Enhancement of electrode performance by a simple casting method using sonochemically exfoliated graphene.

We demonstrate within this paper a method for modifying commercial screen-printed electrodes with aqueous graphene suspensions to enhance their electrochemical activity. The graphene suspensions are synthesized by a simple ultrasonic exfoliation method from graphite, where reaggregation is prevented by the addition of common cationic or anionic surfactants, thereby avoiding the use of organic solvents or harsh chemical procedures. These suspensions can then be simply cast onto the screen-printed electrodes. Cyclic voltammetry with a number of redox active species such as phenols, as well as impedance measurements, were made to characterize these systems. The modified electrodes are shown to demonstrate significantly enhanced electrochemical activity and greatly lowered electron transfer resistances compared to the unmodified electrodes. Initial proof of concept applications of these electrodes, including the detection of heavy metals by absorptive stripping voltammetry, are also shown.

Disposable screen-printed sensors for the electrochemical detection of TNT and DNT.

Due to the heightened level of national security currently prevalent due to the possibility of terrorist incidents, highly portable, miniaturised and sensitive monitoring devices for trace levels of injurious materials, such as explosives are now of the upmost importance. One method that offers a possible route for the development of a detection system for such species is via an electrochemical regime, coupled to the use of disposable sensor technology. Within this study, the use of carbon screen-printed sensors for the detection and analysis of the classical explosive trinitrotoluene (TNT) and the related dinitrotoluene (DNT) is described, with the eventual objective to develop an inexpensive, accurate and sensitive detection system for trace quantities of explosives in field settings. Commercially available screen-printed carbon sensors have been used as the base platform for this investigation and the electrochemistry of both TNT and DNT studied at these surfaces. Two reductive peaks and one oxidative peak were observed for both analytes. The best linear fits and sensitivities were obtained using the reductive peak at -0.72 V vs. Ag/AgCl. A linear range from 1 to 200 µM could be obtained for TNT and DNT in pH 7.0 phosphate buffer with limits of detection as low as 0.4 µM (TNT) and 0.7 µM (DNT). A second system which utilised the addition of the enzyme, nitroreductase, and the coenzyme, NADPH, into the solution matrix prior to electrochemical interrogations with screen-printed carbon electrodes was found to increase the resulting signal magnitude at the oxidation peak at +0.3 V, improving the performance of the sensor at these values.

Construction and interrogation of enzyme microarrays using scanning electrochemical microscopy - optimisation of adsorption and determination of enzymatic activity.

Scanning electrochemical microscopy (SECM) has been used to image and study the catalytic activity of horseradish peroxidase (HRP) immobilised in a patterned fashion onto glass slides. Microarrays of HRP islands could be deposited on amino-modified glass slides using glutaraldehyde crosslinking combined with the SECM being used as a micro-deposition device. The enzymatic activity of the immobilised enzyme on the surface was in the presence of its substrate observed to give rise to substantial positive feedback between the slide and the SECM microelectrode tip. Conversely when either blank slides - or slides coated with HRP which had been subsequently thermally denatured were utilised, these showed negative feedback effects. Various conditions such as enzyme concentration, incubation time and substrate concentration were systematically varied to optimise sensitivity. Regular arrays of HRP could be assembled and when imaged, displayed lower limits of detection of 1.2 × 10(-12) mol ml(-1) of benzoquinone.

Sonochemically fabricated microelectrode arrays for use as sensing platforms.

The development, manufacture, modification and subsequent utilisation of sonochemically-formed microelectrode arrays is described for a range of applications. Initial fabrication of the sensing platform utilises ultrasonic ablation of electrochemically insulating polymers deposited upon conductive carbon substrates, forming an array of up to 70,000 microelectrode pores cm(-2). Electrochemical and optical analyses using these arrays, their enhanced signal response and stir-independence area are all discussed. The growth of conducting polymeric "mushroom" protrusion arrays with entrapped biological entities, thereby forming biosensors is detailed. The simplicity and inexpensiveness of this approach, lending itself ideally to mass fabrication coupled with unrivalled sensitivity and stir independence makes commercial viability of this process a reality. Application of microelectrode arrays as functional components within sensors include devices for detection of chlorine, glucose, ethanol and pesticides. Immunosensors based on microelectrode arrays are described within this monograph for antigens associated with prostate cancer and transient ischemic attacks (strokes).

Sonochemically fabricated microelectrode arrays for biosensors. Part III. AC impedimetric study of aerobic and anaerobic response of alcohol oxidase within polyaniline.

