Characterization of Quantum Dot Bioconjugates for the Development of Fret Based Biosensor Formats

Arjun, Rastogi (2009) Characterization of Quantum Dot Bioconjugates for the Development of Fret Based Biosensor Formats. [Student Project Report]

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Abstract

This Dissertation / Report is the outcome of investigation carried out by the creator(s) / author(s) at the department/division of Central Food Technological Research Institute (CFTRI), Mysore mentioned below in this page.

Item Type:	Student Project Report
Additional Information:	Quantum Dots are luminescent semiconductor nanocrystals whose unique photophysical properties are helping to create a new generation of robust fluorescent biosensors. QD properties of interest for biosensing include high quantum yields, broad absorption spectra coupled to narrow sizetunable photoluminescent emissions and exceptional resistance to both photobleaching and chemical degradation. The greatest potential for these novel particles for biosensor applications lies in their use for FRET based assays. Interpreting the biosensor response through FRET gives the freedom to conduct biosensing in a solution based, immobilization-free, separation-free, reagent-free mode. The development of such a QD based biosensing format would entail the attachment of QD to bioactive material. The conjugation of Quantum dots with biomolecules creates hybrid particles possessing unique photophysical properties along with biological activity. Being novel constructs, such conjugates need to be fully characterized to understand the full range of their modified properties before they can be utilized for biosensor applications. The present study is aimed at characterizing the QD bioconjugates in terms of their optical and biological properties and using these conjugates for the development of FRET based biosensor formats. The project was conducted in two parts. Firstly, QD of different sizes were conjugated to different proteins by the EDC-NHS protocol to synthesize soluble, stable and aggregate-free hybrids. The effect of bioconjugation on the photophysical properties of QD was probed through absorbance spectroscopy, fluorescence spectroscopy and 5 gel electrophoresis. The photophysical properties of QD protein conjugates underwent remarkable change from the properties of the individual particles. A new absorption peak was observed for the hybrid pointing towards an effective coupling of the electronic orbitals of the constituents. This finding also gave a means to quantify the conjugation between QD and proteins. The attachment of proteins to QD was observed to enhance the fluorescence of QD by almost 200 %. Conjugation also resulted in a decrease in the effective stokes shift for Forster Resonance Energy Transfer. Experiments performed to probe the structural stability and bioactivity of the biomolecules showed notable retention of these parameters. The thus synthesized and characterized bioconjugates were used for the development of a FRET based biosensor format. Antibodies were raised against a proof of concept analyte, 2,4-D. QD pair capable of participating in FRET were conjugated to the antibody and antigen. A competitive immunoassay was set up using the labeled antibody and the labeled antigen in competition with the analyte. The specific binding of the QD bound antibody with the QD bound gave a response in the form of FRET which was used to quantify the analyte concentration. The FRET based format so developed had a large dynamic range and a low limit of quantification and a higher sensitivity as compared with conventional methods. This format can thus be used for the development of a new generation of robust biosensors.
Uncontrolled Keywords:	Quantum Dot; Biosensor; Forster Resonance Energy Transfer; Bioconjugates; Immunosensor
Subjects:	600 Technology > 05 Chemical engineering
Divisions:	Fermentation Technology and Bioengineering
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	01 Dec 2009 06:51
Last Modified:	28 Dec 2011 10:12
URI:	http://ir.cftri.res.in/id/eprint/9292

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