Intracellular redox potential is a delicately balanced property in cells. It plays an important role in the regulation of cellular processes and dysfunction of the redox state is believed to get involved in initiation of many kinds of diseases. However, lack of suitable techniques for quantitatively monitoring the redox potential in cells with a wide range is a significant challenge. My project aims to develop SERS nanosensors for measuring intracellular redox state quantitatively and applying them to quantify hypoxia, which is generally described by the cell having a reducing environment and defined as a form of “reductive stress”. Four redox active probe molecules have been synthesised and characterised. Their Raman spectra all change as a function of local redox potential. Since these probe molecules can be assembled on gold nanoshells which are able to enhance the Raman Effect significantly, we can calculate the redox potential from simple optical SERS measurements. Transmission Electron Microscopy was used to investigate the cellular delivery of nanosensors. TEM images confirmed that either single nanosensor or small aggregates can be taken up controllably by cells and translocated in the cytoplasm. The introduction of nanosensors was also found not to be toxic to the cells. The nanosensor has been used to monitor the redox potential in resting cells as well as the redox changes when cells responded to pharmacologically induced hypoxia and oxidative stress. These measurements demonstrated that the SERS based nanosensor developed is able to monitor the intracellular redox change in a reversible, noninvasive way and respond to cellular hypoxia quantitatively.

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.

Consensus opinion suggests that apoptosis occurs when the intracellular redox potential reaches its oxidative range, i.e. when the balance between oxidants and reductants is disturbed. An understanding of the links between redox potential and the induction of apoptosis in cells could improve our understanding of the process and help to predict therapeutic responses. This study investigates the changes in redox potential at distinct stages of apoptosis induced in the human cervical cancer cell line, HeLa. Stages of the apoptotic process were defined by loss of mitochondrial membrane polarisation (ΔΨm), membrane phosphatidyl serine exposure, caspase-3 activation, and nuclear fragmentation. To measure real-time redox potential change in apoptotic cells two independent methods were used: (1) expression of redox-responsive green fluorescent protein (roGFP2) measured by flow cytometry and (2) redox-responsive nanosensors detected by surface enhanced Raman spectroscopy (SERS). roGFP2 measurements showed that HeLa cells demonstrate a shift towards an oxidative redox state during the later stages of apoptosis and this was preceded by loss of ΔΨm. The relationship between these two events was investigated by transient inhibition of mitochondrial permeability transition pore opening using the inhibitor bongkrekic acid (BKA) pre-treatment. At the cell population level, transient exclusion of the mitochondrial contribution delayed two key events of apoptosis in the first two hours measured by nuclear fragmentation and loss of ΔΨm. However, BKA treatment did not affect redox potential, reported by roGFP2, when compared with controls. Therefore, this suggests that mitochondria do not contribute towards the overall redox potential change in apoptosis. To gain insight into the significance of redox change at the earliest stages of apoptosis, single cell studies were performed. SERS, employing simultaneous redox potential and intracellular pH measurements using two synthetic nanosensors AQ-NS and MBA-NS, showed that BKA pre-treatment resulted in increased alkalinity and the cells were consequently protected from induction of apoptosis in the first thirty minutes of the kinase inhibitor staurosporine treatment. Measurements with SERS nanosensors allowed for adjustment for pH, which provides a clearer insight into redox potential dynamics, with consideration of the environment, and accurate quantitative assessment of redox at early stages of apoptosis. Together these data suggest that while roGFP2 is a valid method to use at a population level, SERS is a more sensitive method for measuring the redox potential of the cell at the early stages of apoptosis.

No description available.

Redox regulation and homeostasis are critically important in the regulation of cell function; however, there are significant challenges in quantitatively measuring and monitoring intracellular redox potentials. The work in this thesis details a novel approach to intracellular redox monitoring. The approach is based on the use of nanosensors, which comprise molecules capable of sensing the local redox potential, assembled on gold nanoshells. Since the Raman spectra of the sensor molecules change depending on their oxidation state, and since the nanoshells allow a large enhancement of the Raman scattering, intracellular potential can be calculated by simple optical measurements. A full description of the design, fabrication and characterisation (spectroscopic and electrochemical) of the nanosensors is provided within. The ability to deliver nanosensors into cells in a controllable fashion was confirmed using electron microscopy. Results from a range of assays are also presented which reveal that introduction of nanosensors does not result in any cytotoxicity. Sensor utility in monitoring redox potentials as cells responded to physiological and superphysiological oxidative and reductive stimuli was investigated. Importantly, the capability of the nanosensors in monitoring intracellular potentials in a reversible, non-invasive manner, and over a previously unattainable potential range, is demonstrated.

