The importance of glucose monitoring for in vivo as well as for ex vivo applications has driven a vast number of scientific groups to pursue the development of an advanced glucose sensor. Such a sensor must be robust, versatile, and capable of the long-term, accurate and reproducible detection of glucose levels in various testing media. Among the different configurations and signal transduction mechanisms used, fluorescence-based glucose sensors constitute a growing class of glucose sensors represented by an increasing number of significant contributions to the field over the last few years. This manuscript reviews the progress in the development of fluorescence based glucose sensors resulting from the advances in the design of new receptor systems for glucose recognition and the utilization of new fluorescence transduction schemes.

The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug target in the design of a competitive binding fluorescence resonance energy transfer assay for pharmacological screening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminal cysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalene sulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensity of the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting as a donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representative of MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRET signal was monitored as a function of the concentration of calmodulin antagonists in solution. Calibration curves for both a selection of small molecules and a series of peptides based upon known CaM-binding domains were obtained using this system. The assay demonstrated dose-dependent antagonism by analytes known to hinder the biological activity of CaM. These data indicate that the presence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading to a concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assay can serve as a general model for the development of other protein binding assays intended to screen for molecules with preferred binding activity.

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A s the drug discovery process evolves and demands more challenging and relevant assays, we have experienced a recent and significant escalation in the number of cell-based high-throughput assays for both small molecule and target identification screens. This has resulted in an increased need for the reproducible production of high quality cells in large quantities. Historically, manual cell culture was the only option available for providing cells in sufficient numbers for small molecule ultra-high-throughput screens (uHTSs), representing a technology gap in our automated cell culture process.Recently, we have applied an automated solution in the form of a novel 10-layer tissue culture flask, the HYPERFlask (Corning, Lowell, MA). This technology, when introduced as an upgrade to the SelecT (an automated cell culture system manufactured by The Automation Partnership, Ltd., Royston, England), provides a new approach to automating production of the high number of cells required for uHTS and consequently a highly desirable alternative to manual cell culture.The HYPERFlask has a surface area of 1720 cm 2 and can yield up to 3 Â 10 8 cells after 3 days in culture. This can be compared to a typical yield of 2 Â 10 7 cells from a T175 flask (with a surface area of 175 cm 2 ), the current standard flask type for automated cell culture on the SelecT. Cells grown in both flask types are of comparable quality, as demonstrated by equivalent cell viability, yield per cm 2 , functional response, and pharmacology.

Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NO X ) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.