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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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Related in: MedlinePlus

Electrochemical detection of HBsAg. (a) EIS characterization and (b) CV analysis of the gold electrode fabrication process for the sequential binding of the samples. Dotted line, bare gold; Frame diamond, after 50 μg/mL 6HGBP-ScFv binding; Solid circle, after 0.5 mg/mL BSA binding; Solid line, after 50 μg/mL HBsAg binding.
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f3-sensors-12-10097: Electrochemical detection of HBsAg. (a) EIS characterization and (b) CV analysis of the gold electrode fabrication process for the sequential binding of the samples. Dotted line, bare gold; Frame diamond, after 50 μg/mL 6HGBP-ScFv binding; Solid circle, after 0.5 mg/mL BSA binding; Solid line, after 50 μg/mL HBsAg binding.

Mentions: For the characterization of the electrode fabrication process, EIS and CV were performed each time after binding of each reagent. As shown in Figure 3(a), the dotted line is the impedance spectrum obtained on the bare gold electrode. Frame circle line is an impedance spectrum of 50 μg/mL 6HGBP-ScFv fusion protein modified electrode, and solid circle line is that of after BSA blocking. Finally, solid line represents an impedance spectrum obtained after HBsAg binding. The result reveals the resistance of the electron transfer at the electrode surface increasing step by step because of the insulating effect of the binding proteins. This electron transfer resistance at the interface on the electrode surface, and solution can be determined by the diameter of the semi-circle of the curves in EIS spectra. After the gold electrodes were immobilized with GBP-ScFv, the peak current decreased dramatically with an increase of the peak-to-peak potential separation (ΔEp) for the bare gold electrode. When HBsAg was bound on the gold chip surface, the peak current more decreased and ΔEp (approximately 110 mV) increased comparing with those of the gold electrode and the GBP-ScFv immobilized chip, resulting from the electron transfer resistance of bound HBsAg molecules. BSA was used as a negative control. It can also be observed that the current responses in CV spectra (Figure 3(b)) are decreasing in the process of electrode fabrication, which coincides with a conclusion drawn from impedance assay. These results were due to the insulating characteristics of the protein.


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)

Electrochemical detection of HBsAg. (a) EIS characterization and (b) CV analysis of the gold electrode fabrication process for the sequential binding of the samples. Dotted line, bare gold; Frame diamond, after 50 μg/mL 6HGBP-ScFv binding; Solid circle, after 0.5 mg/mL BSA binding; Solid line, after 50 μg/mL HBsAg binding.
© Copyright Policy
Related In: Results  -  Collection

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

f3-sensors-12-10097: Electrochemical detection of HBsAg. (a) EIS characterization and (b) CV analysis of the gold electrode fabrication process for the sequential binding of the samples. Dotted line, bare gold; Frame diamond, after 50 μg/mL 6HGBP-ScFv binding; Solid circle, after 0.5 mg/mL BSA binding; Solid line, after 50 μg/mL HBsAg binding.
Mentions: For the characterization of the electrode fabrication process, EIS and CV were performed each time after binding of each reagent. As shown in Figure 3(a), the dotted line is the impedance spectrum obtained on the bare gold electrode. Frame circle line is an impedance spectrum of 50 μg/mL 6HGBP-ScFv fusion protein modified electrode, and solid circle line is that of after BSA blocking. Finally, solid line represents an impedance spectrum obtained after HBsAg binding. The result reveals the resistance of the electron transfer at the electrode surface increasing step by step because of the insulating effect of the binding proteins. This electron transfer resistance at the interface on the electrode surface, and solution can be determined by the diameter of the semi-circle of the curves in EIS spectra. After the gold electrodes were immobilized with GBP-ScFv, the peak current decreased dramatically with an increase of the peak-to-peak potential separation (ΔEp) for the bare gold electrode. When HBsAg was bound on the gold chip surface, the peak current more decreased and ΔEp (approximately 110 mV) increased comparing with those of the gold electrode and the GBP-ScFv immobilized chip, resulting from the electron transfer resistance of bound HBsAg molecules. BSA was used as a negative control. It can also be observed that the current responses in CV spectra (Figure 3(b)) are decreasing in the process of electrode fabrication, which coincides with a conclusion drawn from impedance assay. These results were due to the insulating characteristics of the protein.

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