Dissertations / Theses on the topic 'Surface Functionalisation'

Colloidally-stable inorganic nanocrystals have a wide range of envisaged applications in biological environments. To reach their potential, the nanocrystals need to be stable in aqueous environments and have pendant functionality available for attachment of biomolecules. In this thesis, new methods for the transfer of nanocrystals from organic to aqueous media are developed and the interaction of aqueous stabilised particles with serum proteins is investigated. In Chapter 3, a new method for the synthesis of a thin silica layer upon the surface of nanocrystals is demonstrated. The method uses the hydrophobic interaction between an amphiphilic polymer and nanocrystal ligands to provide a foundation for growth of a silica layer. The coated nanocrystals are characterised using a wide range of techniques confirming that the presence and location of the silica shell. In Chapter 4, custom-synthesised amphiphilic polymers for water transfer and functionalisation of nanocrystals are synthesised, characterised and tested. Commercially-available polymers used for this purpose are examined, leading to a rationale for custom-design. Partial water transfers were achieved using activated ester copolymers with styrene but no transfers were achieved the octadecylacrylate copolymers. Poly(ethylene glycol) containing monomers were also used but yielded no transfers. This suggests that behaviour of the polymer during the coating procedure is intimately linked to the structure of the polymer. In Chapter 5, small-angle neutron scattering is used to elucidate structural information for the protein corona formed on nanocrystals and silica nanoparticles. Information on the packing of ligands on colloidal nanocrystals without a amphiphilic polymer coating was determined. The fitting of the protein corona upon silica nanoparticles was explored using core-shell form factors but was hampered by complexities within the scattering profiles which were not accounted for using simple form factors.

Pulsed plasmas were investigated as a means of controlling the composition of the surfaces generated via plasma polymerisation. A variety of precursors were studied under a range of plasma conditions using both continuous wave and pulsed plasmas. Surface and bulk analytical techniques were used to characterise the deposited plasma polymers whilst deposition rate measurements aided in understanding the effects of altering the various plasma parameters. Continuous wave plasma polymerisation of saturated cyclic fluorocarbons yielded plasma polymers with high fluorine/carbon ratios. Plasma instability at low powers limits the extent to which continuous wave power can be used to achieve good selectivity in the polymerisation process. Pulsed plasma polymerisation of perfluoroallylbenzene was studied in detail to investigate the influence of pulsing parameters on the surface composition. Highly aromatic surfaces were obtained through retention of the perfluorophenyl group from the precursor. Deposition rate experiments confirmed polymerisation was taking place in the off-portion of the duty cycle for precursors with a functional group susceptible to radical initiated reactions. A cyclic siloxane precursor with vinyl substituents was used to generate surface consisting of siloxane rings in an organic matrix. The monomer structure was retained through the reaction of the vinyl groups in the off-portion of the duty cycle. For low duty cycle pulsed plasma polymers the Si:0 ratio of the plasma polymers was identical to that of the monomer, indicating successflil retention of monomer structure using pulsed plasmas. Preliminary investigations into the pulsed plasma polymerisation of styrene oxide yielded a range of polymer compositions with varying oxygen contents. The properties of the surfaces varied with oxygen content. The results indicate that pulsed plasmas can give significant enhancements over continuous wave plasmas in controlling surface composition and properties.

Chapter 1 includes a review on dendrimers, their synthesis and applications, with a particular focus on urea-linked dendritic species. The synthetic strategy utilised in this body of work was based on the preparation of a number of branched synthetic building blocks possessing differing terminal functionality. These branched dendrons, bearing three terminal residues and based on the cheap starting material tris(hydroxymethyl)aminomethane (TRIS) 23, involved the coupling of 3.3 equivalents of an appropriately para-substituted benzoic acid chloride with BOC protected TRIS 24 in DCM in the presence of triethylamine. The p-nitro, p-methoxy and p-methyl benzoyl chloride starting materials were obtained commercially, whilst N-(4-carboxyphenyl)maleimide was synthesised according to literature procedures. The BOC protected dendrons (25–27, 34) were synthesized in yields ranging from 50–92%. Deprotection of the BOC protected dendrons 25 and 26 in DCM with TFA, followed by the addition of 1M Na2CO3 afforded the TFA salts 35 and 36, respectively. The corresponding free base amines 37 and 38 were obtained on further treatment of the TFA salts with sodium carbonate. Deprotection of the BOC protected dendrons 27 and 34 afforded the free amines 39 and 48 directly after treatment with sodium carbonate. Synthesis of functionalised branched molecules containing 6- and 9-peripheral functionalities was achieved by refluxing 2 or 3 equivalents of the free amine dendrons with the bi- or tri- functional isocyanate cores, 15 and 45, in refluxing DCM, in most cases the products precipitated from the reaction mixture after 18 h and were isolated simply by filtration, otherwise the removal of the solvent from the reaction mixture afforded the spectroscopically pure product. Conversion of the peripheral nitro functionalised species 14 and 21 to the corresponding amines occurred smoothly via hydrogenation using 5% Pd/C under elevated temperature and pressure (DMF, 55 ºC, 600 psi) and afforded the polyamine 6-mer 51 in 92% yield and the 9-mer 50 in 90% yield, respectively. Similarly, conversion of the methoxy coated 9-mer 42, to the corresponding phenolic compound (AlBr3, dodecane thiol, DCM) afforded the 9-mer polyphenol 52 in an 87% yield. All compounds prepared were fully characterised and crystal structures were obtained for 26 and 35. Chapter 2 includes a review on self-assembled monolayers of organosulfur compounds on gold, applications, patterning techniques and techniques for the characterisation of these surfaces. A number of surface monomers were successfully synthesized, to be used for various surface functionalisations, including the formation of an amine reactive N-hydroxysuccinimide (NHS) disulfide 53, via the DCC coupling of 11,11’-dithiobisundecanoic acid 54 with N-hydroxysuccinimide with an isolated yield of 30%. A novel protein-resistant monomer 58 was also synthesized from 11-undecanoic acid 55 via an acid chloride coupling with triethylene glycol monomethyl ether 58, and isolated in a 72% yield. A number of attempts were made to produce an acyl azide SAM monomer 59, with success finally achieved via the acid chloride coupling of 11,11’-dithiobisundecanoic acid 54 with 5-amino-1,3-benzenedicarbonyl diazide 62 to produce 59 with an isolated yield of ~ 30%. Gold surfaces were prepared on atomically flat silicon wafers using an argon-ion sputterer. SAM films were formed on the gold surfaces via traditional solution based self-assembly methodology. A UV patterning protocol was developed, and a successful patterning trial using the NHS terminated monomer to backfill the UV exposed areas of a dodecane thiol monolayer was achieved and visualized using AFM and fluorescence microscopy after treating the surface with aminofluorescein. The covalent attachment of green fluorescent protein to the monolayer surface via reaction with the NHS terminated monolayer was demonstrated. The fluorescence of the biomolecule was preserved. The formation of a monolayer using the acyl azide monomer 59, was characterised by contact angle and XPS analysis. However, preliminary studies into the activation of the acyl azide surface into the reactive isocyanate were unsuccessful. There is however, significant scope for further investigations into this interesting surface technology. Chapter 3 includes a review on heterobifunctional linker technology with a particular focus on amine and thiol reactive moieties and literature examples of heterobifunctional linkers of this type. Synthesis of heterobifunctional reagents such as 71 and 74 via a two step synthetic methodology involving the coupling of maleic anhydride with the parent amino-acids in acetic acid, followed by a one pot cyclisation and NHS esterification using DCC in DMF were successful, with overall yields of 9% and 32% respectively for the two reaction steps. The one pot extension of 74 with 6-aminohexanoic acid, followed by DCC, facilitated NHS esterification was achieved successfully in a yield of 30%. Attempts to extend 74 with the synthesised amino acid 88 were unsuccessful due to the insolubility of 88 in organic solvents. A different synthetic strategy was devised towards the synthesis of 85 with the coupling of 74 and mono BOC protected ethylene diamine 91 in DCM to give 93 in an isolated yield of 60%. Deprotection of the terminal amine was achieved via reaction with TFA in DCM however all attempts to prepare the free amine were unsuccessful. Subsequent attempts to couple 94 with both succinic anhydride and 92 were unsuccessful. A maleimide functionalized crown ether was synthesised as a molecule for protein modification via the reaction of 74 with 4’-aminobenzo-15-crown-5 97 to produce 98 in an 80% yield. All compounds were fully characterised with crystal structures obtained for 74, 79 and 89.

