Abstract
We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.
Highlights
In recent years, various types of biosensors have been increasingly becoming practical and useful tools in a wide variety of analytical devices [1,2]
Compared with the wild-type E. coli BL21(DE3) strain as a negative control, E. coli BL21(DE3) harboring pET-6histidine-fused GBP (6HGBP)-ScFv expresses a thick band between marker bands of size 27.5 kDa and 37 kDa
To prove that Gold-binding polypeptide (GBP)-fusion proteins could be functionally immobilized on the gold surface, the 6HGBP-ScFv fusion protein was flown over the gold sensor chip to monitor its binding affinity by combining the GBP-fusion approach with SPR
Summary
Various types of biosensors have been increasingly becoming practical and useful tools in a wide variety of analytical devices [1,2]. The resulting receptor layer seems to form heterogeneous and random orientation Another method can be constructed by crosslinking functional reagents by a certain number of functional groups due to its simple procedure and strong chemical bond of proteins. This method has disadvantages as follows: the difficulty in controlling the crosslinking reaction, the gelatinous nature of the proteins and the relatively low activity of the proteins due to the specific structural features [3,4] In each of these methods used for the favorable performance of biosensors, a number of factors deserve careful consideration for strong binding forces between the solid surface and recognition elements, exposure of active sites on the target sample, conservation of biological activity after immobilization process, and simple protocol [3,4]. Electrical signal-based detection methods for HBV such as electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were developed on the gold electrode surface, which has been a very versatile material in the field of biosensors
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