Thesis (M.Sc.Eng.) / Sepsis is a serious medical condition in which a person becomes infected with bacteria in his or her bloodstream. The symptoms of sepsis are a result of the immune system’s interaction with the infecting agent. Currently, to diagnose a patient with sepsis, a blood sample must be collected and cultured for 24-48 hours before the infection can be confirmed. In the meantime, a broad-scope antibiotic is administered which may or may not be effective in treating the patient. If the antibiotic is ineffective, a different antibiotic must be chosen. When the results of the blood culture are available, a narrow scope antibiotic, appropriate to treat the infection is administered. However, sepsis has a mortality rate of 18-30% depending on the infecting agent and the treatment is highly time sensitive. Within 24 hours, the syndrome may progress to septic shock and mortality rates reach 50%. Therefore, it is important to quickly and correctly identify the infecting agent and provide immediate targeted treatment. Surface Enhanced Raman Spectroscopy (SERS) can be used to quickly identify and distinguish between different bacterial strains; however it requires higher bacterial concentrations than are present in the blood during the early stages of sepsis. A microfluidic evaporator chip has been developed to concentrate bacteria samples from 4μl to 100nl; the chip has been evaluated for concentration efficiency on Escherichia coli and methicillin-sensitive Staphylococcus aureus. Various blocking methods using bovine serum albumin (BSA) have been tested to reduce bacterial adhesion to the chip and have improved bacterial recovery to around 70% for both strains tested. Ongoing tests are being performed to improve bacterial recovery and sample purity for identification. / 2031-01-01

Η παρούσα διατριβή εξειδίκευσης αποτελεί το προοίμιο μιας μακρόπνοης εμπλοκής του εργαστηρίου υλοποίησής της στη μελέτη ενδεχόμενης μετανάστευσης ουσιών που χρησιμοποιούνται ως ενισχυτικά φραγής ή/και ως χημικοί αισθητήρες σε βιοπολυμερικές συσκευασίες τροφίμων και αποδέσμευσής τους σε προσομοιωτές τροφίμων. Στο πλαίσιο αυτό, η εργασία αυτή αποτελεί μια προσπάθεια ανάδειξης της μεθόδου επιφανειακής ενίσχυσης της σκέδασης Raman (Surafce Enhanced Raman Scattering) ως κατάλληλης για τον ποσοτικό προσδιορισμό μικρού μοριακού βάρους ενώσεων που ενδεχομένως αποδεσμεύονται σε υδατικά διαλύματα ή/και συγκεκριμένους προσομοιωτές τροφίμων. Η μελέτη εστιάστηκε στην ελεγχόμενη αποδέσμευση φαρμακευτικών ουσιών από μια βιοπολυμερική μήτρα κυρίως κατά το πρώιμο στάδιο της μελέτης και τις παραμέτρους που επηρεάζουν το φαινόμενο αυτό. Βασικός στόχος της μελέτης ήταν η κατά το δυνατό μείωση του ορίου ανίχνευσης με SERS της αποδεσμευόμενης ουσίας με την εμβάπτιση της βιοπολυμερικής μήτρας που την εμπεριέχει σε πρότυπα υδατικά διαλύματα. Πραγματοποιήθηκε μια διεξοδική μελέτη των υποστρωμάτων που χρησιμοποιούνται στο SERS και συγκεκριμένα του νανοκολλοειδούς αργύρου (Ag). Για τον σκοπό αυτό, πραγματοποιήθηκαν πειράματα τα οποία έδειξαν την εξάρτηση της έντασης SERS από τη συσσωμάτωση των υποστρωμάτων Ag συναρτήσει του χρόνου και του παράγοντα συσσωμάτωσης, NaCl. Η εφαρμογή του SERS σε μελέτες ουσιών εξαιρετικά χαμηλών συγκεντρώσεων αναδεικνύεται ως ένα πολύ ενδιαφέρον πεδίο έρευνας. Επίσης, ως πρότυπο πείραμα, παρουσιάζεται μεθοδολογία μελέτης με την τεχνική SERS της αποδέσμευσης του αντικαρκινικού φαρμάκου Μitoxantrone (ΜΤΧ) από εμπορικά ράμματα Maxon. Για το σκοπό αυτό, παρασκευάστηκαν πολυμερικά υμένια με εγκλωβισμένη τη δραστική ουσία και η μελέτη της αποδέσμευσης της σε νερό και PBS (phosphate buffered saline) πραγματοποιήθηκε με SERS και UV-Vis, αντίστοιχα. Η φασματοσκοπία UV-Vis χρησιμοποιήθηκε συμπληρωματικά. Στηριζόμενοι στη μεθοδολογία που αναπτύξαμε εξάγαμε ποσοτικά αποτελέσματα από τρία διαφορετικά εργαστηριακά δείγματα, τα οποία προήλθαν από ανάμιξη εμπορικών ραμμάτων Maxon με 1% κ.