Abstracts of the 13th international symposium on bioluminescence and chemiluminescence

S 129 Copyright © 2004 John Wiley & Sons, Ltd. Luminescence 2004;19:129–192 Luminescence 2004; 19: 129–192 ABSTRACTS Abstracts of the 13th International Symposium ons of the 13th International Symposium on Bioluminescence and Chemiluminescence Homogeneous chemiluminescent assays: selective quenching of acridinium tracers M. Adamczyk, J. R. Fino, P. G. Mattingly, D. D. Johnson, J. A. Moore and Y. Pan Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA Chemiluminescent N-sulphonyl-acridinium-9-carboxamide reporter groups have been successfully commercialized on high-throughput, automated, in vitro diagnostic analysers that utilize a heterogeneous assay format, i.e. the bound and free labelled material is physically separated before the signal is generated. Homogeneous chemiluminescent assays offer significant advantages in terms of assay simplicity, cost and manufacturability of reagents. We describe here a homogeneous chemiluminescent assay concept that exploits an acridinium label designed to achieve signal modulation. Specifically, acridinium labels equipped with hapten-derivatizable linkers were designed to provide tethered or released acridone species upon chemiluminescent triggering. The labels were conjugated to derivatives of biotin, folic acid and vitamin B12. The resulting conjugates were characterized in terms of chemiluminescent output and signal modulation upon specific binding to protein receptors (avidin, folate binding protein and intrinsic factor, respectively). The tethered and released acridone conjugates exhibited markedly different chemiluminescent outputs relative to each other in the presence of a specific binding protein. Signals from conjugates that generated a tethered acridone species upon chemiluminescent triggering were selectively quenched in the presence of a specific binding protein. This signal modulation was exploited in homogeneous chemiluminescent assays for biotin, folic acid and vitamin B12. Tandem bioluminescent enzyme immunoassay for BDNF and NT-4/5 S. Akahane, K. Ito, H. Arakawa and M. Maeda School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan We have developed a highly sensitive simultaneous bioluminescent assay of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) using a firefly luciferase–luciferin reaction, and applied this assay to a tandem bioluminescent enzyme immunoassay (BL-EIA). Recently, Nelson et al. reported that the neonatal blood concentrations of VIP, CGRP, BDNF and NT-4/5 were higher in the autistic spectrum than in control children (1). Therefore, measurements of these four factors in neonatal blood are made possible to diagnose and care for autism at an early stage. In this study, we established a highly sensitive tandem BL-EIA for BDNF and NT-4/5. In the proposed assay, we added 50 μL standard or sample solutions Abstracts are in alphabetical sequence of first author On the CIEEL mechanism of triggerable dioxetanes: does the electron jump or is it charge transfer? W. Adam and A. V. Trofimov Department of Chemistry, University of Puerto Rico, Rio Piedras, PR 00931, USA, and Institute of Organic Chemistry, University of Würzburg, Würzburg D-97074, Germany Institute of Biochemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia 1,2-Dioxetanes are of particular value for the chemical generation of excited states, a process known as chemiluminescence, in view of the accompanying light emission. This may be achieved either thermally or by electron transfer; the latter mode is known as chemically initiated electron-exchange luminescence (CIEEL). A prominent and well-studied biological prototype of the CIEEL phenomenon is firefly bioluminescence, in which the light emission is triggered enzymatically. The mechanistic elucidation of this triggering process requires an in-depth understanding of both the dioxetane cleavage and the chemi-excitation process. Computational work supports full electron transfer and reveals the following salient mechanistic features: the electron transfer initially requires the elongation of the O–O bond in the triggered dioxetane and, once the electron is transferred, the activation energy for the rupture of the peroxide ring is significantly lowered. Furthermore, for the chemiexcitation step, a concerted cleavage of the dioxetane ring may operate, with concomitant excited-state generation through charge transfer (CT) from the electron donor (the phenolate functionality released on triggering) to the electron acceptor (the peroxide bond in the dioxetane). Alternatively, a stepwise process may apply, in which initially one-electron transfer to the peroxide bond takes place and causes dioxetane cleavage into a radical-ion pair, followed by excited-state generation through electron backtransfer (BET). The chemi-excitation efficiency depends decisively on the location (meta vs. para position) of the triggerable phenolate functionality. A detailed electronic analysis of this triggered light emission is consistent with the BET mechanism. The efficiency of the BET process depends on the distribution of the electrons between the two oxygen atoms of the ringopened dioxetane to which the electron is transfered from the triggerable functionality, i.e. the unpaired electron may be localized on the oxygen atom proximate to the phenolate functionality or placed onto the remote one. The latter mechanistic alternative is supported by the observed viscosity dependence of the light efficiency, since a solvent-cage effect operates in the CIEEL process.