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This study presents the combination of electrochemical cell based biosensor and fluorescence microscopy on a single platform to track biomolecular processes contributing to the morphology changes of the cells. Initial experiments demonstrate that using interdigitated electrodes where the gold microfingers were comparable in width and spacing to a single cell have the optimal sensitivity in the final electrochemical cell based‐sensing device. This was determined by measuring the cell index, based on the impedance analysis of bare and cell‐covered microelectrodes. The fabrication of the electrodes on a glass substrate enabled the capture of high‐resolution fluorescence microscopy images of single cells and related intracellular calcium release inside the HeLa cells via a window incorporated into the gold microelectrode design. As an illustration of the enhanced capability of the combined approach over traditional impedance cellular assay, the opto‐electric assay was utilized as a functional readout for G protein couple receptor activation. The simultaneous examinations of cells stimulated with histamine demonstrated an association between time courses of changes in cytosolic calcium concentration and reductions in cell‐cell adhesions. [ABSTRACT FROM AUTHOR]

Herein the development of cellular impedance biosensors, electrochemical impedance spectroscopy, and the general principles and terms associated with the cell–electrode interface is reviewed. This family of techniques provides quantitative and sensitive information into cell responses to stimuli in real-time with high temporal resolution. The applications of cell-based impedance biosensors as a readout in cell biology is illustrated with a diverse range of examples. The current state of the field, its limitations, the possible available solutions, and the potential benefits of developing biosensors are discussed. [ABSTRACT FROM AUTHOR]

Herein is explored a dual optical and electrical cell based biosensor that can provide insights into cellular events. The fabrication steps along with the electrical characterization of the microelectrodes is described. Initial experiments show that the ability of indium tin oxide (ITO) to detect biological cells at the electrode‐cell layer interface mainly depends on the size of sensing area. Following this, the impact of conductivity was also explored to assess the real time impedance signal during the adhesion of a non‐confluent layer of the cells on different substrates. Electrodes with a higher conductivity than ITO gave much higher senstivity of impedance changes which allowed the dynamics of subtle cellular morphology changes to be monitored at densities far lower than a confluent layer of cells. Finally, the capability of ITO and gold microelectrode as a functional readout for G protein couple receptor activation were determined. These set of analyses highlighted the challenges and opportunities of ITO substrate as a dual cell‐based assay for quantitative analysis of subtle changes in cell morphology. [ABSTRACT FROM AUTHOR]

A generic fill-and-flow channel biosensor with upstream electrodes to determine the extent of interferences in the sample is described. A pair of upstream electrodes poised at a suitable potential allows both the calculation of the extent of removal of interfering agents and the effect of interfering agents at the detector electrode. A model was developed and tested that predicts the concentrations of all species throughout the channel and, hence, the current at each electrode due to each species. This enables correction of the detector electrode current and a more accurate determination of the analyte concentration. The concept was applied to a biosensor for the determination of glucose in the presence of ascorbic acid, acetamidophenol, and uric acid, as well as glucose in wine samples containing polyphenolic interfering agents.

Recent advances in CRISPR based biotechnologies have greatly expanded our capabilities to repurpose CRISPR for the development of biomolecular sensors for diagnosing diseases and understanding cellular pathways. The key attribute that allows CRISPR to be widely utilized is the programmable and highly selective mechanism. In this Minireview, we first illustrate the molecular principle of CRISPR functioning process from sensing to actuating. Next, the CRISPR based biosensing strategies for nucleic acids, proteins and small molecules are summarized. We highlight some of recent advances in applications for in vitro detection of biomolecules and in vivo imaging of cellular networks. Finally, the challenges with, and exciting prospects of, CRISPR based biosensing developments are discussed. [ABSTRACT FROM AUTHOR]

Measurement science has been converging to smaller and smaller samples, such that it is now possible to detect single molecules. This Review focuses on the next generation of analytical tools that combine single-molecule detection with the ability to measure many single molecules simultaneously and/or process larger and more complex samples. Such single-molecule sensors constitute a new type of quantitative analytical tool, as they perform analysis by molecular counting and thus potentially capture the heterogeneity of the sample. This Review outlines the advantages and potential of these new, quantitative single-molecule sensors, the measurement challenges in making single-molecule devices suitable for analysis, the inspiration biology provides for overcoming these challenges, and some of the solutions currently being explored. [ABSTRACT FROM AUTHOR]