The surface functionalisation of polyethylene and polypropylene by industrial and laboratory scale corona treatments and by laboratory flame treatment was studied. The surface sensitive techniques of X-ray photoelectron spectroscopy (XPS), attenuated total reflection infra-red spectroscopy (FTIR-ATR), contact angle measurement and electron microscopy (SEM and TEM) were employed. Corona and flame treatments resulted in incorporation of oxygen only into the surfaces of both polyethylene and polypropylene, resulting in improved surface wettabilities. A variety of oxygen functional groups were introduced by the two treatments. The industrial and laboratory scale treatments of both polymers were found to be similar in terms of the oxygen concentrations incorporated and surface wettabilities achieved. The presence of significant amounts of chain scission products were indicated on corona treated surfaces, while only minimal quantities were indicted on flame treated surfaces. This was attributed to their volatilisation during flame treatment. Introduction of sulfur dioxide into the flame and corona regions during treatment resulted in significant improvements in surface wettability. Incorporation of sulfur and nitrogen resulted from the presence of sulfur dioxide. A possible mechanism involving the formation of sulfonic acid groups and ammonium sulfonate groups was suggested. An oxidation depth model developed for use with variable take-off angle XPS showed that significantly deeper oxidation occurred in the presence of sulfur dioxide. Corona treatment was more effective in improving surface wettabilities than flame treatment, this being attributed to heat induced functional group reorientation during flame . treatment for polyethylene and to differences in surface chemistry resulting from the two treatments in the case of polypropylene. The surface wettability of poly ethylene was more readily improved than the surface wettability of polypropylene after all the treatments investigated. A method for estimating functional group concentrations using chemical derivatisation and contact angle measurement was developed. Functional group estimates for flame treated polyethylene were found to be in good agreement with chemical derivatisation used in conjunction with XPS measurements.

Understanding human mesenchymal stem cell (hMSC) adhesion and differentiation in three-dimensional matrices in vitro is important for potential regenerative medicine and stem cell therapies. One of the key requirements for the use of scaffolds is that they correctly display the physicochemical properties mimicking those of the native extracellular matrix (ECM), in particular adhesive heterogeneity. Previous studies in 2D provide evidence for the effects of matrix properties such as stiffness, topography and surface chemistry. Yet, there are very few examples to date where ECM heterogeneity has been recapitulated and its effects on hMSCs investigated. The main aim of this research was to design topologically defined three-dimensional porous scaffolds. This was achieved by exploiting the self-assembly of amphiphilic diblock copolymers confined at an interface. Two methods of scaffold fabrication were used in these studies; high internal phase emulsion (HIPE) templating and electrospinning. In both studies, mixtures of two amphiphilic block copolymers were used to induce phase separation between the dissimilar hydrophilic blocks thereby creating distinct copolymer domains in the nanometer length scales on the scaffold surface. In both scaffold fabrication methods the amphiphilic block copolymers used were a combination of cell inert and cell adhesive chemistries, thereby generating matrices with distinct cell binding sites. The functionality and adhesive heterogeneity of these materials were characterised using varying techniques including x-ray photoelectron spectroscopy, chemical force spectroscopy mapping and contact angle measurements. The effect of adhesive heterogeneity of such matrices on human mesenchymal progenitor adhesion and differentiation based on block copolymer domains were investigated. It was found that hMSCs adhered in a block copolymer dependent manner to scaffolds that most closely mimicked the adhesive heterogeneity in native extracellular matrix.

