Large-amplitude Fourier transformed high-harmonic alternating current cyclic voltammetry: kinetic discrimination of interfering Faradaic processes at glassy carbon and at boron-doped diamond electrodes

Significant advantages of Fourier transformed large-amplitude ac higher (second to eighth) harmonics relative to responses obtained with conventional small-amplitude ac or dc cyclic voltammetric methods have been demonstrated with respect to (i) the suppression of capacitive background currents, (ii) the separation of the reversible reduction of [Ru(NH(3))(6)](3+) from the overlapping irreversible oxygen reduction process under conditions where aerobic oxygen remains present in the electrochemical cell, and (iii) the kinetic resolution of the reversible [Ru(NH(3))(6)](3+/2+) process in mixtures of [Fe(CN)(6)](3-) and [Ru(NH(3))(6)](3+) at appropriately treated boron-doped diamond electrodes, even when highly unfavorable [Fe(CN)(6)](3-) to [Ru(NH(3))(6)](3+) concentration ratios are employed. Theoretical support for the basis of kinetic discrimination in large-amplitude higher harmonic ac cyclic voltammetry is provided.     

Electrochemical performance of diamond thin-film electrodes from different commercial sources

The electrochemical properties of two commercial (Condias, Sumitomo) boron-doped diamond thin-film electrodes were compared with those of two types of boron-doped diamond thin film deposited in our laboratory (microcrystalline, nanocrystalline). Scanning electron microscopy and Raman spectroscopy were used to characterize the electrode morphology and microstructure, respectively. Cyclic voltammetry was used to study the electrochemical response, with five different redox systems serving as probes (Fe(CN)(6)(3)(-)(/4)(-), Ru(NH(3))(6)(3+/)(2+), IrCl(6)(2)(-)(/3)(-), 4-methylcatechol, Fe(3+/2+)). The response for the different systems was quite reproducibile from electrode type to type and from film to film for electrodes of the same type. For all five redox systems, the forward reaction peak current varied linearly with the scan rate(1/2) (nu), indicative of electrode reaction kinetics controlled by mass transport (semi-infinite linear diffusion) of the reactant. Apparent heterogeneous electron-transfer rate constants, k degrees (app), for all five redox systems were determined from deltaE(p)-nu experimental data, according to the method described by Nicholson (Nicholson, R. S. Anal. Chem. 1965, 37, 1351.). The rate constants were also verified through digital simulation (DigiSim 3.03) of the voltammetric i-E curves at different scan rates. Good fits between the experimental and simulated voltammograms were found for scan rates up to 50 V/s. k degrees (app) values of 0.05-0.5 cm/s were observed for Fe(CN)(6)(3)(-)(/4)(-), Ru(NH(3))(6)(3+/2+), and IrCl(6)(2)(-)(/3)(-) without any extensive electrode pretreatment (e.g., polishing). Lower k degrees (app) values of 10(-)(4)-10(-)(6) cm/s were found for 4-methylcatechol and Fe(3+/2+). The voltammetric responses for Fe(CN)(6)(3)(-)(/4)(-) and Ru(NH(3))(6)(3+/2+) were also examined at all four electrode types at two different solution pH (1.90, 7.35). Since the hydrogen-terminated diamond surfaces contain few, if any, ionizable carbon-oxygen functionalities (e.g., carboxylic acid, pK(a) approximately 4.5), the deltaE(p), i(p)(ox), and i(p)(red) values for the two systems were, for the most part, unaffected by the solution pH. This is in contrast to the typical behavior of oxygenated, sp(2) carbon electrodes, such as glassy carbon.     

Determination of heterogeneous electron transfer kinetics in the presence of ultrasound at microelectrodes employing sampled voltammetry

The technique of sampled voltammetry at microelectrodes irradiated with ultrasound is demonstrated for the first time. This technique is used to determine the heterogeneous electron transfer rate constants for the redox couples ferrocene/ferrocenium, Ru(NH(3))(6)(3+/2+), and IrCl(6)(3)(-)/IrCl(6)(2)(-). Determination of the heterogeneous rate constants is also achieved for comparison purposes by analysis of fast sweep rate voltammetry of the redox systems studied at microelectrodes and comparison of the results obtained to the theory developed by Nicholson and Shain. The heterogeneous rate constants determined using sampled voltammetry were 1.0, 0.6, 1.23, and 0.18 cm s(-)(1) for the ferrocene/ferrocenium (0.1 mol dm(-)(3) TEATFB, CH(3)CN), Ru(NH(3))(6)(3+/2+) (0.1 mol dm(-)(3) KCl), IrCl(6)(3)(-) (1 mol dm(-)(3) KCl), and IrCl(6)(3)(-)/IrCl(6)(2)(-) (1 mol dm(-)(3) NaCl), respectively, in agreement with those obtained in the absence of ultrasound.     