Immobilisation of enzymes onto surfaces with defined orientation is of particular importance in order to optimise their activity and capacity to dock electron-transfer partners. Local characterisation methods are thus crucial to understanding the activity-orientation relationship of immobilised enzymes for long-term storage and applications. Herein, cytochrome c (cyt c) is chemoselectively immobilised through a cysteine functional group with a maleimide-functionalised gold surface, thus controlling cyt c orientation. X-ray photoelectron spectroscopy confirmed successful cyt c immobilisation. Scanning electrochemical microscopy methodologies were developed to quantify the activity of immobilised cyt c and cytochrome c peroxidase (C cP). The results suggest optimal enzymatic activities and docking ability for surface-tethered cyt c are obtained through the cysteine-maleimide linkage, in comparison to conventional indiscriminate lysine-carboxyl linkages. This study reveals the strong influence of cyt c orientation, as a result of linking strategy, upon the docking ability of redox-electron partner C cP. [ABSTRACT FROM AUTHOR]

The adoption of plasmonic nanomaterials in optical sensors, coupled with the advances in detection techniques, has opened the way for biosensing with single plasmonic particles. Single nanoparticle sensors offer the potential to analyse biochemical interactions at a single-molecule level, thereby allowing us to capture even more information than ensemble measurements. We introduce the concepts behind single nanoparticle sensing and how the localised surface plasmon resonances of these nanoparticles are dependent upon their materials, shape and size. Then we outline the different synthetic approaches, like citrate reduction, seed-mediated and seedless growth, that enable the synthesis of gold and silver nanospheres, nanorods, nanostars, nanoprisms and other nanostructures with tunable sizes. Further, we go into the aspects related to purification and functionalisation of nanoparticles, prior to the fabrication of sensing surfaces. Finally, the recent developments in single nanoparticle detection, spectroscopy and sensing applications are discussed. [ABSTRACT FROM AUTHOR]

Biofouling on surfaces can deleteriously affect the function of electrochemical sensor used in a biological fluid. The demand is thus to develop a sensor operatable in complex matrices. Herein, we concisely review the technologies developed to date to minimize fouling of electrochemical interfaces by blood-based fluids with an emphasis on affinity biosensing. The methods toward this sensor fabrication that are discussed here include approaches where; chemically modified surfaces with antifouling layers such as ethylene oxides, zwitterionics and some alternative antifouling agents; physical forces such as fluidic systems; electrode surface topography, prevent biofouling, as well as recent approaches where biofouling does not interfere with the response (aptasensors). Furthermore, the important emerging field of implantable biosensors, originated from such fabrication procedures is discussed. [ABSTRACT FROM AUTHOR]

Porous silicon photonics is the ideal platform for high sensitivity, high selectivity monitoring of biological molecules in a complex fluidic environment. The potential of this technology was identified almost 15 years ago, however, it has taken considerable advances in porous silicon surface chemistry, photonics, and micro-fabrication to create truly effective devices that can provide new insights into the behaviour of biological systems. In this review we provide a critical assessment of the development of porous silicon optical biosensors from the early demonstrations of affinity based sensing to the current trends in monitoring single cell activity and perspectives in the use of photonic microparticles for biomedical applications. [ABSTRACT FROM AUTHOR]

