Luminescent bioassays using immobilized bioconjugated quantum dots within fluidics systems

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We are introducing fluorescence methods for analysis of gene fragments, peptides and proteins that offer the exciting capability to selectively determine low quantities of multiple markers in only seconds to minutes. We are learning how to attain advantages in analytical functionality by combining optical properties of nanoparticles with physical properties that are dependent on non-turbulent fluid flow in channels of micrometer dimension. Our work has been exploring quantum dots (QDs) decorated with single-stranded oligonucleotides (termed “QD-probes”) as the basis for bioassays that operate using fluorescence resonance energy transfer (FRET). A fluorescence intensity increase occurs when the QD excites a nearby dye that is associated with the formation of double-stranded DNA that results from hybridization of probe and target strands. Mixtures of different QDs luminescing at multiple wavelengths can be excited using a single blue or ultraviolet wavelength. We have characterized such multiplexed schemes combining QD-FRET for detection of mixtures of oligonucleotide targets. We propose to continue to explore modifications of the surface of QDs, and to build ensembles of immobilized solid-phase QD-probe films, to impart improved functionality and attain new levels of analytical performance. The goal of the proposed work is to learn how surface coatings can be assembled, and how closely packed immobilized QD-probes can offer highly efficient selective binding while blocking non-specific adsorption, in a configuration that makes use of near-field optical interactions to achieve signal enhancement, and a fluidics flow environment to achieve stringency control and sample processing. Three specific objectives are described in this proposal: 1. Exploration of microfluidic channels as platforms for QD-probes 2. Development of paper-based platforms for QD-probes 3. Signal-to-noise and selectivity improvements, including implementation of aptamers using QD-FRET We are introducing a new approach to achieve rapid target oligonucleotide quantification by using microfluidic technology. The method makes use of in-channel spatial distribution of luminescence from solid-phase QD-FRET processes for quantification. By design of conjugation on QDs, selection of QD-probe density, and constraints on channel structures, we intend to achieve sub-femtomole detection limits with results in seconds to minutes. Extension of what is learned will be used to guide the immobilization of QD-probes within paper-substrates, providing for a different form of fluidics system that facilitates practical implementation when using electronic cameras found on cell phones and digital tablets as detectors. We will make use of hybridization-sensitive fluorescent nucleobases that are integrated into oligonucleotide probes so that labeling of target oligonucleotide is unnecessary. Implementation of aptamers will extend the QD-FRET transduction technology to assays for peptide and protein targets. An advantage of solid-phase assembly of QD-probe films is a substantial amplification of signal magnitude due to interaction of more than one acceptor with one donor by FRET. We wish to establish a model to better understand how to improve signal enhancement from such solid-phase ensembles. Further improvement in signal magnitude will be achieved by implementation of upconverting nanoparticles (UCNPs) that are coated with QD-probes. UCNPs that are excited by near infrared (NIR) lasers can luminesce to optically excite the QDs. This will reduce background fluorescence and optical scattering, while maintaining cooperative effects between the QDs to achieve signal amplification.