Biosensing under an applied voltage using optical waveguide lightmode spectroscopy

An applied dc voltage offers a means of controlling immobilization during biosensor fabrication and detection during biosensing application. We present a method to directly and continuously measure the adsorption of biomacromolecules or other polyelectrolytes, under an applied potential difference, based on optical waveguide lightmode spectroscopy (OWLS). An indium tin oxide (ITO) film of thickness ca. 10 nm coated onto a silicon titanium oxide (STO) waveguiding film serves as the working (sensing) electrode. We observe the effective refractive index of the 0th transverse electric guided mode to increase significantly in the presence of an applied potential due to charging of the interfacial double layer and, possibly, modest electrochemical oxidation. Adsorption from solution onto the ITO electrode is detected by a further increase in the effective refractive index. We achieve accurate detection by employing an optical model in which the STO and ITO layers are combined into a single waveguiding film. No improvement is found using models treating the ITO as a separate layer, either dielectric or conducting. Using this method, we find the adsorption of human serum albumin and horse heart cytochrome c to be considerably enhanced in the presence of an applied potential exceeding 1 V. We attribute this behavior to adsorption at positions on the protein molecules of complementary charge.     

Real-time study of the effect of different stress factors on lactic acid bacteria by electrochemical optical waveguide lightmode spectroscopy

Lactic acid bacteria play an important role in the fermentation of different food products. During the fermentation processes, lactobacilli are confronted with many inhibitor factors. These factors by themselves or in combination can influence the growth of lactic acid bacteria and their acidification capacity. The subject of our study was to monitor with a newly developed biosensing technique how the different chemical stress factors influence the survival of lactic acid bacteria. Electrochemical optical waveguide lightmode spectroscopy combines evanescent-field optical sensing with electrochemical control of surface adsorption processes. For optical sensing, a layer of indium tin oxide served as a high refractive index waveguide and as a conductive electrode, as well. Lactobacillus plantarum 2142 suspended in Jerusalem artichoke syrup was used in the experiments. Electrochemical optical waveguide lightmode spectroscopy measurements were undertaken by using OW 2,400c indium tin oxide coated waveguide sensors (MicroVacuum, Budapest, Hungary) and were performed in a flow-injection analyzer system. The bacterial cells were adsorbed in native form without any chemical binding on the surface of the sensor by ensuring polarizing potential (1V) and were exposed to different concentration of acetic acid/Jerusalem artichoke syrup, lactic acid/Jerusalem artichoke syrup and hydrogen peroxide/Jerusalem artichoke syrup solution for 1h, respectively, and the effect on bacteria cells was monitored. Results were compared to the traditional micro-assay method, and it can be assumed that after further investigations this new technique could be used in real-time application.     

Locally Addressable Electrochemical Patterning Technique (LAEPT) applied to poly(L-lysine)-graft-poly(ethylene glycol) adlayers on titanium and silicon oxide surfaces

The protein-resistant polycationic graft polymer, poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), was uniformly adsorbed onto a homogenous titanium surface and subsequently subjected to a direct current (dc) voltage. Under the influence of an ascending cathodic and anodic potential, there was a steady and gradual loss of PLL-g-PEG from the conductive titanium surface while no desorption was observed on the insulating silicon oxide substrates. We have implemented this difference in the electrochemical response of PLL-g-PEG on conductive titanium and insulating silicon oxide regions as a biosensing platform for the controlled surface functionalization of the titanium areas while maintaining a protein-resistant background on the silicon oxide regions. A silicon-based substrate was micropatterned into alternating stripes of conductive titanium and insulating silicon oxide with subsequent PLL-g-PEG adsorption onto its surfaces. The surface modified substrate was then subjected to +1800 mV (referenced to the silver electrode). It was observed that the potentiostatic action removed the PLL-g-PEG from the titanium stripes without inducing any polyelectrolyte loss from the silicon oxide regions. Time-of-flight secondary ions mass spectroscopy and fluorescence microscopy qualitatively confirmed the PLL-g-PEG retention on the silicon oxide stripes and its absence on the titanium region. This method, known as "Locally Addressable Electrochemical Patterning Technique" (LAEPT), offers great prospects for biomedical and biosensing applications. In an attempt to elucidate the desorption mechanism of PLL-g-PEG in the presence of an electric field on titanium surface, we have conducted electrochemical impedance spectroscopy experiments on bare titanium substrates. The results showed that electrochemical transformations occurred within the titanium oxide layer; its impedance and polarization resistance were found to decrease steadily upon both cathodic and anodic polarization resulting in the polyelectrolyte desorption from the titanium surface.     

Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides

Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.     

Interaction of poly(L-lysine)-g-poly(ethylene glycol) with supported phospholipid bilayers

Interactions between the graft copolymer poly(L-lysine)-g-poly(ethylene glycol), PLL-g-PEG, and two kinds of surface-supported lipidic systems (supported phospholipid bilayers and supported vesicular layers) were investigated by a combination of microscopic and spectroscopic techniques. It was found that the application of the copolymer to zwitterionic or negatively charged supported bilayers in a buffer of low ionic strength led to their decomposition, with the resulting formation of free copolymer-lipid complexes. The same copolymer had no destructive effect on a supported vesicular layer made up of vesicles of identical composition. A comparison between poly(L-lysine), which did not induce decomposition of supported bilayers, and PLL-g-PEG copolymers with various amounts of PEG side chains per backbone lysine unit, suggested that steric repulsion between the PEG chains that developed upon adsorption of the polymer to the nearly planar surface of a supported phospholipid bilayer (SPB) was one of the factors responsible for the destruction of the SPBs by the copolymer. Other factors included the ionic strength of the buffer used and the quality of the bilayers, pointing toward the important role defects present in the SPBs play in the decomposition process.     

Electrochemical Behaviour of Copper-Containing Nitrite Reductase from Alcaligenes Faecalis Strain-6

Indirect and direct electrochemical reactions of copper containing nitrite reductase (NIR) from Alcaligenes faecalis strain-6 are described. The reactivity of mediators, including blue protein from the same organism (the native redox partner of NIR, AfBP), in electrocatalytic reactions (EC') involving a mediator, NIR and nitrite was investigated. Several types of EC were observed and AfBP was found to be an effective mediator in spite of its high redox potential. Direct electrochemistry was observed at an Indium Tin Oxide electrode (ITO) and an edge plane oriented pyrolytic graphite electrode (PGE). Observation of the redox activity of NIR at an ITO in an optically transparent thin layer electrode cell (OTTLE) showed that it underwent reversible changes in absorbance that corresponded to the applied potential. The electrochemically adsorbed NIR at PGE showed fast electrochemical kinetics in cyclic voltammetry. It is suggested that the weak affinity of NIR to the PGE electrode may prevent complete denaturation of NIR in the adsorbed state.     

Fabrication of sucrose biosensor based on single mode planar optical waveguide using co-immobilized plant invertase and GOD