Abstract: A label-free immunosensor based on the modulation of the electrochemistry of a surface bound redox species, to detect the presence of antibodies, is demonstrated. In this proof of concept study the model epitope was biotin and the model antibody was anti-biotin IgG. Glassy carbon (GC) electrode surfaces were first modified with 4-nitrophenyl groups by electrochemical reductive adsorption of the corresponding aryl diazonium salt. Subsequently, the nitro group was reductively converted into an amine, giving 4-aminophenyl groups. Oxidatively shortened single walled carbon nanotubes (SWNTs) were then covalently attached to the electrode via self-assembly; a procedure that has previously been shown to give SWNTs aligned normal to the surface. 1,1-Di(aminomethyl)ferrocene was attached to the carboxylic acid terminated SWNTs followed by attachment of biotin to the remaining free amine of the ferrocene derivative. Binding of anti-biotin IgG to the surface bound epitope resulted in attenuation of the ferrocene electrochemistry. This label-free immunosensor was successfully able to detect anti-biotin between 30 and 450ngmL−1. [Copyright &y& Elsevier]

The ease of fabricating high quality photonic crystals from porous silicon and its biocompatibility have inspired the conception of various biosensing schemes using this material. However, the instability of porous silicon has significantly slowed progress in this area. Here we discuss the potential of different porous silicon photonic crystals for biosensing in the context of its surface chemistry and nanostructure, both of which need to be optimized to obtain sensitive and stable devices. Of particular promise are recent approaches that use porous silicon as sensors for enzymatic activity, for cell capture and concentration devices. [ABSTRACT FROM AUTHOR]

The charge-transfer properties of DNA duplexes are exploited to produce a fast, simple, sensitive, and selective DNA biosensor by exposing the DNA recognition interface to a sample containing target DNA and the redox-active intercalator, anthraquinonemonosulfonic acid (AQMS). Electrochemistry from electron transfer through the DNA to AQMS intercalated into DNA duplexes can be differentiated from electrochemistry due to direct access of the AQMS to the electrode surface due to the difference in the environment of the AQMS giving a shift in the potential at which the molecule is reduced. The ability to distinguish between the two electrochemical signals enables DNA hybridization to be monitored in real time. This in situ detection scheme has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing either C-A or G-A singlebase mismatches. The concentration detection limit of the biosensor is 0.5 nM (1 pmol) with an assay time of 1 h. The fact that the end user is only required to simultaneously add the sample containing the target DNA and AQMS gives a DNA biosensor that is highly compatible with PCR on chip technologies. [ABSTRACT FROM AUTHOR]

Abstract: This review paper outlines the important issues with regards to the development of biosensors for the monitoring of biological warfare agents (BWAs) starting with the basic components of biosensors and the features of BWAs, which are compatible with detection using biological recognition molecules. The advantages and limitations of biosensors are discussed followed by the current state of the art in biosensors for detecting BWAs. Finally the developments required to enable biosensors to become more effective at providing early warning of possible biological attack and advances towards these developments are covered. [Copyright &y& Elsevier]

Abstract: Carbon nanotubes have begun to attract enormous interest in electrochemistry because of their small size and good electrochemical properties. The vast majority of studies thus far have used ensembles of carbon nanotubes to nanostructure macroscopic electrodes either with randomly dispersed nanotubes or with aligned carbon nanotubes. The resultant nanotube modified electrodes have most frequently been used for electro-analytical purposes such as the development of biosensors. This review introduces carbon nanotubes and approaches to nanostructuring electrodes with carbon nanotubes, discusses what is known of their electrochemical properties and briefly outlines some of the exciting applications to which they are being targeted for use. [Copyright &y& Elsevier]

Presents an alternative design for an amperometric biosensor for lactate. Examination of descriptions of biosensors; Description of the geometry for this biosensor; Information on experiments undertaken in this research.

Two different interfaces prepared via UV-hydrosilylation of undecylenic acid and 1,8-nonadiyne on silicon(111) have been explored to develop a robust electrochemical DNA sensor. Electrodes modified with undecylenic acid were found to stably immobilise DNA but could not resist the growth of insulating oxides, whereas 1,8-nonadiyne modified electrodes satisfy both requirements. [ABSTRACT FROM AUTHOR]

Review: [71 refs. [ABSTRACT FROM AUTHOR]