Scanning electrochemical microscopy. 32. Gallium ultramicroelectrodes and their application in ion-selective probes

Gallium ultramicroelectrodes for amperometric measurements in scanning electrochemical microscopy were fabricated by introduction of liquid Ga into drawn glass micropipets. Cyclic voltammetry of Ru(NH(3))(6)(3+) and the use of this species for SECM imaging is described. Double-barrel micropipet tips with a Ga amperometric electrode and an ion-selective (K(+)) potentiometric probe can also be constructed. This probe was used to image the K(+) activity near a 20-μm-diameter lumen of a glass capillary.     

Conductive supports for combined AFM-SECM on biological membranes

Four different conductive supports are analysed regarding their suitability for combined atomic force and scanning electrochemical microscopy (AFM-SECM) on biological membranes. Highly oriented pyrolytic graphite (HOPG), MoS(2), template stripped gold, and template stripped platinum are compared as supports for high resolution imaging of reconstituted membrane proteins or native membranes, and as electrodes for transferring electrons from or to a redox molecule. We demonstrate that high resolution topographs of the bacterial outer membrane protein F can be recorded by contact mode AFM on all four supports. Electrochemical feedback experiments with conductive cantilevers that feature nanometre-scale electrodes showed fast re-oxidation of the redox couple Ru(NH(3))(6)(3+/2+) with the two metal supports after prolonged immersion in electrolyte. In contrast, the re-oxidation rates decayed quickly to unpractical levels with HOPG or MoS(2) under physiological conditions. On HOPG we observed heterogeneity in the re-oxidation rate of the redox molecules with higher feedback currents at step edges. The latter results demonstrate the capability of conductive cantilevers with small electrodes to measure minor variations in an SECM signal and to relate them to nanometre-scale features in a simultaneously recorded AFM topography. Rapid decay of re-oxidation rate and surface heterogeneity make HOPG or MoS(2) less attractive for combined AFM-SECM experiments on biological membranes than template stripped gold or platinum?supports.     

Electrochemical DNAzyme sensor for lead based on amplification of DNA-Au bio-bar codes

An electrochemical DNAzyme sensor for sensitive and selective detection of lead ion (Pb(2+)) has been developed, taking advantage of catalytic reactions of a DNAzyme upon its binding to Pb(2+) and the use of DNA-Au bio-bar codes to achieve signal enhancement. A specific DNAzyme for Pb(2+) is immobilized onto an Au electrode surface via a thiol-Au interaction. The DNAzyme hybridizes to a specially designed complementary substrate strand that has an overhang, which in turn hybridizes to the DNA-Au bio-bar code (short oligonucleotides attached to 13 nm gold nanoparticles). A redox mediator, Ru(NH3)6(3+), which can bind to the anionic phosphate of DNA through electrostatic interactions, serves as the electrochemical signal transducer. Upon binding of Pb(2+) to the DNAzyme, the DNAzyme catalyzes the hydrolytic cleavage of the substrate, resulting in the removal of the substrate strand along with the DNA bio-bar code and the bound Ru(NH3)6(3+) from the Au electrode surface. The release of Ru(NH3)6(3+) results in lower electrochemical signal of Ru(NH3)6(3+) confined on the electrode surface. Differential pulse voltammetry (DPV) signals of Ru(NH3)6(3+) provides quantitative measures of the concentrations of Pb(2+), with a linear calibration ranging from 5 nM to 0.1 microM. Because each nanoparticle carries a large number of DNA strands that bind to the signal transducer molecule Ru(NH3)6(3+), the use of DNA-Au bio-bar codes enhances the detection sensitivity by five times, enabling the detection of Pb(2+) at a very low level (1 nM). The DPV signal response of the DNAzyme sensor is negligible for other divalent metal ions, indicating that the sensor is highly selective for Pb(2+). Although this DNAzyme sensor is demonstrated for the detection of Pb(2+), it has the potential to serve as a general platform for design sensors for other small molecules and heavy metal ions.     