β. MTX: (α) στην άμορφη φάση έπειτα από ταχεία ψύξη του τήγματος, (β) στην ημικρυσταλλική φάση με σχετικά χαμηλό ποσοστό κρυσταλλικότητας, που λάβαμε έπειτα από ανόπτηση της άμορφης φάσης για περιορισμένο χρόνο στη θερμοκρασία κρυστάλλωσης και (γ) σε μια επίσης ημικρυσταλλική φάση με αρκετά μεγάλο ποσοστό κρυστάλλωσης (όσης και τα εμπορικά ράμματα). Τα αποτελέσματα δείχνουν πως υπάρχει συσχέτιση μεταξύ κρυσταλλικότητας και αποδέσμευσης του φαρμάκου, με τα μικρότερα ποσά αποδέσμευσης στην περίπτωση του άμορφου δείγματος. Αυτό που παρατηρήθηκε στα πρώιμα στάδια της αποδέσμευσης από τις μετρήσεις SERS φαίνεται να επαληθεύεται από αντίστοιχα αποτελέσματα σε μεταγενέστερα στάδια αποδέσμευσης που λάβαμε με εφαρμογή της συμβατικής τεχνικής απορρόφησης ορατού – υπεριώδους (UV-Vis). Ωστόσο, οι ποσοτικές μετρήσεις με τη χρήση του SERS σε πολύ μικρές συγκεντρώσεις έδειξαν μεγαλύτερη ανιχνευτική ευαισθησία σε σχέση με αυτές που πραγματοποιήθηκαν με την απορρόφηση UV-Vis. Συμπερασματικά, το SERS δείχνει ικανό στον ποσοτικό προσδιορισμό ενεργών ουσιών που αποδεσμεύονται από βιοσυμβατά πολυμερικά συστήματα μεταφοράς δραστικών ουσιών σε πολύ μικρές συγκεντρώσεις. / This thesis of specialization is the precursor of a long-term involvement of the laboratory of Applied Molecular Spectroscopy of FORTH/ICE-HT in the implementation of the study of the migration of substances used as barrier and/or as chemical sensors in biopolymer based food packaging and their release into food simulants. In this context, this work attempts to highlight the method of surface enhanced Raman scattering (SERS) as appropriate for quantifying low molecular weight compounds that may be released in aqueous solutions and/or specific food simulants. The study focused on the controlled release of pharmaceuticals from a biopolymeric matrix mainly during the early stage of the study and the parameters affecting this phenomenon. The main objective of the study was to reduce SERS detection limit of the released substance by emerging the substance-incorporated biopolymeric matrix in standard aqueous solutions. In this context, we developed methods to maximize SERS enhancement and consequently reduce the limit of detection of an active substance, Mitoxantrone (MTX). This was achieved by a thorough study of the substrates used in SERS, namely nanocolloidal silver (Ag) suspensions. For this purpose, we contacted experiments which show the dependence of the SERS intensity on the aggregation of Ag substrates as a function of both time and the aggregating agent, NaCl. Also, as a standard experiment, present SERS methodology was applied in the study of the release of the anticancer drug Mitoxantrone (MTX) from commercially available sutures, Maxon. For this purpose, polymeric films prepared with the encapsulated active substance were immersed either in water or/and in PBS (phosphate buffered saline) and the release of MTX was probed by both SERS and UV-Vis. Based on the developed methodology we obtained quantitative results from three different laboratory samples produced by mixing commercial Maxon sutures with 1 wt% MTX: (a) an almost completely amorphous mixture produced by quenching from the melt, (b) a semi-crystalline one possessing low crystallinity that was produced by annealing the amorphous sample at the temperature close to the crystallization one and (c) a semi-crystalline one possessing high crystallinity similar to the commercial product. The results indicate a correlation between crystallinity and drug release rate; the more amorphous the sample is the less quantity of the drug is released. SERS was able to probe the active agent at the early state of release; UV-Vis has supported these results at a later state of the release process. In conclusion, SERS may enable low concentration quantitative assessment of controlled release of drugs from biopolymer-based delivery systems.

