Limits...
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.

Show MeSH

Related in: MedlinePlus

SPR sensorgrams. (a) Optimization of the concentration of 6HGBP-ScFv fusion protein. (b) SPR detection of target antigen, HBsAg, with 6HGBP-ScFv as a receptor.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3472818&req=5

f2-sensors-12-10097: SPR sensorgrams. (a) Optimization of the concentration of 6HGBP-ScFv fusion protein. (b) SPR detection of target antigen, HBsAg, with 6HGBP-ScFv as a receptor.

Mentions: To confirm the successful binding of a series of molecules, the detection of HBV was first performed using commercial SPR system. At first, 6HGBP-ScFv concentration was optimized as follows: 100, 50 and 10 μg/mL of 6HGBP-ScFv fusion protein was flowed over the gold surface, respectively. The fusion protein 6HGBP-ScFv was immobilized onto the gold surface via its intrinsic affinity with the gold as shown in Figure 2. Interestingly, coverage of 6HGBP-ScFv on the gold surface didn't always increase with increasing protein concentrations, a maximal RU change appearing at concentrations of 50 μg/mL. From this result, 6HGBP-ScFv concentration of 50 μg/mL was chosen for subsequent experiments.


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)

SPR sensorgrams. (a) Optimization of the concentration of 6HGBP-ScFv fusion protein. (b) SPR detection of target antigen, HBsAg, with 6HGBP-ScFv as a receptor.
© Copyright Policy
Related In: Results  -  Collection

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

f2-sensors-12-10097: SPR sensorgrams. (a) Optimization of the concentration of 6HGBP-ScFv fusion protein. (b) SPR detection of target antigen, HBsAg, with 6HGBP-ScFv as a receptor.
Mentions: To confirm the successful binding of a series of molecules, the detection of HBV was first performed using commercial SPR system. At first, 6HGBP-ScFv concentration was optimized as follows: 100, 50 and 10 μg/mL of 6HGBP-ScFv fusion protein was flowed over the gold surface, respectively. The fusion protein 6HGBP-ScFv was immobilized onto the gold surface via its intrinsic affinity with the gold as shown in Figure 2. Interestingly, coverage of 6HGBP-ScFv on the gold surface didn't always increase with increasing protein concentrations, a maximal RU change appearing at concentrations of 50 μg/mL. From this result, 6HGBP-ScFv concentration of 50 μg/mL was chosen for subsequent experiments.

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