Centre for Research in Biomolecular Interactions presents a guest speaker, Dr. Ulrich Krull from Department of Chemical and Physical Sciences University of Toronto, Mississauga, who will give a talk about the use of Quantum Dots in optical biosensing.

Abstract:

Optical Biosensing at the Nanoscale: Multiplexed Nucleic Acid Biosensors Based on Quantum Dots for Microfluidic Devices

The optical properties and available surface area of QDs have made them an interesting material for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) have been developed for the selective detection of nucleic acids. QDs were immobilized on optical fibers and conjugated with probe oligonucleotides. This represents a convenient platform to investigate spectral properties and biosensor response. Hybridization with acceptor labeled target oligonucleotides generated FRET-sensitized acceptor fluorescence that was used as the analytical signal. A sandwich assay was also introduced and avoided the need for target labeling. Green and red emitting CdSe/ZnS QDs were used as donors with Cy3 and Alexa Fluor 647 acceptors, respectively. Quantitative measurements were made via spectrofluorimetry and fluorescence microscopy. Mixed films of the two colors of QD and two probe oligonucleotide sequences were prepared for multiplexed solid-phase hybridization assays. It was possible to simultaneously detect two target sequences with retention of selectivity, including SNP discrimination. A two-plex assay is also possible using a single green emitting QD donor with Cy3 and Rhodamine Red‐X acceptor dyes. Further development has resulted in various strategies for the simultaneous and selective detection of three and four target nucleic acid sequences based on mixed films of immobilized quantum dots (QDs) and oligonucleotide probes, i.e. three-plex and four-plex assays. An assembly of unique QD-probe systems on a surface therefore allows for multi-colour emission so that multiplexing to simultaneously determine different nucleic acid targets becomes possible without spatial registration. The typical challenges associated with such detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. Our work has considered immobilization of mixtures of different QD-probes onto glass microfluidic channels using various chemistries. Microfluidic flow can be used to deposit and remove QD-probes. The ability to dynamically control stringency by adjustment of the potential in an electroosmotic based microfluidics experiment is advantageous. The shearing force, Joule heating and the competition between electroosmotic and electrophoretic mobility provide optimization of hybridization conditions, amelioration of adsorption, and regeneration of the sensing surface. As a specific example, bi-conjugated QDs can be used where one oligonucleotide sequence on the QD is available for immobilization by hybridization with complementary oligonucleotide on a glass surface, and a different oligonucleotide on the QD serves as a probe to transduce hybridization with target in a sample solution. A Cy3 label that can be excited by FRET using green emitting CdSe/ZnS QDs provides for analytical signal to explore this detection strategy.

 

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Refreshments will be served.