NSF: NIRT (BES 0507036); NSF-GRAS (BES 0541661) (Lee)

1. A CS ys B C IME ime cs YS C Correlation and Characterization 3D Morphologically Dependent LSPR Spectra of Single Ag NPs CHL Introduction A iii i A iv ii v b c a B B iv ii iii Fig 3: Correlation of single Ag NPs using micro-patterned windows: (A): (a) AFM images, (b) optical true-color images and (c) CCD images of single Ag NPs in a hexagon micro-patterned window, respectively; (B) Zoom-in images of individual NPs squared in (A), Scale bar is 5 μm in (A: a-c) and 0.5 μm in (B: a-c). Results and Discussion B A a b c Transport and Accumulation of Au NPs in Zebrafish Embryos c b a d (A)Extracellularand(B) IntracellularAg NPs Single-molecule Nanoparticle Biosensors (SMNOBS) Fig 5: Optical images of the gastrulation-stage embryos in (a) egg water alone and incubated with (b) 0.05, (c) 0.20, and (d) 1.20 nM Au NPs for 4 h since its cleavage stage. The embryos in (b-d) show the reddish and darken burgundy color of Au NPs, suggesting that the amount of accumulated Au NPs in the embryos increases with concentration. Scale bar = 500 µm. i (D): Image of a cleavage-stage embryo (C) Illustration of membrane transport of NPs in and out of a living cell v Fig 10: Synthesis of photostable SMNOBS for sensing single protein molecules. B A Summary Fig 6: Illustration of transport of single Au NPs (1.20 nM) into a cleavage stage zebrafish embryo, leading to 74% normally developed, 2% deformed and 24% dead zebrafish. B A A References Fig 7: Biocompatibility and toxicity of Ag NPs show high dependence on NP concentration. (A) Histogram of distribution of normally developed (red) and dead (green) zebrafish, vs Ag NP concentration; (B) Histogram of distribution of five representative types of deformities of the zebrafish vs Ag NP concentration: finfold abnormality (FF), tail and spinal cord flexure and truncation (TF), cardiac malformation (CM), yolk sac edema (YSE), head edema (HA), and eye abnormality (no eys). Fig 2: Characterization of photostability of single Ag NPs: (A) optical images of single Ag NPs and (B) plots of scattering intensity of (i) a single NP and (ii) background vs illumination time, showing Ag NPs resist photobleaching and blinking. A d a b c Diffusion and Transport of Single NPs in Zebrafish Embryos B A C B a c d e b Fig 8: Normal and deformed Zebrafish resulted from being treated chronically with NPs since their cleavage stage: (A) Optical images of normally developed zebrafish show the normal development of (a) finfold; (b) tail/spinal cord; (c) cardiac and yolk sac; and (d) head and eyes. (B) optical images of zebrafish treated with given concentrations of NPs show: (a) finfold abnormality; (b-c) tail/spinal cord flexure and truncation; (c-d) cardiac malformation and yolk sac edema; and (e) acephaly and no tail. Fig 4: Real-time imaging and probing of nano-environments of a segmentation stage (21 hpf) zebrafish living embryo. Scale bar = 100 µm. Design of Biocompatible Nanoparticles for Probing Living Cellular Functions and their Potential Environmental ImpactsNSF NIRT Grant BES 0507036X. Nancy Xu (PI)*, C. Osgood, H. Elsayed-Ali,P. Nallathamby, K. Lee, L. Browning, T. Huang, Y. Song, Old Dominion University, Norfolk, VA 23529; *E-mail: xhxu@odu.edu; http://www.odu.edu/sci/xu/xu.html New Method for Imaging and Characterization of Single NPs Embedded in Tissues Recent advances in nanoscience and nanotechnology suggest a wide variety of potential applications of nanoparticles (NPs), such as nanosensors and nanophotonic probes for in vivo imaging, diagnosis and smart drug delivery. Noble metal NPs have unique optical properties, which depend on their size, shape, surrounding environment, and