This dissertation comprises experimental studies on the synthesis and applications of metallic nanoshells. These are a class of nanoparticles composed of a dielectric core and a thin metallic shell. Metallic nanoshells play an important role in nanotechnology, particularly in nanomedicine, due to their peculiar optical properties. The overall objectives of the dissertation were to improve the fabrication of these nanoparticles, and to demonstrate new applications of these materials in cancer research and spectroscopy. The fabrication of nanoshells is a multi-step process. Previously to our work, the procedures for the synthesis of nanoshells reported in the literature lacked systematic characterization of the various steps. The procedure was extremely time-consuming and the results demonstrated a high degree of size variation. In Chapter 3, we have developed characterization tools that provide checkpoints for each step of the synthesis. We demonstrated that it is possible to control the degree of coverage on the shell for a fixed amount of reagents, and also showed important differences on the shell growth phase for gold and silver. The synthetic optimization presented in Chapter 3 led to an overall faster protocol than those previously reported. Although the improvements presented in Chapter 3 led to a higher degree of control on the synthesis of nanoshells, the variations in the resulting particle population were still too large for applications in single particle spectroscopy and imaging. In Chapter 4, the synthesis was completely reformulated, aiming to narrow the size distribution of the nanoshell colloids. Through the use of a reverse microemulsion, we were able to fabricate ultramonodisperse silica (SiO2) cores, which translate into nanoshell colloids with narrow extinction bands that are comparable to those of a single nanoshell. We then fabricate a library of colloids with different core sizes, shell thicknesses and composition (gold or silver). The localized surface plasmon resonance (LSPR) of these colloids span across the visible range. From this library, two nanoshells (18nm silver on a 50nm SiO2 core, and 18nm gold on a 72nm SiO2 core) were selected for a proof of principle cell imaging experiment. The silver nanoshells were coated with a nuclear localization signal, allowing it to target the nuclear membrane. The gold nanoshells were coated with an antibody that binds to a receptor on the plasma membrane of MCF-7 human breast cancer cells. The nanoshells were easily distinguishable by eye in a dark field microscope and successful targeting was demonstrated by hyperspectral dark field microscopy. A comparison was made between fluorescent phalloidin and nanoshells, showing the superior photostability of the nanoparticles for long-term cell imaging. The results from Chapter 4 suggest that the nanoshells obtained by our new synthetic route present acceptable particle-to-particle variations in their optical properties that enables single particle extinction spectroscopy for cell imaging. In Chapter 5 we explored the use of these nanoshells for single-particle Surface-enhanced Raman spectroscopy (SERS). Notice that particle-to-particle variations in SERS are expected to be more significant than in extinction spectroscopy. This is because particle-to-particle SERS variabilities are driven by subtle changes in geometric parameters (particle size, shape, roughness). Two types of gold nanoshells were prepared and different excitation wavelengths (λex) were evaluated, respective to the LSPR of the nanoshells. Individual scattering spectra were acquired for each particle, for a total of 163 nanoshells, at two laser excitation wavelengths (632.8 nm and 785 nm). The particle-to-particle variations in SERS intensity were evaluated and correlated to the efficiency of the scattering at the LSPR peak. Chapter 6 finally shows the application of gold nanoshells as a platform for the direct visualization of circulating tumor cells (CTCs). 4T1 breast cancer cells were transduced with a non-native target protein (Thy1.1) and an anti-Thy1.1 antibody was conjugated to gold nanoshells. The use of a transduced target creates the ideal scenario for the assessment of nonspecific binding. On the in vitro phase of the study, non-transduced cells were used as a negative control. In this phase, parameters such as incubation times and nanoshell concentration were established. A murine model was then developed with the transduced 4T1 cells for the ex vivo portion of the work. Non-transduced cells were implanted in a control group. Blood was drawn from mice in both groups over the course of 29 days. Antibody-conjugated nanoshells were incubated with the blood samples and detection of single CTCs was achieved in a dark field microscope. Low levels of nonspecific binding were observed in the control group for non-transduced cells and across different cell types normally found in peripheral blood (e.g. lymphocytes). All positive and negative subjects were successfully identified. Chapter 7 provides an outlook of the work presented here and elaborates on possible directions to further develop the use of nanoshells in bioapplications and spectroscopy. / Graduate / 2019-05-03