Stable tris(2,2'-bipyridine)ruthenium(III) for chemiluminescence detection

Two exceedingly stable [Ru(bipy)(3)](3+) reagents were prepared by dissolving either [Ru(bipy)(3)](ClO(4))(2) in acetonitrile (containing 0.05 M HClO(4)) or [Ru(bipy)(3)]Cl(2)·6H(2)O in 95:5 glacial acetic acid-acetic anhydride (containing 0.05 M H(2)SO(4)) followed by oxidation with PbO(2). These conveniently prepared solutions provide highly reproducible chemiluminescence detection over long periods of analysis, avoiding the need for recalibration or preparation of fresh reagent solutions and without the complications associated with online chemical or electrochemical oxidations. The reagent prepared in acetonitrile produced much greater signal intensities with a range of analytes and was deemed most suitable for high-performance liquid chromatography (HPLC) with postcolumn chemiluminescence detection.     

Bioassembled nanocircuits of Mo6S9-xIx nanowires for electrochemical immunodetection of estrone Hapten

We demonstrate a novel and highly sensitive electrochemical detection of estrone based on an immunosensor platform, composed of bioassembled nanocircuits of Mo 6S 9- x I x nanowires (MoSI NWs) covalently connected to anti-estrone antibodies. The one-step, label-free, and quantitative detection of estrone is realized by employing the [Ru(NH 3) 6] (3+/2+) redox ions to sense anti-estrone antibody and estrone interactions. The MoSI NWs/anti-estrone antibody nanocircuit architectures provide an amplification and conductive pathway for the specific electrochemical sensing of estrone hapten. A detection limit of 1.4 pg x mL (-1) was achieved in contrast to previous electrochemical techniques in which the sensitivity was limited to the nanomolar range.     

Characterization of batch-microfabricated scanning electrochemical-atomic force microscopy probes

A procedure for the batch microfabrication of scanning electrochemical-atomic force microscopy (SECM-AFM) probes is described. The process yields sharp AFM tips, incorporating a triangular-shaped electrode (base width 1 microm, height 0.65 microm) at the apex. Microfabrication was typically carried out on (1)/(4) 3-in. wafers, yielding 60 probes in each run. The measured spring constant of the probes was in the range 1-1.5 N m(-1). To date, processing has been carried out twice successfully, with an estimated success rate for the fabrication process in excess of 80%, based on field emission-scanning electron microscopy imaging of all probes and current-voltage measurements on a random selection of approximately 30 probes. Steady-state voltammetric measurements for the reduction of Ru(NH(3))(6)(3+) in aqueous solution indicate that the electrode response is well-defined, reproducible, and quantitative, based on a comparison of the experimental diffusion-limited current with finite element simulations of the corresponding mass transport (diffusion) problem. Topographical imaging of a sputtered Au film with the SECM-AFM probes demonstrates lateral resolution comparable to that of conventional Si(3)N(4) AFM probes. Combined electrochemical-topographical imaging studies have been carried out on two model substrates: a 10-microm-diameter disk ultramicroelectrode (UME) and an array of 1-microm-diameter UMEs, spaced 12.5 microm apart (center to center). In both cases, an SECM-AFM probe was first employed to image the topography of the substrates. The tip was then moved back a defined distance from the surface and use to detect Ru(NH(3))(6)(2+) produced at the substrate, biased at a potential to reduce Ru(NH(3))(6)(3+), present in bulk solution, at a diffusion-controlled rate (substrate generation-tip collection mode). These studies establish the success of the batch process for the mass microfabrication of SECM-AFM tips.     

Bioinorganic composites for enzyme electrodes

Sparingly soluble redox salts were combined with a model enzyme, glucose oxidase, in a host matrix of a biopolymer chitosan to form bioinorganic composite films on the surface of glassy carbon electrodes. Four redox salts, each containing the Ru(NH3)6(3+) cation and a selected anion, such as Ru(CN)6(4-), Fe(CN)6(4-), Co(CN)6(3-) or IrCl6(3-), were studied. The composition and catalytic properties of such composite materials toward glucose oxidation were investigated by spectroscopic and electrochemical methods. The composite films provided an oxygen-independent electrical communication between the enzyme's redox centers and a glassy carbon surface at a potential as low as -0.10 V vs Ag/AgCl(3 M Cl-). The nature of the electrical communication is discussed in terms of redox mediation by the Ru(NH3)6(3+)-containing ion pairs formed inside the biocomposites. The kinetic significance of the mediator's charge is considered by postulating that neutral ion pairs are more efficient redox mediators of the enzymatic reaction than those negatively charged. The low operating potential of enzyme electrodes based on the bioinorganic composites allows for an interference-free determination of glucose. The design of the biocomposites is generic and can incorporate oxidoreductase enzymes other than glucose oxidase to provide a host of biosensors for biologically and environmentally important analytes.     

