Enzyme catalysis is broadly used in various fields but generally applied in media with high ion strength. Here, we propose the exploitation of enzymatic catalysis in ultra-low ion strength media to induce ion strength increase for developing a novel impedance biosensing method. Avian influenza virus H5N1, a serious worldwide threat to poultry and human health, was adopted as the analyte. Magnetic beads were modified with H5N1-specific aptamer to capture the H5N1 virus. This was followed by binding concanavalin A (ConA), glucose oxidase (GOx), and Au nanoparticles (AuNPs) to create bionanocomposites through a ConA-glycan interaction. The yielded sandwich complex was transferred to a glucose solution to trigger an enzymatic reaction to produce gluconic acid, which ionized to increase the ion strength of the solution, thus decreasing the impedance on a screen-printed interdigitated array electrode. This method took advantages of the high efficiency of enzymatic catalysis and the high susceptibility of electrochemical impedance on the ion strength and endowed the biosensor with high sensitivity and a detection limit of 8 × 10(-4) HAU in 200 μL sample, which was magnitudes lower than that of some analogues based on biosensing methods. Furthermore, the proposed method required only a bare electrode for measurements of ion strength change and had negligible change on the surficial properties of the electrode, though some modification of magnetic beads/Au nanoparticles and the construction of a sandwich complex were still needed. This helped to avoid the drawbacks of commonly used electrode immobilization methods. The merit for this method makes it highly useful and promising for applications. The proposed method may create new possibilities in the broad and well-developed enzymatic catalysis fields and find applications in developing sensitive, rapid, low-cost, and easy-to-operate biosensing and biocatalysis devices.
In this study, a cost-effective nanowell structure was fabricated and utilized for the development of a nanowell-based quartz crystal microbalance (QCM) aptasensor for rapid, sensitive, and label-free detection of H5N1 avian influenza virus (AIV). A nanoporous gold film with a thickness of 120 nm and a pore size of ~20 nm was prepared using a metallic corrosion method. Then, the nanoporous gold film was immobilized onto a gold electrode surface using a self-assembled monolayer to form a nanowell-based electrode. A specific H5N1 AIV ssDNA aptamer with a NH 2 conjugated 5'-terminal was used in the fabrication of the QCM aptasensor through covalent bonding. The stepwise assembly of the aptasensor was characterized by means of QCM. The result showed that the binding of target AIV H5N1 onto the immobilized aptamers decreased the sensor's resonant frequency, and the frequency change correlated to the virus titer. We demonstrated that the developed nanowell-based QCM aptasensor could dramatically reduce detection time down to 10 min using a label-free assay. The detection range of 2-4 to 2 4 hemagglutination units (HAUs) /50 μl was obtained with a detection limit of 2-4 HAU/50 μl for AIV H5N1. The binding of target H5N1 virus onto the nanowell-based electrode surface was further confirmed by scanning electron microscopy (SEM). No interference was observed from non-target AIV subtypes of H1N1, H2N2, H7N2 and H5N3. The aptasensor using H5N1 aptamer was validated for the detection of AIV H5N1 in chicken tracheal swab samples. The developed aptasensor could be adopted for detection of other viruses.
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