A sonochemically fabricated alcohol oxidase enzyme micro-electrode array is reported. Sensors of this type were fabricated by first depositing an insulating polydiaminobenzene film on supporting gold electrodes. Sonication and subsequent ablation exposed discrete areas of the underlying conducting electrode, which collectively act as a microelectrode array. Electropolymerisation of aniline has been used to generate in situ polyaniline containing entrapped alcohol oxidase. The physical and electrochemical properties of these films were studied and reported within this paper. The final composites were shown to behave with microelectrode performance characteristics for the detection of aqueous ethanol concentrations.

Sonochemically fabricated microelectrode arrays for biosensors offering widespread applicability: Part I.

A novel and patented procedure is described for the sonochemical fabrication of a new class of microelectrode array based sensor with electrode element populations of up to 2 x 10(5) cm(-2). For some years it has been accepted that microelectrode arrays offer an attractive route for lowering minimum limits of detection and imparting stir (convectional mass transport) independence to sensor responses; despite this no commercial biosensors, to date, have employed microelectrode arrays, largely due to the cost of conventional fabrication routes that have not proved commercially viable for disposable devices. Biosensors formed by our sonochemical approach offer unrivalled sensitivity and impart stir independence to sensor responses. This format lends itself for mass fabrication due to the simplicity and inexpensiveness of the approach; in the first instance impedimetric and amperometric sensors are reported for glucose as model systems. Sensors already developed for ethanol, oxalate and a number of pesticide determinations will be reported in subsequent publications.

Label-free impedimetric immunosensors for psoriasin--increased reproducibility and sensitivity using an automated dispensing system.

We describe within this paper the construction of a label-free immunosensor for the protein psoriasin (S100A7), which is associated with a number of clinical conditions such as skin diseases or cancer. Antibodies to psoriasin were immobilised onto screen-printed carbon electrodes that had been pre-modified with the conductive polymer polyaniline. We compared and contrasted a number of different methods of assembly to optimise the construction and properties of the immunosensor. Immunosensors were fabricated using both manual liquid handling (pipette) and an automated liquid dispensing platform, the BioDot AD3200(TM). Two immobilisation methods were also utilised; simple electrostatic binding of the antibody to polyaniline as well as a more complex procedure using a biotin-neutravidin bridge. The optimum results in terms of sensitivity and reproducibility were obtained utilising the automated system and the biotin-avidin assembly procedure. The resultant immunosensors could be interrogated using AC impedance without the need for any labelling and demonstrated quantification of psoriasin from 250 pg ml(-1) to 10 ng ml(-1)-a concentration range suitable for determining physiological levels of psoriasin.

Detection and imaging the expression of the trans-membrane protein CD44 in RT112 cells by use of enzyme-labeled antibodies and SECM.

The deposition of human RT112 cells in a patterned fashion onto glass substrates and subsequent imaging of the expression of the trans-membrane protein CD44 have been studied using scanning electrochemical microscopy (SECM). Patterns of RT112 cells derived from a transitional cell carcinoma of the bladder could be deposited on amino-modified glass substrates by cytospinning. These were then treated with horseradish peroxidase (HRP) labeled secondary antibodies to the trans-membrane protein CD44. Expression of CD44 protein by the cells directly leads to immobilisation of the labeled antibodies. The presence of the enzyme substrate (hydrogen peroxide) along with a hydroquinone mediator then allowed an enzymatic reaction to proceed, generating benzoquinone. Reduction of benzoquinone gave rise to positive feedback between the substrate and the SECM microelectrode tip. Control samples such as blank slides or slides not treated with HRP-labeled antibody showed negative feedback effects. Patterns of RT112 cells could be assembled and their expression of the target protein imaged whereas control samples showed minimal activity.

Template and catalytic effects of DNA in the construction of polypyrrole/DNA composite macro and microelectrodes.

