Quantitative detection of a biological affinity reaction, the biotin/avidin recognition, was achieved using our newly developed photoelectrochemical analytical system. The system is based on the operation mechanism of the well-developed dye-sensitized photoelectrochemical solar cells and comprises a ruthenium tris(2,2'-bipyridine) (Ru-bipy) derivative as the photoelectrochemical signal-generating molecule, oxalate as the sacrificial electron donor, and tin oxide nanoparticle as the semiconductor electrode material. To perform the affinity reaction, avidin was immobilized on SnO(2) electrode by passive adsorption. Biotin-linked bovine serum albumin (BSA) was labeled with an NHS-ester derivative of Ru-bipy. After binding of BSA to the surface-immobilized avidin through biotin, photoelectrochemical measurement was carried out in the presence of oxalate. Anodic photocurrent was turned on and off repeatedly by control of incidental light. The action spectrum of the photocurrent resembled the absorption spectrum of Ru-bipy, proving the photocurrent was generated from the metal complex. A linear relationship between photocurrent and BSA concentration was obtained in the range of 1-100 microg/mL. This is the first case of quantitative photoelectrochemical detection of a biological affinity interaction.
A newly developed chemically amplified electrochemical detection system was applied to the quantitation of DNA in solution. The system employed Ru(bpy) 2 dppz (bpy = 2,2 0 -bipyridine, dppz = dipyrido[3,2-a :2 0 ,3 0 -c]phenazine), a high-affinity DNA intercalator, as the electrochemical indicator, oxalate as the sacrificial electron donor to chemically amplify the electrochemical signal, and tin-doped indium oxide as the working electrode to suppress background current. Intercalation of Ru(bpy) 2 dppz into calf thymus DNA in solution led to a reduction in the oxalate-amplified electrochemical current as compared to a DNA-free solution. The degree of reduction was a function of the concentration of DNA, thus forming the basis for DNA detection. To illustrate the advantages of the new system, a direct comparison was made between amplified (with oxalate) and non-amplified (without oxalate) DNA detection. In the presence of 100 mM oxalate, anodic current of Ru(bpy) 2 dppz was amplified by more than 60 folds, resulting in substantial improvement in signal-to-background ratio. Furthermore, as the DNA concentration was increased, the amplified current decayed much faster than the non-amplified signal, giving rise to higher detection sensitivity. The steeper decay was attributed to slower redox reaction between DNA-intercalated Ru(bpy) 2 dppz and oxalate, as the negatively charged phosphate groups on DNA repelled the anions. Effect of ionic strength was investigated to provide support for the interpretation. As expected, the decay of the amplified response with increasing concentration of DNA became less steep when more NaCl was added into the solution. The opposite effect was observed when tri-propylamine, a cationic electron donor, was used instead of oxalate. With an optimized concentration of 30 mM oxalate and 5 lM Ru(bpy) 2 dppz, calf thymus DNA of as low as 1 pM was detected in solution, which was close to the detection limit of some fluorescence measurements.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.