The ability to directly monitor biomolecular interactions (e.g. DNA-DNA, RNA-DNA, protein-protein) in real-time is of great importance to many areas of biology and medicine. At the cellular level, very few molecules can be responsible for inducing a significant biological response and there remains an urgent need for highly sensitive optical methods able to both identify and spatially track multiple target biomolecules simultaneously in complex and dynamic biological environments. To address this challenge we propose to develop a unique multi-imaging platform capable of monitoring large numbers of individual, freely moving nanoparticles and monitoring their interactions with target molecules and other nanoparticles. This new technology will initially be applied to the multiplexed detection of microRNAs with the distinct advantage of not requiring either target pre-modification or subsequent amplification steps to achieve the sensitivities necessary for the direct analysis of genomic RNA samples. The research takes advantage of the electronic properties of metallic nanoparticles that are associated with greatly enhancing the intensity of various types of spectroscopic signals such as scattering, Raman and fluorescence. These signals are highly responsive to changes in the immediate environment around each nanoparticle with Raman in particular providing a molecular fingerprint useful for identification. However, typical investigations involve applying only one of these spectroscopic modalities and either looking at select individual particles immobilised on a surface or acquiring an ensemble-averaged spectrum of the bulk sample. Imaging is a particularly powerful and intuitive approach for investigating complex systems. The radically different multi-spectroscopic methodology proposed here enabling the high-throughput visualisation of individual particles along with rapid optical discrimination between different particles sizes and clusters is expected to have a far-reaching impact. In addition to creating a powerful tool for bioanalytical investigation, this research will open up significant new opportunities to physicists, chemists and engineers interested in the functionalisation and assembly of nanoparticles to create next generation materials and devices.
The central aim of the research was to develop novel nanoparticle-enhanced imaging methodologies which could be used for the ultrasensitive detection of disease biomarkers. This involved successfully working towards achieving several objectives. In particular: (i) the construction of a novel imaging platform capable of the high-throughput tracking of freely moving particles via more than one optical imaging modality (e.g. scattering, fluorescence, Raman) simultaneously; (ii) the design of a novel class of gold nanoparticle-dye conjugate that enables multimodal imaging, and (iii) the biofunctionalisation and application of these new nanomaterials.
The first major accomplishment of the project using equipment purchased with the grant was to publish a proof-of-principle study (J. Phys. Chem. C, 2010, 18115) demonstrating an imaging approach capable of dynamically tracking and sizing freely suspended individual nanoparticle clusters based on their surface-enhanced Raman scattering (SERS) signal. This is the first time such an approach has been demonstrated since the SERS signal is often relatively weak. It also provided the basis for further instrument development with the integration of a second highly sensitive CCD for the wide-field monitoring of nanoparticles via two different spectroscopic signals (e.g. Rayleigh scattering and SERS) at the same time. Also added to the instrument platform was a microspectroscopy setup to acquire spectra of individual nanoparticles.
Another objective successfully achieved was to demonstrate the potential of the imaging platform to monitor interactions between DNA-functionalised nanoparticles in the presence of a specific target. A novel approach was to look at DNA triplex formation and control the interparticle distance and correlate this between the plasmonic and SERS responses. This work was achieved in collaboration with Prof Duncan Graham and published in the RSC flagship journal (Chemical Science, 2012, 2262).
A separate research theme running concurrently was the design of novel gold nanorod-dye nanostructures as promising nanotags for multimodal imaging. The ability to control the self-assembly of these hybrid materials has featured in a series of high-quality journal publications (Chem. Comm. 2011, 3757; PCCP 2013, 18835 and ACS Nano 2014, 8600). The main achievement of these papers is that we were able to develop a new design of functionalised nanorod particles that are optically much brighter than equivalent spherical particles thus allowing individual nanorods to be imaged and tracked at the single particle level via Raman and Rayleigh scattering.
As envisioned in the proposal, successful completion of the main objectives would pave the way for exploring new opportunities long after the expiry date of the grant itself. For example, the ACS Nano 2014 paper mentioned above was also partially supported by a “Bridging The Gap" grant with colleagues in biology and engineering at Strathclyde which enabled us to translate our research to cellular measurements and demonstrate that our surface functionalisation chemistries resulted in non-cytotoxic nanoparticles as well as being able to perform multimodal imaging measurements across a wide range of incident wavelengths. A number of other exciting opportunities are also currently being explored utilising the unique dynamic imaging platform established using this funding.