Integrating loop-mediated isothermal amplification (LAMP) with capacitively coupled contactless conductivity detection (C4D), we have developed an electrical sensor for the simultaneous amplification and detection of specific sequence DNA. Using the O26-wzy gene as a model, the amount of initial target gene could be determined via the threshold time obtained by monitoring the progression of the LAMP reaction in real time. Using the optimal conditions, a detection limit of 12.5 copy/μL can be obtained within 30 min. Monitoring the LAMP reaction by C4D has not only all the advantages that existing electrochemical methods have, but also additional attractive features including being completely free of carryover contamination risk, high simplicity and extremely low cost. These benefits all arise from the fact that the electrodes are separated from the reaction solution, that is C4D is a contactless method. Hence in proof of principle, the new strategy promises a robust, simple, cost-effective and sensitive method for quantitative determination of a target gene, that is applicable either to specialized labs or at point-of-care. [ABSTRACT FROM AUTHOR]

Abstract: A simple method is described to determine sulfite in beer samples using a fill and flow channel biosensor. A droplet of sample is placed into the inlet of a rectangular flow cell and begins to flow through the channel by capillarity. The flow is maintained and controlled by a porous outlet plug of defined porosity. In a rectangular flow cell, the sample solution flows through three consecutive zones: over a predictor electrode, an enzyme layer and a detector electrode. Together these three zones enable the differentiation between current due to sulfite and current due to other electroactive species in the sample. The predictor electrode is located upstream, and on the opposite channel wall to the enzyme layer and detector electrode, and is poised at the same potential (+0.65V versus Ag/AgCl) as the detector electrode. On this electrode, the current contribution from all species in the sample solution that are oxidized at that potential is determined. The enzyme layer contains sulfite oxidase, which, in the process of oxidizing sulfite, produces hydrogen peroxide, which itself is reduced by excess sulfite. The current at the downstream detector electrode is therefore different from that at the predictor electrode as a result of the enzyme reaction and the difference of the currents, corrected for the dimensions of the electrodes, is proportional to the concentration of sulfite. The method enables a straightforward correction of the interfering current at the detector electrode and a determination of the analyte concentration. The effect of interferences from ascorbic acid, ethanol, sorbic acid and tartaric acid in the detection of sulfite is efficiently removed. The concentration of sulfite in a sample of beer measured by the biosensor is equivalent to that measured using a reference method based on the AOAC-recommended Monier–Williams method. [Copyright &y& Elsevier]

Abstract: An electrochemical sensor for the detection of lead ions is described which is made by modifying a gold electrode substrate with self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) or thioctic acid (TA) followed by covalent attachment of a lead binding peptide, human angiotensin I. Cyclic voltammetry of MPA–angiotensin modified gold electrodes complexed with lead displayed voltammograms with prominent lead peaks at E 0′, −0.29V. A detection limit of 1nM was achieved using Osteryoung square wave voltammetry. However, the electrodes were not stable over repeated electrochemical cycles due to partial electrochemical desorption of the SAM. The TA–angiotensin modified gold electrode showed greater stability and were able to be regenerated several times. Using Osteryoung square wave voltammetry for TA–angiotensin modified electrodes, lead concentrations down to 1.9nM were detected. Although the detection limit of the TA–angiotensin modified electrode is higher than achieved with MPA–angiotensin, it is still well below Australian drinking water guidelines. Studies of interference effects on the Pb2+ current showed Hg2+ as a significant interferent but only at levels significantly greater than those found in natural waters. [Copyright &y& Elsevier]

Abstract: An electrochemical sensor for cadmium ions is described which is made by modifying a gold electrode with 3-mercaptopropionic acid followed by covalently coupling the cadmium-selective hexapeptide His–Ser–Gln–Lys–Val–Phe. The resultant electrode had significantly superior analytical performance compared with a glutathione (GSH) modified electrode for the detection of Cd2+, having a greater sensitivity, broader detectable concentration range and a five times lower detection limit of 0.9 nM. Interference from Pb2+ was observed which is an improvement from the GSH modified electrode which had Pb2+ and Cu2+ as interfering ions. The effects of Cu2+, Ni2+, Zn2+, Cr3+ and Ba2+were insignificant. [Copyright &y& Elsevier]

