Synthesis and characterisation of hybrid gold/polymer nanoparticles for bioassay application

A bioassay technique, based on surface-enhanced Raman scattering (SERS) tagged gold nanoparticles encapsulated with a biotin functionalised polymer, has been demonstrated through the spectroscopic detection of a streptavidin binding event. A methodical series of steps preceded these results: synthesis of nanoparticles which were found to give a reproducible SERS signal; design and synthesis of polymers with RAFT-functional end groups able to encapsulate the gold nanoparticle. The polymer also enabled the attachment of a biotin molecule functionalised so that it could be attached to the hybrid nanoparticle through a modular process. Finally, the demonstrations of a positive bioassay for this model construct using streptavidin/biotin binding. The synthesis of silver and gold nanoparticles was performed by using tri-sodium citrate as the reducing agent. The shape of the silver nanoparticles was quite difficult to control. Gold nanoparticles were able to be prepared in more regular shapes (spherical) and therefore gave a more consistent and reproducible SERS signal. The synthesis of gold nanoparticles with a diameter of 30 nm was the most reproducible and these were also stable over the longest periods of time. From the SERS results the optimal size of gold nanoparticles was found to be approximately 30 nm. Obtaining a consistent SERS signal with nanoparticles smaller than this was particularly difficult. Nanoparticles more than 50 nm in diameter were too large to remain suspended for longer than a day or two and formed a precipitate, rendering the solutions useless for our desired application. Gold nanoparticles dispersed in water were able to be stabilised by the addition of as-synthesised polymers dissolved in a water miscible solvent. Polymer stabilised AuNPs could not be formed from polymers synthesised by conventional free radical polymerization, i.e. polymers that did not possess a sulphur containing end-group. This indicated that the sulphur-containing functionality present within the polymers was essential for the self assembly process to occur. Polymer stabilization of the gold colloid was evidenced by a range of techniques including, visible spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and Raman spectroscopy. After treatment of the hybrid nanoparticles with a series of SERS tags, focussing on 2-quinolinethiol the SERS signals were found to have comparable signal intensity to the citrate stabilised gold nanoparticles. This finding illustrates that the stabilization process does not interfere with the ability of gold nanoparticles to act as substrates for the SERS effect. Incorporation of a biotin moiety into the hybrid nanoparticles was achieved through a =click‘ reaction between an alkyne-functionalised polymer and an azido-functionalised biotin analogue. This functionalized biotin was prepared through a 4-step synthesis from biotin. Upon exposure of the surface-bound streptavidin to biotin-functionalised polymer hybrid gold nanoparticles, then washing, a SERS signal was obtained from the 2-quinolinethiol which was attached to the gold nanoparticles (positive assay). After exposure to functionalised polymer hybrid gold nanoparticles without biotin present then washing a SERS signal was not obtained as the nanoparticles did not bind to the streptavidin (negative assay). These results illustrate the applicability of the use of SERS active functional-polymer encapsulated gold nanoparticles for bioassay application.

A profluorescent azaphenalene nitroxide for nitroxide-mediated polymerization

A novel nitroxide-mediated polymerization (NMP) control agent; 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO), was used in the free-radical polymerization of styrene. The conversion of styrene during NMP was studied using FT-Raman spectroscopy and the effectiveness of TMAO as a NMP control agent was assessed by GPC analysis. Fidelity of the TMAO-alkoxyamine end-group on the synthesized polymers was confirmed by GPC, UV-Vis and fluorescence spectroscopic analyses. Comparison to the well known NMP control agent, TEMPO was made. TMAO showed control of molecular weight approaching that of TEMPO. Attempts to improve the properties of TMAO as an NMP control agent by synthesizing an analogue with bulkier substituents around the nitroxide did not generate the target molecule but demonstrated some of the interesting chemistry of the azaphenalene ring system

Glycosaminoglycan and growth factor mediated murine calvarial cell proliferation

Understanding the complex mechanisms underlying bone remodeling is crucial to the development of novel therapeutics. Glycosaminoglycans (GAGs) localised to the extracellular matrix (ECM) of bone are thought to play a key role in mediating aspects of bone development. The influence of isolated GAGs was studied by utilising in vitro murine calvarial monolayer and organ culture model systems. Addition of GAG preparations extracted from the cell surface of human osteoblasts at high concentrations (5 microg/ml) resulted in decreased proliferation of cells and decreased suture width and number of bone lining cells in calvarial sections. When we investigated potential interactions between the growth factors fibroblast growth factor-2 (FGF2), bone morphogenic protein-2 (BMP2) and transforming growth factor-beta1 (TGFbeta1) and the isolated cell surface GAGs, differences between the two model systems emerged. The cell culture system demonstrated a potentiating role for the isolated GAGs in the inhibition of FGF2 and TGFbeta1 actions. In contrast, the organ culture system demonstrated an enhanced stimulation of TFGbeta1 effects. These results emphasise the role of the ECM in mediating the interactions between GAGs and growth factors during bone development and suggest the GAG preparations contain potent inhibitory or stimulatory components able to mediate growth factor activity.