1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Development of a nano biosensor for detection of bract mosaic virus in banana (Musa spp.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Saurav Saha; Abida, P SBanana 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.Item Development of a nanobiosensor for detection of banana bunchy top virus(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Vinusri, S; Shylaja, M RThe investigations on ‘Development of a nanobiosensor for detection of Banana bunchy top virus’ were conducted at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur and Centre for Materials for Electronics Technology(C-MET), Thrissur during August, 2014 to June, 2016. The objective of the study was to develop a nanobiosensor for easy and quick detection of Banana bunchy top virus (BBTV). Biosensors based on Localised Surface Plasmon Resonance (LSPR) have gained much attention now-a-days. Gold nanoparticles are ideal for optical sensing due to their better light absorption and scattering properties, chemical stability, high surface to volume ratio and high surface energy to provide stable immobilization of large amount of biomolecules. In the present study, a solution phase LSPR biosensor using gold nanorods (GNRs) was developed for detection of BBTV. Gold nanorods were synthesized using seed mediated growth method. The concentration of silver nitrate and volume of seed solution required to synthesize stable GNRs were optimized in the present study. The characterization of GNRs was done using UV-Vis spectrophotometry and Transmission Electron Microscopy. Two absorption bands were observed in the absorption spectrum of GNRs. The longitudinal plasmon band was observed at 679 nm and the transverse plasmon band was observed at 515 nm. The mean length, width and aspect ratio of the synthesized GNRs recorded using transmission electron microscopy were 35.84 nm, 13.57 nm and 2.64 respectively. Surface modification of CTAB capped GNRs was done by ligand exchange method. Functionalization of GNRs with BBTV specific antibody was undertaken by conjugating the antibody with GNRs to make a GNR probe. Dilution of antibody required for conjugation with GNRs was standardised as 1:100. Development of LSPR based biosensor using GNRs was attempted as chip based and solution phase based. As immobilization of GNRs on glass surface was not successful, studies on chip based biosensor could not be continued and further focus was made on solution phase LSPR biosensor. The BBTV antigen was isolated from the BBTV infected samples of banana collected from Banana Research Station of Kerala Agricultural University. The isolated antigen was allowed to interact with GNR probe and it was found that LPB was more sensitive to the interaction. Different concentrations of antigen were allowed to interact with GNR probe solution to identify the minimum detection limit. The efficacy of solution phase LSPR biosensor in detecting the antigen was further checked with BBTV infected leaf samples of six different banana cultivars viz. Yangambi Km5, Chenkadali, Nendran, Palayamkodan, Grand Naine and Kunnan. The kinetics of interaction of GNR probe with antigen from different banana cultivars and characterization of the interaction with UV-Vis spectrophotometry revealed that the developed biosensor was able to detect very low concentration of antigen (0.02 mg/ml) within a period of five to ten minutes. Colour change was also noticed due to interaction of GNR probe with antigen. In the infected samples, colour change from pinkish red to pale grey was evident while no such colour change was noticed in healthy samples. Due to the colour change, the developed solution phase sensor as such can be used for field level applications. The efficacy of developed solution phase LSPR based GNR biosensor was compared with Enzyme Linked Immuno Sorbent Assay (ELISA) for detecting BBTV antigen. The detection limit of antigen in ELISA was 0.08 mg/ml (80 ppm) while in the developed biosensor the detection limit was 0.02 mg/ml (20 ppm). The solution phase LSPR based GNR biosensor developed in the present study is thus effective for easy and quick detection of BBTV. Conversion of solution phase based GNR biosensor to chip based sensor will open up more applications and will help for easy commercial fabrication. Multiplexing is also possible in chip based sensor by controlling the aspect ratios of GNRs and functionalising with different antibodies specific to different pathogens so that the sensor can detect multiple pathogens using a single chip. The technology generated from the present investigations for the development of LSPR based GNR biosensor could be applied in other crops/ other pathogens with modifications