An oxygen-rich fill-and-flow channel biosensor has been developed for the measurement of glucose in wine. Glucose oxidase (GOD), immobilised in carbon paste (CP), was located in a well adjacent to a downstream detector electrode. When the analyte solution flows, hydrogen peroxide produced in the enzyme reaction is swept down to the detector electrode. Mineral oil and Kel-F oil (poly(chlorotrifluorethylene)) were used to prepare an enzyme layer of GOD within a CP. The hydrophobicity of the CP confined the reaction between the enzyme and its substrate to the surface of the enzyme layer. The oxidation current of hydrogen peroxide was sensitive to the enzyme loading but insensitive to mass transport variations such as flow rate. This response was, therefore, limited by the kinetics of the reaction between the enzyme and the substrate. For Kel-F oil, which can support a high concentration of dissolved oxygen, good reproducibility and greater dynamic range was obtained and the response did not decrease after degassing for 40 min with argon. Analysis of wine samples showed good agreement with the values obtained by spectrophotometric enzyme assay. [Copyright &y& Elsevier]

Describes a generic fill-and-flow channel biosensor with upstream electrodes to determine the extent of interferences in the sample. Development of a model that predicts the concentrations of all species; Application of the concept to a biosensor for the determination of glucose; Presence of glucose in wine samples containing polyphenolic interfering agents.

Affinity biosensors play a crucial role in clinical diagnosis, pharmaceuticals, immunology, and other areas of human health. Affinity biosensors rely on the specific binding between target analytes and biological ligands such as antibodies, nucleic acids, aptamers, or other receptors to primarily generate electrochemical or optical signals. Considerable effort has been put into improving the performance of the affinity technologies to make them more sensitive, efficient and reproducible, of the many approaches electrokinetic phenomena are a viable option. In this perspective, studies that combine electrokinetic phenomena with affinity biosensor are discussed about their promise for achieving higher sensitivity and lower detection limit. [ABSTRACT FROM AUTHOR]

The integration of plasmonic nanoparticles into biosensors has the potential to increase the sensitivity and dynamic range of detection, through the use of single nanoparticle assays. The analysis of the localized surface plasmon resonance (LSPR) of plasmonic nanoparticles has allowed the limit of detection of biosensors to move towards single molecules. However, due to complex equipment or slow analysis times, these technologies have not been implemented for point-of-care detection. Herein, we demonstrate an advancement in LSPR analysis by presenting a technique, which utilizes an inexpensive CMOS-equipped digital camera and a dark-field microscope, that can analyse the λ max of over several thousand gold nanospheres in less than a second, without the use of a spectrometer. This improves the throughput of single particle spectral analysis by enabling more nanoparticles to be probed and in a much shorter time. This technique has been demonstrated through the detection of interleukin-6 through a core-satellite binding assay. We anticipate that this technique will aid in the development of high-throughput, multiplexed and point-of-care single nanoparticle biosensors. [ABSTRACT FROM AUTHOR]

Electrochemical DNA biosensors composed of a redox marker modified nucleic acid probe tethered to a solid electrode is a common experimental construct for detecting DNA and RNA targets, proteins, inorganic ions, and even small molecules. This class of biosensors generally relies on the binding-induced conformational changes in the distance of the redox marker relative to the electrode surface such that the charge transfer is altered. The conventional design is to attach the redox species to the distal end of a surface-bound nucleic acid strand. Here we show the impact of the position of the redox marker, whether on the distal or proximal end of the DNA monolayer, on the DNA interface electrochemistry. Somewhat unexpectedly, greater currents were obtained when the redox molecules were located on the distal end of the surface-bound DNA monolayer, notionally furthest away from the electrode, compared with currents when the redox species were located on the proximal end, close to the electrode. Our results suggest that a limitation in ion accessibility is the reason why smaller currents were obtained for the redox markers located at the bottom of the DNA monolayer. This understanding shows that to allow the quantification of the amount of redox labeled target DNA strand that hybridizes to probe DNA immobilized on the electrode surface, the redox species must be on the distal end of the surface-bound duplex. [ABSTRACT FROM AUTHOR]

Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis. [ABSTRACT FROM AUTHOR]