A nanofluidic biosensor using surface-enhanced Raman scattering (SERS) was developed to detect the β-amyloid (Aβ) protein, one of the biomarkers of Alzheimer’s disease (AD). Recent studies have indicated that investigating changes in relative concentrations of structure specific Aβ oligomers in cerebral spinal fluid (CSF) during the progression of AD could be important indicators for diagnosing AD pre-mortem. However, there is no definitive pre-mortem diagnosis of AD thus far because of the lack of technology available for sensitive Aβ detection. Hence, the development of a system for detecting the structure specific Aβ oligomers, along with the concentrations of these oligomers in CSF, would be useful in the investigation of the molecular mechanisms of Aβ cytotoxicity associated with AD. In this thesis, a nanofluidic trapping device trapping system for detecting biomolecules at sub-picomolar concentrations was developed for using SERS. The device, with a microchannel leading to a nanochannel, carries out dual functions: encouraging sizedependent trapping of gold nanoparticles (60nm) at the entrance of the nanochannel as well as restricting the target molecules between the gaps created by the aggregated nanoparticles. Initially, the trapping capability of the nanofluidic device was tested using fluorescent polystyrene and gold nanoparticles. UV-vis absorption spectroscopy was used to characterize the gold nanoparticle clusters at the entrance to the nanochannel. The device established controlled, reproducible, SERS active sites within the interstices of gold nanoparticle clusters and shifted the plasmon resonance to the near infrared, in resonance with incident laser light. Two strongly Raman active molecules, adenine and Congo red, were used to test the feasibility of the SERS nanofluidic device as a platform for the detection of multiple analytes. The results showed that strong SERS signals were obtained from the nanoparticle clusters at the nanochannel entrance. Once the feasibility of the approach was determined with strong Raman molecules, Aβ was detected using this nanofluidic SERS platform. Distinct surface-enhanced Raman spectra of Aβ was observed in different conformational states as a function of concentration and structure (monomer versus oligomer form) due to Aβ refolding from α-helical to a predominantly β-pleated sheet form. The sensor was also shown to potentially distinguish Aβ from insulin and albumin, confounder proteins in cerebral spinal fluid. Thus, a novel platform was developed to detect picomoler levels of Aβ with the ultimate goal of facilitating the diagnosis and understanding of Alzheimer’s disease by means of detecting structure specific oligomers of Aβ.

This thesis explores the use of Raman Spectroscopy to study the battery cathode material LiFePO4. Surface Enhanced Raman Spectroscopy (SERS) was incorporated into the study due to fluorescence that traditionally plagues Raman. By imaging LiFePO4 nanoparticles, an understanding can be gained of the complex chemistry taking place when the material is lithiated and delithiated at the nanoscale level and the phase changes of the material that occur during this process. The use of bimetallic (Au/Ag) SERS substrates allowed for more stable substrates with longer shelf life compared single metal Ag substrates. Further tuning of these substrates can be applied to the ever evolving science of energy storage material technology as a way to track phase changes in the material. / text

Pathogenic microbial contamination of the environment poses a significant threat to human health. Accordingly, microbial surveillance is needed to ensure safe drinking water and air quality. Current analytical methods for microbes are generally either culture-based, gene amplification-based, or sequencing-based. However, these approaches require centralized facilities, well-trained personnel, and specialized instruments that result in high costs and long turnaround times. Surface-enhanced Raman spectroscopy (SERS)-based techniques have been proposed to overcome these limitations. In this dissertation, we discuss work conducted to develop novel SERS-based methods to enable both sensitive microbial quantification and analysis of the interactions of pathogens, their hosts, and the surrounding environment. We first developed a labeled SERS-based lateral flow test for virus quantification. Optimization of the lateral flow design and digital signal analysis enabled high sensitivity towards SARS-CoV-2. To elicit a comprehensive