Chapter 1 includes a review on dendrimers, their synthesis and applications, with a particular focus on urea-linked dendritic species. The synthetic strategy utilised in this body of work was based on the preparation of a number of branched synthetic building blocks possessing differing terminal functionality. These branched dendrons, bearing three terminal residues and based on the cheap starting material tris(hydroxymethyl)aminomethane (TRIS) 23, involved the coupling of 3.3 equivalents of an appropriately para-substituted benzoic acid chloride with BOC protected TRIS 24 in DCM in the presence of triethylamine. The p-nitro, p-methoxy and p-methyl benzoyl chloride starting materials were obtained commercially, whilst N-(4-carboxyphenyl)maleimide was synthesised according to literature procedures. The BOC protected dendrons (25–27, 34) were synthesized in yields ranging from 50–92%. Deprotection of the BOC protected dendrons 25 and 26 in DCM with TFA, followed by the addition of 1M Na2CO3 afforded the TFA salts 35 and 36, respectively. The corresponding free base amines 37 and 38 were obtained on further treatment of the TFA salts with sodium carbonate. Deprotection of the BOC protected dendrons 27 and 34 afforded the free amines 39 and 48 directly after treatment with sodium carbonate. Synthesis of functionalised branched molecules containing 6- and 9-peripheral functionalities was achieved by refluxing 2 or 3 equivalents of the free amine dendrons with the bi- or tri- functional isocyanate cores, 15 and 45, in refluxing DCM, in most cases the products precipitated from the reaction mixture after 18 h and were isolated simply by filtration, otherwise the removal of the solvent from the reaction mixture afforded the spectroscopically pure product. Conversion of the peripheral nitro functionalised species 14 and 21 to the corresponding amines occurred smoothly via hydrogenation using 5% Pd/C under elevated temperature and pressure (DMF, 55 ºC, 600 psi) and afforded the polyamine 6-mer 51 in 92% yield and the 9-mer 50 in 90% yield, respectively. Similarly, conversion of the methoxy coated 9-mer 42, to the corresponding phenolic compound (AlBr3, dodecane thiol, DCM) afforded the 9-mer polyphenol 52 in an 87% yield. All compounds prepared were fully characterised and crystal structures were obtained for 26 and 35. Chapter 2 includes a review on self-assembled monolayers of organosulfur compounds on gold, applications, patterning techniques and techniques for the characterisation of these surfaces. A number of surface monomers were successfully synthesized, to be used for various surface functionalisations, including the formation of an amine reactive N-hydroxysuccinimide (NHS) disulfide 53, via the DCC coupling of 11,11’-dithiobisundecanoic acid 54 with N-hydroxysuccinimide with an isolated yield of 30%. A novel protein-resistant monomer 58 was also synthesized from 11-undecanoic acid 55 via an acid chloride coupling with triethylene glycol monomethyl ether 58, and isolated in a 72% yield. A number of attempts were made to produce an acyl azide SAM monomer 59, with success finally achieved via the acid chloride coupling of 11,11’-dithiobisundecanoic acid 54 with 5-amino-1,3-benzenedicarbonyl diazide 62 to produce 59 with an isolated yield of ~ 30%. Gold surfaces were prepared on atomically flat silicon wafers using an argon-ion sputterer. SAM films were formed on the gold surfaces via traditional solution based self-assembly methodology. A UV patterning protocol was developed, and a successful patterning trial using the NHS terminated monomer to backfill the UV exposed areas of a dodecane thiol monolayer was achieved and visualized using AFM and fluorescence microscopy after treating the surface with aminofluorescein. The covalent attachment of green fluorescent protein to the monolayer surface via reaction with the NHS terminated monolayer was demonstrated. The fluorescence of the biomolecule was preserved. The formation of a monolayer using the acyl azide monomer 59, was characterised by contact angle and XPS analysis. However, preliminary studies into the activation of the acyl azide surface into the reactive isocyanate were unsuccessful. There is however, significant scope for further investigations into this interesting surface technology. Chapter 3 includes a review on heterobifunctional linker technology with a particular focus on amine and thiol reactive moieties and literature examples of heterobifunctional linkers of this type. Synthesis of heterobifunctional reagents such as 71 and 74 via a two step synthetic methodology involving the coupling of maleic anhydride with the parent amino-acids in acetic acid, followed by a one pot cyclisation and NHS esterification using DCC in DMF were successful, with overall yields of 9% and 32% respectively for the two reaction steps. The one pot extension of 74 with 6-aminohexanoic acid, followed by DCC, facilitated NHS esterification was achieved successfully in a yield of 30%. Attempts to extend 74 with the synthesised amino acid 88 were unsuccessful due to the insolubility of 88 in organic solvents. A different synthetic strategy was devised towards the synthesis of 85 with the coupling of 74 and mono BOC protected ethylene diamine 91 in DCM to give 93 in an isolated yield of 60%. Deprotection of the terminal amine was achieved via reaction with TFA in DCM however all attempts to prepare the free amine were unsuccessful. Subsequent attempts to couple 94 with both succinic anhydride and 92 were unsuccessful. A maleimide functionalized crown ether was synthesised as a molecule for protein modification via the reaction of 74 with 4’-aminobenzo-15-crown-5 97 to produce 98 in an 80% yield. All compounds were fully characterised with crystal structures obtained for 74, 79 and 89.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Full Text

In this thesis work nanoscrystalline optical properties of diamond and two recent new NCD functionalisation techniques involving UV light (one step method and photochemical oxidation) have been investigated. Firstly the oxidation of diamond surface caused by the irradiation of the surface with UV-light in oxygen atmosphere was considered. Two different experiments in situ were realized in order to understand the physic-chemistry of this method. The chemical bonds between oxygen and surface carbon atoms were investigated by firstly performing an annealing treatment in ultra hight vacuum of a oxidized UV surface and then comparing the obtained result with annealing treatments of two different oxygenated diamond surfaces using other two techniques: plasma oxidation and piranha solution oxidation. An other interesting aspect on which clarity has to be made deal with amination process of diamond surface. As a first fundamental step, the efficiency on hydrogenated diamond surface was investigate. Successively the role of oxygen in the chemistry of amination process was studied performing in situ experiments using different terminated diamond surface (hydrogenated, chemically oxidized, UV oxidized) and different gaese (pure NH3 or NH3 + O2).

In this thesis work nanoscrystalline optical properties of diamond and two recent new NCD functionalisation techniques involving UV light (one step method and photochemical oxidation) have been investigated. Firstly the oxidation of diamond surface caused by the irradiation of the surface with UV-light in oxygen atmosphere was considered. Two different experiments in situ were realized in order to understand the physic-chemistry of this method. The chemical bonds between oxygen and surface carbon atoms were investigated by firstly performing an annealing treatment in ultra hight vacuum of a oxidized UV surface and then comparing the obtained result with annealing treatments of two different oxygenated diamond surfaces using other two techniques: plasma oxidation and piranha solution oxidation. An other interesting aspect on which clarity has to be made deal with amination process of diamond surface. As a first fundamental step, the efficiency on hydrogenated diamond surface was investigate. Successively the role of oxygen in the chemistry of amination process was studied performing in situ experiments using different terminated diamond surface (hydrogenated, chemically oxidized, UV oxidized) and different gaese (pure NH3 or NH3 + O2).

In this work, the modification of conducting substrates with thin (nanometer thick) aryl films via reaction with aryldiazonium salts was investigated. Two methods were used: modification by electro-reduction of the aryldiazonium salts and modification by spontaneous reaction of aryldiazonium the salts with the surface at open circuit potential. The majority of the studies were undertaken using p-nitrobenenze diazonium salt, which gives electro-active nitrophenyl (NP) films at the surface that can be detected and characterized by cyclic voltammetry. Films prepared spontaneously on carbon and gold electrodes at open circuit potential were characterized by electrochemistry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. At both carbon and gold, spontaneous modification proceeds via electron transfer from the surface to the diazonium salt.Furthermore, on both types of surface, spontaneously prepared NP films were found to be loosely packed multilayers of less than 5 nm in thickness. The spontaneous reaction was utilized for the patterning of carbon, gold, silicon and copper surfaces by microcontact printing (μCP) with diazonium salts. The presence of spontaneously formed films upon printing was confirmed by cyclic voltammetry and AFM. The films were demonstrated to be useful for the tethering of further molecules to the surface. Patterns prepared by μCP were imaged using scanning electron microscopy (SEM) and condensation figures. The preparation of two-component systems, with different chemical functionalities attached to different, well-defined, regions of the surface, was demonstrated. The optimization of the long term activity of glucose oxidase hydrogels by covalent attachment of the hydrogels to modified carbon electrodes was investigated. Covalent attachment was demonstrated, but the resulting electrode-hydrogel surfaces did not show long-term activities superior to those for physisorbed hydrogels. It is suggested that the limiting factor for long-term hydrogel activity is not adhesion of the hydrogel to the surface, but degradation of enzymatic activity by H2O2.

