Toward multiplexed electrogenerated chemiluminescent detection

Shijun Wang

Abstract

The main objective of this dissertation is to understand and study the principle of electrogenerated chemiluminescence (ECL) and its applications to detect biomolecules simultaneously. Four aspects of ECL were studied. In order to carry out multiplexed ECL detection, both classical and several novel ECL systems have been investigated. In the first aspect, significant effect of chloride ions on the ECL behavior of the tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)32+ )/tri- n -propylamine (TPrA) system at Au electrode was investigated. At low concentrations (e.g., [Cl- ] < 5 mM), the ECL was enhanced; at relatively high concentrations, however, the ECL intensity decreased with the increase of the [Cl- ]. At [Cl- ] = 90 mM, ∼ 50% and 100% ECL inhibition was observed for the first and the second ECL wave, respectively. The electrogenerated chloroaurate anions (AuCl2 - and AuCl4- ) which were verified using an electrochemical quartz-crystal microbalance (EQCM) method were found to be responsible for the ECL inhibition. This study suggests that care must be taken when Au working electrode is used for ECL studies in chloride-containing buffer solutions (widely used in DNA probes) and/or with the commonly used chloride-containing reference electrodes since in these cases the ECL behavior may significantly disagree with that obtained using other electrodes and reaction media. In the second aspect, the electrochemical behavior of a trinuclear ruthenium(II)-containing complex, [((phen)2 Ru(dpp))2 RhCl2 ] 5+ (where phen = 1,10-phenanthroline, dpp = 2,3-bis-2-pyridylpyrazine), was studied in acetonitrile (MeCN) and aqueous solutions. In MeCN containing 0.10 M tetra-n -butylammonium perchlorate (TBAP), the complex displayed a reversible, overlapping RuII/III redox process with E1/2 = +1.21 V vs Ag/Ag+ (10 mM), an irreversible reduction of RhIII/I at -0.73 V vs Ag/Ag + , and two quasi-reversible dpp/dpp- couples with E 1/2 = -1.11 V and -1.36 V vs Ag/Ag+ at a Pt electrode with a scan rate of 50 mV s-1 . In 0.20 M Tris buffer solution (pH 7.4), an irreversible, overlapping RuII/III oxidation at +1.48 V vs Ag/AgCl (3 M KCl), and an irreversible reduction of RhIII/II at -0.78 V vs Ag/AgCl were observed at a glassy carbon electrode with a scan rate of 50 mV/s. In the third aspect, ECL behavior of core/shell semiconductor CdSe/ZnS nanocrystals coated with a carboxyl polymer layer (quantum dot, Qdot, or QDs) was studied in aqueous solutions using TPrA and DBAE as ECL coreactant. Upon the anodic potential scanning, strong ECL emissions were observed at glassy carbon (GC) electrode within the potential range of ∼0.75 to 1.5 V vs Ag/AgCl (3.0 M KCl) when DBAE was used as the coreactant. The ECL behavior of the Qdot was found to be strongly dependent on the types and concentrations of ECL coreactants as well as the nature of the working electrode. The ECL emission measured with the Qdot/DBAE/GC electrode system has a peak value of ∼625 nm, which matched well with its fluorescence. The Qdot as a label for ECL-based C-reactive protein (CRP) immunoassays was realized by covalent binding of avidin on its surface, which allowed biotinalyted antibodies to be attached and interacted with antigens and the antibodies linked to micro-sized magnetic beads. In the fourth aspect, the ECL behavior of Ru(bpy)3 2+ , 9,10-diphenylanthracene (DPA), and rubrene (RUB) with DBAE or TPrA as the coreactant was studied in acetonitrile solution. The ECL emission spectra of the mixed solution including the above three ECL labels were investigated. The ECL maximum emissions at ∼440 nm for DPA, ∼560 nm for RUB, and ∼630 nm for Ru(bpy) 32+ were linearly proportional to the concentration of each individual ECL labels in mixed solutions, suggesting that multiplexing detection and quantification of biomolecules with ECL technology is feasible. (Abstract shortened by UMI.)