Microchip HPLC of peptides and proteins.

Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.

Microfluidic routing of aqueous and organic flows at high pressures: fabrication and characterization of integrated polymer microvalve elements.

This paper presents the first systematic engineering study of the impact of chemical formulation and surface functionalization on the performace of free-standing microfluidic polymer elements used for high-pressure fluid control in glass microsystems. System design, chemical wet-etch processes, and laser-induced polymerization techniques are described, and parametric studies illustrate the effects of polymer formulation, glass surface modification, and geometric constraints on system performance parameters. In particular, this study shows that highly crosslinked and fluorinated polymers can overcome deficiencies in previously-reported microvalve architectures, particularly limited solvent compatibility. Substrate surface modification is shown effective in reducing the friction of the polymer-glass interface and thereby facilitating valve actuation. A microchip one-way valve constructed using this architecture shows a 2 x 10(8) ratio of forward and backward flow rates at 7 MPa. This valve architecture is integrated on chip with minimal dead volumes (70 pl), and should be applicable to systems (including chromatography and chemical synthesis devices) requiring high pressures and solvents of varying polarity.

On-chip high-pressure picoliter injector for pressure-driven flow through porous media.

A high-pressure (> 3 MPa) on-chip injector has been developed for microchip applications including HPLC. The mechanical injector is implemented using in situ photopolymerization of fluorinated acrylates inside wet-etched silica microchips. The injector allows reproducible injections as small as 180 pL with < 250 ms duration. The injector operated robustly over 60 days and over 1000 injections. The injector is unique among polymer-based valves as it functions in aqueous, acetonitrile, and mixed buffers at high pressures without detectable leakage.

Microchip dialysis of proteins using in situ photopatterned nanoporous polymer membranes.

Chip-level integration of microdialysis membranes is described using a novel method for in situ photopatterning of porous polymer features. Rapid and inexpensive fabrication of nanoporous microdialysis membranes in microchips is achieved using a phase separation polymerization technique with a shaped UV laser beam. By controlling the phase separation process, the molecular weight cutoffs of the membranes can be engineered for different applications. Counterflow dialysis is used to demonstrate extraction of low molecular weight analytes from a sample stream, using two different molecular weight cutoff (MWCO) membranes; the first one with MWCO below 5700 for desalting protein samples, and the second one with a higher MWCO for size-based fractionation of proteins. Modeling based on a simple control volume analysis on the microdialysis system is consistent with measured concentration profiles, indicating both that membrane properties are uniform, well-defined, and reproducible and that diffusion of subcutoff analytes through the membrane is rapid.

Programmable modification of cell adhesion and zeta potential in silica microchips.

Spatial patterning of thin polyacrylamide films bonded to self-assembled monolayers on silica microchannels is described as a means for manipulating cell-adhesion and electroosmotic properties in microchips. Streaming potential measurements indicate that the zeta potential is reduced by at least two orders of magnitude at biological pH, and the adhesion of several kinds of cells is reduced by 80-100%. Results are shown for cover slides and in wet-etched silica microchannels. Because the polyacrylamide film is thin and transparent, this film is consistent with optical manipulation of cells and detection of cell contents. The spatial patterning technique is straightforward and has the potential to aid on-chip analysis of single adherent cells.

Voltage-addressable on/off microvalves for high-pressure microchip separations.

We present a microchip-based, voltage-addressable on/off valve architecture that is fundamentally consistent with the pressures and solvents employed for high-pressure liquid chromatography. Laser photopatterning of polymer monoliths inside glass microchannels is used to fabricate mobile fluid control elements, which are opened and closed by electrokinetic pressures. The glass substrates and crosslinked polymer monoliths operate in water-acetonitrile mixtures and have been shown to hold off pressures as high as 350 bar (5000 p.s.i.). Open/closed flow ratios of 10(4) to 10(6) have been demonstrated over the pressure range 1.5-70 bar (20-1000 p.s.i.), and the pressure-leak relationship shows the potential for valving control of flow through packed or monolithic chromatography columns. We expect that this valve platform will enable multiplexing of multiple chromatographic separations on single microchips.

High-pressure microfluidic control in lab-on-a-chip devices using mobile polymer monoliths.

We have developed a nonstick polymer formulation for creating moving parts inside of microfluidic channels and have applied the technique to create piston-based devices that overcome several microfluidic flow control challenges. The parts were created bycompletely filling the channels of a glass microfluidic chip with the monomer/ solvent/initiator components of a nonstick photopolymer and then selectively exposing the chip to UV light in order to define mobile pistons (or other quasi-two-dimensional shapes) inside the channels. Stops defined in the substrate prevent the part from flushing out of the device but also provide sealing surfaces so that valves and other flow control devices are possible. Sealing against pressures greater than 30 MPa (4,500 psi) and actuation times less than 33 ms are observed. An on-chip check valve, a diverter valve, and a 10-nL pipet are demonstrated. This valving technology, coupled with high-pressure electrokinetic pumps, should make it possible to create a completely integrated HPLC system on a chip.

Electrochromatography in microchips: reversed-phase separation of peptides and amino acids using photopatterned rigid polymer monoliths.

A microfabricated glass chip containing fluidic channels filled with polymer monolith has been developed for reversed-phase electrochromatography. Acrylate-based porous polymer monoliths were cast in the channels by photopolymerization to serve as a robust and uniform stationary phase. UV light-initiated polymerization allows for patterning of polymer stationary phase in the microchip, analogous to photolithography, using a mask and a UV lamp for optimal design of injection, separation, and detection manifolds. The monoliths are cast in situ in less than 10 min, are very reproducible with respect to separation characteristics, and allow easy manipulation of separation parameters such as charge, hydrophobicity, and pore size. Moreover, the solvent used to cast the polymer enables electroosmotic flow, allowing the separation channel to be conditioned without need for high-pressure pumps. The microchip was used for separation of bioactive peptides and amino acids labeled with a fluorogenic dye (naphthalene-2,3-dicarboxaldehyde) followed by laser-induced fluorescence detection using a Kr+ ion laser. The microchip-based separations were fast (six peptides in 45 s), efficient (up to 600,000 plates/m), and outperformed the capillary-based separations in both speed and efficiency. We have also developed a method for complete removal of polymer from the channels by thermal incineration to regenerate the glass chips.