Os filmes moleculares constituídos por complexos polipiridínicos de rutênio, clusters trigonais de acetato de rutênio e porfirinas polimetaladas, tem sido investigados de forma sistemática, proporcionando uma ampla variedade de interfaces funcionais, para uso em dispositivos eletrônicos, sensores, células electrocatalíticas e de fotoconversão de energia. Tais filmes tem sido gerados por meio de métodos conhecidos como dip coating, drop casting, e deposição química/eletroquímica, apresentando aspectos morfológicos extremamente ricos, os quais foram investigados nesta tese, por meio de microscopia de varredura de sonda. Estudos de microscopia de força atômica foram dirigidos para os filmes moleculares gerados por dip coating. As imagens foram analisadas com bons resultados através do Programa da Gwyddion, permitindo a avaliação dos parâmetros de rugosidade envolvidos nas formas e distribuição dos grãos superficiais. Os filmes obtidos por drop casting foram investigados por meio da microscopia de força atômica, MACMode, apresentando uma grande variedade morfológica, associada principalmente com o processo de dewetting, sob a influência de interações moleculares específicas. A eletropolimerização dos complexos foi monitorada por meio de AFM condutivo, permitindo sondar as características condutoras dos filmes poliméricos. Filmes de DNA e nanopartículas magnéticas ancoradas sobre superfície de ouro foram investigados através de AFM MACMode, e AFM magnético, respectivamente. Finalmente, uma nova abordagem de sondas ultrassensitivas, foi explorada através da imobilização de nanopartículas magnéticas sobre MACLevers, ampliando os limites de detecção gravimétrica até sub-picograma. / Molecular films constituted by ruthenium polypyridine complexes, triangular ruthenium acetate clusters, and polymetallated porphyrins have been systematically investigated, providing a wide range of functional interfaces for electronic devices, sensors, photoconversion and electrocatalytical cells. Such films have been generated by dip coating, drop casting and electrochemical deposition, displaying very rich morphological aspects, which have been investigated by means of scanning probe microscopy. AFM studies have been carried out for the molecular films generated by dip coating. The images were successfully analysed using the Gwyddion software, allowing the evaluation of the rugosity parameters determining the grain shapes and distribution over the films. The films obtained by drop casting have been investigated by means of MACMode SFM, exhiting a wide variety of morphologies, mainly associated with the dewetting of the samples, under the influence of specific molecular interactions. Electropolymerization of molecular complexes have been monitored by SFM techniques, including the electroconducting mode to probe the conductivity of the polymeric films. Gold anchored films of DNA and magnetic nanoparticles have also been successfully investigated by MACMode SFM and magnetic SFM techniques, respectively. Finally, a new approach for ultra-sensitive probes, based on the immobilization of magnetic nanoparticles onto the MACLevers has been devised, allowing the detection of sub-picogram amounts of analytes

Os filmes moleculares constituídos por complexos polipiridínicos de rutênio, clusters trigonais de acetato de rutênio e porfirinas polimetaladas, tem sido investigados de forma sistemática, proporcionando uma ampla variedade de interfaces funcionais, para uso em dispositivos eletrônicos, sensores, células electrocatalíticas e de fotoconversão de energia. Tais filmes tem sido gerados por meio de métodos conhecidos como dip coating, drop casting, e deposição química/eletroquímica, apresentando aspectos morfológicos extremamente ricos, os quais foram investigados nesta tese, por meio de microscopia de varredura de sonda. Estudos de microscopia de força atômica foram dirigidos para os filmes moleculares gerados por dip coating. As imagens foram analisadas com bons resultados através do Programa da Gwyddion, permitindo a avaliação dos parâmetros de rugosidade envolvidos nas formas e distribuição dos grãos superficiais. Os filmes obtidos por drop casting foram investigados por meio da microscopia de força atômica, MACMode, apresentando uma grande variedade morfológica, associada principalmente com o processo de dewetting, sob a influência de interações moleculares específicas. A eletropolimerização dos complexos foi monitorada por meio de AFM condutivo, permitindo sondar as características condutoras dos filmes poliméricos. Filmes de DNA e nanopartículas magnéticas ancoradas sobre superfície de ouro foram investigados através de AFM MACMode, e AFM magnético, respectivamente. Finalmente, uma nova abordagem de sondas ultrassensitivas, foi explorada através da imobilização de nanopartículas magnéticas sobre MACLevers, ampliando os limites de detecção gravimétrica até sub-picograma. / Molecular films constituted by ruthenium polypyridine complexes, triangular ruthenium acetate clusters, and polymetallated porphyrins have been systematically investigated, providing a wide range of functional interfaces for electronic devices, sensors, photoconversion and electrocatalytical cells. Such films have been generated by dip coating, drop casting and electrochemical deposition, displaying very rich morphological aspects, which have been investigated by means of scanning probe microscopy. AFM studies have been carried out for the molecular films generated by dip coating. The images were successfully analysed using the Gwyddion software, allowing the evaluation of the rugosity parameters determining the grain shapes and distribution over the films. The films obtained by drop casting have been investigated by means of MACMode SFM, exhiting a wide variety of morphologies, mainly associated with the dewetting of the samples, under the influence of specific molecular interactions. Electropolymerization of molecular complexes have been monitored by SFM techniques, including the electroconducting mode to probe the conductivity of the polymeric films. Gold anchored films of DNA and magnetic nanoparticles have also been successfully investigated by MACMode SFM and magnetic SFM techniques, respectively. Finally, a new approach for ultra-sensitive probes, based on the immobilization of magnetic nanoparticles onto the MACLevers has been devised, allowing the detection of sub-picogram amounts of analytes

