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Studies on Bioconjugation of CdTequantum dot for monitoring Enterotoxin B producing Staphylococcus aureus.

Vinayaka, A. C. (2012) Studies on Bioconjugation of CdTequantum dot for monitoring Enterotoxin B producing Staphylococcus aureus. PhD thesis, University of Mysore.

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Abstract

Rapid detection of foodborne pathogens and their toxins is one of the thrust areas of research. Keeping potentials of nanoparticles for biosensing in mind, quantum dot (QD) based biosensor system as an integrated technique was attempted considering „staphylococcal enterotoxin B‟ (SEB) as a model bio-toxin. CdTe QDs having 2.2 to 3.06 nm size and band gap in the range 2.74 to 2.26 eV respectively with 33% quantum yield were synthesized by aqueous phase. Bioconjugation of these QDs with proteins resulted in unique photophysical properties demonstrating an effective coupling of electronic orbitals of constituents. A proportionate quenching in protein fluorescence followed by an enhancement of QD fluorescence point towards Förster Resonance Energy Transfer (FRET) based nonradiative dipolar interactions. Further, possible applications of CdTe QDs were studied for developing biosensing techniques based on immuno-affinity reactions. Anti-SEB antibodies were generated in avian system with highest yield of 51.1 mg/egg having affinity constant of 1.81 x 1010 M-1. In the first approach of FRET based two QD fluorescence, the mutual affinity between CdTe523 bioconjugated anti- SEB antibody and CdTe601 bioconjugated SEB were used to obtain efficient energy transfer between respective QDs. Presence of SEB in the range 1000 ng to 50 ng varied the rate of fluorescence quenching of CdTe523 with the limit of detection at 33.44 ng/mL. In the second approach, CdTe523 and GNP bioconjugated to SEB and anti-SEB IgY respectively were used as donoracceptor pair during FRET in an immunocomplex. Here SEB was detected in the range 1000 ng to 20 ng with the limit of detection at 8.53 ng/mL. Finally, a competitive fluoroimmunoassay developed in an immunoreactor column by ii immobilizing anti-SEB antibodies made it possible to quantify SEB from 1000 ng to 10 ng with a limit of detection at 8.15 ng/mL. Standard deviation (SD) and coefficient of variation percentage (CV %) was satisfactory for inter- and intra-assay precision in immunoreactor based competitive fluoroimmunoassay for spiked SEB samples in milk. The CV calculated for both intra- and interassay were 3.4% and 6.18% respectively. Thus, integration of QD with immuno-affinity reactions revealed versatility of nanoparticles demonstrating a new approach for monitoring “Enterotoxin B” producing Staphylococcus aureus at sensitive level.

Item Type: Thesis (PhD)
Uncontrolled Keywords: foodborne pathogens, quantum dot (QD) based biosensor system, staphylococcal enterotoxin B
Subjects: 600 Technology > 01 Medical sciences > 07 Food borne disease
600 Technology > 05 Chemical engineering > 01 Biotechnology and Bioengineering
Divisions: Fermentation Technology and Bioengineering
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 04 Mar 2014 05:00
Last Modified: 04 Mar 2014 05:00
URI: http://ir.cftri.com/id/eprint/11359

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