Fluorigenic enzyme assays using long-wavelength substrates

Investigations into the use of long-wavelength fluorigenic substrates, which are non- or weakly fluorescent but are converted by an appropriate enzyme to a highly fluorescent product, have been carried out in order to determine several different enzymes and their inhibitors. Certain nonfluorescent compounds based on long-wavelength xanthenes and oxazines dyes were synthesised. Amongst these, naphthofluorescein diphosphate and naphthofluorescein mono-phosphate has been demonstrated to be new fluorigenic substrates for alkaline phosphatase. The hydrolysis product of these substrates can be used for trace analyses using diode laser based fluorescence detection. [Continues.

A new long-wavelength fluorigenic substrate for alkaline phosphatase: synthesis and characterisation

Naphthofluorescein diphosphate has been synthesised from the parent dye, and shown to be an attractive longwavelength alternative to other fluorigenic substrates for the determination of alkaline phosphatase. Its application to the determination of theophylline, an inhibitor of this enzyme, has been demonstrated. The optimum excitation wavelength of the hydrolysis product naphthofluorescein has been found to depend on the presence of additives such as cyclodextrins and (3-[3-cholamidopropyl]-dimethylamino)-1-propane sulfonate (CHAPS): such effects can be used to raise the excitation wavelength to match the output of a 635 nm diode laser in a simple and sensitive fluorescence detector

A compact long-wavelength fluorescence detection system and its application to flow injection immunoassay

A compact and robust long-wavelength fluorescence detection system (LWFDS) has been constructed using a pulsed diode laser as the light source and a photodiode detector. The detection limits for naphthofluorescein (NF) and the cyanine dyes Cy5 and Cy5.5 at pH 8.8, determined using the LWFDS, were 1.0 × 10-10, 2.3 × 10-12 and 6.2 × 10-11M, respectively. These values compared favourably with data from a commercial research grade fluorescence spectrometer. The LWFDS instrument was used as the detector for a competitive heterogeneous flow injection immunoassay for α-interferon with Cy5 as the label. A calibration graph usable over the range 0.5-10 μg ml-1 α-interferon was obtained. The instrument demonstrated its potential for rapid ( <200s per sample) multi-analyte assays and for field monitoring