Real-time study of the transport and biocompatibility of nanomaterials in early embryonic development at single-nanoparticle resolution can offer new knowledge about the delivery and effects of nanomaterials in vivo and provide new insights into molecular transport mechanisms in developing embryos. In this study, we directly characterized the transport of single silver nanoparticles into an in vivo model system (zebrafish embryos) and investigated their effects on early embryonic development at single-nanoparticle resolution in real time. We designed highly purified and stable (not aggregated and no photodecomposition) nanoparticles and developed single-nanoparticle optics and in vivo assays to enable the study. We found that single Ag nanoparticles (5-46 nm) are transported into and out of embryos through chorion pore canals (CPCs) and exhibit Brownian diffusion (not active transport), with the diffusion coefficient inside the chorionic space (3 x 10(-9) cm(2)/s) approximately 26 times lower than that in egg water (7.7 x 10(-8) cm(2)/s). In contrast, nanoparticles were trapped inside CPCs and the inner mass of the embryos, showing restricted diffusion. Individual Ag nanoparticles were observed inside embryos at each developmental stage and in normally developed, deformed, and dead zebrafish, showing that the biocompatibility and toxicity of Ag nanoparticles and types of abnormalities observed in zebrafish are highly dependent on the dose of Ag nanoparticles, with a critical concentration of 0.19 nM. Rates of passive diffusion and accumulation of nanoparticles in embryos are likely responsible for the dose-dependent abnormalities. Unlike other chemicals, single nanoparticles can be directly imaged inside developing embryos at nanometer spatial resolution, offering new opportunities to unravel the related pathways that lead to the abnormalities.

We have synthesized and characterized stable (non-aggregation, non-photobleaching and nonblinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10 -11 to 1.3×10 -8 cm 2 /s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in-vivo assays to screen their biocompatibility.

We report here, for the first time, the use of a simple washing approach to reduce the ionic strength of the solution, which increased thickness of electric double layer on the surface of silver (Ag) nanoparticles, and thereby enhanced their surface zeta potential. This approach allowed us to prepare optically uniform (75-99%) and purified Ag nanoparticles (11.3 ± 2.3 nm) that are stable (non aggregation) in solution for months, permitting them to become robust and widely-used single nanoprobes for in vivo optical imaging. These Ag nanoparticles show remarkable photostability and serve as single nanoparticle photonic probes for continuous imaging nano-environments of segmentation-stage zebrafish embryos for hours. Unlike other particle tracking experiments, we utilized size-dependent localized-surface-plasmon-resonance-spectra (LSPRS) (colors) of single Ag nanoparticles to determine given colored (sized) nanoparticles in situ and used the monodisperse color (size) of nanoparticles to simultaneously measure viscosities and flow patterns of multiple proximal nano-environments in segmentation-stage zebrafish embryos in real-time. We found new interesting counterclockwise flow patterns with rates ranging from 0.06 to 1.8 μm/s and stunningly high viscosity gradients spanning two-orders of magnitude in chorion space of the embryos, with the highest viscosity observed around the center of chorion space and the lower viscosity at the interfacial areas near the surface of both chorion layers and inner mass of the embryos. This study demonstrates the possibility of using individual monodisperse nano-photonics to probe the roles of embryonic fluid dynamics in embryonic development.

We synthesized tiny stable silver nanoparticles (2.6 ± 1.1 nm) and used its small surface area and functional groups to control single molecule detection (SMD) volumes on single nanoparticles. These new approaches allowed us to develop intrinsic single molecule nanoparticle optical biosensors (SMNOBS) for sensing and imaging of single human cytokine molecules, recombinant human tumor necrosis factor-α (TNFα), and probing its binding reaction with single monoclonal antibody (MAB) molecules in real-time. We found that SMNOBS retained their biological activity over months and showed exceptionally high photostability. Our study illustrated that smaller nanoparticles exhibited higher dependence of optical properties on surface functional groups, making it a much more sensitive biosensor. Localized surface plasmon resonance spectra (LSPRS) of SMNOBS showed a large red shift of peak wavelength of 29 ± 11 nm, as single TNFα molecules bound with single MAB molecules on single nanoparticles. Utilizing its LSPRS, we quantitatively measured its binding reaction in real time at SM level, showing stochastic binding kinetics of SM reactions with binding equilibrium times ranging from 30 to 120 min. SMNOBS exhibited extraordinarily high sensitivity and selectivity, and a notably wide dynamic range of 0-200 ng/mL (0-11.4 nM). Thus, SMNOBS is well suited for the fundamental study of biological functions of single protein molecules and SM interactions of chemical functional groups with the surface of nanoparticles, as well as development of effective disease diagnosis and therapy.

At the cellular level, a small number of protein molecules (receptors) can induce significant cellular responses, emphasizing the importance of molecular detection of trace amounts of protein on single living cells. In this study, we designed and synthesized silver nanoparticle biosensors (AgMMUA-IgG) by functionalizing 11.6 +/- 3.5-nm Ag nanoparticles with a mixed monolayer of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (1:3 mole ratio) and covalently conjugating IgG with MUA on the nanoparticle surface. We found that the nanoparticle biosensors preserve their biological activity and photostability and can be utilized to quantitatively detect individual receptor molecules (T-ZZ), map the distribution of receptors (0.21-0.37 molecule/microm(2)), and measure their binding affinity and kinetics at concentrations below their dissociation constant on single living cells in real time over hours. The dynamic range of detection is 0-50 molecules per cell. We also found that the binding rate (2-27 molecules/min) is highly dependent upon the coverage of receptors on living cells and their ligand concentration. The binding association and dissociation rate constants and affinity constant are k1 = (9.0 +/- 2.6) x 10(3) M(-1) s(-1), k(-1) = (3.0 +/- 0.4) x 10(-4) s(-1), and KB = (4.3 +/- 1.1) x 10(7) M(-1), respectively.