No abstract

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.

No abstract

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.

No abstract

No abstract

a b s t r a c tFacile electrical communication between redox-active labeling molecules and electrode is essential in the electrochemical detection of bio-affinity reactions. In this report, nanometer-sized indium tin oxide (ITO) particles were employed in the fabrication of porous thick film electrodes to enhance the otherwise impeded electrochemical activity of redox labels in multi-layered protein films, and to enable quantitative detection of avidin/biotin binding interaction. To carry out the affinity reaction, avidin immobilized on an ITO electrode was reacted with mouse IgG labeled with both biotin and ruthenium Tris-(2,2 -bipyridine) (Ru-bipy). The binding reaction between avidin and biotin was detected by the catalytic voltammetry of Ru-bipy in an oxalate-containing electrolyte. On sputtered ITO thin film electrode, although a single layer of Ru-bipy labeled avidin exhibited substantial anodic current, attaching the label to the outer IgG layer of the avidin/biotin-IgG binding pair resulted in almost complete loss of the signal. However, electrochemical current was recovered on ITO film electrodes prepared from nanometer-sized particles. The surface of the nanoparticle structured electrode was found by scanning electron microscopy to be very porous, and had twice as much surface binding capacity for avidin as the sputtered electrode. The results were rationalized by the assumption of different packing density of avidin inner layer on the two surfaces, and consequently different electron transfer distance between the electrode and Ru-bipy on the IgG outer layer. A linear relationship between electrochemical current and IgG concentration was obtained in the range of 40-4000 nmol L −1 on the nanoparticle-based electrode. The approach can be employed in the electrochemical detection of immunoassays using non-enzymatic redox labels.

a b s t r a c tA new electrochemiluminescence (ECL) sensor was developed for 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) quantification and Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) activity assay. The sensor employed a novel spermine conjugated ruthenium tris-(bipyridine) derivative (spermine-Ru) which binds specifically with 8-oxodGuo through a one-step reaction and also acts as an ECL signal reporter. In the sensor, an 8-oxodGuo-containing ds-DNA film was first immobilized on a gold electrode by self-assembly. The DNA film was then incubated with spermine-Ru under oxidative condition for 8-oxodGuo labeling. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Addition of FPG resulted in some loss of the signal due to the excision of 8-oxodGuo by the enzyme. An inverse relationship between ECL intensity and FPG concentration was observed in a range from 0 to 4.0 U/mL, demonstrating that this sensor could be used for FPG activity assay. A number of metal ions were screened by the sensor for their inhibition effect on FPG activity. Among them, Hg 2 þ and methyl Hg(II)shown very potent inhibition, with IC 50 values of 4.04 mM and 4.34 nM respectively. The result may suggest that interference on the DNA repair system could be another mechanism for the high toxicity of MeHg.