understanding of pathogen infection, label-free living-cell SERS sensors were engineered by incubating host cells with nanoparticles. SERS spectral changes in host cellular components and metabolites during infection were used for viral quantification and offered inherent insights into the temporal and spatial molecular-level mechanisms of infection. These biosensors were validated using bacteriophage Phi6 and then developed for infectious H1N1 influenza. To understand microbial survival in the environment, living-cell SERS methods were applied under various conditions. Results showed cell inactivation and antibiotic treatment induced significant cellular and metabolic responses in the living whole-cell sensors, implying their potential applicability to various environmental conditions. Our research achieves rapid and on-site pathogen quantification and infection mechanism identification. / Doctor of Philosophy / Pathogenic microbes, such as the SARS-CoV-2 virus, can spread through air and water and are potentially harmful to human health. Monitoring the concentrations of these microbes in the environment is crucial to track their presence and provide an early warning of their spread. Unfortunately, current microbial detection methods are often expensive and take a long time since they typically require professional facilities and expert elicitation. Our research relies on a technique called surface-enhanced Raman spectroscopy (SERS) to address these challenges. SERS enables identification and quantification of microbes by analyzing specific features (i.e., peak position, peak intensity) in the spectra. We first applied this technique by modifying a commercial SARS-CoV-2 antigen test kit with a label molecule that provides SERS signals. We achieve accurate and sensitive quantification, even in the presence of high levels of environmental interference. To better understand how these harmful microbes interact with our bodies, we developed sensors that can measure SERS signal changes in host cells before and after infection. These sensors were tested using the bacteriophage virus Phi6 that infects bacteria and infectious H1N1 influenza virus. Furthermore, we applied these sensors to study how bacteria respond to different environmental conditions, providing valuable insights into their survival and behavior under various conditions. In summary, our research introduces methods that are more accessible to identify and quantify harmful microbes that can be potentially used by the general public. The methods provide us with molecular level understanding of pathogen interactions with humans and the environment.

The interaction between nanoscience and biomedicine is one of the important developing areas of modern science. The usage of functional nanoparticles with biological molecules provides sensitive and selective detection, labeling and sensing of biomolecules. Until today, several novel types of tagging materials have been used in bioassays, such as plasmon-resonant particles, quantum dots (QDs), and metal nanoshells. However, nowadays, Surface enhanced raman scattering (SERS) tags have been attracting considerable attention as a tagging system. SERS-tags provide high signal enhancement, and they enable multiplex detection of biomolecules due to high specificity. This thesis is focused on the designing proper SERS nanotags for DNA studies. SERS nano-tags are nanostructures consisting of core nanoparticle generally silver, Raman reporter molecule for labeling, and shell to make surface modifications and to prevent deterioration arising from environmental impact. Based on this information, silver core synthesized by thermal decomposition and chemical reduction methods. Thermal decomposition method provides synthesis of silver nanoparticles in hydrophobic medium, resulting in proper silica coating by reverse microemulsion method. On the other hand, silver nanoparticles sythesized by chemical reduction method exhibit hydrophilic property. Due to capping reagents, negatively charged silver nanoparticles could easily attach with positively charged Raman dye which is brilliant cresyl blue (BCB). After addition of Raman active molecule, silica coating process was done by using modified St&ouml / ber method. The resulting particles were characterized by Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX) ,UV-vis Spectrometry (UV-vis) and Surface-Enhanced Raman Spectroscopy (SERS). In recent years, DNA detection has gained importance for cancer and disease diagnosis and the detection of harmful microorganisms in food and drink. In this study, gene sequences were detected via SERS. For this, probe sequences were labelled with Raman reporter molecule, BCB,and SERS nano-tags and were called as SERGen probes. Then, after hybridization of DNA targets to complementary probe sequences onto gold substrate, SERS peak was followed.