High-sensitivity, label-free DNA sensors using electrochemically active conducting polymers

Label-free oligonucleotide sensors that use a change in the electrode kinetics of the redox reaction of the negatively charged Fe(CN)(6)(3-/4-) redox couple to signal the formation of a DNA duplex with a surface-conjugated probe nucleotide are investigated. Electrochemically active conducting polymers (ECPs) can advantageously be used both as the active electrode and as the means of surface conjugation of the probe nucleotide. Here, we demonstrate that the sensitivity of the detection of the surface-complementary oligonucleotide can significantly be improved, into the low nanomolar range, by forming the ECP as a highly porous, very rough layer by growing it using electrochemical polymerization on a microelectrode. In comparison, smoother surfaces formed on macroelectrodes had detection sensitivity in the low micromolar range. We propose Donnan exclusion of the redox couple from small pores as the reason for the enhanced sensitivity. We discuss the effects using a simple patch model for the electrochemical kinetics and use the model to derive the equilibrium binding constant and binding kinetic rate constants for the surface hybridization reaction. We use the electrochemically active copolymer of pyrrole (Py) and 3-pyrrolylacrylic acid (PAA) [poly(Py-co-PAA)] as the sensing electrode and binding surface and measure the surface hybridization-induced changes in electrode kinetics of Fe(CN)(6)(3-/4-) by electrochemical impedance spectroscopy.     

Microfabricated recessed microdisk electrodes: characterization in static and convective solutions

Construction and characterization of microfabricated recessed microdisk electrodes (RMDs) of 14- and 55-μm diameters and 4-μm depth are reported. For evaluation of electrode function, both faradaic current in Ru(NH(3))(6)(3+)/KNO(3) solution and charging current in KNO(3) solution were measured with cyclic voltammetry. The experimental maximum current was measured and compared to calculated values, assuming radial and linear diffusion. A model for diffusion to a RMD best matches the behavior of the 14-μm RMD, which has a larger depth-to-diameter ratio than the 55-μm RMD. At fast scan rates (204 V s(-)(1)), where linear diffusion should dominate, there are large deviations from the linear diffusion model. Uncompensated resistance and overcorrection for background current contribute to this deviation. The dependence of capacitance on scan rate of the RMDs was found to be similar to that of a macroelectrode, indicating good adhesion between the insulator and the electrode. Chronoamperometry of Ru(NH(3))(6)(3+) in KNO(3) in both static and stirred solutions was performed using the RMDs and the current is compared to those from a 10-μm-diameter planar microdisk electrode (PMD). The signal-to-noise ratio of the 14-μm RMDs compared to the PMD is on average 4 times greater for stirred solutions. The 55-μm RMD exhibited no protection to convection of the stirred solution.     

Multilabeling biomolecules at a single site. 1. Synthesis and characterization of a dendritic label for electrochemiluminescence assays

Ultrasensitive bioanalytical assays are of great value for early detection of human diseases and pathogens. The sensitivities of immunoassays and DNA probing can be enhanced by multilabeling the biorecognition partner used for affinity-based assays. However, the bioreactivity of biomolecules is affected by a high degree of multilabeling at multiple functional sites. It is proposed that dendritic scaffoldings be used to link multiple signal-generating units to a single site with potentially minimum impact on the bioaffinity. A prototype label, a zeroth-generation dendron, bearing three [Ru(bpy)(3)](2+) units for electrochemiluminescence (ECL) assays was synthesized and characterized preliminarily by spectroscopic, electrochemical, and ECL methods. No evidence of interaction between the neighboring [Ru(bpy)(3)](2+) units in the label molecule was found from these characterizations. Both the photoluminescence and ECL of the prototype label have features very similar to those of mononuclear [Ru(bpy)(3)](2+) compounds. Labeling a model protein with a triad of [Ru(bpy)(3)](2+) at one NH(2) position was demonstrated. The results reported here provide support to applying the proposed multilabeling strategy to affinity-based bioanalytical assays.     