Work is reported on composites comprising either epoxy resin or crosslinked polyethylene (XLPE) filled with silica nanoparticles (surface functionalisated and unfunctionalisated). Measurements were made of the dielectric spectra, charging and discharging currents under high electric fields, and space charge dynamics using the pulsed electroacoustic (PEA) technique. Considerable studies were made of the effect of humidity on epoxy nanocomposites. It was found that the epoxy composites filled with nanoparticles could absorb up to 60% more water by weight than the unfilled epoxy. For composites filled with microparticles, nearly all the water was absorbed by the resin. The glass transition temperature (Tg) for all epoxy samples, measured by both differential scanning calorimetry (DSC) and dielectric spectroscopy, showed a monotonic reduction with increase of hydration resulting in a 20K decrease for fully hydrated samples. This led to the conclusion that the extra hydration found in the nanocomposites was not in the bulk resin but was likely to be located on the surface of the nanoparticles. This is further supported by measurement of the hydration isotherms at room temperature and the resultant swelling as a function of humidity. A "water shell" model is developed in which there is an inner layer of approximately 5 – 10 bound water molecules on the surface of the nanoparticles, a further layer, approximately 25nm thick, in which water is in sufficient concentration to allow conduction, and an outer layer, approximately 50nm thick, which cannot support true conduction (i.e. the continuous movement of charge carriers.) This model is used to explain the sub-hertz dielectric results (in terms of percolation limited conduction) as well as those at around 1 – 10Hz that indicate the presence of bound or free water.

Nerve injury is a very common trauma affecting 300,000 people in Europe every year. Although autografts are currently the gold standard in surgery, they can cause loss of sensation and scar tissue formation. Artificial nerve conduits are a valid alternative for peripheral nerve repair. They can provide a confined environment during the regeneration process, enabling axons sprouting from the proximal to the distal nerve segments as well as reducing scar tissue formation. Poly-e-caprolactone (PCL) is a biocompatible and biodegradable polymer suitable for the fabrication of nerve guidances. In particular, previous works demonstrated that neural cells are able to adhere and proliferate on micropitted PCL films obtained through solvent casting. Also, short term studies showed that axons were able to bridge 1cm injury gap. In this work a 18 weeks long term in vivo experiment using a rat model was performed to investigate the reinnervation of end organ skin and muscle. PCL conduits were compared to autografts, with no significant differences in terms of regeneration and reinnervation. However, Schwann cells (SCs), the most important glial cells in the peripheral nervous system, showed poor attachment in vitro on PCL scaffolds; hence, surface modification was carried out in order to improve the material biocompatibility. The effect of both hydrophilicity and functional groups on SCs was first investigated. PCL films were then hydrolysed and aminolysed to modify the surface with carboxylic and amino groups respectively. Hydrolysed films increased remarkably the surface hydrophilicity, although topography and mechanical properties were not affected. Conversely, the tensile modulus and strength were significantly reduced by aminolysis, but still suitable for the desired application. The two treatments influenced also the morphology of SCs. It was demonstrated that cell elongation was induced by hydrophilic surfaces, whilst cells preferred cell-cell interaction when cultured on aminolysed films. However, cell proliferation was remarkably increased on the latter surfaces, confirming previous results obtained on substrates characterised by amino groups. These results confirmed that a good balance between hydophilicity and surface chemistry is necessary to guarantee the best cell response. In order to enhance both proliferation and morphology of SCs, arg-gly-asp (RGD) sequences were immobilised on the PCL film surface using two different reaction mechanisms. Carbodiimide chemistry was compared to a new mechanism developed in the present study based on the Thiol chemistry. Biological tests performed on these modified films demonstrated the improvement of SC response after the peptide immobilisation using the novel approach. Cell attachment and proliferation were three times higher compared to untreated PCL films. It was also observed that the presence of peptides on the film surface induced the formation of focal adhesion plaques by SCs, important for the perception of cellular signals when in contact with a particular substrate. Hence, a good balance between focal adhesion and adhesion forces was achieved after peptide immobilisation. Overall the results of this study showed that material functionalisation is very important for SC response and it will be fundamental for the production of artificial nerve conduits.

Recently, a novel diffractive-based encoded system has been developed for various multiplexed suspension biological assays. These microparticles, which are manufactured using photolithography of a commercial epoxy-based negative photoresist (SU-8), contain micrometre-sized diffractive elements and can encode millions of unique codes. In this thesis, the preparation and surface modification of different diffractive microparticles; the attachment of a range of biological molecules (e.g. proteins and peptides) onto the functionalised surfaces; and different on-bead analytical techniques for surface characterisation are described. From a thermodynamic study of a multiplexed immunoassay for immunoglobulins (Ig, MW ~150 kDa), the immobilised probe molecules exhibit high affinity (Kd = 9 ± 3 nM) and excellent specificity (S/N >36:1) for the target analytes. The suspension assay resembles solution-like reaction kinetics allowing detection of multiple target proteins in <20 min. The encoded microparticles can also be used for quantifying small proteins, such as cytokines (MW ~20 kDa). In a particle-based sandwich suspension immunoassay, human tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) are detected and compared with conventional enzyme-linked immunosorbent assays (ELISA). Correlation coefficients (R2) for the two cytokines are found to be >0.96. Intra-assay variability (%CV) is determined to be <25%. The sensitivity of the multiplexed immunoassay, expressed as Lowest Detection Limit (LDL), is found to be 379 fM and 1.47 pM for TNF-α and IL-6 respectively, and are comparable to the corresponding ELISAs as demonstrated by the suppliers