The propagation of contaminants in the environment continues to threaten public health and safety. Conventional analytical techniques for environmental detection require centralized facilities and intensive resources for operation. An effective implementation of a wide network of field deployable point-of-use (POU) sensors can potentially enable real-time monitoring of water quality parameters and inform decision making on public health outbreaks. The use of nanotechnology and field-deployable analytical tools can potentially advance the development of POU sensors for future field application. In this dissertation, we developed environmental sensing techniques that utilize nanocomposites made of low-cost, biocompatible, and sustainable nanomaterials combined with Raman spectroscopy. First, a technology pre-assessment was performed that included a comprehensive evaluation of cellulose-derived nanocomposites and nanobiotechnology enabled techniques for their sustainable long-term environmental application. Furthermore, to contribute to the better understanding of the potential environmental implications of nanomaterial production and application, life cycle assessment (LCA) was used to evaluate the environmental impacts of six iron precursors and seven iron oxide nanoparticle synthesis methods. Secondly, in the technology development step, gold (Au) and iron oxide (Fe3O4) nanoparticles were incorporated onto bacterial cellulose nanocrystals and nanoscale magnetite were synthesized. As proof-of-concept environmental applications, the Au@Fe3O4@BCNCs were applied for the magnetic separation and surface-enhanced Raman scattering (SERS) detection of malachite green isothiocyanate (MGITC), and nanoscale magnetite were applied for phosphate (PO43-) removal and recovery from synthetic urine matrices. Finally, in the technological application step, three environmental sensing applications are presented that use nanomaterial-based sensor platforms and/or Raman spectroscopic techniques. The first application involved using Lectin-modified BCNCs coupled SERS and machine learning for discrimination of bacterial strains. The second application presents a simple Raman-stable isotope labeling approach for the study of viral infection of bacteria. The third application involved use of SERS pH nanoprobes for measuring pH in droplets of complex matrices (e.g., DMEM cell culture media, human saliva). / Doctor of Philosophy / The current generation of analytical tools for environmental detection rely upon centralized facilities and intensive resources for operation. The combination of nanotechnology and field deployable analytical tools can aid in the development of point-of-use (POU) sensors for field monitoring of environmental contaminants. In this dissertation, we combined low-cost, biocompatible, and sustainable nanomaterials with Raman spectroscopy-based techniques to develop potentially field-deployable environmental sensing techniques. First, a technology pre-assessment was performed which involved a comprehensive evaluation of cellulose-derived nanocomposites and nanobiotechnology enabled techniques for their sustainable long-term environmental application. Furthermore, life cycle assessment (LCA) was used to evaluate the environmental impacts of iron oxide nanoparticle synthesis methods to better understand environmental impacts of nanoparticle production. Secondly, in the technology development step, we developed the nanocomposites: Au and Fe3O4 nanoparticles incorporated bacterial cellulose nanocrystals and nanoscale magnetite. As proof-of-concept environmental applications, the Au@Fe3O4@BCNCs were used for the detection of malachite green isothiocyanate (MGITC), and the nanoscale magnetite were used for phosphate (PO43-) removal and recovery from synthetic urine. Finally, in the technological application step, (1) selective detection of bacteria was performed using lectin-modified BCNCs as SERS biosensors coupled with SERS and machine learning. (2) Viral infection of bacteria was evaluated using Raman spectroscopy and Deuterium isotope labeling, and (3) pH in micro-droplets of DMEM cell culture media and human saliva were observed using SERS pH nanoprobes.