In present studies, the new optical sensing platform based on optical planar waveguide (OPWG) for sucrose estimation was reported. An evanescent-wave biosensor was designed by using novel agarose–guar gum (AG) biopolymer composite sol–gel with entrapped enzymes (acid invertase (INV) and glucose oxidase (GOD)). Partially purified watermelon invertase isolated from Citrullus vulgaris fruit (specific activity 832 units mg⁻¹) in combination with GOD was physically entrapped in AG sol–gel and cladded on the surface of optical planar waveguide. Na⁺–K⁺ ion-exchanged glass optical waveguides were prepared and employed for the fabrication of sucrose biosensor. By addressing the enzyme modified waveguide structure with, the optogeometric properties of adsorbed enzyme layer (12 μm) at the sensor solid–liquid interface were studied. The OPWG sensor with short response time (110 s) was characterized using the 0.2 M acetate buffer, pH 5.5. The fabricated sucrose sensor showed concentration dependent linear response in the range 1 × 10⁻¹⁰ to 1 × 10⁻⁶ M of sucrose. Lower limit of detection of this novel AG–INV–GOD cladded OPWG sensor was found to be 2.5 × 10⁻¹¹ M sucrose, which indicates that the developed biosensor has higher sensitivity towards sucrose as compared to earlier reported sensors using various transducer systems. Biochips when stored at room temperature, showed high stability for 81 days with 80% retention of original sensitivity. These sucrose sensing biochips showed good operational efficiency for 10 cycles. The proper confinement of acid invertase and glucose oxidase in hydrogel composite was confirmed by scanning electron microscopy (SEM) images. The constructed OPWG sensor is versatile, easy to fabricate and can be used for sucrose measurements with very high sensitivity.     

Bacterial protein patterning by micro-contact printing of PLL-g-PEG

Biomimetic micro-patterned surfaces of three S-layer (fusion) proteins, wild type (SbpA), enhanced green fluorescence protein (SbpA-EGFP) and streptavidin (SbpA-STV), were built by microcontact printing of poly-L-lysine grafted polyethylene glycol (PLL-g-PEG). The functionality of the adsorbed proteins was studied with atomic force microscopy and fluorescence microscopy. Atomic force microscopy (AFM) measurements showed that wild-type SbpA recrystallized on PLL-g-PEG free areas, while fluorescent properties of SbpA-EGFP and the interaction of SbpA-streptavidin heterotetramers with biotin were not affected due to the adsorption on the micro patterned substrates.     

Electrochemically induced oxidative damage to double-stranded calf thymus DNA adsorbed on gold electrodes

Electrochemically induced oxidative damage to DNA was studied with double-stranded calf thymus DNA immobilized directly on a gold electrode surface. Pre-polarization of the DNA-modified electrodes at +0.5 V versus Ag/AgCl reference electrode, in a free from DNA blank buffer solution, pH 7.4, allowed for subsequent detection of direct electrochemical oxidation of adsorbed on gold DNA, in the potential range from +0.7 to +0.8 V. The redox potential of the process corresponded to the potentials of the oxidation of guanine bases in DNA. It is shown that with increasing potential scan rate, v, the mechanism of electrochemical oxidation of DNA changes from the irreversible 4e^– oxidative damage of DNA at low v to reversible 1e^– oxidation at high v, keeping the electrochemical activity of the adsorbed DNA layer virtually the same.     

Controlled electrophoretic deposition of multifunctional nanomodules for bioelectrochemical applications

The design of an electrochemical glucose sensing device formed by the electrodeposition of multifunctional Au nanoparticles is reported here as a novel concept for an enhanced generic sensing platform. Initially gold nanoparticles (Au) were alternatively coated with a layer of positively charged redox polymer (ORP) and a negatively charged glucose oxidase (GOX) layer alternatively using layer-by-layer methodology to form multifunctional Au/ORP/GOX/ORP particles. The modification and stability of the Au nanoparticles was monitored by using UV-vis spectroscopy and zeta-potential measurements. The modified Au nanoparticles were electrophoretically deposited onto an electrode to produce an electrochemical glucose sensing device. A considerable influence of electrophoretic deposition time and potential was found on the sensing platform response. Preliminary responses to glucose addition showed an enhanced performance by applying an electrophoretic deposition potential of +1.2V vs. Ag/AgCl for 30min. The observed response in the case of microelectrode geometry was in the range of mAcm(2). This work also shows that the presence of a second outer ORP layer on the functionalised Au nanoparticles improved the response.     

Study of human serum albumin-TiO(2) nanocrystalline electrodes interaction by impedance electrochemical spectroscopy.