The ability to provide mass customised and biocompatible implants is increasingly important to improve the quality of life. Additive manufacturing (AM) techniques have obtained increasing popularity and selective laser melting (SLM), a metal-based AM technique with an ability to build complex and well defined porous structures, has been identified as a route to fabricate customised biomedical implants. Surface modification of an implant with a biomolecule is used to improve its biocompatibility and to reduce post-implant complications. In this thesis, the potential of a novel approach to use self-assembled monolayers to modify SLM fabricated surfaces with therapeutic drugs has been evaluated. Although there are numerous studies on the material development, process optimisation and mechanical testing of SLM fabricated parts, the surface chemistry of these parts is poorly understood. Initially, the surface chemistry of SLM as-fabricated (SLM-AF), SLM fabricated and mechanically polished (SLM-MP) and forged and mechanically polished (FGD-MP) parts made of Ti6Al4V was determined using an X-ray photoelectron spectrophotometer (XPS). Later the impact of laser power on the surface chemistry of the parts was also studied. A non-homogeneous surface chemistry was observed due to a change in the distribution of the alloying elements titanium, aluminium and vanadium on the surface oxide layer. Surface modification of the SLM fabricated component would be beneficial to obtain a homogenous surface chemistry, especially for biomedical application. Coating of self-assembled monolayers (SAMs) onto SLM fabricated Ti6Al4V structures was performed to modify their surface chemistry. 16-phosphanohexadecanoic acid monolayers (16-PhDA) were used to modify SLM-AF and SLM-MP surfaces. XPS and static water contact angle measurements confirmed the chemisorption of monolayers on these surfaces. The obtained results confirmed that SAMs were stable on the Ti6Al4V surface for over 28 days before its desorption. It was also witnessed that the stability of monolayers on the rough SLM-AF and smooth SLM-MP surfaces were not significantly different. Later, the 16-PhDA SAM coated Ti6Al4V SLM-MP surface was functionalised with a model drug, Paracetamol. An esterification reaction was performed to functionalise the phosphonic acid monolayers with Paracetamol. Surface characterisation revealed the sucessful attachment of Paracetamol to the SAMs. Bacterial infections from biomedical implants and surgical devices are reported to be a major problem in orthopaedic, dental and vascular surgery. Hence, to further explore the potential of the proposed method, Ciprofloxacin® a broad spectrum antibiotic was immobilised to the SAMs, previously adsorbed on the SLM-MP Ti6Al4V surfaces. Using the proposed approach, approximately 1.12 µg/cm2 of the drug was coated to the surface. Results showed that Ciprofloxacin® is highly stable under the oxidative conditions used in this study. Under in vitro condition, the drug was observed to release in a sustained manner. Antibacterial susceptibility tests revealed that the immobilised Ciprofloxacin® was therapeutically active upon its release. Thus, a novel methodology to fabricate customised and functionalised implants has been demonstrated for an improved biocompatibility and reduced post-implant complications.

A considerable amount of effort has been directed to develop porous materials as drug delivery systems (DDSs) – one of the most promising emerging applications in healthcare, as most anticancer therapeutics have toxic dose dependence due to a lack of tumour selectivity – as their hierarchical porosity can be used to store and release challenging drugs. Among them, Metal-Organic Frameworks (MOFs) – emerging hybrid, highly porous crystalline structures – offer several advantages compared to other available DDS, as they combine desirable features from both organic (biocompatibility, e.g. porous polymers) and inorganic (high loadings, e.g. mesoporous silica) porous materials. MOFs are highly amenable to functionalisation, meaning fine control over their physical properties can be achieved, and thus they have experienced tremendous development during the past decade in many applications. Despite surface engineering being advantageous for diverse fields – in biomedicine, it can both improve stability and dispersion, and provide the possibility of targeted carriers, decreasing the immune system recognition – surface functionalization of MOFs is underdeveloped. The multiple synthetic steps – synthesis, drug loading and surface modification – and the lack of orthogonality between them hinder their industrial manufacturing as DDSs. This thesis focuses on the development of surface functionalisation protocols of Zirconium MOFs, particularly UiO-66, a Zr-terephthalate MOF, the study of their cell internalisation fate and routes and the correlation with their therapeutic activity. During Chapter 1, an introduction to the use of DDSs in anticancer therapy, followed by examples of the most relevant MOFs from a coordination chemistry point of view, is given, in which zirconium MOFs and their synthesis are highlighted. Particular focus is given to the coordination modulation process, in which monodentate modulators are introduced to the MOFs synthesis to compete with the multidentate linkers during nucleation, enhancing properties such as porosity through the induction of defects. Then, the most relevant examples of surface functionalization of Zr MOFs for drug delivery are discussed with respect to the effects on properties such as colloidal dispersion in aqueous solvents, physiological stability, and drug release kinetics. In Chapter 2 different functionalised modulators (i.e p-functionalised benzoic acids, folic acid or biotin) are introduced to UiO-66 synthesis to obtain surface-functionalised UiO-66 with the appropriate size for drug delivery by one-pot synthesis. Full characterisation of the materials shows them to be remarkably porous due to the defects formed when modulators attach to available zirconium positions in the pores and on the surfaces of the MOFs. Furthermore, the use of a carboxylate-containing anticancer metabolic target (dichloroacetic acid, DCA) as a modulator of UiO-66 synthesis is explored, and co-modulated samples, in which both DCA and functionalised modulators are introduced to UiO-66 synthesis, are synthesised and fully characterised, resulting in drug-containing (ca. 20% w/w) surface-functionalised MOFs by one pot syntheses. Importantly, DCA modulation induces a high number of defects, and consequently highly charged nanoparticles which are colloidally stable in aqueous solvents. Particle size control in the DCA modulated synthesis of the UiO family of isoreticular MOFs – including UiO-66 and its bromo, amino and nitro derivatives, and extended structures Zr-Naphthalenedicarboxylate (NDC) and Zr-Biphenyldicarboxylate (BPDC) – is achieved, obtaining ca. 100 nm particles of UiO-66 derivatives and microcrystals of Zr-NDC and Zr-BPDC when ZrCl4 is the metal precursor, and mesoporous < 20 nm UiO-66 derivatives and ca. 200 nm Zr-NDC and Zr-BPDC when ZrOCl2 is used as the metal precursor. The high porosity of the DCA modulated samples, due to DCA attachment to the inner and outer surface at defect sites, allows the loading of a second drug, the well-known anticancer drug 5-fluorouracil (5-FU), into the pores of the isoreticular MOFs to create dual DDSs. Different postsynthetic modes of surface coating, based in both coordination and covalent chemistry, are studied during Chapter 3. The functionalities of the p-functionalised benzoic acid modulators, introduced to UiO-66 structure during Chapter 2, are used to covalently attach short-chain alkanes and long-chain polymers to UiO-66 surface through copper-catalysed azide-alkyne cycloaddition. Exhaustive characterisation confirms that the attachment occurs through covalent chemistry and not through surface adhesion or electrostatic forces. Folic acid and biotin, which are introduced to UiO-66 surface as synthetic modulators during Chapter 2, are also introduced to UiO-66 surface postsynthetically. Colloidal dispersion and stability towards phosphates are investigated and compared to bare MOFs, in order to gain insights into the effect of both surface chemistry and mode of attachment on physical properties. A comprehensive overview of in vitro studies of cellular internalisation of zirconium MOFs is given in Chapter 4, focussing on the relevance of the endocytosis internalisation routes, which are strictly correlated with therapeutic efficacy. The postsynthetic surface functionalisation protocols investigated in Chapter 3 are applied to analogous calcein-loaded UiO-66 samples. Calcein is a fluorescent molecule not able to efficiently cross the cell membrane by itself, and hence serves as an in ideal probe of MOFs cellular internalisation. Its release from bare and poly(ethylene glycol) coated UiO-66 into phosphate buffered saline at pH 7.4 and 5.5, in order to simulate extracellular and intracellular conditions, is found to be pH responsive (more pronounced at 5.5) for all MOFs, but an ideal decrease in calcein release at pH 7.4 occurs only for PEGylated MOFs. Internalisation of calcein-loaded MOFs by HeLa cervical cancer cells is studied by fluorescence assisted cell sorting, highlighting the effects of surface chemistry on endocytosis efficiencies and internalisation mechanisms. A discussion of in vitro studies into anticancer drug delivery from Zr MOFs is provided in Chapter 5, alongside a summary of the therapeutic effects of DCA and approaches to enhance its anticancer efficacy. Experimental assessment of the in vitro anticancer performance towards MCF-7 breast cancer cells of the DCA-containing MOFs of the UiO family of different sizes (ca. 100 nm and <20 nm), synthesised by coordination modulation during Chapter 2, is given. The effect of dual-drug containing MOFs (DCA and 5-FU) is also examined, to investigate the possible synergic effect of the drug combination. Then, the cytoxicity of bare and surface functionalised, DCA-loaded and empty UiO-66 MOFs is studied at first upon incubation with HeLa cells, for which the cellular routes of internalisation were elucidated in Chapter 4. The most promising MOFs are then tested for selective anticancer activity against a series of cancerous and healthy cells lines, and their macrophage uptake and ROS production is also analysed, to determine the effect of surface functionalization. The selective anticancer cytotoxicity of folate-coated MOFs is attributed to a combination of cancer cell targeting and optimal cell internalisation routes. To summarise, the one-pot synthesis of drug-loaded, surface functionalised UiO-66 has been successfully performed, resulting in porous, crystalline MOFs with the appropriate size for drug delivery. The use of a carboxylate-containing anticancer metabolic target as a modulator has been explored for the UiO family of isoreticular MOFs, resulting in well-dispersed nanoMOFs with enhanced anticancer activity, into which a second drug can be loaded, enabling the creation of dual DDSs.
A series of postsynthetic surface modifications are performed, enabling the study of the MOF’s properties (colloidal dispersion, physiological stability and biocompatibility) with respect to their surface chemistry and coating mode, but more importantly providing valuable insights into correlations between surface chemistry, routes of cellular internalisation and therapeutic effect.