abstract: In today's world there is a great need for sensing methods as tools to provide critical information to solve today's problems in security applications. Real time detection of trace chemicals, such as explosives, in a complex environment containing various interferents using a portable device that can be reliably deployed in a field has been a difficult challenge. A hybrid nanosensor based on the electrochemical reduction of trinitrotoluene (TNT) and the interaction of the reduction products with conducting polymer nanojunctions in an ionic liquid was fabricated. The sensor simultaneously measures the electrochemical current from the reduction of TNT and the conductance change of the polymer nanojunction caused from the reduction product. The hybrid detection mechanism, together with the unique selective preconcentration capability of the ionic liquid, provides a selective, fast, and sensitive detection of TNT. The sensor, in its current form, is capable of detecting parts per trillion level TNT in the presence of various interferents within a few minutes. A novel hybrid electrochemical-colorimetric (EC-C) sensing platform was also designed and fabricated to meet these challenges. The hybrid sensor is based on electrochemical reactions of trace explosives, colorimetric detection of the reaction products, and unique properties of the explosives in an ionic liquid (IL). This approach affords not only increased sensitivity but also selectivity as evident from the demonstrated null rate of false positives and low detection limits. Using an inexpensive webcam a detection limit of part per billion in volume (ppbV) has been achieved and demonstrated selective detection of explosives in the presence of common interferences (perfumes, mouth wash, cleaners, petroleum products, etc.). The works presented in this dissertation, were published in the Journal of the American Chemical Society (JACS, 2009) and Nano Letters (2010), won first place in the National Defense Research contest in (2009) and has been granted a patent (WO 2010/030874 A1). In addition, other work related to conductive polymer junctions and their sensing capabilities has been published in Applied Physics Letters (2005) and IEEE sensors journal (2008). / Dissertation/Thesis / Ph.D. Electrical Engineering 2012

The Warburg effect describes the survival advantage of cancer cells in that they can proliferate under low oxygen/hypoxic conditions via a less efficient pathway known as glycolysis. It has not yet been documented at which point, in an oxygen gradient, phenotypic changes occur. Measuring the intracellular redox potential (IRP) and its impact on cellular dynamics would provide greater insight into how disruption of redox homeostasis caused by changes in oxygen concentration leads to aberrant cell signalling and diseases such as cancer. Current techniques in measuring IRP include redox-sensitive fluorescent proteins such as roGFP which is glutathione-specific. Measuring the concentration of one redox couple is, however, not an accurate representation of IRP as it does not necessarily inform about the state of other redox couples. Furthermore, fluorescent biosensors can suffer from photobleaching and may interact with other oxidants. The IRP was measured, in this work, using our newly developed novel-class of surface enhanced Raman scattering nanoparticles which can quantitatively measure the redox potential of cells in vitro. A 'homemade' device was created to keep the cells under fixed pO2 whilst obtaining measurements. The IRP was correlated with the transcriptomic and downstream metabolic profiles of MCF7 breast cancer cells, under perturbed pO2, using 1H NMR spectroscopy (NMR), mass spectrometry (MS) and RNA-sequencing. Discriminatory metabolites were all associated with energy and glucose metabolism. Discriminatory microRNAs were all affiliated with the hallmarks of cancer; the regulation of some is controlled by transcription factors containing redox-sensitive motifs in their DNA binding domains. Multivariate analysis techniques were used to analyse the different data streams in a holistic way that allows the correlation of redox potential, metabolism and transcription.

Current methods to diagnose and treat cancer often involve expensive, time-consuming equipment and materials that may lead to unwanted side effects and may not even increase a patient’s chance of survival. Thus, for a while now, a large part of the research community has focused on developing improved methods to detect, diagnose, and treat cancer on the molecular scale. One of the most recently discovered methods of cancer therapy is targeted therapy. These targeted therapies have potential to provide a patient with a form of personalized medicine because these therapies are biological molecules that specifically target other molecules involved with a cancer’s growth. Past trials using these therapeutic molecules, however, have led to controversial results, where certain patients responded better than others to the therapy for unknown reasons. Elucidating the reason behind these mixed results can be accomplished using metal nanoparticle technologies which could provide a bright signal to monitor the path that these therapeutic molecules take in vivo as well as enhance the molecule’s efficacy. Literature has shown that presenting targeting molecules in a dense manner to their target will increase these molecules’ binding affinity. This concept has been explored here to increase binding affinity of therapeutic molecules by attaching these molecules in a dense manner on the surface of gold nanoparticles, and correlating this increased affinity with therapeutic efficacy. Additionally, gold nanoparticles provide an easy surface for molecules to be functionalized on and have shown to be effective imaging, x-ray, and photothermal therapy agents. A major roadblock to using these gold nanoparticles clinically is their non-degradability and thus potential to cause long-term negative side effects in vivo. A platform for developing biodegradable gold nanoparticles is also explored here to take advantage of the gold nanoparticles’ excellent imaging and drug delivery capabilities while still allowing them to be used safely in the long term. / text