The adsorption of human serum albumin (HSA) onto nanocrystalline TiO(2) electrodes was studied by electrochemical impedance spectroscopy (EIS) in function of pH and electrode potential. The characterization and physico-chemical properties of the TiO(2) electrode were investigated by scanning electron microscopy (SEM), UV-photoelectron spectroscopy (UPS), cyclic voltammetry and capacitance measurements. The impedance response of the particulate TiO(2) electrode/protein interface was fitted using an equivalent circuit model to describe the adsorption process. The adsorbed protein layer, which is formed as soon as the protein is injected into the solution and becomes in contact with the electrode, was investigated as a function of electrode potential and solution pH. The measurements were performed under pseudo-steady-state and steady-state conditions, which gave information about the different states of the system. With the pseudo-steady state measurements, it was possible to determine two rate constants of the protein adsorption process, which correspond to two different states of the protein. The shortest one was associated with the first contact between the protein and the substrate and the second relaxation time, with the protein suffering an structural rearrangement due to the interaction with the TiO(2) electrode. It was detected that at sufficiently long times (approx. 1 h, where the system was under steady state conditions), a quasi-reversible protein adsorption mechanism was established. The measurements performed as a function of frequency under steady-state conditions, an equivalent circuit with a Warburg element gave the better fitting to data taken at -0.585 V closer to the oxide flat band potential and it was associated with protein diffusion. Experimental results obtained at only one frequency as a function of potential could be fitted to a model that takes into account non-specific and probable specific protein adsorption, which renders to be potential- and pH-dependent. Low capacity values were obtained in the whole potential range, which were measured in the presence and in the absence of the protein layer. The capacity dependence on potential and pH were associated with the generation of surface states on TiO(2). A surface state concentration of 4.1x10(18) cm(-2) was obtained by relating the parallel capacitance with oxide surface states arising from the protein-oxide interaction.     

In-situ FTIR study on adsorption and oxidation of native and thermally denatured calf thymus DNA at glassy carbon electrodes

In-situ Fourier transform infra-red (FTIR) spectra of native and thermally denatured calf thymus DNA (CT DNA) adsorbed and/or oxidized at a glassy carbon (GC) electrode surface are reported. The adsorption of native DNA occurs throughout the potential range (- 0.2 approximately 1.3 V) studied, and the adsorbing state of DNA at electrode surface is changed from through the C=O band of bases and pyrimidine rings to through the C=O of cytosine and imidazole rings while the potential shifts negatively from 1.3 V to -0.2 V. An in-situ FTIR spectrum of native CT DNA adsorbed at GC electrode surface is similar to that of the dissolved DNA, indicating that the structure of CT DNA is not distorted while it is adsorbed at the GC electrode surface. In the potential range of -0.2 approximately1.30 V, the temperature-denatured CT DNA is adsorbed at the electrode surface first, then undergoes electrochemical oxidation reaction and following that, diffuses away from the electrode surface.     

A novel hydrogen peroxide biosensor based on the immobilization of hemoglobin on three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-doped titanium dioxide (GTD) film

The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film was modified on the indium-tin oxide (ITO) electrode surface. Hemoglobin (Hb) has been successfully immobilized on the 3DOM GTD film and the fabrication process was characterized by Raman and UV-vis spectra. The results indicated that the Hb immobilized on the film retained its biological activity and the secondary structure of Hb was not destroyed. The direct electrochemistry and electrocatalysis of Hb immobilized on this film have been investigated. The Hb/3DOM GTD/ITO electrode exhibited two couples of redox peaks corresponding to the Hb intercalated in the mesopores and adsorbed on the external surface of the film with the formal potential of -0.20 and -0.48 V in 0.1M PBS (pH7.0), respectively. The Hb/3DOM GTD/ITO electrode exhibits an excellent eletrocatalytic activity, a wide linear range for H(2)O(2) from 5.0 μM to 1.0mM with a limit of detection of 0.6μM, high sensitivity (144.5 μA mM(-1)), good stability and reproducibility. Compared with the TiO(2) nanoneedles modified electrode, the GTD modified electrode has higher sensitivity and response peak current. The 3DOM GTD provided a good matrix for bioactive molecules immobilization, suggesting it has the potential use in the fields of H(2)O(2) biosensors.     

