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.

Single bead-based electrochemical biosensor

“…The behavior depicted in Figure 5 is consistent with first order kinetics which predicts the number of bound complexes to be proportional to $R{\left(1+\frac{1}{K_{A}C}\right)}^{-1}$, where R is the number of binding sites (likely reduced due to steric hindrance), K A is the affinity constant, and C is the analyte concentration. Similar experimental results were reported by Yean et al, who developed a streptavidin-treated, screen-printed carbon electrode for the detection of PCR amplicons (Yean et al, 2008). Figure 5 demonstrates that the SA-BMP biosensor can detect the amplicons of 0.001ng (1 pg) DNA template of B. Cereus , which exceeds the detection ability of the conventional gel electrophoresis by a factor of ~10.…”

“…Numerous approaches have been used for biomolecular immobilization such as physical adsorption (Chen and Pardue, 2000), entrapment in a porous matrix (Yu and Ju, 2003), covalent binding (Yabuki et al, 2000), and copolymerization (Liu et al, 2007; Qu et al, 2007). One of the most commonly used strategies for effective immobilization of biomaterials to different substrates is based on the avidin-biotin affinity reaction (LaGier et al, 2007; Lermo et al, 2008; Luppa et al, 2001; Yean et al, 2008; Zacco et al, 2006). …”

DETEKSI URUTAN OLIGONUKLEOTIDA Mycobacterium tuberculosis SECARA VOLTAMMETRI MENGGUNAKAN SCREEN PRINTED CARBON ELECTRODE (SPCE)

“…Berbagai jenis masalah yang dihadapi dalam melakukan diagnosis TB dalam sampel klinis, dua diantaranya adalah penggunaan senyawa kimia berbahaya seperti cahaya UV dan etidium bromida yang bersifat mutagen selama melakukan elektroforesis gel agarosa (pada saat karakterisasi amplikon PCR). Selain itu, penggunaan instrumen yang mahal seperti real time-PCR memerlukan keahlian khusus dalam pengoperasiannya, dan juga membutuhkan reagen kimia yang cukup mahal seperti pewarna hijau SYBR dan Taqman (Yean et al 2008;Hamdan et al 2012).…”

One-step green synthesis of graphene/ZnO nanocomposites for non-enzymatic hydrogen peroxide sensing

“…A planar screen-printed carbon electrode (SPCE) was fabricated (ScrintTechnology, Malaysia) based on the design described by Chan et al 14 2 μL of the as-prepared graphene/ZnO nanocomposite dispersion was dropped onto the surface of the working SPCE and dried at ambient temperature.…”