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Electrochemical detection of plasma membrane cholesterol at the surface of excised mouse trachea tissue is reported. Cholesterol oxidase is covalently linked to an 11-mercaptoundecanoic acid submonolayer on the platinum electrode surface. The cholesterol oxidase-modified electrodes show steady-state responses for cholesterol in solution at physiological temperatures. Experiments for direct contact between the cholesterol oxidase-modified electrode and the surface of excised trachea tissue at 37 [degrees]C indicate steady-state responses that are largely independent of the position of contact on the tissue surface. Tissue samples are mounted on a quartz crystal microbalance electrode to gauge contact force between the electrode and the tissue surface, and the steady-state electrode response for tissue cholesterol is shown to be largely independent of the contact force. Trachea tissue excised from a mouse model of cystic fibrosis, which is known to exhibit evaluated cholesterol in airway cells, shows an electrode response that is ~40% larger than the response observed at wild-type mouse trachea tissue.

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from [cftr.sup.-/-] mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of [cftr.sup.-/-] mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.

Platinum microelectrodes are modified with a lipid bilayer membrane incorporating cholesterol oxidase. Details for electrode surface modification are presented along with characterization studies of electrode response to cholesterol solution and to cholesterol contained in the lipid bilayer membrane of vesicles. Ferrocyanide voltammetric experiments are used to track deposition of a submonolayer of a thiol-functionalized lipid on the platinum electrode surface, vesicle fusion for bilayer formation on the thiolipid-modified surface, and incorporation of cholesterol oxidase in the electrode-supported thiolipid/lipid bilayer membrane. The data are consistent with formation of a lipid bilayer structure on the electrode surface that contains defects. Experiments for detection of cholesterol solubilized in cyclodextrin solution show steady-state current responses that correlate with cholesterol concentration. Direct contact between the electrode and a vesicle lipid bilayer membrane shows a response that correlates with vesicle membrane cholesterol content.

A capacitive immunoassay based on antibody-embedded ultrathin [gamma]-alumina sol--gel films (~20 to 40 nm) was successfully prepared in this work. The nanofilms greatly increased the capacitance change initiated by the recognition between the immobilized antibody and the target antigen, which allowed capacitive measurements capable of directly determining the antigen more sensitive than that of thick films. Meanwhile, the inorganic films with high permittivity significantly increased the time constant (i.e., RC value) of the films, which rendered the potentiostatic step method with acceptable S/N ratio. These two advantages enabled the immunosensor to be readily employed in a multichannel capacitance analysis system. An eight-channel hIgG capacitive sol--gel-derived immunoassay based on this system was constructed to illustrate the application. Compared with the detection limits of Si[O.sub.2] sol--gel-derived hIgG capacitive immunosensors or the conventional ELISA immunoassay, the immunoassay based on thin alumina gel film showed a lower detection limit of 1 ng [mL.sup.-1]. The novel immunoassay was employed to co-determine two liver fibrosis markers (hyaluronan and laminin) in mixed samples from ~0.5 to 50 ng [mL.sup.-1]. The little derivation caused by the interfered antigen indicated that the sensitive, specific, low-cost sol--gel-derived multichannel immunosensors might be a promising approach in the application of screening disease markers.