Electrodes modified with organic layers and nanoparticles are promising tools for analytical devices. A major challenge however is how to make electrodes that resist the nonspecific protein adsorption from biological media? Herein, we report gold electrode–organic layer-gold nanoparticle constructs as a platform for electrochemical biosensing. This study focuses on the fabrication of antifouling electrodes with the organic layers formed by two different approaches; self-assembled monolayer (SAM) procedure and electrochemical reduction of aryl diazonium salts, followed by the attachment of gold nanoparticle (AuNP) to allow the electrochemistry to proceed. The results show that the electrodes modified with ethylene oxide-based SAM enable a better antifouling behaviour with less adsorption of human serum albumin (HSA) as compared to the electrodes modified with ethylene oxide-based aryl diazonium-derived layer. Furthermore, findings suggest such distinction could be due to the surface packing and/or conformation of organic film on Au electrodes. [ABSTRACT FROM AUTHOR]

Controlling the composition of an interface is very important in tuning the chemical and physical properties of a surface in many applications including biosensors, biomaterials, and chemical catalysis. Frequently, this requires one molecular component to a minor component in a mixed layer. Such subtle control of composition has been difficult to achieve using aryldiazonium salts. Herein, aryldiazonium salts of carboxyphenyl (CP) and phenylphosphorylcholine (PPC), generated in situ from their corresponding anilines, are electrografted to form molecular platform that are available for further functionalization. These two components are chosen because CP provides a convenient functionality for further coupling of biorecognition species while PPC offers resistance to nonspecific adsorption of proteins to the surface. Mixed layers of CP and PPC were prepared by grafting them either simultaneously or consecutively. The latter strategy allows an interface to be developed in a controlled way where one component is at levels of less than 1% of the total layer. [ABSTRACT FROM AUTHOR]

We investigate the analytical performance of label free porous silicon Bloch surface wave (BSW) devices as protease sensors. Protease detection is based on the digestion of gelatin, which was covalently immobilized onto the surface modified PSi structure. Exposure to the protease enzyme, subtilisin initiates catalytic degradation of the gelatin gel network. The degradation of this network which is present in the top layer of the sensor, causes a blue shift in the spectral position of the Bloch surface mode. The magnitude of the resonance shift is directly proportional to the concentration of subtilisin and the digestion time. The lowest concentration of subtilisin detected was 370 pM. Secondary spectral features, such as band-edge modes, are relatively insensitive to refractive index changes in the superficial layers and thus can be utilised as an internal reference to exclude any nonspecific adsorption and bonding that may occur through the bulk of the film. The advantages of the system here include fast diffusion of digested product and self-referencing capability. [ABSTRACT FROM AUTHOR]

The ability to impart discrete surface chemistry to theinsideand outside of mesoporous silicon is of great importance for a rangeof biomedical applications, from selective (bio)sensing to tissue-specificdrug delivery. Here we present a generic strategy toward achievingdepth-resolved functionalization of the external and internal poroussurfaces by a simple change in the wavelength of the light being usedto promote surface chemical reactions. UV-assisted hydrosilylation,limited by the penetration depth of UV light, is used to decoratethe outside of the mesoporous structure with carboxylic acid molecules,and white light illumination triggers the attachment of dialkyne moleculesto the inner porous matrix. [ABSTRACT FROM AUTHOR]

Abstract: This paper studies the analytical performance and characterization of a peptide modified biosensor towards detection of Cu2+ ions. The peptide modified biosensor is developed by carbodiimide attachment of the Gly-Gly-His tripeptide onto a 3-mercaptopropionic acid (MPA) modified gold surface. X-ray photoelectron spectroscopy (XPS) was used to characterize the steps in the biosensor fabrication. Detection of Cu2+ ions are determined by cyclic and Osteryoung square wave voltammetry. The analytical performance of this biosensor is found to be affected by the pH, salt concentration, temperature and accumulation time of the test solution. Interference studies and investigations of the reusability of this biosensor have also been performed. [Copyright &y& Elsevier]

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described. [Copyright &y& Elsevier]