Nonblinking plasmonic quantum dot assemblies for multiplex biological detection.

Optical labels enable visualization and detection of molecules, cells, and tissues. These labels have limited multiplexing capabilities, blink on/off at the single-molecule level, and, in many cases, they have low brightness. We address each of these issues through the development of a sub-50 nm barcoded optical label system by coating fluorescent quantum dots onto the surface of plasmonic nanoparticles using a layerby-layer polyelectrolyte deposition strategy. We denote these gold nanoparticle–quantum dot hybrid structures as GQHs. This probe design strategy has the potential to create thousands of uniquely emitting nonblinking fluorescent probes. These barcodes can be further conjugated to biorecognition molecules, target cells, and have prolonged intracellular retention with minimal toxicity. Our results demonstrate that plasmonic nanoparticles are ideal templates for designing nanoscale barcodes. Quantum dots are semiconductor nanocrystals with a broad excitation range and tunable emission spectra, enabling spectral multiplexing at a single excitation wavelength. Their narrow and symmetric emission profile simplifies color discrimination. Quantum dots are also brighter and more photostable than conventional organic fluorophores. Quantum dots have been used as labels for staining diseased cells and tissues, single molecule analysis in live cells, and as contrast agents for in vivo cancer detection, but there is a need to increase the multiplexing capabilities of quantum dots to address the detection needs in biology and medicine. Despite the sizeand shape-tunable emission of quantum dots, a maximum of only seven different emitting quantum dots have been distinguished in cell experiments. To address this problem, researchers have started to encapsulate or dope quantum dots inside 200 nm to 10 mm polymer beads for protein or nucleic acid detection. This detection platform has improved the efficiency and accuracy of molecular analysis and detection in vitro. The use of these barcodes for studying cells or molecules within cells is limited by their inefficient cellular uptake. Rejman and co-workers showed that 500 nm beads are taken up 8–10 times less efficiently than 50 nm beads. Strategies have been attempted to encapsulate quantum dots inside sub-100 nmsized beads, but it can be difficult to control the number and ratio of different emitting quantum dots inside nanometersized beads. Such limitation leads to inaccurate coding. Size reduction of the barcode to the nanoscale is further constrained by the intrinsic fluorescence intermittency, or blinking behavior, of quantum dots. This property can complicate the acquisition, detection, and analysis of the emission from single quantum dots, as blinking may lead to false-negative signals. This blinking behavior is not a problem for microscale barcodes because they contain thousands of quantum dots loaded inside. Suppression of blinking has been demonstrated by coupling quantum dots to plasmonic gold films or by placing these particles near each other on a flat surface. Plasmonic quantum dots have been recently developed with a quantum dot core encapsulated in a gold shell, but this design showed reduced fluorescence, and the blinking behavior was not characterized. These plasmonic quantum dots may not be feasible for barcoding as each nanocomposite only contained a single quantum dot. Finally, non-blinking ZnSe capped CdZnSe quantum dots can be prepared by gradually alloying the Zn in the core, but their emission spectra is difficult to control. We created nanobarcodes by using a layer-by-layer deposition strategy to deposit a combination of quantum dots onto the surface of a gold nanoparticle. The gold nanoparticles enhanced the fluorescence and reduced the blinking of the quantum dots. The size of these hybrid structures is approximately 50 nm, which is small enough to enter cells and interact with biological molecules. To develop these nanobarcodes, gold nanoparticles were coated with different emitting quantum dots with a layer-bylayer polyelectrolyte deposition strategy. By carefully controlling the amount of the polyelectrolyte deposition, we were able to control the distance between the gold nanoparticles and quantum dots. It has been demonstrated that the maximum enhancement of quantum dot fluorescence can be achieved when the quantum dot is 11 nm away from the metal surface. To obtain maximum fluorescence enhancement, citrate-coated gold nanoparticles were first coated with an amphiphilic diblock copolymer (polystyrene-block-poly([*] Dr. F. Song, P. Tang, Prof. Dr. W. Chan Institute of Biomaterials and Biomedical Engineering Donnelly Centre for Cellular and Biomolecular Research Chemical Engineering, Chemistry, Materials Science and Engineering, University of Toronto Toronto, ON, M5S 3G9 (Canada) E-mail: [email protected]