title: Enzyme-linked amperometric electrochemical genosensor assay for the detection of PCR amplicons on a streptavidin-treated screen-printed carbon electrode.
abstract: A general purpose enzyme-based amperometric electrochemical genosensor assay was developed wherein polymerase chain reaction (PCR) amplicons labeled with both biotin and fluorescein were detected with peroxidase-conjugated antifluorescein antibody on a screen-printed carbon electrode (SPCE). As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. Factors affecting immobilization, hybridization, and nonspecific binding were optimized to maximize sensitivity and reduce assay time. On the basis of the background amperometry signals obtained from nonspecific organisms and positive signals obtained from known V. cholerae, a threshold point of 4.20 microA signal was determined as positive. Under the optimum conditions, the limit of detection (LOD) of the assay was 10 CFU/mL of V. cholerae. The overall precision of this assay was good, with the coefficient of variation (CV) being 3.7% using SPCE and intermittent pulse amperometry (IPA) as an electrochemical technique. The assay is sensitive, safe, and cost-effective when compared to conventional agarose gel electrophoresis, real-time PCR, and other enzyme-linked assays for the detection of PCR amplicons. Furthermore, the use of a hand-held portable reader makes it suitable for use in the field.
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Malaysia.
authors.forename: Chan Yean
authors.initials: CY
authors.lastname: Yean
authors.forename: Balqis
authors.initials: B
authors.lastname: Kamarudin
authors.forename: Dilsat Ariksoysal
authors.initials: DA
authors.lastname: Ozkan
authors.forename: Lee Su
authors.initials: LS
authors.lastname: Yin
authors.forename: Pattabhiraman
authors.initials: P
authors.lastname: Lalitha
authors.forename: Asma
authors.initials: A
authors.lastname: Ismail
authors.forename: Mehmet
authors.initials: M
authors.lastname: Ozsoz
authors.forename: Manickam
authors.initials: M
authors.lastname: Ravichandran
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods; D001710:Biotin / Q000737:chemistry; D002244:Carbon / Q000737:chemistry*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004563:Electrochemistry / Q000379:methods*; D004583:Electrons; D019793:Fluorescein / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry*; D014734:Vibrio cholerae / Q000235:genetics
chemical_list: D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D001710:Biotin; D002244:Carbon; D019809:Streptavidin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D019793:Fluorescein