Intracellular redox potential (IRP) is a measure of how oxidising or reducing the environment is within a cell. It is a function of numerous factors including redox couples, antioxidant enzymes and reactive oxygen species. Disruption of the tightly regulated redox status has been linked to the initiation and progression of cancer. However, there is very limited knowledge about the quantitative nature of the redox potential and pH gradients that exist in cancer tumour models. Multicellular tumour spheroids (MTS) are three-dimensional cell cultures that possess their own microenvironments, similar to those found in tumours. From the necrotic core to the outer proliferating layer there exist gradients of oxygen, lactate, pH and drug penetration. Tumours also have inadequate vasculature resulting in a state of hypoxia. Hypoxia is a key player in metabolic dysregulation but can also provide cells with resistance against cancer treatments, particularly chemotherapy and radiation therapy. The primary hypoxia regulators are HIFs (Hypoxia Inducible Factors) which under low O2 conditions bind a hypoxia response element, inhibiting oxidative phosphorylation and upregulating glycolysis which has two significant implications: the first is an increase in levels of NADPH/NADH, the main electron donors found in cells which impacts the redox state, whilst the second is a decrease in intracellular pH (pHi) because of increased lactate production. Thus, redox state and intracellular pHi can be used as indicators of metabolic changes within 3D cultures and provide insight into cellular response to therapy. Surface-Enhanced Raman Spectroscopy (SERS) provides a real-time, high resolution method of measuring pHi and IRP in cell culture. It allows for quick and potentially portable analysis of MTS, providing a new platform for monitoring response to drugs and therapy in an unobtrusive manner. Redox and pH-active probes functionalised to Au nanoshells were readily taken up by prostate cancer cell lines and predominantly found to localise in the cytosol. These probes were characterised by density functional theory and spectroelectrochemistry, and their in vitro behaviour modelled by the chemical induction of oxidative and reductive stress. Next, targeting nanosensors to different zones of the MTS allowed for spatial quantification of redox state and pHi throughout the structure and the ability to map the effects of drug treatments on MTS redox biology. The magnitude of the potential gradient can be quantified as free energy (ΔG) and used as a measurement of MTS viability. Treatment of PC3 MTS with staurosporine, an apoptosis inducer, was accompanied by a decrease in free energy gradients over time, whereas treatment of MTS with cisplatin, a drug to which they are resistant, showed an increase in viability indicating a compensatory mechanism and hence resistance. Finally, using this technique the effects of ionising radiation on IRP and pHi in the tumour model was explored. Following exposure to a range of doses of x-ray radiation, as well as single and multi-fractionated regimes, IRP and pHi were measured and MTS viability assessed. Increased radiation dosage diminished the potential gradient across the MTS and decreased viability. Similarly, fractionation of a single large dose was found to enhance MTS death. This novel SERS approach therefore has the potential to not only be used as a mode of drug screening and tool for drug development, but also for pre-clinical characterisation of tumours enabling clinicians to optimise radiation regimes in a patient-specific manner.

Η χρήση των πολυμερών στον τομέα της ιατρικής βιομηχανίας κερδίζει ολοένα και μεγαλύτερο έδαφος τα τελευταία χρόνια έχοντας ήδη κάνει ισχυρή την παρουσία τους σε ένα ευρύ πεδίο κλάδων της βιοϊατρικής όπως στη μηχανική ιστών, στην εμφύτευση ιατρικών συσκευών και τεχνητών οργάνων, στην προσθετική και την οφθαλμολογία, στην οδοντιατρική και την αποκατάσταση οστών, στη χημειοθεραπεία και σε ποικιλία άλλων ιατρικών εφαρμογών. Με τη χρήση πολυμερικών συστημάτων μεταφοράς δραστικών ουσιών καθίσταται ικανή η ελεγχόμενη αργή αποδέσμευση φαρμάκων στο σώμα καθώς και η στοχευμένη απελευθέρωσή τους σε σημεία όπου υπάρχουν φλεγμονές ή όγκοι. Τοιουτοτρόπως, χημειοθεραπείες με χρήση βιοπολυμερών ως διαμεσολαβητές, προβάλλουν ως δυνητικές υποψήφιοι στην αντιμετώπιση του καρκίνου του εγκεφάλου με ενθαρρυντικά αποτελέσματα. Συγκρινόμενη με την τυπική συστημική χημειοθεραπεία, η ενδοογκική απελευθέρωση φαρμάκου με τη χρήση βιοπολυμερών θεωρητικώς παρουσιάζει αρκετά πλεονεκτήματα: τα βιοπολυμερή μπορούν να μεταφέρουν το φάρμακο απευθείας στον όγκο-στόχο αυξάνοντας τη