Avec une mortalité après pancréatectomie de l'ordre de 5 % et une morbidité aux alentours de 50 %, la chirurgie pancréatique est l'une des plus délicates des interventions digestives. L'une des complications les plus graves est l'apparition de fistules pancréatiques (FP), c'est-à-dire un défaut d'imperméabilisation au niveau des sutures, impliquant une fuite enzymatique dans le milieu péritonéal qui peut engager le pronostic vital du patient. A ce jour, aucun dispositif médical n'est indiqué dans la prévention de ces FPs. Ainsi, ce projet a pour ambition de concevoir et de valider un biomatériau constitué d'une matrice absorbante qui assurera la double fonction d'éponge et de réservoir d'agent antibactérien, sur laquelle un greffage chimique devrait conférer des propriétés bioadhésives thermorégulées. La première partie de ce travail est consacrée à l'optimisation de la matrice absorbante, à base d'alginate et de chitosane, déjà développée lors d'une précédente thèse. Trois types de procédés de séchages ont été comparés : le séchage par évaporation, la lyophilisation et le séchage en milieu CO2 supercritique. Ces différents procédés conduisent à des matériaux de structures internes et de porosités différentes. L'impact de ces différences de structure a été évalué en termes de capacité de gonflement dans différents milieux, dont un milieu pancréatique simulé, mais aussi en termes de résistance enzymatique et de libération de principe actif. En tenant compte des résultats obtenus, le séchage par évaporation a été identifié comme le plus approprié pour la suite de l'étude. Dans une deuxième partie, du poly(N-isopropylacrylamide) (PNIPAM) a été synthétisé par polymérisation contrôlée (RAFT) afin d'être greffé sur la matrice absorbante. Le PNIPAM est un polymère thermosensible dont les propriétés bioadhésives sont directement dépendantes de la température. Ce polymère est généralement bioadhésif au-dessus de sa " lower critical solution température " (LCST), située aux alentours de 32 °C. Dans cette étude la masse molaire et la densité de greffage du PNIPAM sont les deux principaux paramètres étudiés pour son greffage sur les matrices. Enfin, les propriétés de surface des matrices greffées ont été caractérisées. Les matériaux ont montré, in vitro, des propriétés bioadhésives thermosensibles, avec une bioadhésion cellulaire observée principalement au-delà de la LCST. Les essais ex vivo ont cependant montré une bioadhésion sur organe plus importante en dessous de celle-ci. Cette étude a permis de mettre au point des biomatériaux absorbants aux propriétés de surface thermorégulées. Une compréhension plus fine des relations propriétés de surface/propriétés d'usage permettrait d'optimiser les propriétés de bioadhésion thermorégulée
Pancreatic surgery, which leads to 5 % of mortality and around 50 % of morbidity, is one of the most critical digestive operations. The most serious complication is the appearance of pancreatic fistulas (PFs), i.e. enzymatic leaks from the surgical sutures to the peritoneal environment that can lead to the life threatening of the patient. To date, no medical device is indicated for the prevention of these FPs. The aim of this project is to design and validate a biomaterial constituted of a matrix that will ensure the dual function of absorbent and antibacterial agent reservoir, on which a chemical grafting should confer thermoregulated bioadhesive properties. The first part of this work is devoted to the optimisation of the absorbent matrix, based on alginate and chitosan, already developed during a previous thesis. Three types of drying processes were compared: drying by evaporation, lyophilisation and drying in supercritical CO2 medium. These different processes led to materials with different internal structures and porosities. The impact of these structures was evaluated in terms of swelling capacity in various media, including a simulated pancreatic environment, but also in terms of enzymatic resistance and release of an active molecule. Taking into account the obtained results, drying by evaporation was identified as the most appropriate process. In a second part, poly (N-isopropylacrylamide) (PNIPAM) was synthesised by controlled polymerisation (RAFT) in order to be grafted onto the absorbent matrix surfaces. PNIPAM is a thermosensitive polymer with bioadhesive properties which depend on the temperature. This polymer is usually bioadhesive above its lower critical solution temperature (LCST), around 32 ° C. In this study, the molar mass and the grafting density of PNIPAM are the two main parameters studied for the surface modifications. Finally, the surface properties of the grafted matrices were characterised. In vitro, the materials showed thermosensitive bioadhesive properties, with a cellular bioadhesion mainly observed above the LCST. However, ex vivo tests exhibited higher bioadhesion on porcine organs at lower temperatures. This study led to the development of absorbent biomaterials with thermoregulated surface properties. Further understanding of the relationship between surface properties and in vivo bioadhesion would allow the optimisation of the thermoregulated surface properties

This research reports a sophisticated avenue for the fabrication of bio-inspired, multifunctional and substrate-independent polymer coatings for versatile applications. Of particular interest was the design of nitroxide functional surfaces as a new strategy to combat bacterial biofilm formation on medical devices and to reduce biofilm-related infections in hospitals worldwide. Furthermore, nitroxide-coated surfaces served as a versatile platform for the precision design of reprogrammable surfaces with additional spatial control over the surface properties applying photolithography.

