Developing ruthenium-based complexes as redox labels for electrochemical biosensors

Abstract

Ferrocene-based redox labels have been used extensively for biosensing applications. However, the oxidised form of ferrocene, the ferricenium ion, is shown to decompose in solutions containing chloride salts or other strongly nucleophilic reagents. This thesis describes the synthesis and electrochemical studies on ruthenium-based complexes bearing 2,2’-bipyridine (bpy) and β-diketonato ligands in order to develop ruthenium-based redox labels for sensing applications.

Two series of ruthenium complexes, [Ru(bpy)2(β-diketonato)](PF6) (Series I) and Ru(β-diketonato)2(bpy) (Series II) complexes were prepared. The complexes in Series I were prepared by displacing the Cl ligand in Ru(bpy)2Cl2•2H2O with respective β-diketonato ligand while the complexes in Series II were synthesised by three major routes: Route 1: via Ru(β-diketonato)2(MeCN)2, Route 2: via Ru(bpy)(Cl)4 and Route 3: via Ru(β diketonato)2(diene) (MeCN = acetonitrile, diene = 1,5-cyclooctadiene (COD) or norbornadiene (NBD)). The complexes of both Series I and II were studied electrochemically to examine the effects of ligand substituents on the half-wave potentials (E1/2) of the complexes and showed that the use of β-diketonato ligands allowed the fine-tuning of E1/2 of these complexes. The electron-withdrawing and electron-donating groups on the β-diketonato ligands shifted the E1/2 in the anodic and the cathodic directions, respectively. In addition, the positioning of these groups on the β-diketonato ligands relative to the ruthenium centre also affected the extent of change in E1/2. The E1/2 values of the complexes in Series I and II correlated well with Hammett constant, electrochemical ligand parameter and UV-Vis data.

Of the complexes studied, the E1/2 of Ru(acac)2(bpy) and Ru(dbm)2(bpy) fell in the desired range of -0.3 V to +0.5 V (vs Ag/AgCl) (acac = 2,4-pentanedionato, dbm = dibenzyolmethanato). To enable immobilisation of redox label onto gold electrodes and attachment of bioreceptor onto redox label, the bpy ligand was functionalised with primary amine groups (bpy-NH2). Redox label Ru(acac)2(bpy-NH¬2) was prepared via Route 1 and immobilised on gold electrodes modified by 6-mercaptohexanoic acid (MHA) and thioctic acid (TA). The redox-label bound surfaces were characterised by cyclic voltammetry and X-ray photoelectron spectroscopy. The redox label was shown to be far more stable than the ferrocene-based label when subjected to repetitive redox cycling in chloride-containing buffer. To evaluate the performance of the redox label, a sensing surface was constructed by immobilising N-glycosylated VHLTP (GPP) onto the redox label on MHA-modified gold electrode for the detection of Hb1Ac monoclonal antibody. The voltammetry response of the redox label upon the binding of Hb1Ac antibody to GPP was studied by square wave voltammetry, where it was shown that the signal was not affected by the buffer the antibody was in. The current of the redox label was attenuated when the sensing surface was incubated in the antibody solution: the longer the incubation time, the larger the decrease in current due to the binding of more antibodies to the peptides. This indicates that the redox label, Ru(acac)2(bpy-NH2) has the potential to be applied in sensing applications.