RESUMEN
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.
Asunto(s)
Electroquímica/instrumentación , Grafito/química , Ondas Ultrasónicas , Impedancia Eléctrica , Electrodos , Oxidación-Reducción , Tensoactivos/químicaRESUMEN
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.
Asunto(s)
Técnicas Biosensibles/instrumentación , Inmunoensayo/instrumentación , Proteínas S100/análisis , Adsorción , Compuestos de Anilina/química , Anticuerpos Inmovilizados/química , Avidina/química , Biotina/química , Carbono/química , Impedancia Eléctrica , Electrodos , Humanos , Proteína A7 de Unión a Calcio de la Familia S100 , Sensibilidad y Especificidad , Electricidad EstáticaRESUMEN
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.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas de Química Analítica/instrumentación , Dinitrobencenos/análisis , Equipos Desechables , Impresión , Trinitrotolueno/análisis , Técnicas Biosensibles/economía , Calibración , Técnicas de Química Analítica/economía , Dinitrobencenos/química , Electroquímica , Escherichia coli/enzimología , Nitrorreductasas/metabolismo , Oxígeno/química , Trinitrotolueno/químicaRESUMEN
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.
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Conductometría/métodos , Peroxidasa de Rábano Silvestre/química , Receptores de Hialuranos/metabolismo , Microscopía Electrónica de Rastreo/métodos , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Línea Celular Tumoral , Humanos , Coloración y Etiquetado/métodosRESUMEN
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.
Asunto(s)
Bioensayo/instrumentación , Conductometría/instrumentación , ADN/análisis , ADN/genética , Microelectrodos , Impresión Molecular/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Polímeros/química , Pirroles/química , Catálisis , ADN/química , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Anticuerpos Inmovilizados/inmunología , Antígenos/análisis , Antígenos/inmunología , Técnicas Biosensibles/instrumentación , Electroquímica/instrumentación , Inmunoensayo/instrumentación , Microscopía/instrumentación , Animales , Anticuerpos Inmovilizados/química , Biotinilación , Calibración , Bovinos , Ciprofloxacina/análisis , Ciprofloxacina/inmunología , Colágeno Tipo I/análisis , Colágeno Tipo I/inmunología , Péptidos/análisis , Péptidos/inmunología , Polietileneimina/química , Antígeno Prostático Específico/análisis , Antígeno Prostático Específico/inmunologíaRESUMEN
The number of Adenovirus (Ad) infections detected in immunocompromised people has increased due to the number of patients receiving transplants, as well as the HIV pandemic. Ads cause life-threatening diseases specific to the infected organs of immunocompromised hosts, with discontinuation of immunosuppressive agents necessary to prevent morbidity. The methodology in this paper has been employed to develop a novel impedimetric based assay platform to detect and quantify human Ads, which is comparable in performance to current methods, such as ELISA and PCR, but is also less expensive and faster. Novel immunosensors have been fabricated using polyclonal antibodies raised against a human Ad (Ad5) capsid protein, which were selectively cleaved into antibody fragments by 2-mercaptoethylamine. The fragments were immobilized onto a functionalized conducting copolymer matrix comprising polyaniline and 2-aminobenzylamine. Fully fabricated sensors were incubated with two immunologically distinct serotypes of Ad, Ad5 and Ad3, with between 10 and 10(12)virus particles/mL prior to sensor interrogation. Electrochemical impedance spectroscopy was used to measure the charge transfer resistance of the sensors over a range of frequencies from 25 kHz to 0.1 Hz. Our data demonstrate that the immunosensors specifically detect, and differentiate between, closely related human Ad serotypes with a limit of detection of 10(3)virus particles/mL. In addition, atomic force microscopy was applied to study the sensor surface nanostructure. Future work looks to test virus containing clinical samples but this could be a viable and valuable alternative for point-of-care virus detection and quantification.
Asunto(s)
Infecciones por Adenoviridae/diagnóstico , Adenoviridae/inmunología , Adenoviridae/aislamiento & purificación , Anticuerpos Inmovilizados/inmunología , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica/métodos , Infecciones por Adenoviridae/inmunología , Compuestos de Anilina/química , Anticuerpos Inmovilizados/química , Bencilaminas/química , Proteínas de la Cápside/inmunología , Humanos , Inmunoensayo/métodos , Límite de Detección , Propiedades de SuperficieRESUMEN
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.