Alfred Pollak

A new europium chelate for protein labelling and time-resolved fluorometric applications

Synthetic procedures are presented for a new chelator that forms stable and highly fluorescent complexes with Eu3+. This chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) is synthesized in a high-yield three-step procedure. BCPDA can be covalently incorporated into proteins under relatively mild conditions, and when complexed with Eu3+ forms a fluorescent product that has a lifetime in the range of 0.4 to 0.7 ms. Thus, it is useful for time-resolved fluorescence immunoassay applications.

TIME-RESOLVED DETECTION OF LANTHANIDE LUMINESCENCE FOR ULTRASENSITIVE BIOANALYTICAL ASSAYS

The principles and practice of the application of time-resolved lanthanide chelate luminescence (or fluorescence) as a detection method for ultrasensitive bioanalytical assays such as immunoassays and nucleic acid hybridization assays are reviewed. The various lanthanide chelate-based detection systems which have been developed for use in heterogeneous and homogeneous assay formats are described, including reagents, assay methods, and instrumentation, along with recent improvements in these methods. Detection systems described include those based on dissociative enhancement of lanthanide ions, direct labeling with luminescent chelates, enzyme-amplified lanthanide luminescence, lanthanide luminescence quenching, and energy transfer.

Ultrasensitive bioanalytical assays using time-resolved fluorescence detection

This article reviews the use of time-resolved fluorimetric detection of lanthanide chelate luminescence as a detection method for ultrasensitive bioanalytical assays. Assay formats and detection methods, and the principle of time-resolved fluorimetric detection, are described. Detection systems, assay formats, reagents, and instrumentation for time-resolved fluorimetric detection are outlined. A review of published and commercially available immunoassays and DNA hybridization assays using time-resolved fluorimetric detection of lanthanide chelate luminescence is given.

Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays

Enzyme-amplified lanthanide luminescence (EALL) is a new method which has been developed for enzymatically amplified signal detection in ultrasensitive bioanalytical assays where an enzyme is used as label or is itself the analyte of interest. Signal generation is performed by enzymatically transforming a substrate into a product which forms a luminescent lanthanide chelate; the product chelate can then be detected using time-resolved or normal fluorescence methods. Alkaline phosphatase substrates have been developed and demonstrated in a model immunoassay in microwell format. The method has also been demonstrated for detection of a variety of other hydrolytic and oxidative enzymes. Thus the EALL method shows promise for use in a wide variety of bioanalytical applications.

Alkyl- and aryl-substituted salicyl phosphates as detection reagents in enzyme-amplified fluorescence DNA hybridization assays on solid support

Nine salicyl phosphate esters with hydrophobic substituents (5-phenyl, 5-(2,4-difluorophenyl), 5-tert-octyl, 5-cumyl, 5-(4-tert-butylphenyl, 5-(1-adamantyl), 5-(n-dodecyl), 5-(1,1-diphenylethyl, and 5-trityl) were synthesized and found to be good substrates for calf intestinal alkaline phosphatase. The enzymatic hydrolysis produced the corresponding salicylates, which were strongly fluorescent when excited by ultraviolet light around 300 nm with maximum emission at 420-435 nm. The salicylates were less soluble and/or more adhesive than the nonfluorescent salicyl phosphate substrates, resulting in localization of fluorescence signal, which is a requirement for membrane-based assays. The salicyl phosphates bearing 8-14 carbon substitutents were found to be suitable detection reagents for dot-blot DNA hybridization assays on nylon membrane using a biotinylated probe, allowing the detection of 125 pg of target pBR322 plasmid DNA using a simple apparatus consisting of a transilluminator, a camera. and a 455-nm cutoff optical filter.