συγκέντρωση τοπικά και παράλληλα μειώνοντας τη συστημική τοξικότητα· μπορούν έτσι να χρησιμοποιούνται στη θεραπεία ανοσοκατασταλμένων ασθενών που δεν μπορούν να υποβληθούν σε συστημική χημειοθεραπεία. Από τη στιγμή που είναι απαραίτητη η ποσοτικοποίηση των φαρμάκων για τον χαρακτηρισμό των συστημάτων αποδέσμευσης και για μελέτες φαρμακοκινητικής, θα πρέπει να επιλέγεται η καταλληλότερη μέθοδος ποσοτικοποίησης παρέχοντας υψηλή ευαισθησία και ακρίβεια, εξασφαλίζοντας μεγάλη ανιχνευτική ικανότητα ακόμη και για πολύ χαμηλές συγκεντρώσεις. Στην παρούσα εργασία δύο αναλυτικές τεχνικές, η απορρόφηση υπεριώδους-ορατού και η επιφανειακή ενίσχυση της σκέδασης Raman (Surface Enhanced Raman Scattering, SERS), χρησιμοποιήθηκαν για την ποσοτική εκτίμηση του αντινεοπλασματικού φαρμάκου Mitoxantrone και του αντιμυκητιακού παράγοντα Ambisome (Αμφοτερισίνη Β) που αποδεσμεύτηκαν από βιοσυμβατές πολυμερικές μήτρες συμπολυμερούς αιθυλενίου-οξικού βινυλεστέρα, συμπολυμερούς γλυκολικού-γαλακτικού οξέος και πολυπροπυλενίου. Το SERS είναι ένα νέο, εναλλακτικό, ταχύ και μη καταστροφικό εργαλείο που μπορεί να βρεί εφαρμογή και στην ποσοτική εκτίμηση ουσιών πάρα πολύ χαμηλών συγκεντρώσεων. Χάρις στην ενίσχυση που παρέχεται στο σήμα Raman από τα νανο-εκτραχυμένα υποστρώματα ευγενών μετάλλων ή τα νανο-συσσωματώματα κολλοειδών διαλυμάτων ευγενών μετάλλων, έχει αναφερθεί ακόμη και συλλογή φάσματος SERS από ένα μόνο μόριο. Συνεπώς, η εφαρμογή του SERS σε μελέτες ουσιών εξαιρετικά χαμηλών συγκεντρώσεων φαίνεται να είναι πολύ ενδιαφέρουσα. Κατασκευάστηκαν πολυμερικά υμένια με εγκλωβισμένες τις δραστικές ουσίες και η μελέτη αποδέσμευσης πραγματοποιήθηκε σε νερό. Ποσοτικές μετρήσεις με τη χρήση του SERS σε πολύ μικρές συγκεντρώσεις έδειξαν μεγαλύτερη ανιχνευτική ευαισθησία σε σχέση με αυτές που πραγματοποιήθηκαν με την απορρόφηση UV-Vis. Συμπερασματικά, το SERS δείχνει ικανό στον ποσοτικό προσδιορισμό ενεργών ουσιών που αποδεσμεύονται από βιοσυμβατά πολυμερικά συστήματα μεταφοράς δραστικών ουσιών σε πολύ μικρές συγκεντρώσεις. / The application of polymeric materials for medical purposes is growing very fast. Polymers have found applications in such diverse biomedical fields as tissue engineering, implantation of medical devices and artificial organs, prosthesis, ophthalmology, dentistry, bone repair, chemotherapy and many other medical fields. Polymer-based delivery systems enable controlled slow release of drugs into the body and also they make possible targeting of drugs into sites of inflammation or tumors. Thus, biopolymer-mediated chemotherapy has shown promising results in the treatment of brain tumors. When compared to conventional systemic chemotherapy, intratumoral biopolymer-mediated drug delivery has several theoretical advantages: Biopolymers can deliver drugs into the tumor bed, thus maximizing local concentration while minimizing systemic toxicity. They may therefore be employed in the treatment of immunodepressed patients etc. Since drugs need to be quantified for drug delivery system characterization, intracellular distribution studies, free or vehicular, and for pharmacokinetic assays, the most suitable quantification method must be chosen. It should have a high sensitivity, specificity and reproducibility and should be capable of measuring at very low concentration range, as well. In the present study, two analytical techniques are utilized to quantitatively evaluate the antineoplastic drug Mitoxantrone and the antifungal agent Ambisome (Amphotericin b) released from active agents-loaded biocompatible polymer matrices poly(propylene), poly(ethylene-co-vinyl acetate), poly(lactic-co-glycolic acid); the UV-Vis absorption and the Surface Enhance Raman Scattering (SERS). SERS is a new, versatile, fast and non destructive tool for the estimation of extremely small amounts of substances. Due to the enhancement provided to the Raman signal by the nano-rough noble-metal substrates or the nano-structured colloidal clusters of noble metals, even single molecule detection has been reported. Therefore, applying SERS to extremely low concentration measurements proves to be challenging. Drug loaded polymer specimens were prepared and the in vitro drug release was determined in water. Fast SERS quantitative measurements showed enhanced sensitivity compared to the UV-Vis absorption; SERS may enable low concentration quantitative assessment of controlled release of drugs from biopolymer-based delivery systems.