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Tosar J.P.,University of the Republic of Uruguay | Holmes J.L.,Cranfield University | Collyer S.D.,Microarray Ltd | Davis F.,Cranfield University | And 2 more authors.
Biosensors and Bioelectronics | Year: 2013

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. © 2012 Elsevier B.V.

Roberts W.S.,Cranfield University | Davis F.,Cranfield University | Holmes J.L.,Cranfield University | Collyer S.D.,Microarray Ltd | And 3 more authors.
Biosensors and Bioelectronics | Year: 2013

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. © 2012 Elsevier B.V.

Holford T.R.J.,Cranfield University | Holmes J.L.,Cranfield University | Collyer S.D.,Microarray Ltd | Davis F.,Cranfield University | Higson S.P.J.,Cranfield University
Biosensors and Bioelectronics | Year: 2013

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 AD3200TM. 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 250pgml-1 to 10ngml-1-a concentration range suitable for determining physiological levels of psoriasin. © 2012 Elsevier B.V.

Collyer S.D.,Microarray Ltd | Davis F.,Cranfield University | Higson S.P.J.,Cranfield University
Sensors | Year: 2010

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{norm of matrix} 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). © 2010 by the authors.

Holmes J.L.,Cranfield University | Davis F.,Cranfield University | Collyer S.D.,Microarray Ltd | Higson S.P.J.,Cranfield University
Analytica Chimica Acta | Year: 2012

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. © 2012 Elsevier B.V.

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