a biological system. In this paper, we developed a method to realize a nanofluidic preconcentrator on a poly(dimethylsiloxane) (PDMS)-based microfluidic channel. The originality of our preconcentration device is the simple nanogap formation using the junction gap breakdown phenomenon between two PDMS microchannels, without using any photolithography and etching techniques. From the dc current measurement, we confirm that the nanogap formed between two microchannel junctions with approximately 80 nm depth. Using this device, we achieve the concentration volume off [beta]-phycoerythrin protein as high as 70 pL, which is 120-fold larger than that from our previous reports, with a concentration factor as high as [10.sup.4] within 1 h. Also we show the availability of protein preconcentration under several different buffers (phosphate, acetate) at several different pH values (pH 5 to pH 9).

We developed a drop-on-demand microdroplet generator for the discrete dispensing of biosamples into a bioanalytical unit. This disposable PDMS microfluidic device can generate monodisperse droplets of picoliter volume directly out of a plane sidewall of the microfluidic chip by an electrohydrodynamic mechanism. The droplet generadon was accomplished without using either an inserted capillary or a monolithically built-in tip. The minimum droplet volume was ~4 pL, and the droplet generation was repeatable and stable for at least 30 min, with a typical variation of less than 2.0% of drop size. The Taylor cone, which is usually observed in electrospray, was suppressed by controlling the surface wetting property of the PDMS device as well as the surface tension of the sample liquids. A modification of the channel geometry right before the opening of the microchannel also enhanced the continuous droplet generation without applying any external pumping. A simple numerical simulation of the droplet generation verified the importance of controlling the surface wetting conditions for the droplet formation. Our microdroplet generator can be effectively applied to a direct interface of a microfluidic chip to a biosensing unit, such as AMS, MALDI-MS or protein microarray-type biochips.

Efficient sample preparation tools are the key to measuring molecular signals in a complex biological system. A novel continuous-flow isoelectric point (pI)-based sorting technique has been developed for proteins and peptides in a microfluidic chip format. It can sort biomolecules at a relatively high flow rate of up to 10 [micro]L/min and does not require carrier ampholytes, which can create molecular backgrounds for subsequent analysis. Furthermore, the electrophoretic field required to run the pI-based sorting is generated by the diffusion of buffer ions in situ, at the liquid junction between two laminar flows within the microfluidic channel. Utilizing the diffusion potential in combination with a pH difference between the buffers, we demonstrated a separation of binary mixtures of pI markers and proteins without applying any external field. The sorting resolution and its efficiency are sufficiently high for sample preparation and could be further improved by optimizing buffers or with an additional transverse electric field. Once fully developed, it can potentially be a pI-based sample fractionation tool for proteomic analysis of complex biomolecule samples.

We have developed a highly efficient microfluidic sample preconcentration device based on the electrokinetic trapping mechanism enabled by nanofluidic filters. The device, fabricated by standard photolithography and etching techniques, generates an extended space charge region within a microchannel, which was used to both collect and trap the molecules efficiently. The electrokinetic trapping and collection can be maintained for several hours, and concentration factors as high as [10.sup.6]-[10.sup.8] have been demonstrated. This device could be useful in various bioanalysis microsystems, due to its simplicity, performance, robustness, and integrabilty to other separation and detection systems.

Methods are described to achieve more efficient multidimensional protein separation in a microfluidic channel. The new methods couple isoelectric focusing (IEF) with high ionic strength electrophoretic separations by active microvalve control in a microchip. Several experiments demonstrating independent 2D separation were performed, and critical parameters for optimal chip performance were identified, including channel passivation, electroosmosis control, and IEF linearity control. This strategy can be used for integration of different heterogeneous separation techniques, such as IEF, capillary electrophoresis, and liquid chromatography. This new device can be ideal for preseparation and preconcentration of complex biomolecule samples for a streamlined biomolecule analysis using mass spectrometry.

Recently, a microfabricated entropic trap array was demonstrated to be useful in separating large (5-200 kbp) DNA molecules efficiently (within ~30 min), by dc electrophoresis, on a microchip platform without a sieving matrix. This paper reports further development of the technique, with emphasis on optimizing separation selectivity and resolution. The interaction of DNA molecules with regularly spaced entropic barriers was modeled in order to predict the effect of changing various structural parameters. The selectivity (differential mobility) was shown to be dependent on the depth of deep and shallow channel regions, applied electric field, and number of entropic barriers. Experimental data were compared with the prediction of the model. It was expected from the model that, in the low-field (severe trapping) limit, separation resolution should depend only on the number of entropic traps. However, in reality, resolution did depend on the applied field because the relaxation of DNA is not achieved at high fields. The requirement and feasibility of megabase pair DNA separation with the entropic trap array device was discussed.