Regenerable biosensor platform: a total internal reflection fluorescence cell with electrochemical control.

A new biosensor platform that provides simultaneous fluorescence detection and electrochemical control of biospecific binding has been developed and investigated using antibody-antigen and streptavidin-biotin interactions. Specifically, biotin was covalently bound to a transparent indium-tin oxide (ITO) working electrode, which also served as an integral part of a total internal reflection fluorescence (TIRF) flow cell. TIRF was used to monitor biospecific interactions, while electrochemical polarization was employed to control interactions between biotin and streptavidin or between biotin and anti-biotin antibodies. Both streptavidin and polyclonal anti-biotin antibodies bound kinetically irreversibly to the biotinylated surface. In the absence of electrochemical control, the assay exhibited an extremely slow release of the bound analytes, causing poor regeneration ability of the biosensor surface. However, electrochemical polarization was found to stimulate dissociation of kinetically irreversibly bound biotin-streptavidin and antibody-antigen complexes. A "square wave" polarization function stimulated dissociation more effectively than a "saw tooth" function over the same voltage range. Application of the square wave polarization resulted in regeneration of an active biotinylated surface. Electrochemical polarization also modified affinity and kinetics of protein adsorption, which could likely be used to promote biospecific interactions and/or to suppress nonspecific adsorption.

Thermal modulation of channel catfish intestinal dimensions, BBM fluidity, and glucose transport.

In light of the direct influence of temperature on metabolic rates and dietary loads of ectotherms, intestinal responses were evaluated by measuring 1) dimensions, 2) transapical initial rates of transport using intact tissues and brush-border membrane vesicles (BBMV), and 3) BBMV fluidity using two size groups of channel catfish (Ictalurus punctatus) acclimated to different water temperatures. Intestines of larger fish at 15 degrees C were 23% longer and 61% heavier than those at 30 degrees C. Regardless of assay temperature, rates of glucose uptake by fish held at 30 degrees C relative to those at 15 degrees C were twofold higher for intact tissues and over fivefold higher for BBMV. Although rates of PBMV transport were higher for smaller fish, adaptive responses were greater for larger fish. Temperature coefficients (Q10S) for BBMV transport were higher between 5 and 15 degrees C (3.5-4.5) relative to 15 to 35 degrees C (1.9-2.0) and may be partly related to the inability of catfish held at low temperatures to adjust apical membrane fluidity. Our findings indicate that 1) cold-acclimated catfish maintain transport capacities by increasing intestinal dimensions, 2) high acclimation temperatures increase rates of uptake by as yet unknown mechanisms, 3) thermal modulation of transport varies among species and nutrients, and 4) adaptive responses of ectotherms are different from those of homeotherms).