Development of impedimetric immunosensors for the detection of a range of antigens of biological or biomedical significance

This PhD project was funded by the European Union Framework 6 ELISHA programme as part of the ELISHA programme. The aim of the research was focused towards the development of point-of-care, simple, cost-efficient and reliable impedimetric immunosensors for the rapid detection of important antigens such as ciprofloxacin and digoxin. Through the cooperation of the 9 involved partners a number of protocols have been developed for sensor fabrication and sample testing that allow both rapid and reliable detection of a range of antigens. This work describes in depth, the use of polymers and cyclic voltammetry for electrode surface modification, the use of the avidin-biotin system for antibody immobilisation and finally the use of Electrochemical Impedance Spectroscopy for antigen detection. We report here the successful development of electrochemical impedimetric carbon based immunosensors for the detection of free-form and chelated ciprofloxacin (both in laboratory buffer and milk), digoxin and green fluorescent protein. It was observed during this work that unavailability of sufficient quantities of the monoclonal antibodies could lead to early sensor saturation and hence a less extended antigen detection range, while very low quantities of immobilised antibodies could also give rise to erroneous results. The immunosensors towards ciprofloxacin detected the respective antigen when this was present between 1 ng ml-1 and 10 μg ml-1 in PBS buffer and 0.1 ng ml-1 to 10 μg ml-1 when the antigen was added to milk samples. The developed digoxin immunosensors could detect digoxin between the concentrations of 0.1 ng ml-1 and 10 μg ml-1. Lower detection limits where observed for the sensors targeting GFP which could detect their respective antigen at concentrations as low as 100 pg ml-1. The tested concentration range of the latter sensors was extended up to 100 ng ml-1

Development of impedimetric immunosensors for the detection of a range of antigens of biological or biomedical significance

This PhD project was funded by the European Union Framework 6 ELISHA programme as part of the ELISHA programme. The aim of the research was focused towards the development of point-of-care, simple, cost-efficient and reliable impedimetric immunosensors for the rapid detection of important antigens such as ciprofloxacin and digoxin. Through the cooperation of the 9 involved partners a number of protocols have been developed for sensor fabrication and sample testing that allow both rapid and reliable detection of a range of antigens. This work describes in depth, the use of polymers and cyclic voltammetry for electrode surface modification, the use of the avidin-biotin system for antibody immobilisation and finally the use of Electrochemical Impedance Spectroscopy for antigen detection. We report here the successful development of electrochemical impedimetric carbon based immunosensors for the detection of free-form and chelated ciprofloxacin (both in laboratory buffer and milk), digoxin and green fluorescent protein. It was observed during this work that unavailability of sufficient quantities of the monoclonal antibodies could lead to early sensor saturation and hence a less extended antigen detection range, while very low quantities of immobilised antibodies could also give rise to erroneous results. The immunosensors towards ciprofloxacin detected the respective antigen when this was present between 1 ng ml-1 and 10 μg ml-1 in PBS buffer and 0.1 ng ml-1 to 10 μg ml-1 when the antigen was added to milk samples. The developed digoxin immunosensors could detect digoxin between the concentrations of 0.1 ng ml-1 and 10 μg ml-1. Lower detection limits where observed for the sensors targeting GFP which could detect their respective antigen at concentrations as low as 100 pg ml-1. The tested concentration range of the latter sensors was extended up to 100 ng ml-1.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Labeless Immunosensor Assay for Fluoroquinolone Antibiotics Based Upon an AC Impedance Protocol.

This paper describes the construction of a labeless immunosensor for the antibiotic ciprofloxacin and its interrogation using an AC impedance protocol. Commercial screen-printed carbon electrodes were used as the basis for the sensor. Polyaniline was electrodeposited onto the sensors and then utilized to immobilize a biotinylated antibody for ciprofloxacin using classical avidin- biotin interactions. Electrodes containing the antibodies were exposed to solutions of antigen and interrogated using an AC impedance protocol. The faradaic component of the impedance of the electrodes was found to increase with increasing concentration of antigen. Control samples containing a non-specific IgG antibody were also studied and calibration curves obtained by subtraction of the responses for specific and non-specific antibody-based sensors, thereby eliminating the effects of non-specific adsorption of antigen

Labeless AC impedimetric antibody-based sensors with pg ml-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 μm, containing entrapped anti- BS