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Intracellular redox potential affects cellular function and its dysregulation is associated with disease. Current methods of monitoring intracellular redox potential are limited because they typically only report potentials of the redox buffer glutathione. Our group has developed redox-active probe molecules that change bond order depending on the probe oxidation state, and are instead sensitive to overall redox potential within the cell. Gold nanoshells coated with the probe form a novel intracellular redox nanosensor, and spectral discrimination of the oxidised and reduced states by Surface-Enhanced Raman Scattering (SERS) allows calculation of redox potential. Prior work by the group provided basic proof-of-principle for its use in measuring intracellular redox potential. The aim of this project, therefore, was to develop the tools and techniques to enable its application to meaningful biological questions, and extend the method into a pathologically relevant cell line. The initial stages of the project standardised the functionalisation of gold nanoshells with the NQ probe molecule and the application of the nanosensors to the A549 human lung cancer cell line. Toxicity tests confirmed the nanosensor was non-toxic. A protocol was then developed for rapidly obtaining SERS maps to enable localisation of nanosensors within the cell. This was successful, and the protocols can be applied to any combination of adherent cell type and nanosensor. A bespoke piece of software was created to determine redox potential and pH from SERS maps to produce a colourmap showing spatial variation of redox potential and pH with subcellular resolution. This software enables more rapid and precise calculation of redox potential or pH than manual processing. As a test case, changes in intracellular redox potential in response to treatment with toxic metal nanoparticles were studied and shown to correlate with other measures of oxidative stress. Hypoxia (abnormally low oxygen levels) is relevant in disease. Investigating redox potential in hypoxic cells requires precise control of the oxygen concentration during the acquisition of SERS spectra. To facilitate such experiments, a specialised imaging chamber was designed, constructed and tested. Such environmental control enables experiments to be carried out at various oxygen concentrations as well as under optimal cellular physiological conditions, enabling not only the response to alterations in oxygen levels to be studied but also extending the biological model system to more closely reflect animal physiology. Finally, a device was constructed that allowed the acquisition of SERS spectra from both intracellular and extracellular nanosensors in the same experiment, as the relationship between intracellular and extracellular redox potential is incompletely understood. The intracellular and extracellular nanosensors are spatially separated, allowing clear discrimination of the SERS spectra obtained simply by changing the orientation of the device. This device enables the effect of quantitative modification of extracellular redox potential on intracellular redox potential to be investigated. In summary, the work has greatly extended a method of measuring intracellular redox potential. It was taken from the proof-of-principle stage to being a robust method, capable of providing useful quantitative biological information. Improvements have been made in production and toxicity testing of the nanosensors, robustness of SERS data acquisition and analysis, environmental control during SERS data acquisition and application to disease-relevant cell culture models. The result is that we are now able to rapidly and reproducibly determine intracellular redox potential in single cells. |