Dans une opération de mise en forme, les propriétés volumiques des matériaux ne sont pas les seules garantes de la réussite du procédé. Les aspects surfaciques, en lien notamment avec les propriétés tribologiques des corps en contact (tôle, lubrifiant, outils) se révèlent tout aussi importants. Il convient en ce sens de maîtriser et optimiser autant que faire se peut les conditions de contact lors d’un glissement entre la tôle et l’outillage afin d’assurer la réussite de l’opération de formage.Ces travaux de thèse portent sur la caractérisation d’une solution alternative de lubrification, nommée AFULudine, par fonctionnalisation de surface à base de molécules organiques ; solution ayant pour ambition de se substituer aux huiles minérales chlorées, performantes dans le travail des aciers inoxydables mais particulièrement nocives pour l’environnement et difficiles d’application.Les caractérisations physico-chimiques de surface (IR, électrochimie, MEB) montrent le greffage effectif d’une couche de molécules chimisorbées en surface de l’oxyde métallique, avec une cinétique de l’ordre de quelques secondes. Celle-ci est recouverte d’amas cristallisés et formés en un second temps pendant la phase d’évaporation du solvant. Des analyses morphologiques des cristaux sont réalisées montrant que leur densité et leur structure sont intimement liées au bon comportement tribologique de la surface fonctionnalisée.Le comportement tribologique des tôles (substrat en acier inoxydable ferritique K41 – 1.4509) ainsi traitées a été investigué à la fois grâce à un tribomètre d’étirage-plan permettant de découpler les influences de différentes conditions de sollicitations, telles que la pression de contact, la montée en température des outils ou encore l’influence de la déformation plastique à coeur du matériau, mais aussi en conditions réelles, à l’aide d’une presse d’emboutissage semi-industrielle. De manière plus fondamentale, la formation et la stabilité du tribofilm créé à l’interface a été analysé par l’utilisation d’un micro-tribomètre linéaire en géométrie sphère/plan couplé à de la microscopie électronique et des analyses EDS
During forming processes, bulk properties of material are not the only guarantee in a process achievement. Surfacicalaspects, mainly coupled with tribological properties of bodies in contact (metal sheet, lubricant, tool) prove to be alsosignificant. It is therefore necessary to control and optimize the contact conditions during a sheet/tool sliding in order toguarantee the success of a forming process.The present work concerns a green lubricant solution characterization, named AFULudine, produced by thefunctionalization of the metallic surface with organic molecules. This solution was developed in substitution to the usualmineral oils, effective in stainless steel stamping but deterious for environment.Physicochemical surface characterization (IR, electrochemistry, SEM) show that the grafting of chemisorbed molecules ontothe surface occures in few seconds. This layer is covered by physisorbed and crystallised species which appear during thesolvent evaporation time. Morphological analyses of these crystals show that both density and structure determine thetribological behavior of the functionalized surface.Sheets tribological behavior (stainless steel ferritic grade, 1.4509 - K41) after treatment, has been investigated thanks to astrip drawing tribometer, allowing to dissociate the influence of various sollicitations conditions (such as contact pressure,tools temperature or plastic deformation of the material) and on an industrial scale with an Erichsen press. Fundamentally,tribo-layerʹs formation and stability have been analysed with the use of a linear micro-tribometer (ball on plane type)coupled with electronic microscopy and EDS analyses

Ce travail s’inscrit dans le cadre général du développement de biomatériaux ostéoinducteurs pour la réparation de grands défauts osseux. L’étude est une contribution à la compréhension des interactions physiques et chimiques entre des céramiques phosphocalciques et deux protéines d’intérêt : la fibronectine, protéine d’adhésion cellulaire, et le VEGF (pour Vascular Endothelial Growth factor) qui est impliqué dans la vascularisation et l’amélioration de la formation osseuse.Les interactions physiques fibronectine/biocéramique ont été étudiées par spectroscopie de force afin d’évaluer l’influence de la topographie et de la composition chimique de céramiques phosphocalciques en hydroxyapatite (HA), hydroxyapatite silicatée (SiHA) et hydroxyapatite carbonatée (CHA) sur l’adhésion de la fibronectine. Les résultats obtenus par cartographie de forces mettent en évidence une absence d’incidence de la chimie des céramiques polies sur la répartition en surface et l’intensité des forces d’adhésion. En revanche ces dernières sont plus fortes au niveau des joints de grains des céramiques non polies mettant en avant une influence de la topographie de surface des matériaux modulée par la chimie.Le protocole de fonctionnalisation par le VEGF consiste en trois étapes : silanisation, addition du SM(PEG)6 et immobilisation du VEGF. Les interactions chimiques VEGF/biocéramique ont été étudiées principalement par imagerie Raman pour suivre ces étapes successives de la fonctionnalisation par le VEGF de céramiques polies en hydroxyapatite (HA) et hydroxyapatite carbonatée (CHA). Cette approche a permis de cartographier l’évolution chimique de la surface des matériaux et de mettre en évidence la distribution spatiale ainsi que les réactions préférentielles entre les molécules intermédiaires et le VEGF en fonction de la nature du substrat
This work is ascribed within the framework of the development of osteoinductive biomaterials for the repair large bone defects. It is a contribution to the understanding of the physical and chemical interactions between phosphocalcic ceramics and two proteins of interest: fibronectin (Fn), a cell adhesion protein, and Vascular Endothelial Growth Factor (VEGF) which is involved in vascularisation and improvement of bone formation.Fibronectin/bioceramic physical interactions were studied by force spectroscopy to evaluate the influence of the topography and the chemical composition of phosphocalcic ceramics made of hydroxyapatite (HA), silicated hydroxyapatite (SiHA) and carbonated hydroxyapatite (CHA) on fibronectin adhesion. The results obtained in terms of force cartography do not indicate any impact of the polished ceramics chemistry on the surface distribution and intensity of adhesion forces. However, these forces are more intense at the level of the grain boundaries of unpolished ceramics, highlighting an influence of the topography modulated by the chemical composition.The protocol for functionalisation by VEGF consists of three steps: silanisation, addition of SM(PEG)6 and immobilisation of VEGF. VEGF/bioceramic chemical interactions were studied mainly by Raman imaging in order to follow the successive steps of the functionalisation by VEGF of the polished surface of ceramics made of hydroxyapatite (HA) and carbonated hydroxyapatite (CHA). This approach allowed to map the surface chemical changes and to point out the spatial distribution as well as the preferential reactions between the intermediate molecules and VEGF depending of the substrate

