Enzyme-linked amperometric electrochemical genosensor assay for the detection of PCR amplicons on a streptavidin-treated screen-printed carbon electrode.
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.
Yean
Chan Yean
CY
Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Malaysia.
Kamarudin
Balqis
B
Ozkan
Dilsat Ariksoysal
DA
Yin
Lee Su
LS
Lalitha
Pattabhiraman
P
Ismail
Asma
A
Ozsoz
Mehmet
M
Ravichandran
Manickam
M
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
D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D001710:Biotin; D002244:Carbon; D019809:Streptavidin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D019793:Fluorescein