An electrochemical RNA aptamer-based biosensor for rapid and label-free detection of the bronchodilator theophylline was developed. The 5'-disulfide-functionalized end of the RNA aptamer sequence was immobilized on a gold electrode, and the 3'-amino-functionalized end was conjugated with a ferrocene (Fc) redox probe. Upon binding of theophylline the aptamer switches conformation from an open unfolded state to a closed hairpin-type conformation, resulting in the increased electron-transfer efficiency between Fc and the electrode. The electrochemical response, which was measured by differential pulse voltammetry, reaches saturation within a few minutes after addition of theophylline, and the dynamic range for detecting theophylline is 0.2-10 muM. The electrode displays an inhibited response when applied directly in serum samples treated with RNase inhibitors; however a full response to the theophylline serum concentration was obtained by transferring the electrode to blank serum-free buffer solutions. It was demonstrated that theophylline is detected with high selectivity in the presence of caffeine and theobromine.

A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

The electrodeposition method was used for modification of a nanostructured hematite photoanode with Ti and Zn to improve the photoelectrocatalytic performance of hematite in the water splitting reaction. The photoelectrocatalytic activity of the hematite electrodes modified with Ti 4+ and Zn 2+ was optimized through the controlled variation of the dopant ion concentration in the electrodeposition solution. Under optimized conditions, for standard illumination of AM 1.5G (100 mW cm −2 ), the photocurrent density at the Ti/Zn-modified hematite anode reached 1.5 mA cm −2 at 1.23 V vs RHE that was 2.5-times higher than that observed with the pristine hematite electrode, the photoelectrocatalysis onset potential being 63 mV reduced. Effects of Ti and Zn doping on the photoelectrochemical activity of pristine hematite were studied by scanning electron microscopy, UV−vis spectroscopy, elemental analysis, and electrochemical impedance spectroscopy. On the basis of the obtained results, the improved performance of the Ti/Zn-modified hematite stemmed from the combination of the enhanced electrical conductivity along with the facilitated charge transport in the bulk phase and at the surface of hematite. The effect of Zn-doping decreasing the overpotential of the reaction by 218 mV (solely Zn-doped compared to that of the pristine hematite) was correlated with the Zn contribution to the interfacial catalysis of water oxidation.

Direct heterogeneous electron transfer (ET) of sulfite oxidase (SOx), a heme- and molybdopterin cofactor-containing intermembrane enzyme, was studied on alkanethiol-modified Au electrodes both with SOx entrapped between the modified Au electrode and a permselective membrane and with SOx adsorbed at the electrode surface, in the absence of any membrane. SOx in direct electronic communication with the electrode surface gave a quasi-reversible electrochemical signal with a midpoint potential of--120 mV vs Ag/AgCl corresponding to the redox transformations of the heme domain of SOx and with a heterogeneous ET constant in the order of 15 s(-1). The efficiency of the bioelectrocatalytic 2e- oxidation of sulfite catalyzed by SOx in direct ET exchange with the electrode was shown to depend essentially on the nature of the alkanethiol layer. Adsorption and orientation of SOx on an 11-mercapto-1-undecanol (MuD-OH) self-assembled monolayer, i.e., terminally functionalized with OH groups, provided efficient catalytic oxidation of sulfite, contrary to nonfunctionalized alkanethiols, e.g., 1-decanethiol, or alkanethiol layers terminally functionalized with NH2 groups. Comparative studies with short-chain alkanethiols, e.g., cysteamine and 2-mercaptoethanol, revealed an evidently different mode of adsorption of SOx on these layers, onto which SOx was not catalytically active. Coadsorption of MuD-OH and 11-mercapto-1-undecanamine improved the surface properties of the SAM, resulting in a higher surface coverage with bioelectrocatalytically active SOx but not in an increased apparent catalytic rate constant, kcat, ranging in the order of 18-24 s(-1) at pH 7.4. The achieved efficiency of SOx bioelectrocatalysis in direct ET reaction between the modified electrode and the enzyme approached the rates characteristic for the catalysis mediated by cytochrome c, the natural redox partner of SOx, thus implying the retention of the biological function of SOx under the heterogeneous electrode reaction conditions. Results obtained enable the development of a third-generation biosensor for sulfite monitoring.

The inherent redox activity of dopamine enables its direct electrochemical in vivo analysis ( Venton , B. J.; Wightman, M. R. Anal. Chem. 2003, 75, 414A). However, dopamine analysis is complicated by the interference from other electrochemically active endogenous compounds present in the brain, including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine-modified Au electrode, and further electrochemical oxidation of dopamine. Specific recognition of dopamine by the aptamer allowed a selective amperometric detection of dopamine within the physiologically relevant 100 nM to 5 μM range in the presence of competitive concentrations of catechol, epinephrine, norepinephrine, 3,4-dihydroxy-phenylalanine (L-DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), methyldopamine, and tyramine, which gave negligible signals under conditions of experiments (electroanalysis at 0.185 V vs Ag/AgCl). The interference from ascorbic and uric acids was eliminated by application of a Nafion-coated membrane. The aptasensor response time was <1 s, and the sensitivity of analysis was 62 nA μM(-1) cm(-2). The proposed design of the aptasensor, based on electrostatic interactions between the positively charged cysteamine-modified electrode and the negatively charged aptamer, may be used as a general strategy not to restrict the conformational freedom and binding properties of surface-bound aptamers and, thus, be applicable for the development of other aptasensors.

Electron transfer (ET) between gold electrodes and redox-labeled DNA duplexes, immobilized onto the electrodes through the alkanethiol linker at the 3'-end and having internal either methylene blue (MB) or anthraquinone (AQ) redox labels, was shown to depend on the redox label charge and the way the redox label is linked to DNA. For loosely packed DNA monolayers, the conjugation of the positively charged MB to DNA through the long and flexible alkane linker provided ET whose kinetics was formally governed by the diffusion of the redox label to the negatively charged electrode surface. For the uncharged AQ label no ET signal was detected. The conjugation of AQ to DNA through the short and more conductive acetylene linker did not provide the anticipated DNA-mediated ET to the AQ-moiety: ET appeared to be low-efficient if any in the studied system, for which no intercalation of AQ within the DNA duplex occurred. The ET communication between the electrode and AQ, built in DNA through the acetylene linker, was achieved only when Ru(NH(3))(6)(3+) molecules were electrostatically attached to the DNA duplex, thus forming the electronic wire. These results are of particular importance both for the fundamental understanding of the interfacial behavior of the redox labeled DNA on electrodes and for the design of biosensors exploiting a variation of ET properties of DNA in the course of hybridization.

No abstract

The role of the electrode material in the efficiency of direct (non-mediated) bioelectrocatalytic reduction of H 2 O 2 catalyzed by horseradish peroxidase (HRP) is studied and discussed. The variations in direct peroxidase bioelectrocatalysis when coming from carbon/graphite to metal electrodes and oxides, as well as modified electrodes, are analyzed regarding the variations in adsorption/orientation of peroxidase at the electrodes, interfacial electron transfer rates and mechanism of catalysis.