Absorbance based measurements are extremely sensitive to bubbles and volume/meniscus variations. One approach to enable highly miniaturized absorbance assays is to construct the assay using an epi-absorbance read-out. This can be achieved by using the intrinsic fluorescence properties of the plastic used to construct solid white microtiter plates (Zuck et al., 2005). Quenching of plate fluorescence by the enzymatic product can

provide a higher signal-to-background as the both the quenching of the light through the sample (either excitation or emission light) as well as the light reflected off the plate plastic results MDV3100 research buy in increased path length in the sample. This mode of detection has been used for inorganic phosphate detection derived from enzyme Z-VAD-FMK in vivo assays with malachite green-based detection of the free phosphate. In this case the white 1536-well plates were excited at 530 nm and fluorescence was measured at 630 nm – with phosphate production the malachite green turns into a blue solution which absorbs the 630 nm light emission light (Zuck et al., 2005). Proteases are a well-established class of drug targets (Leung et al., 2000) and have received considerable coverage in terms of assay formats and reagent kits. Proteases are typically measured using a peptide labeled with a FRET pair or a pro-fluorescent substrate. The use of 5-(2-aminoethyl)aminonaphthalene-1-sulphonyl (EDANS)

and 4-(-4-dimethylaminophenylazo)benzoyl (DABCYL) has been applied to endoproteases using FRET for detection (λex=340 nm/λem=475 nm) but suffers from compound interference and solubility issues. Another simpler fluorogenic substrate incorporates an aminomethyl coumarin (AMC) moiety at the carboxy terminus of a short peptide. The AMC group is dark when conjugated to the rest of the peptide but when liberated as a result of protease-catalyzed hydrolysis, exhibits strong fluorescence in the UV region (λex=360 nm, λem=450 nm). This approach is widely used to assay proteases

and has numerous advantages such as allowing real-time monitoring of reaction progress. They are extremely Liothyronine Sodium simple to configure as only one addition step is required to start the reaction. The AMC-containing substrates are generally stable, easy to synthesize, and widely available in a variety of sequence contexts from different vendors. A drawback of this approach is that the fluorogenic substrate, being an extremely truncated version of the biologically relevant substrate, cannot serve as a probe for the entire enzymatic pocket. As a number of studies aim to target proteases׳ extended binding sites ( Schechter and Berger, 1967), different types of substrates are being developed. Primarily, these are longer peptides (7–12 amino acids long) in which the scissile bond is around the middle of the sequence. In order to generate a detectable signal, a FRET donor pair is incorporated.