Electrocatalytic four-electron reduction of oxygen at the cytochrome c3-adsorbed electrode

The electrocatalytic activity of cytochrome c3 for the reduction of molecular oxygen was characterized from the studies of the adsorption of cytochrome c3 and the co-adsorption of cytochrome cs with cytochrome c on the mercury electrode by the a.c. polarographic technique. The adsorption of cytochrome c3 on the mercury electrode is irreversible and is diffusion-controlled. The maximum amount of cytochrome c3 absorbed was 0.92 . 10(-11) mol . cm-2 at -0.90 V. The amount of cytochrome c3 in the mixed adsorbed layer with cytochrome c was determined from the differential capacitance measurement. It was shown that the fractional coverage of cytochrome c3 can be estimated from its bulk concentration and the diffusion coefficient (1.05 . 10(-6) cm2 . s-1). Cytochrome c3 catalyzes the electrochemical reduction of molecular oxygen from the two-electron pathways via hydrogen peroxide to the four-electron pathway at the mercury electrode in neutral phosphate buffer solution. The catalytic activity varies with the bulk concentration of cytochrome c3. The highest catalytic activity for the oxygen reduction (no hydrogen peroxide formation) is attained when one-half of the mercury electrode surface is covered by cytochrome c3. The addition of cytochrome c or bovine serum albumin to the cytochrome c3 solution inhibits the catalytic activity of cytochrome c3. The reversible polarographic behavior of cytochrome c3 through the mixed adsorbed layer of cytochrome c3 and cytochrome c was also investigated.     

High sensitivity waveguide grating sensor based on radiative losses

A scheme of a waveguide sensor utilizing an output grating coupler with the grating length less than the coupling length is analyzed. This is the new method for the evaluation of average thickness of the adsorbed layer. This method, based on measurements of radiation loss coefficients, is proposed. Sensitivity limits of such a scheme are discussed. The new scheme has strong response on formation on the adsorbed layer and relatively weaker response on the fluctuations of temperature and analyte concentration. New possibilities for multiparameter sensing are shown.     

Application of europium(III) chelate-dyed nanoparticle labels in a competitive atrazine fluoroimmunoassay on an ITO waveguide

We have demonstrated the use of an optical indium tin oxide (ITO) (quartz) waveguide as a new platform for immunosensors with fluorescent europium(III) chelate nanoparticle labels (Seradyn) in a competitive atrazine immunoassay. ITO as a solid surface facilitated the successful use of particulate labels in a competitive assay format. The limit of detection in the new nanoparticle assay was similar to a conventional ELISA. The effect of particle size on bioconjugate binding kinetics was studied using three sizes of bioconjugated particle labels (107, 304, and 396nm) and a rabbit IgG/anti-IgG system in a 96-well plate. A decrease in particle size resulted in faster binding but did not increase the assay sensitivity. Flux calculations based on the particle diffusivity prove that faster binding of the small particles in this study was primarily due to diffusion kinetics and not necessarily to a higher density of antibodies on the particle surface. The results suggest that ITO could make a good platform for an optical immunosensor using fluorescent nanoparticle labels in a competitive assay format for small molecule detection. However, when used in combination with fluorescent particulate labels, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Different regeneration methods tested in this study showed that repeated washings with 0.1 M glycine-HCl facilitated the reuse of the ITO waveguide.     

