title: Novel impedimetric immunosensor for the detection and quantitation of Adenovirus using reduced antibody fragments immobilized onto a conducting copolymer surface.
abstract: 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.
authors.affiliation: Biosensors and Biocatalysis Laboratory, Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire, LS2 9JT, UK.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Caygill
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Hodges
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Holmes
authors.forename: Séamus P J
authors.initials: SP
authors.lastname: Higson
authors.forename: G Eric
authors.initials: GE
authors.lastname: Blair
authors.forename: Paul A
authors.initials: PA
authors.lastname: Millner
mesh_terms: D000256:Adenoviridae / Q000276:immunology* / Q000302:isolation & purification*; D000257:Adenoviridae Infections / Q000175:diagnosis* / Q000276:immunology; D000814:Aniline Compounds / Q000737:chemistry; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology*; D001596:Benzylamines / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D036022:Capsid Proteins / Q000276:immunology; D058266:Dielectric Spectroscopy / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D057230:Limit of Detection; D013499:Surface Properties
chemical_list: D000814:Aniline Compounds; D055910:Antibodies, Immobilized; D001596:Benzylamines; D036022:Capsid Proteins; C416807:polyaniline