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

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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.

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Transmission Electron Microscope images of GO (A) and MB surface confined GO (B).
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f5: Transmission Electron Microscope images of GO (A) and MB surface confined GO (B).

Mentions: To further characterize the immobilisation of methylene blue on graphene oxide, comparative FTIR spectroscopy was carried out with GO@MB, GO and MB as in Fig. 4C. A vibration signal due to C = C (1732 cm−1, 1728 cm−1, 1737 cm−1) and > C = O (1569 cm−1 and 1602 cm−1) were qualitatively noticed with all the samples (No > C = O signal with MB); but with a shift in the wavenumbers. In addition, specific vibrational band at 3412 cm−1 corresponds to the N-H stretching of MB was also noticed with GO@MB (3431 cm−1) ascribing MB immobilization and its GO interaction features. The morphological structure of GO and GO@MB was investigated using TEM Fig. 5(A and B). A transparent sheet like structure can be seen clearly with the GO as that of previously reported literature22. For MB modified GO case, large number of black spots of average size ~10 nm on the GO sheets was observed. This observation can be taken as a proof for the immobilization of MB on GO.


An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform
Transmission Electron Microscope images of GO (A) and MB surface confined GO (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Transmission Electron Microscope images of GO (A) and MB surface confined GO (B).
Mentions: To further characterize the immobilisation of methylene blue on graphene oxide, comparative FTIR spectroscopy was carried out with GO@MB, GO and MB as in Fig. 4C. A vibration signal due to C = C (1732 cm−1, 1728 cm−1, 1737 cm−1) and > C = O (1569 cm−1 and 1602 cm−1) were qualitatively noticed with all the samples (No > C = O signal with MB); but with a shift in the wavenumbers. In addition, specific vibrational band at 3412 cm−1 corresponds to the N-H stretching of MB was also noticed with GO@MB (3431 cm−1) ascribing MB immobilization and its GO interaction features. The morphological structure of GO and GO@MB was investigated using TEM Fig. 5(A and B). A transparent sheet like structure can be seen clearly with the GO as that of previously reported literature22. For MB modified GO case, large number of black spots of average size ~10 nm on the GO sheets was observed. This observation can be taken as a proof for the immobilization of MB on GO.

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.


Related in: MedlinePlus