dielectric constant of the embedding medium. Unlike fluorescent probes and QDs, these noble metal NPs do not suffer photodecomposition and do not blink under dark-field optical illumination, and silver (Ag) NPs possess exceptionally high quantum yield of Raleigh scattering that are orders of magnitude higher than fluorophors (e.g., R6G). Recently, we have used these intrinsic optical properties of NPs for real-time imaging nanoenvironments of zebrafish embryos,1-3 for sensing single receptor molecules on single living cells,4,5 analysis of single protein molecules, probing size and transport kinetics of individual membrane transporters of single living cells with sub-100 nm spatial resolution and millisecond time resolution for better understanding membrane transport mechanisms and their potential environmental impacts.6-8 C Fig 9:Characterization of individual Ag NPs embedded inside a fully developed (120 hpf) zebrafish using DFOMS: (A) optical image of a fixed normally developed zebrafish. The rectangles (i-v) highlight representative areas: (i) retina, (ii) brain (mesencephalon cavity), (iii) heart, (iv) gill arches, and (v) tail. (B) Zoom-in optical images of single Ag NPs embedded in those tissue sections outlined in (A). Dashed circles outline the representative embedded individual Ag NPs. Scale bar = 400 µm in (a); 4 µm in (b). (C) LSPR spectra of single Ag NPs. Synthesis and Characterization of Single Ag NPs C In summary, we have developed photostable (non-photodecomposition and non-blinking) NP photonic probes for continuous and simultaneous imaging multiple nano-environments in zebrafish embryos and for probing kinetics of single membrane transporters of single living cells in real-time, aiming to better understand developmental biology and membrane transporters at the molecular level. We have developed effective in vivo assays for characterization of biocompatibility and toxicity of NPs. We found that Au NPs with the same size, shape and surface charges as Ag NPs, are much more biocompatible (less toxic) to the embryos than Ag NPs, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of NPs highly depend on their chemical properties, and the embryos can serve as effective in-vivo assays to screen their biocompatibility and toxicity. We have functionalized the photostable NPs and developed photostable single NP biosensors for detecting and sensing single protein molecules, and for imaging and mapping single receptor molecules and single ligand-receptor interactions on single living cells, aiming to better understand their cellular function in real-time and for molecular imaging and diagnosis. Fig 1: (A) dark field optical and (B) HR-TEM images of single Ag NPs, and (C) Correlation of the size and color of single NPs. Scale bar = 5 nm. c B 1. P. Nallathamby, K. Lee, X. Xu*, ACS Nano,2, 1371 (2008). 2. K. Lee, P. Nallathamby, L. Browning, C. Osgood, X. Xu*, ACS Nano, 1, 133 (2007). 3. L. Browning, K. Lee, T. Huang, P. Nallathamby, J. Lowman, X. Xu*, Nanoscale, (2009). 4. Y. Song, P. Nallathamby, T. Huang, H. Elsayled-Ali, X. Xu*, J. Phys. Chem. C. (2009) 5. T. Huang, P. Nallathamby, X. Xu*, J. Am. Chem. Soc. 130, 17095 (2008). 6. T. Huang, P. Nallathamby, D. Gillet, X. Xu*, Anal. Chem. 79, 7708 (2007). 7. X. Xu*, W. Brownlow, S. Kyriacou, Q. Wan, J. Viola, Biochemistry 43, 10400 (2004). 8. S. Kyriacou, W. Brownlow, X. Xu*, Biochemistry 43, 140 (2004). 9. X. Xu*, J. Chen, R. Jeffers, S. Kyriacou, Nano Letters, 2, 175 (2002). Acknowledgements • NSF: NIRT (BES 0507036); NSF-GRAS (BES 0541661) (Lee) • NSF-NNIN: UM-HR TEM, CNF of Cornell University • NIH: R01GM076440; R01 GM076440S1(Browning) • Old Dominion Fellowships (Nallathamby)