Graphene nanoplatelets outperforming platinum as the electrocatalyst in co-bipyridine-mediated dye-sensitized solar cells

Graphene nanoplatelets (GNP) in the form of thin semitransparent films on F-doped SnO2 (FTO) exhibit high electrocatalytic activity for the Co(bpy)3(3+/2+) redox couple in acetonitrile electrolyte solution. The GNP film is superior to the traditional electrocatalyst, that is, platinum, both in charge-transfer resistance (exchange current) and in electrochemical stability under prolonged potential cycling. The good electrochemical performance of GNP is readily applicable for dye-sensitized solar cells with Y123-sensitized TiO2 photoanodes and Co(bpy)3(3+/2+) as the redox shuttle. The dye-sensitized solar cell with GNP cathode is superior to that with the Pt-FTO cathode particularly in fill factor and in power conversion efficiency at higher illumination intensity.     

Simple template-free solution route for the synthesis of Ni(SO(4))(0.3)(OH)(1.4) nanobelts and their thermal degradation

Nanobelts of nickel hydroxyl sulfate have been prepared on a large scale via a simple template-free hydrothermal reaction on the basis of a complex [Ni(NH(3))(6)](2+) formed with Ni(2+) and ammonia in an ethanol-water solution. The as-synthesized nanobelts were single crystals, with several tens of microns in length and 50-150?nm in width. The nanobelts were enclosed by top surfaces (100) and side surfaces (001) and their growth direction was parallel to [010]. The function of aqueous ammonia and ethanol was discussed. Furthermore, nanostructures of a mixture of crystralline NiO and amorphous nickel sulfate with various morphologies, such as nanobelts, porous nanobelts, and nanoparticles, were obtained by the thermal treatment of the as-synthesized Ni(SO(4))(0.3)(OH)(1.4) nanobelts at different temperatures.     

Aptamer-based biosensors for label-free voltammetric detection of lysozyme

This paper reports a simple electrochemical approach for the detection of the ubiquitous protein lysozyme using aptamer-modified electrodes. Anti-lysozyme DNA aptamers were immobilized on gold surfaces by means of self-assembly, for which the surface density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH3)6]3+) bound to the surface via electrostatic interaction with the DNA phosphate backbone. Upon incubation of the electrode with a solution containing lysozyme, the CV response of surface-bound [Ru(NH3)6]3+ changed substantially, and the relative decrease in the integrated charge of the reduction peak can be tabulated as a quantitative measure of the protein concentration. It is significant that the on-chip protein/aptamer binding constant and the optimized surface density to achieve the best detection limit can be evaluated. This biosensor is label-free and offers an alternative, sensitive, and versatile method for protein detection, which is beneficial to the ever-growing interests of fabricating portable bioanalytical devices with simple electrical readout protocols.     

Voltammetric procedure for examining DNA-modified surfaces: quantitation, cationic binding activity, and electron-transfer kinetics

To examine DNA-modified surfaces, we have developed a simple, convenient, and reliable procedure based on the voltammetric response of multiply charged transition metal cations (such as [Ru(NH3)6]3+) bound electrostatically to the DNA probes. At micromolar concentrations of the redox molecules in the electrolyte, the reduction and oxidation waves resulting from the immobilized cations on DNA-modified electrodes are well defined, stable, and reproducible. The surface densities of both single- and double-stranded oligonucleotides were accurately determined by integration of the peak for reduction of [Ru(NH3)6]3+ to [Ru(NH3)6]2+. In addition, the binding constant and electron-transfer rate constant of [Ru(NH3)6]3+ on DNA-modified electrodes were evaluated with the help of classical models. The present research provides not only an applicable and simple protocol for the quantitation of DNA probes on chips but also a versatile and powerful tool for the investigation of the binding activity and electron-transfer kinetics of cationic analytes on DNA-modified surfaces.     