Facile fabrication of gelatin‐based biopolymeric optical waveguides

The rapid development in optical detection techniques for sensing applications has led to an increased need for biocompatible, biodegradable, and disposable optical components. We present a controllable fabrication technique for an entirely biopolymeric planar optical waveguide via simple spin‐coating. The refractive index difference, thermal responsive properties, and inherent biocompatibility of gelatin and agarose were exploited in the fabrication of thin, stacked films that efficiently guide light in a core layer with higher index of refraction. These planar waveguides were fabricated using a simple spin‐coating technique, which resulted in controllable layer thicknesses and smooth layer interfaces. This technique, therefore, offers a path for routine engineering of biopolymer structures with contrasting refractive indices. The thermal stability of the gelatin core layer was improved using two crosslinkers; glutaraldehyde or microbial Transglutaminase. Light guiding in the core layer of the waveguide was demonstrated using a simple He–Ne laser setup. Guiding efficiency was further illustrated by directly embedding fluorescent markers within the core layer and detecting their spectral signature. Combined with the biopolymers' inherent biocompatibility and biodegradability, our simple strategy to fabricate disposable optical components holds the potential for the development of applications in biological sensing and implantable biomedical devices. Biotechnol. Bioeng. 2009;103: 725–732. © 2009 Wiley Periodicals, Inc.     

Towards a biosensor based on anti resonant reflecting optical waveguide fabricated from porous silicon

Recently, we demonstrated that Anti Resonant Reflecting Optical Waveguide (ARROW) based on porous silicon (PS) material can be used as a transducer for the development of a new optical biosensor. Compared to a conventional biosensor waveguide based on evanescent waves, the ARROW structure is designed to allow a better overlap between the propagated optical field and the molecules infiltrated in the porous core layer and so to provide better molecular interactions sensitivity. The aim of this work is to investigate the operating mode of an optical biosensor using the ARROW structure. We reported here an extensive study where the antiresonance conditions were adjusted just before the grafting of the studied molecules for a given refractive index range. The interesting feature of the studied ARROW structure is that it is elaborated from the same material which is the porous silicon obtained via a single electrochemical anodization process. After oxidation and preparation of the inner surface of porous silicon by a chemical functionalization process, bovine serum albumin (BSA) molecules, were attached essentially in the upper layer. Simulation study indicates that the proposed sensor works at the refractive index values ranging from 1.3560 to 1.3655. The experimental optical detection of the biomolecules was obtained through the modification of the propagated optical field and losses. The results indicated that the optical attenuation decreases after biomolecules attachment, corresponding to a refractive index change Δn(c) of the core. This reduction was of about 2 dB/cm and 3 dB/cm for Transverse Electric (TE) and Transverse Magnetic (TM) polarizations respectively. Moreover, at the detection step, the optical field was almost located inside the core layer. This result was in good agreement with the simulated near field profiles.     

Cholesterol biosensors prepared by layer-by-layer technique

The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet–visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.     

Electrochemical DNA sensing using osmium complexes as hybridization indicators

A surface-based method for the study of the interactions of DNA with redox-active osmium complexes is described. The study was carried out using gold electrodes modified with DNA by adsorption and [Os(bpy)3]3+/2+ (bpy=2,2'-bipyridyl) or [Os(phen)3]3+/2+ (phen=1,10-phenantroline) as electrochemical indicators. The method, which is simple and reagent saving, allows the accumulation of osmium complexes on the DNA layer. The amount of osmium complex bound by the layer of double-stranded (dsDNA) or single-stranded DNA (ssDNA) adsorbed at gold electrodes was estimated from the cyclic voltammetric (CV) peak charge of osmium complex reduction. The dissociation constants (K) for the oxidized and reduced forms of a bound species are also estimated. [Os(phen)3]3+/2+ was applied to a probe for electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 70 pmol/cm2 on a gold electrode. Following hybridization with the complementary DNA, the osmium complex was electrochemically accumulated on the double-stranded DNA layer and the differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of [Os(phen)3]3+/2+ that was bound to the double-stranded DNA on the electrode.