This proposal involves development of novel fluorogenic substrates and their bioanalytical applications. Support is specifically requested for synthesis of fluorogenic enzyme substrates that yield intensely fluorescent precipitates near the site of enzymatic activity. Work in progress has demonstrated a significant potential for these reagents for ultrasensitive detection in histochemistry, molecular biological techniques including DNA hybridization, detection of low abundance cellular receptors and detection of enzymes in living cells. Enzymatic amplification of the signal results in fluorescence that is orders of magnitude greater than that possible with fluorescent dye conjugates and significantly greater than that possible with substrates that yield colored precipitates. Since the fluorescent precipitates represent """"""""pure"""""""" dye with no carrier protein, the substrates can potentially be used to detect enzymatic activity with high spatial resolution whether the enzyme is intrinsically present in cells or is used as a tool in a conjugated form such as in ELISA techniques.
| Paragas, V B; Kramer, J A; Fox, C et al. (2002) The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets. J Microsc 206:106-19 |
| Haugland, R P (1995) Detecting enzymatic activity in cells using fluorogenic substrates. Biotech Histochem 70:243-51 |
| Larison, K D; BreMiller, R; Wells, K S et al. (1995) Use of a new fluorogenic phosphatase substrate in immunohistochemical applications. J Histochem Cytochem 43:77-83 |
