Amphiphilic block copolymer nanotubes and vesicles stabilized by photopolymerization.

We report on a new method to stabilize nanotube and vesicle structures created from amphiphilic diblock copolymers by means of photopolymerization. Cross-linking with UV light exposure minimizes fluid disruption during stabilization. Additionally, the spatial control afforded by focusing or masking the initiating light source enables stabilization of distinct segments of individual nanostructures. This contribution demonstrates (1) that vesicles and nanotubes formed from poly(ethylene oxide)-block-polybutadiene are stabilized by exposure to UV light in the presence of a water-soluble photoinitiator and (2) that new nanotube geometries can be constructed by means of spot-curing, and (3) it reveals an application for photopolymerized nanotubes by showing electrophoresis of DNA through a UV-stabilized nanotube.

Room-temperature imprinting method for plastic microchannel fabrication

A new plastic imprinting method using a silicon template is demonstrated. This new approach obviates the necessity of heating the plastic substrate during the stamping process, thus improving the device yield from approximately 10 devices to above 100 devices per template. The dimensions of the imprinted microchannels were found to be very reproducible, with variations of less than 2%. The channel depths were dependent on the pressures applied and the materials used. Rather than bonding the open channels with another piece of plastic, a flexible and adhesive poly(dimethylsiloxane) film is used to seal the microchannels, which offers many advantages. As an application, isoelectric focusing of green fluorescence protein on these plastic microfluidic devices is illustrated.

Measurement of electroosmotic flow in plastic imprinted microfluid devices and the effect of protein adsorption on flow rate.

Several commercially available plastic materials were used as substrates in the fabrication of microfluid channels for biochemical analysis. Protocols for fabrication using the wire-imprinting method are reported for polystyrene, polymethylmethacrylate and a copolyester material. Channel sealing was accomplished by low-temperature bonding of a substrate of similar material; therefore, each channel was composed of a single material on all sides. The electroosmotic flow in 25-microm imprinted channels was evaluated for each substrate material. The copolyester material exhibited the highest electroosmotic flow mobility of 4.3 x 10(-4) cm2 V(-1) s(-1) which is similar to that previously reported for fused-silica capillaries. Polystyrene exhibited the lowest electroosmotic flow mobility of 1.8 x 10(-4) cm2 V(-1) s(-1). Plots of linear velocity versus applied electric field strength were linear from 100 V cm(-1) to 500 V cm(-1) indicating that heat dissipation is effective for all substrates in this range. Electroosmotic flow was reevaluated in the plastic channels following incubation in antibody solution to access the non-specific binding characteristics of a common biochemical reagent onto the substrate materials. All materials tested showed a high degree of non-specific adsorption of IgG as indicated by a decrease in the electroosmotic flow mobility in post-incubation testing.

Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye.

A technique is described for the measurement of fluid temperatures in microfluidic systems based on temperature-dependent fluorescence. The technique is easy to implement with a standard fluorescence microscope and CCD camera. In addition, the method can be used to measure fluid temperatures with micrometer spatial resolution and millisecond time resolution. The efficacy of the method is demonstrated by measuring temperature distributions resulting from Joule heating in a variety of microfluidic circuits that are electrokinetically pumped. With the equipment used for these measurements, fluid temperatures ranging from room temperature to 90 degrees C were measured with a precision ranging from 0.03 to 3.5 degrees C-dependent on the amount of signal averaging done. The spatial and temporal resolutions achieved were 1 microm and 33 ms, respectively.

Imaging of electroosmotic flow in plastic microchannels.

We have characterized electroosmotic flow in plastic microchannels using video imaging of caged fluorescent dye after it has been uncaged with a laser pulse. We studied flow in microchannels composed of a single material, poly(methyl methacrylate) (acrylic) or poly(dimethylsiloxane) (PDMS), as well as in hybrid microchannels composed of both materials. Plastic microchannels used in this study were fabricated by imprinting or molding using a micromachined silicon template as the stamping tool. We examined the dispersion of the uncaged dye in the plastic microchannels and compared it with results obtained in a fused-silica capillary. For PDMS microchannels, it was possible to achieve dispersion similar to that found in fused silica. For the acrylic and hybrid microchannels, we found increased dispersion due to the nonuniformity of surface charge density at the walls of the channels. In all cases, however, electroosmotic flow resulted in significantly less sample dispersion than pressure-driven flow at a similar velocity.

Laser modification of preformed polymer microchannels: application to reduce band broadening around turns subject to electrokinetic flow.

A pulsed UV excimer laser (KrF, 248 nm) was used to modify the surface charge on the side wall of hot-embossed microchannels fabricated in a poly(methyl methacrylate) substrate. Subablation level fluences, less than 2,385 mJ/cm2, were used to prevent any changes in the physical morphology of the surface. It is shown that the electroosmotic mobility, induced by an electric field applied along the length of the channel, increases by an average of 4% in the regions that have been exposed to UV laser pulses compared to nonexposed regions. Furthermore, application of UV modification to electroosmotic flow around a 90 degrees turn results in a decrease in band broadening, as measured by the average decrease in the plate height of 40% compared to flow around a nonmodified turn. The ability to modify the surface charge on specific surfaces within a preformed plastic microchannel allows for fine control, adjustment, and modulation of the electroosmotic flow without using wall coatings or changing the geometry of the channel to achieve the desired flow profile.

