Hashem Akhavan-Tafti

Comparison Between Acridan Ester, Luminol, and Ruthenium Chelate Electrochemiluminescence

Transparent indium tin oxide (ITO) electrodes are attractive for use in electrochemiluminescence (ECL). In this work the ECL of an acridan ester (2′,3′,6′-trifluorophenyl 10-methylacridan-9-carboxylate), luminol, and a ruthenium chelate (ruthenium tris 2,2′-bipyridyl) are compared using ITO electrodes. The electrochemistry of all the compounds is described and related to their ECL mechanisms; three possible mechanisms for luminol ECL are described, and it is suggested that the currently accepted mechanism involving luminol and superoxide radicals may not always be correct. Maximum light emission in ruthenium chelate ECL occurs at 1.5 V (vs. Ag/AgCl), but potentials of more than 1 V have a corrosive effect on ITO. The effect of this limitation on ruthenium chelate ECL is discussed as part of a wider assessment of the advantages and disadvantages of ITO as an electrode material for ECL. The effects of pH and H2O2 concentration on ECL detection of the acridan ester and luminol in a planar flow cell are described. The limits of detection of the acridan ester and luminol were 65 pM and 72 pM, respectively.

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.

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.

Electrochemiluminescence of 2′,6′-difluorophenyl 10-methyl-9,10-dihydroacridine-9-carboxylate

Electrochemical oxidation of the acridine ester 2′,6′-difluorophenyl 10-methyl-9,10-dihydroacridine-9-carboxylate yields the corresponding acridinium ester which reacts with H2O2 to generate intense chemiluminescence.

Analytical applications of electrochemiluminescence

Electro chemiluminescence is a form of chemiluminescence in which the light emitting reaction is preceded by an electrochemical reaction. The ECL reaction of an acridan ester is described as an example. With this kind of reaction of the advantages of CL are retained, but the electrochemical step allows the time and position of the light emitting reaction to be controlled. By controlling the time of the reaction light emission can be delayed until levels such as immune or enzyme catalyzed reactions have taken place, and by controlling the position light emission can be confirmed to a region that is precisely located with respect to the detector, improving sensitivity by increasing the ratio of signal to noise. The advantages will be highlighted by describing an enzyme ECL immunoassay for small molecules such as dinitrophenol, in which superparamagnetic bead technology is used to concentrate enzyme labeled antibodies on the surface of an electrode, in close proximity to the detector. Finally the future of research in this are will be examined by describing how bipolar electrodes can be used to free ECL from the constraints imposed on it by conventional 2D electrodes.

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.