A two-channel microfluidic sensor that uses anodic electrogenerated chemiluminescence as a photonic reporter of cathodic redox reactions

This paper describes a new approach for sensing electrochemically active substrates in microfluidic systems. This two-electrode sensor relies on electrochemical detection at one electrode and electrogenerated chemiluminescent (ECL) reporting at the other. Each microfabricated indium tin oxide electrode is located in a separate microfluidic channel, but the channels are connected downstream of the electrodes to maintain a complete electrical circuit. Because of laminar flow, there is no bulk mixing of the fluids in the detecting and reporting channels. This approach allows the ECL reaction to be physically and chemically decoupled from the sensing channel of the device, which greatly expands the number of analytes that can be detected. However, because the cathode and anode are connected, electron-transfer processes occurring at the sensing electrode are electrically coupled to the ECL reaction. Charge balance permits the ECL light output to be quantitatively correlated to electrochemical reductions at the cathode. The system is used to detect Fe(CN)6(3-), Ru(NH3)6(3+), and benzyl viologen and report their presence via Ru(bpy)3(2+) (bpy = bipyridine) luminescence. Each different redox target initiates ECL at a unique potential bias related to its standard redox potential. The influence of the concentrations of Ru(bpy)3(2+) and the target analytes is discussed.     

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 2. Demonstration of selectivity in the presence of direct interferences

Three modes of selectivity based on charge-selective partitioning, electrolysis potential, and spectral absorption wavelength were demonstrated simultaneously in a new type of spectroelectrochemical sensor. Operation and performance of the three modes of selectivity for detection of analytes in the presence of direct interferences were investigated using binary mixture systems. These binary mixtures consisted of Fe(CN)(6)(3-) and Ru(bpy)(3)(2+) and of Fe(CN)(6)(4-) and Ru(CN)(6)(4)(-) in aqueous solutions. Results on the Fe(CN)(6)(3-)/Ru(bpy)(3)(2+) binary mixture showed that an anion-exchange coating consisting of PDMDAAC-SiO(2) [where PDMDAAC is poly(dimethyldiallylammonium chloride)] and a cation-exchange coating consisting of Nafion-SiO(2) can trap and preconcentrate analytes with charge selection. At the same time, such coatings exclude interferences carrying the same type of charge as that of the exchange sites in the sensor coating. Using the Fe(CN)(6)(4-)/Ru(CN)(6)(4-) binary mixture, the Fe(CN)(6)(4-) component can be selectively detected by restricting the modulation potential cycled to a range specific to the redox-active Fe(CN)(6)(4-) component and simultaneously monitoring the optical response at the overlapping wavelength of 420 nm. It was also shown that, when the wavelength for optical monitoring was chosen as 500 nm, which is specific to the Ru(CN)(6)(4-) component, interference from the Fe(CN)(6)(4-) component for spectroelectrochemical detection of Ru(CN)(6)(4-) was significantly suppressed, even though the cyclic modulation potential encompassed the redox range for the Fe(CN)(6)(4-) component.     

Electrochemical immunosensor using p-aminophenol redox cycling by hydrazine combined with a low background current

Signal amplification and noise reduction are crucial for obtaining low detection limits in biosensors. Here, we present an electrochemical immunosensor in which the signal amplification is achieved using p-aminophenol (AP) redox cycling by hydrazine, and the noise level is reduced by implementing a low background current. The redox cycling is obtained in a simple one-electrode, one-enzyme format. In a sandwich-type heterogeneous immunosensor for mouse IgG, an alkaline phosphatase label converts p-aminophenyl phosphate into AP for 10 min. This generated AP is electrooxidized at an indium tin oxide (ITO) electrode modified with a partially ferrocenyl-tethered dendrimer (Fc-D). The oxidized product, p-quinone imine (QI), is reduced back to AP by hydrazine, and then AP is electrooxidized again to QI, resulting in redox cycling. Moreover, hydrazine protects AP from oxidation by air, enabling long incubation times. The small amount of ferrocene in a 0.5% Fc-D-modified ITO electrode, where 0.5% represents the ratio of ferrocene groups to dendrimer amines, results in a low background current, and this electrode exhibits high electron-mediating activity for AP oxidation. Moreover, there is insignificant hydrazine electrooxidation on this electrode, which also results in a low background current. The detection limit of the immunosensor using a 0.5% Fc-D-modified electrode is 2 orders of magnitude lower than that of a 20% Fc-D-modified electrode (10 pg/mL vs 1 ng/mL). Furthermore, the presence of hydrazine reduces the detection limit by an additional 2 orders of magnitude (100 fg/mL vs 10 pg/mL). These results indicate that the occurrence of redox cycling combined with a low background current yields an electrochemical immunosensor with a very low detection limit (100 fg/mL). Mouse IgG could be detected at concentrations ranging from 100 fg/mL to 100 microg/mL (i.e., 9 orders of magnitude) in a single assay.