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.
The energetics and kinetics of the interaction of heparin with the Ca +P and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca +P dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a K d of V21 nM for the interaction of annexin V with end-chain immobilized heparin and a K d of V49 nM for the interaction with end-chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The K d of a heparin octasaccharide binding to annexin V was V1 W WM with a binding stoichiometry of 1:1.z 1999 Federation of European Biochemical Societies.
A sensor system based on the optical phenomenon of surface plasmon resonance (SPR), which employs either photothermal deflection spectroscopy (PDS) or a photodiode array (PDA) for detection, was developed to use molecularly imprinted (MI) polymethacrylic acid - ethylene glycol dimethacrylates (PMAA-EDMA) as the sensing element. The MI polymers were first processed by Soxhlet extraction to remove the print molecules (theophylline, caffeine, and xanthine), yielding the specific anti-polymers. Each anti-polymer was layered over a silver film to serve as the analysis surface for the molecularly imprinted sorbent assay (MIA) of one target drug. This surface was exposed for 60 min to an aqueous standard drug solution, dried in air, and the uptake of the print molecule into the anti-polymer was monitored by shifts in the SPR angle θ r (and hence the SPR-PDS signal measured at constant θ ). The linear dynamic range of the MIA was found to extend up to 6 mg/mL, with a concentration detection limit estimated at 0.4 mg/mL for theophylline in aqueous solution. A cross-reactivity study of the anti-theophylline and anti-caffeine polymers, using eight other drugs structurally similar to theophylline and caffeine, showed none or very slight shifts in θ r. This implies that the anti-polymers were selective only for their original print molecules and had no affinity for the other drug molecules. Similar molecular recognition characteristics were observed for the anti-xanthine polymer.Key words: surface plasmon resonance, molecular imprinting, theophylline, caffeine, xanthine, sensor.
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