Detection of viable Cryptosporidium parvum using DNA-modified liposomes in a microfluidic chip.

This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid-sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensities not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 microL of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in mu-total analysis systems (mu-TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.

Integrated plastic microfluidic devices with ESI-MS for drug screening and residue analysis.

For this work, two different plastic microfluidic devices are designed and fabricated for applications in high-throughput residue analysis of food contaminants and drug screening of small-molecule libraries. Microfluidic networks on copolyester and poly(dimethylsiloxane) substrates are fabricated by silicon template imprinting and capillary molding techniques. The first device is developed to perform affinity capture, concentration, and direct identification of targeted compounds using electrospray ionization mass spectrometry. Poly(vinylidene fluoride) membranes sandwiched between the imprinted copolyester microchannels in an integrated platform provide continuous affinity dialysis and concentration of a reaction mixture containing aflatoxin B1 antibody and aflatoxins. The second microfluidic device is composed of microchannels on the poly(dimethylsiloxane) substrates. The device is designed to perform miniaturized ultrafiltration of affinity complexes of phenobarbital antibody and barbiturates, including the sequential loading, washing, and dissociation steps. These microfabricated devices not only significantly reduce dead volume and sample consumption but also increase the detection sensitivity by at least 1-2 orders of magnitude over those reported previously. Improvements in detection sensitivity are attributed to analyte preconcentration during the affinity purification step, limited analyte dilution in the microdialysis junction, minimal sample loss, and the amenability of ESI-MS to nanoscale sample flow rates.

Control of flow direction in microfluidic devices with polyelectrolyte multilayers.

Electroosmotic flow (EOF) is commonly utilized in microfluidics. Because the direction of the EOF can be determined by the substrate surface charge, control of the surface chemical state offers the potential, in addition to voltage control, to direct the flow in microfluidic devices. We report the use of polyelectrolyte multilayers (PEMs) to alter the surface charge and control the direction of flow in polystyrene and acrylic microfluidic devices. Relatively complex flow patterns with simple arrangements of applied voltages are realized by derivatization of different arms of a single device with oppositely charged polyelectrolytes. In addition, flow in opposite directions in the same channel is possible. A positively derivatized plastic substrate with a negatively charged lid was used to achieve top-bottom opposite flows. Derivatization of the two sides of a plastic microchannel with oppositely charged polyelectrolytes was used to achieve side-by-side opposite flows. The flow is characterized using fluorescence imaging and particle velocimetry.

Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification.

An integrated platform is presented for rapid and sensitive protein identification by on-line protein digestion and analysis of digested proteins using electrospray ionization mass spectrometry or transient capillary isotachophoresis/capillary zone electrophoresis with mass spectrometry detection. A miniaturized membrane reactor is constructed by fabricating the microfluidic channels on a poly(dimethylsiloxane) substrate and coupling the microfluidics to a poly(vinylidene fluoride) porous membrane with the adsorbed trypsin. On the basis of he large surface area-to-volume ratio of porous membrane media, adsorbed trypsin onto the poly(vinylidene fluoride) membrane is employed for achieving ultrahigh catalytic turnover. The extent of protein digestion in a miniaturized membrane reactor can be directly controlled by the residence time of protein analytes inside the trypsin-adsorbed membrane, the reaction temperature, and the protein concentration. The resulting peptide mixtures can either be directly analyzed using electrospray ionization mass spectrometry or further concentrated and resolved by electrophoretic separations prior to the mass spectrometric analysis. This microfluidic system enables rapid identification of proteins in minutes instead of hours, consumes very little sample (nanogram or less), and provides on-line interface with upstream protein separation schemes for the analysis of complex protein mixtures such as cell lysates.

Surface characterization of laser-ablated polymers used for microfluidics.

Fabrication of microfluidic devices by excimer laser ablation under different atmospheres may provide variations in polymer microchannel surface characteristics. The surface chemistry and electroosmotic (EO) mobility of polymer microchannels laser ablated under different atmospheres were studied by X-ray photoelectron spectroscopy and current monitoring mobility measurements, respectively. The ablated surfaces of PMMA were very similar to the native material, regardless of ablation atmospheres due to the negligible absorption of 248-nm light by that polymer. The substrates studied that exhibit nonnegligible absorption at this energy, namely, poly(ethylene terephthalate glycol), poly(vinyl chloride), and poly(carbonate), showed significant changes in surface chemistry and EO mobility when the ablation atmospheres were varied. Ablation of these three polymer substrates under nitrogen or argon resulted in low EO mobilities with a loss of the well-defined chemical structures of the native surfaces, while ablation under oxygen yielded surfaces that retained native chemical structures and supported higher EO mobilities.

Fabrication of plastic microfluid channels by imprinting methods.

Microfluidic devices have been fabricated on poly(methyl methacrylate) substrates by two independent imprinting techniques. First-generation devices were fabricated using a small-diameter wire to create an impression in plastics softened by low-temperature heating. The resulting devices are limited to only simple linear channel designs but are readily produced at low cost. Second-generation devices with more complex microchannel arrangements were fabricated by imprinting the plastic substrates using an inverse three-dimensional image of the device micromachined on a silicon wafer. This micromachined template may be used repeatedly to generate devices reproducibly. Fluorescent analtyes were used to demonstrate reproducible electrophoretic injections. An immunoassay was also performed in an imprinted device as a demonstration of future applications.