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Label-free electrochemical diagnosis of viral antigens with genetically engineered fusion protein.

Heo NS, Zheng S, Yang M, Lee SJ, Lee SY, Kim HJ, Park JY, Lee CS, Park TJ - Sensors (Basel) (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea. hns0924@kaist.ac.kr

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.

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Schematic diagram of bio-recognition onto the gold surface by GBP-fusion proteins and detection of targets (Immobilization of single-chain variable fragment antibody via 6HGBP-ScFv fusion protein for the detection of HBsAg).
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f1-sensors-12-10097: Schematic diagram of bio-recognition onto the gold surface by GBP-fusion proteins and detection of targets (Immobilization of single-chain variable fragment antibody via 6HGBP-ScFv fusion protein for the detection of HBsAg).

Mentions: Moreover, immobilization with correct orientation of biological material is a problem of prime importance in biosensors. We employed GBP-fusion proteins in the construction of biosensor for the detection of hepatitis B viral surface antigen (HBsAg) as a model (Figure 1), which is a biomarker for diagnosing the hepatitis B virus (HBV). The strong affinity between the GBP and the gold surface guarantees the stability of this sensor system and orients the sensing parts outward from the solid surface, exposing them directly to the target sample [13,16]. Furthermore, electrochemical detection has attracted considerable interest recently for miniaturized analytical systems [20,21], including remarkable sensitivity (approaching that of fluorescence), inherent miniaturization of both the detector and control instrumentation, independence of optical path length or sample turbidity, low cost, low-power requirements and high compatibility [22,23]. Besides, one of the most attractive points of this method is its potential for portable assays in a variety of point-of-care testing (POCT) environments. We here developed a simple platform biosensor technology by mediating the recognition parts and the solid surface on the gold substrate. SPR analyses were used for optimization of sample concentrations and verification of target sensing. 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.


Label-free electrochemical diagnosis of viral antigens with genetically engineered fusion protein.

Heo NS, Zheng S, Yang M, Lee SJ, Lee SY, Kim HJ, Park JY, Lee CS, Park TJ - Sensors (Basel) (2012)

Schematic diagram of bio-recognition onto the gold surface by GBP-fusion proteins and detection of targets (Immobilization of single-chain variable fragment antibody via 6HGBP-ScFv fusion protein for the detection of HBsAg).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472818&req=5

f1-sensors-12-10097: Schematic diagram of bio-recognition onto the gold surface by GBP-fusion proteins and detection of targets (Immobilization of single-chain variable fragment antibody via 6HGBP-ScFv fusion protein for the detection of HBsAg).
Mentions: Moreover, immobilization with correct orientation of biological material is a problem of prime importance in biosensors. We employed GBP-fusion proteins in the construction of biosensor for the detection of hepatitis B viral surface antigen (HBsAg) as a model (Figure 1), which is a biomarker for diagnosing the hepatitis B virus (HBV). The strong affinity between the GBP and the gold surface guarantees the stability of this sensor system and orients the sensing parts outward from the solid surface, exposing them directly to the target sample [13,16]. Furthermore, electrochemical detection has attracted considerable interest recently for miniaturized analytical systems [20,21], including remarkable sensitivity (approaching that of fluorescence), inherent miniaturization of both the detector and control instrumentation, independence of optical path length or sample turbidity, low cost, low-power requirements and high compatibility [22,23]. Besides, one of the most attractive points of this method is its potential for portable assays in a variety of point-of-care testing (POCT) environments. We here developed a simple platform biosensor technology by mediating the recognition parts and the solid surface on the gold substrate. SPR analyses were used for optimization of sample concentrations and verification of target sensing. 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea. hns0924@kaist.ac.kr

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.

Show MeSH
Related in: MedlinePlus