The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).
We report the development of a hand-held instrument capable of performing two simultaneous microchip separations (gel and zone electrophoresis), and demonstrate this instrument for the detection of protein biotoxins. Two orthogonal analysis methods are chosen over a single method in order to improve the probability of positive identification of the biotoxin in an unknown mixture. Separations are performed on a single fused-silica wafer containing two separation channels. The chip is housed in a microfluidic manifold that utilizes o-ring sealed fittings to enable facile and reproducible fluidic connection to the chip. Sample is introduced by syringe injection into a septum-sealed port on the device exterior that connects to a sample loop etched onto the chip. Detection of low nanomolar concentrations of fluorescamine-labeled proteins is achieved using a miniaturized laser-induced fluorescence detection module employing two diode lasers, one per separation channel. Independently controlled miniature high-voltage power supplies enable fully programmable electrokinetic sample injection and analysis. As a demonstration of the portability of this instrument, we evaluated its performance in a laboratory field test at the Defence Science and Technology Laboratory with a series of biotoxin variants. The two separation methods cleanly distinguish between members of a biotoxin test set. Analysis of naturally occurring variants of ricin and two closely related staphylococcal enterotoxins indicates the two methods can be used to readily identify ricin in its different forms and can discriminate between two enterotoxin isoforms.
In vibrationally resonant sum-frequency generation (VR-SFG) spectra, the resonant signal contains information about the molecular structure of the interface, whereas the nonresonant signal is commonly treated as a background and has been assumed to be negligible on transparent substrates. The work presented here on model chromatographic stationary phases contradicts this assumption. Model stationary phases, consisting of functionalized fused-silica windows, were investigated with VR-SFG spectroscopy, both with and without experimental suppression of the nonresonant response. When samples are moved from CD(3)OD to D(2)O, the VR-SFG spectrum was found to change over time when the nonresonant signal was present but not when the nonresonant signal was suppressed. No effect was seen when the solvent was changed and pressurized to 900 psi. These results suggest that the response to the new solvent manifests primarily in the nonresonant response, not the resonant response. Any structural changes caused by the new solvent environment appear to be minor. The nonresonant signal is significant and must be properly isolated from the resonant signal to ensure a correct interpretation of the spectral data. Curve-fitting procedures alone are not sufficient to guarantee a proper interpretation of the experimental results.
Diffuse reflectance infrared spectroscopy has been used to monitor the changes in functional groups on the surface of two woods (western redcedar and southern pine) when they are subjected to a range of artificial weathering conditions. The effects of light only, water only, and light plus water have been studied for a variety of exposure times. The changes in the chemical composition of the wood caused by light plus water is considerably greater than that caused by light only, whereas water alone causes no permanent chemical changes. The effects of some of these weathering conditions have been investigated for wood protected by varnish, for earlywood and latewood and for tangentially, radially, and cross-sectionally cut wood samples. The application of two coats of varnish was found to considerably reduce the weathering effects caused by UV radiation; earlywood undergoes light-only degradation faster than late wood; and the effect of weathering was found to be greatest for cross-sectional cut samples and least for radially cut samples. The weathering profile with depth of penetration has been studied in some detail for western redcedar, and our results show that, after 300 h of continual artificial weathering, the chemical changes are confined to the first 200 μm.
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