Development of a nano biosensor for detection of bract mosaic virus in banana (Musa spp.)

Abstract

Banana bract mosaic virus (BBrMV) is a recently described virus of banana which contributed to yield reduction by 5 to 36 per cent and is a barrier to international exchange of germplasm. There is a no effective measure to control this virus, only by routine virus indexing of planting material can protect the spreading of this virus. Currently ELISA and real time PCR is effectively used for diagnosis but the protocol is time consuming and expensive. In the recent years biosensor based on the novel metallic nanoparticle gain much importance for industrial applications and efficient detection of viruses. The study entitled ―Development of a nano biosensor for detection of Banana bract mosaic virus in banana (Musa spp.)‖ was carried out in the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture and Centre for Electronics and Materials(C-MET), Thrissur during the academic year 2014-2016. The objective of this study to develop an antibody based nanobiosensor for quick detection of Banana bract mosaic virus. Goldnanorods (GNRs) were fabricated through seed-mediated procedure and UV-Vis spectra of GNRs solution indicated characteristic longitudinal and transverse band at 710 nm and 520 nm respectively. The transmission image of electron microscope revealed that solution contain rod shaped gold nanoparticles with length and diameter (42±3) nm (14±1.9) nm respectively. The aspect ratio of GNRs was measured through ImageJ software and found that aspect ratio of GNRs was 3.03. The effect of silver nitrate solution on the growth of GNRs was studied and found that with increasing silver ion concentration in a growth solution longitudinal peak shift was observed from 710-740 nm and also aspect ratio of GNRs also increased from 3.03 to 3.75. In order to detection of analyte (BBrMV) surface of a GNRs activated with complete replacement with alkalithiol molecule for covalent attachment of an antibody. UV-Vis spectra of activated GNRs indicated that due to formation of SAM (Self assembly monolayer)position of a peak shifted from 710- 716 and also due to binding of an antibody to SAM layer again peak position was changed from 716-727nm. SDS-PAGE and Nanodrop spectrophotometer analysis were carried out for the BBrMV antigen to check the quality and quantity of protein (antigen). The results had shown 38KDa band coat protein specific band of virus in a gel and concentration of antigen was 3mg/ml. Bio-recognition induced gold nanorods aggregation here takes as an analytical tool for detection of a BBrMV. In this case due to addition of antigen to antibody labeled GNRs solution. Colour of the solution changed red to black and notable peak shift of (7- 25) nm was observed both in transverse and longitudinal peak of GNRs in UV-Vis Spectra. Antigen concentration up to 0.25 mg/ml and above shows stability in the Peak shift and colour change in infected sample compared to control sample. In healthy sample no colour changes were observed and with only minimum peak shift was there. The positive result was also obtained in a micro titre plate where ELISA reader clearly differentiated healthy and infected samples with different concentrations of antigen. In case of longitudinal peak shift, the kinetics curve of an infected sample remained relatively flat and after 15min the shift remained stable until the end of the observation and the absorbance of GNRs continuously decreased up to 80th min and after that no changes were observed in the kinetics curves of an absorbance. For determining the accuracy and sensitiveness of a nanobiosensor, results of the different serological techniques (ELISA, DIBA) were compared with the result of the fabricated solution based nanobiosensor and found that nanobiosensor could detect the viral protein at a very low concentration (2-0.02) mg/ml, whereas in the case of other techniques the detection was possible up to 0.12 mg/ml of antigen concentration. The developed gold nano rods based nanobiosensor was evaluated for detection of a BBrMV in five varieties of banana and it was found that cv. Nendran was more affected by BBrMV compared to other varieties of banana due to the high concentration of viral load in the infected samples. The solution based gold nano rod based biosensor is sensitive, cost effective and easy for virus indexing of tissue culture plants and planting materials compared to other methods currently in use. Further investigations and refinement could lead to the fabrication and development of nanobiosensor on a commercial scale.