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An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform

View Article: PubMed Central - PubMed

ABSTRACT

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E1/2), −0.4 V vs Ag/AgCl and mediated H2O2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10−3 to 1.36 × 107 copies μL−1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

No MeSH data available.


CV responses of GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP, prepared with different dilution of antigen (10−1 to 10−10), in 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.
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f6: CV responses of GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP, prepared with different dilution of antigen (10−1 to 10−10), in 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.

Mentions: Next, calibration plot for electrochemical immunosensing of WSSV was constructed by subjecting different concentration of the standard virus (vp28 protein) discreetly on the modified electrode as GCE/GO@MB-Ab1-BSA-Ag-Ab2 and subjected to CV experiment with fixed H2O2 concentration (500 μM) in pH 7 PBS. Figure 6 is the typical calibration response for the virus obtained from different dilutions (10−1 to 10−10) of the stock WSSV, 1.37 × 107 copies μL−1. The sensor showed regular variation in the H2O2 reduction current with respect to dilution. Note that the detection range (1.37 × 10−3–1.37 × 107 copies μL−1) and low-detection concentration (1.37 × 10−3 copies μL−1) obtained in this work are much better than the previous reported procedures like label-free affinity immunosensors (1.6 × 103–1.6 × 106 copies μL−1 and 1.6 × 101–1.6 × 106 copies μL−1) and colorimetric ELISA (1.6 × 103–1.6 × 107 copies μL−1)111230.


An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform
CV responses of GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP, prepared with different dilution of antigen (10−1 to 10−10), in 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385493&req=5

f6: CV responses of GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP, prepared with different dilution of antigen (10−1 to 10−10), in 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.
Mentions: Next, calibration plot for electrochemical immunosensing of WSSV was constructed by subjecting different concentration of the standard virus (vp28 protein) discreetly on the modified electrode as GCE/GO@MB-Ab1-BSA-Ag-Ab2 and subjected to CV experiment with fixed H2O2 concentration (500 μM) in pH 7 PBS. Figure 6 is the typical calibration response for the virus obtained from different dilutions (10−1 to 10−10) of the stock WSSV, 1.37 × 107 copies μL−1. The sensor showed regular variation in the H2O2 reduction current with respect to dilution. Note that the detection range (1.37 × 10−3–1.37 × 107 copies μL−1) and low-detection concentration (1.37 × 10−3 copies μL−1) obtained in this work are much better than the previous reported procedures like label-free affinity immunosensors (1.6 × 103–1.6 × 106 copies μL−1 and 1.6 × 101–1.6 × 106 copies μL−1) and colorimetric ELISA (1.6 × 103–1.6 × 107 copies μL−1)111230.

View Article: PubMed Central - PubMed

ABSTRACT

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E1/2), −0.4 V vs Ag/AgCl and mediated H2O2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10−3 to 1.36 × 107 copies μL−1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

No MeSH data available.