Electrochemical DNA hybridization-based sensors show great promise as portable and automated analytical devices for routine screening of pathogenic or foreign nucleic acid sequences in biological samples. However, current sensor technologies still exhibit some unresolved issues which hampers their direct application into everyday life. Conducting polymers, such as polypyrrole (PPy), are increasingly being adopted as suitable platforms for DNA probe immobilization and signal transduction. Immobilization of DNA probes during pyrrole electropolymerization is a simple and efficient strategy to build composite electrodes suitable for DNA sensing. However, the effects of the probe state and sequence on PPy growth kinetics have not been studied yet. Here, we show that growth of PPy is drastically affected by the presence of guanine in the DNA probes and whether DNA is present in its single-stranded or double-stranded form. We show that some immobilization protocols may provoke irreversible oxidation of guanine moieties in the probe and that this issue deserves careful investigation as it may interfere with hybridization processes. We have also explored new procedures to build microelectrode arrays bearing immobilized DNA molecules, which are known to show beneficial properties in stirred samples. Overall, we present new techniques and concerns regarding the development of DNA-containing PPy-based composite electrodes, which may be taken into consideration for increasing genosensor reproducibility, response and performance.

A new application of scanning electrochemical microscopy for the label-free interrogation of antibody-antigen interactions: Part 2.

Within this paper we describe the use of scanning electrochemical microscopy (SECM) to fabricate a dotted array of biotinylated polyethyleneimine which was then used to immobilise first neutravidin and then a biotinylated antibody towards a relevant antigen of interest (PSA, NTx, ciprofloxacin). These antigens were selected both for their clinical relevance but also since they display a broad range of molecular weights, to determine whether the size of the antigen used effects the sensitivity of this approach. The SECM was then used to image the binding of both complementary and non-complementary antigens in a label-free assay. Imaging of the arrays before and following exposure to various concentrations of antigen in buffer showed clear evidence for specific binding of the complementary antigens to the antibody functionalised dots. Non-specific binding was also quantified by control experiments with other antigens. This demonstrated non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen of interest at the surface of the dots. The binding of ciprofloxacin was investigated both in simple buffer solution and in a more complex media, bovine milk.

A new application of scanning electrochemical microscopy for the label-free interrogation of antibody-antigen interactions.

Within this work we present a 'proof of principle' study for the use of scanning electrochemical microscopy (SECM) to detect and image biomolecular interactions in a label-free assay as a potential alternative to current fluorescence techniques. Screen-printed carbon electrodes were used as the substrate for the deposition of a dotted array, where the dots consist of biotinylated polyethyleneimine. These were then further derivatised, first with neutravidin and then with a biotinylated antibody to the protein neuron specific enolase (NSE). SECM using a ferrocene carboxylic acid mediator showed clear differences between the array and the surrounding unmodified carbon. Imaging of the arrays before and following exposure to various concentrations of the antigen showed clear evidence for specific binding of the NSE antigen to the antibody derivatised dots. Non-specific binding was quantified. Control experiments with other proteins showed only non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen at the surface of the dots. Binding of the antigen was accompanied by a measured increase in current response, which may be explained in terms of protein electrostatic interaction and hydrophobic interactions to the mediator, thereby increasing the localised mediator flux. A calibration curve was obtained between 500 fg mL(-1) to 200 pg mL(-1) NSE which demonstrated a logarithmic relationship between the current change upon binding and antigen concentration without the need for any labelling of the substrate.

Labeless AC impedimetric antibody-based sensors with pgml(-1) sensitivities for point-of-care biomedical applications.

This paper describes the development and characterisation of labeless immunosensors for (a) the cardiac drug digoxin and (b) bovine serum albumin (BSA). Commercial screen-printed carbon electrodes were used as the basis for the sensors. Two methods were used to immobilise antibodies at the electrode surface. Aniline was electropolymerised onto these electrodes to form a thin planar film of conductive polyaniline; the polyaniline film was then utilised as a substrate to immobilise biotinylated anti-digoxin using a classical avidin-biotin affinity approach. As an alternative approach, poly(1,2-diaminobenzene) was electrodeposited onto the carbon electrodes and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerisation step was then used to co-deposit conductive polyaniline along with antibodies for BSA within these pores to produce a microarray of polyaniline protrusions with diameters of several mum, containing entrapped anti-BSA. The resulting antibody grafted electrodes were interrogated using an AC impedance protocol before and following exposure to digoxin or BSA solutions, along with control samples containing a non-specific IgG antibody. The impedance characteristics of both types of electrode were changed by increasing concentrations of antigen up to a saturation level. Calibration curves were obtained by subtraction of the non-specific response from the specific response, thereby eliminating the effects of non-specific adsorption of antigen. Both the use of microelectrode arrays and affinity binding protocols showed large enhancements in sensitivity over planar electrodes containing entrapped antibodies and gave similar sensitivities to our other published work using affinity-based planar electrodes. Detection limits were in the order of 0.1ngml(-1) for digoxin and 1.5ngml(-1) for BSA.