The development of a 'labeless' immunosensor for the detection of Listeria monocytogenes cell surface protein, Internalin B.
Electrochemical impedance spectroscopy (EIS) is a widely used technique for probing bioaffinity interactions at the surfaces of electrically conducting polymers. EIS methods can be employed to investigate 'labeless' detection of analytes via impedimetric transduction. This paper describes the development of a direct immunosensor for the detection of a cell-surface protein on Listeria monocytogenes, an extremely important food-borne pathogen. L. monocytogenes are facultative anaerobic, non-sporing, Gram-positive, motile rods that employ the surface bound protein, Internalin B (InlB), to promote invasion into host cells. A recombinant form of InlB was previously cloned and expressed in Escherichia coli and a panel of antibodies and antibody fragments directed against the protein were also produced. Here, we describe how a portion of the recombinant InlB protein, the F3 fragment, and an anti-InlB polyclonal antibody, were used to develop a platform for the labeless immunosensing of InlB. Sensors were fabricated by electropolymerisation of planar screen-printed carbon electrodes with polyaniline (PANI), to produce a conductive substrate. Polyclonal anti-InlB antibody was subsequently incorporated onto the PANI layer using a biotin-avidin system for site-specific immobilisation. The sensors were then probed with varying concentrations of InlB antigen and the impedimetric response at each concentration was recorded. An anti-IgG antibody was immobilised at the electrode surface, as a control and subsequently exposed to the same concentrations of InlB. Impedimetric data for the control sensors were also recorded. Upon exposure to a range of concentrations of antigen, complex plane impedance analyses were used to relate the differing redox states of the polymer layer, to the possible charge transfer at the surface, with respect to the related mechanisms between the antibody and the polymer. These effects were subsequently monitored to assess the impedance of the polymer thereby determining the amount of bound antigen at the sensor surface. Calibration profiles for both sample (InlB) and control (IgG) sensors were constructed. A limit of detection of 4.1 pg/ml was achieved for Internalin B.
Tully
Elizabeth
E
School of Biotechnology & National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland. Elizabeth.tully2@mail.dcu.ie
Higson
Seamus P
SP
O'Kennedy
Richard
R
D000814:Aniline Compounds / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D002138:Calibration; D017097:Electric Impedance; D004563:Electrochemistry; D007118:Immunoassay / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*
D000814:Aniline Compounds; D001426:Bacterial Proteins; D008565:Membrane Proteins; C095768:inlB protein, Listeria monocytogenes; C416807:polyaniline