High-sensitivity biophotonic detection method for in-vitro and in-vivo applications
Lead Research Organisation:
CARDIFF UNIVERSITY
Department Name: School of Biosciences
Abstract
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Technical Summary
We aim to create a new biophotonic method for quantitative detection of biomolecules with significantly higher sensitivity than existing techniques. This will be achieved by a novel combination of colloidal semiconductor quantum dots (QDs) and fluorescence lifetime detection. The award will be used to bring a new discipline in the research group in the form of a Research Associate from the physical sciences with strong background in chemistry/material sciences and experience in organic and inorganic synthesis. This individual will enter the environment of the School of Biosciences, where the project will be carried out, and will benefit from the expertise in biophotonics of the Principal Investigator and in molecular cell biology of the co-applicants.
Since they were first reported in 1996, quenched oligonucleotide probes, also called molecular beacons (MBs), have been widely used for quantification of DNA. However, for quantitative detection of low amounts of target molecules, especially in-vivo, there are several limiting factors to the traditional MB approach. In fact, signal-to-background ratio still needs to be improved by at least 10-fold to achieve single target molecule detection. The design of a new type of MB combining time-resolved fluorescence detection with QDs will benefit from the brightness, photostability and long lifetimes of QDs and from the accuracy of the lifetime detection method. This combination has the potential to overcome the major limitations of the current MB approach for highly sensitive detection of biomolecule concentrations both in-vitro and in-vivo.
To quantitatively assess the sensitivity of the proposed method, we will investigate a test system using fluorescently-labeled oligonucleotides in solution. We will design our oligonucleotide probes to a series of Dictyostelium genes cloned in the co-applicants research group. Two non-complementary oligonucleotide probes will be synthesized that hybridize to adjacent sequences on their nucleic acid target. One oligonucleotide will be conjugated to a QD donor, the other to a fluorescent organic dye acceptor. When both oligonucleotides anneal to their target, donor and acceptor will be brought into close proximity leading to fluorescence resonant energy transfer. For the time-resolved detection, the expertise in photonics of the PI will be used to construct a simple, compact, and cost-effective home-made apparatus. The properties of the QD-acceptor pair are key to the success of this project. This award should support a full-time postdoctoral Research Associate with expertise in the photophysical and photochemical characteristics of colloidal QDs.
Since they were first reported in 1996, quenched oligonucleotide probes, also called molecular beacons (MBs), have been widely used for quantification of DNA. However, for quantitative detection of low amounts of target molecules, especially in-vivo, there are several limiting factors to the traditional MB approach. In fact, signal-to-background ratio still needs to be improved by at least 10-fold to achieve single target molecule detection. The design of a new type of MB combining time-resolved fluorescence detection with QDs will benefit from the brightness, photostability and long lifetimes of QDs and from the accuracy of the lifetime detection method. This combination has the potential to overcome the major limitations of the current MB approach for highly sensitive detection of biomolecule concentrations both in-vitro and in-vivo.
To quantitatively assess the sensitivity of the proposed method, we will investigate a test system using fluorescently-labeled oligonucleotides in solution. We will design our oligonucleotide probes to a series of Dictyostelium genes cloned in the co-applicants research group. Two non-complementary oligonucleotide probes will be synthesized that hybridize to adjacent sequences on their nucleic acid target. One oligonucleotide will be conjugated to a QD donor, the other to a fluorescent organic dye acceptor. When both oligonucleotides anneal to their target, donor and acceptor will be brought into close proximity leading to fluorescence resonant energy transfer. For the time-resolved detection, the expertise in photonics of the PI will be used to construct a simple, compact, and cost-effective home-made apparatus. The properties of the QD-acceptor pair are key to the success of this project. This award should support a full-time postdoctoral Research Associate with expertise in the photophysical and photochemical characteristics of colloidal QDs.