Richard S. Handley

Chemical and enzymatic triggering of 1,2-dioxetanes. 3: alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane

Abstract We describe herein the preparation of an aryl phosphate-substituted 1,2-dioxetane and the enzymatic cleavage of this chemiluminescent dioxetane in aqueous buffer with alkaline phosphatase.

Chemical and Enzymatic Triggering of 1,2-Dioxetanes.2. Fluoride-Induced Chemiluminescence from tert-Butyldimethylsilyloxy-Substituted Dioxetanes.

Abstract Thermally stable 1,2-dioxetanes bearing tert-butyldimethylsilyloxyaryl groups have been prepared. Reaction of these dioxetanes with fluoride ion at ambient temperature in MeCN and DMSO generates chemiluminescence with efficiencies up to 25%.

Chemical and enzymatic triggering of 1,2-dioxetanes. 1: Aryl esterase-catalyzed chemiluminescence from a naphthyl acetate-substituted dioxetane

Abstract A thermally stable 1,2-dioxetane bearing a naphthyl acetate group is enzymatically cleaved in aqueous buffer to generate chemiluminescence at ambient temperature.

A Homogeneous Chemiluminescent Immunoassay Method

A new homogeneous chemiluminescent immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase is reported. Formation of an immunocomplex brings the chemiluminescent compound and the peroxidase into close proximity. Without any separation steps, a chemiluminescent signal is generated upon addition of a trigger solution, and the intensity is directly correlated to the quantity of the analyte.

Effects of Heteroatom Substituents on the Properties of 1,2-Dioxetanes.

Abstract Nitrogen and sulfur-substituted dioxetanes exhibit dramatically lower activation energies for decomposition compared to the corresponding oxygen-bearing dioxetane. A mechanism involving intramolecular electron-transfer processes is proposed for the cleavage of these unstable dioxetanes.

Characterization of new fluorescent peroxidase substrates.

The reagents Lumigen PS-1 and Lumigen PS-3 were originally developed as chemiluminescent substrates for ultrasensitive detection of horseradish peroxidase (HRP) in homogeneous solution and membrane blotting assays. However, an additional unique feature of these acridan-based reagents is the generation of a fluorescent species on reaction with peroxidase, a property which has been termed as chemifluorescence. These reagents, therefore, represent the first dual-use substrates enabling both chemiluminescent and fluorescent detection. We have developed several additional acridan-based substrates for fluorescent detection of HRP which are capable of subattomole detection sensitivity. By varying several structural parameters within the class of compounds we have produced substrates which either produce fluorescence alone or both chemiluminescence and fluorescence.

Novel chemiluminescent substrate and probe systems for the identification of CFΔF508 genotypes

Background: Chemiluminescence detection systems are rapidly gaining popularity as safer alternatives to isotopic methods in molecular diagnostics with equal sensitivity and specificity. In addition, they offer versatility of detection because of the availability of different haptens for labeling the probes, the antihapten antibodies conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP), and their respective chemiluminescent substrates. A novel dual chemiluminescent substrate (AP and HRP based) and probe systems to distinguish genotypes of cystic fibrosis DeltaF(508) mutation are described. Methods and Results: Two methodologies have been formulated to identify positively the genotypes of the cystic fibrosis DeltaF(508) mutation. In method 1, a pair of oligonucleotides designed to anneal to the fanking regions of DeltaF(508) mutation are differentially labeled with the hapten biotin or fluorescein and ligated using the template DNA of wild-type (N/N), heterozygous (N/DeltaF(508)), and homozygous (DeltaF(508)/DeltaF(508)) genotypes. The ligated product containing both labels is detected by first binding with avidin-HRP and anti-fluorescein-AP followed by reaction with the dual substrate. As expected, the ligation products are detected only in n/DeltaF(508) and DeltaF(508)/DeltaF(508) genotypes but not in N/N, where the ligation is precluded by the presence of three intervening nucleotides. In method 2, the three genotypes are hybridized on a membrane simultaneously with uniquely labeled (biotin or digoxigenin) oligonucleotides each designed to bind either the normal or the mutant allele. On treatment with HRP- and AP-conjugated antibodies followed by reaction with the dual substrate, only the band from N/DeltaF(508) genotype emitted a strong signal because of the binding of both oligonucleotides. Conclusions: The ligation and hybridization methods in conjunction with the dual substrate can detect and differentiate the genotypes with the DeltaF(508) mutation. These formats may be valuable for distinguishing normal individuals from carriers in population screening and fetuses that are heterozygous, from those that are homozygous for cystic fibrosis DeltaF(508) in prenatal and neonatal diagnosis.