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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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(a) EIS detection of different concentrations of HBsAg. 10 μg/mL, 1 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg were detected with 10 μg/mL BSA as a negative control, respectively. Numerical data was fitted with circuit as shown in Figure 3(a) (Inset: an equivalent circuit representing each component at the interface and in the solution during an electrochemical reaction is shown for comparison with the physical components. Cd, double layer capacitor; Rp, polarization resistor; W, Warburg resistor; Rs, solution resistor). Error bars represent standard deviations from 5-time measurements. (b) Linear calibration curve into log scale of EIS data (R2 = 0.97).
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f4-sensors-12-10097: (a) EIS detection of different concentrations of HBsAg. 10 μg/mL, 1 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg were detected with 10 μg/mL BSA as a negative control, respectively. Numerical data was fitted with circuit as shown in Figure 3(a) (Inset: an equivalent circuit representing each component at the interface and in the solution during an electrochemical reaction is shown for comparison with the physical components. Cd, double layer capacitor; Rp, polarization resistor; W, Warburg resistor; Rs, solution resistor). Error bars represent standard deviations from 5-time measurements. (b) Linear calibration curve into log scale of EIS data (R2 = 0.97).

Mentions: As shown in Figure 4, 50 μg/mL, 10 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg were detected by using this method, respectively, and they presented a rough trend of increasing electron transfer resistance with increasing target concentrations. As a negative control, BSA of 10 μg/mL was detected at the same time. Numerical data were drawn by fitting with a circuit model as shown in an inset of Figure 4. Correspondingly, binding of 50 μg/mL, 10 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg and 10 μg/mL BSA caused electron transfer resistance increase of 8,493 Ω, 6,473 Ω, 4,047 Ω, 3,097 Ω, 2,143 Ω, 1,513 Ω and 777 Ω, respectively. Though linearity is not very strict as a function of HBsAg concentration, it demonstrated a rough linear trend into log scale. Target concentrations as low as 0.14 ng/mL of HBsAg calculated via 3-sigma rule was successfully detected compared with the negative control of 10 μg/mL of BSA (Figure 4). Furthermore, a lower limit of detection (LOD) can be further expected if experimental conditions, such as 6HGBP-ScFv concentration, binding time, temperature and washing condition, were optimized. In order to check the nonspecific binding of real blood sample, we tested a fetal bovine serum of 10 μg/mL instead of HBsAg for clinical trials [27,28] and confirmed no effect.


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)

(a) EIS detection of different concentrations of HBsAg. 10 μg/mL, 1 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg were detected with 10 μg/mL BSA as a negative control, respectively. Numerical data was fitted with circuit as shown in Figure 3(a) (Inset: an equivalent circuit representing each component at the interface and in the solution during an electrochemical reaction is shown for comparison with the physical components. Cd, double layer capacitor; Rp, polarization resistor; W, Warburg resistor; Rs, solution resistor). Error bars represent standard deviations from 5-time measurements. (b) Linear calibration curve into log scale of EIS data (R2 = 0.97).
© Copyright Policy
Related In: Results  -  Collection

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

f4-sensors-12-10097: (a) EIS detection of different concentrations of HBsAg. 10 μg/mL, 1 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg were detected with 10 μg/mL BSA as a negative control, respectively. Numerical data was fitted with circuit as shown in Figure 3(a) (Inset: an equivalent circuit representing each component at the interface and in the solution during an electrochemical reaction is shown for comparison with the physical components. Cd, double layer capacitor; Rp, polarization resistor; W, Warburg resistor; Rs, solution resistor). Error bars represent standard deviations from 5-time measurements. (b) Linear calibration curve into log scale of EIS data (R2 = 0.97).
Mentions: As shown in Figure 4, 50 μg/mL, 10 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg were detected by using this method, respectively, and they presented a rough trend of increasing electron transfer resistance with increasing target concentrations. As a negative control, BSA of 10 μg/mL was detected at the same time. Numerical data were drawn by fitting with a circuit model as shown in an inset of Figure 4. Correspondingly, binding of 50 μg/mL, 10 μg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg and 10 μg/mL BSA caused electron transfer resistance increase of 8,493 Ω, 6,473 Ω, 4,047 Ω, 3,097 Ω, 2,143 Ω, 1,513 Ω and 777 Ω, respectively. Though linearity is not very strict as a function of HBsAg concentration, it demonstrated a rough linear trend into log scale. Target concentrations as low as 0.14 ng/mL of HBsAg calculated via 3-sigma rule was successfully detected compared with the negative control of 10 μg/mL of BSA (Figure 4). Furthermore, a lower limit of detection (LOD) can be further expected if experimental conditions, such as 6HGBP-ScFv concentration, binding time, temperature and washing condition, were optimized. In order to check the nonspecific binding of real blood sample, we tested a fetal bovine serum of 10 μg/mL instead of HBsAg for clinical trials [27,28] and confirmed no effect.

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