La formation de biofilms bactériens pathogènes est un problème important, particulièrement dans les secteurs médicaux et agro-alimentaires. La formation contrôlée de biofilms de la bactérie probiotique Lactobacillus rhamnosus GG (LGG) est sélectionnée ici comme méthode potentielle pour prévenir la contamination de surfaces par des bactéries pathogènes. Nous avons étudié le développement de biofilms de LGG ainsi que leur possible contrôle en combinant des approches physico-chimiques et de fonctionnalisation de surface. L’impact des conditions environnementales sur la cinétique de croissance des biofilms et sur leur composition biochimique a été analysé par des mesures in situ et en temps réel par spectroscopie infrarouge à transformée de Fourier en réflexion totale atténuée (ATR-FTIR) sous conditions de flux. Ces données ont été complétées par des images de microscopie en épifluorescence permettant d’obtenir des informations sur la distribution et la forme des cellules bactériennes sur la surface à des étapes clé du développement du biofilm. Compatible avec les mesures ATR-FTIR, un cristal de séléniure de zinc a été choisi comme substrat, nu ou fonctionnalisé avec des monocouches auto-assemblées d’alcane-thiols (SAMs). Différents groupes fonctionnels ont été étudiés : méthyl (-CH3), hydroxyle (-OH) ou amine (-NH2) pour obtenir respectivement des substrats hydrophobe, hydrophile ou chargé positivement. La cinétique d’auto-assemblage des SAMs, leur organisation et l’énergie de surface ont été étudiées en combinant ATR-FTIR, spectroscopie de rétrodiffusion de Rutherford à hautes énergies et mesures d’angles de contact. L’analyse des spectres ATR-FTIR des biofilms de LGG enregistrés in situ et en temps réel pendant 24 heures a montré un rôle important du milieu nutritif sur la composition biochimique et le métabolisme bactériens. Les propriétés du substrat ont un impact faible sur la composition biochimique des biofilms, mais ont un rôle crucial sur leur force d’attachement à la surface. Ce travail pluridisciplinaire a fourni des informations sur l’influence de l’environnement, et particulièrement des caractéristiques du support, sur les propriétés des biofilms aux échelles moléculaire et cellulaire. La méthodologie développée dans ce travail peut notamment être utilisée dans la recherche des conditions les plus favorables à la croissance des biofilms de bactéries probiotiques
Biofilm formation by pathogenic bacteria brings concerns, particularly in food and medical sectors, and is associated with high sanitary risks and economic losses. Biofilms of probiotic bacteria can potentially be used to prevent the surface contamination by pathogenic species. This work was focused on the investigation of the development of biofilms of probiotic Lactobacillus rhamnosus GG (LGG) and the possible control of their formation by combining surface functionalisation and physico-chemical approaches. The effect of different environmental conditions on the kinetics of the biofilm growth and on its biochemical composition was analysed by in situ and real time measurements with infrared spectroscopy in attenuated total reflection mode (ATR-FTIR) under flow conditions. These data were complemented by epifluorescence images providing information on the surface distribution and the shape of the bacterial cells at specific stages of the biofilm development. Compatible with ATR-FTIR measurements, a zinc selenide (ZnSe) crystal was chosen as a substrate, bare or functionalised with self-assembled monolayers (SAMs). SAMs were formed from alkanethiols terminated by methyl (-CH3), hydroxyl (-OH) or amine (-NH2) groups to obtain hydrophobic, hydrophilic and positively charged substrates, respectively. The kinetics of self-assembly of the alkanethiols onto ZnSe, the organisation of the molecules, their areal density and the surface energy of thus obtained surfaces were studied preliminarily to the biofilm cultivation by means of ATR-FTIR spectroscopy, high energy Rutherford backscattering spectrometry, and contact angle measurements. The analysis of the ATR-FTIR spectra of LGG biofilms recorded in situ and in real time during 24 hours revealed an important role of the nutritive medium in the biosynthesis of nucleic acids, phospholipids, polysaccharides and lactic acid. Substrate properties had low impact on the biochemical composition of LGG biofilms, but had a critical role in the strength of attachment of cultivated biofilms. The findings of this multidisciplinary work provide a fundamental understanding of how the direct environment, including a support surface, influences the properties of bacterial biofilms at the molecular and cellular scales, based on which favourable conditions for the enhancement of probiotic biofilm growth and its mechanical stability can be chosen

The present PhD thesis, entitled: "Essential oil components encapsulated in mesoporous silica supports: a bioactive properties evaluation and a toxicological approach" focuses on the study of protection and controlled release of natural bioactive agents, derived from essential oil components (EOCs), encapsulated in mesoporous silica particles (MSPs). In addition, this thesis evaluatesthe silica-based supports to reduceundesirable sensorial propertiesandfor ensuring a low-health risk. The first section of the thesis shows the effect of encapsulation of EOCs in mesoporous silica supports. This study evaluates the efficiency of free and encapsulated EOCs to reduce the viability of cancer colon cell lines. This sectionalso shows the selectivity of encapsulated EOCs against cancer linesandtheir effect onnormal (non-cancer) colon cells. Results indicate that EOCs effect can be enhanced and sustained in time when EOCs are encapsulated. Moreover, EOCs' encapsulation shows promising specificity indices, reaching to double effect on colon cancer cells above normal cells. On the other hand,the encapsulation supports and their surface functionalization allows the odour masking of high volatility EOCs. Therefore, the delivery system based on MSPs represents an excellent alternative to promote controlled EOCs release, taking advance of their bioactive properties and solving the technical disadvantages related to volatility and unpleasant odours. Finally, samples used for garlic components encapsulation were immobilised in nanofibers to provide homogeneous and easy-to-handle hybrid system for controlling delivery.The developed 'composite' has potential applications on food, pharmacology, medical or engineering fields. The second sectionof the thesis evaluates the toxicity of the mesoporous silica supports through in vitro and in vivo assessments. Cell viability allows to identify the cytotoxic impact based on the kind of silica-based support, and their features (doses range, size and surface structure changes). Furthermore, the use of Caednorhabditis elegansmodel,shows the in vivo effects afterMSPs ingestion. The toxicological study confirms that size and surface structure, are decisiveMSPs' featuresfor reducing the toxicity risks for health. In summary, the present thesis evaluates the mesoporous silica-based particles as supports for EOCs encapsulation and identifies the main MSPs' features forreducingthe health-toxicity impact. Results of this thesis show that MSPs improve the EOCs activity and help to solve technical problemsof EOCs' volatility.Moreover, these results open up a suitable and safety option for oral delivery devices.
La presente tesis titulada: "Componentes de aceites esenciales encapsulados en soportes mesoporosos de sílice: una evaluación de sus propiedades bioactivas y un enfoque toxicológico" se centra enla evaluación de las propiedades funcionales y organolépticas de agentes naturales bioactivos, derivados de componentes de aceites esenciales, encapsulados en materiales mesoporosos de sílice; a la vez que evalua la toxicidad de los soportes utilizados,con el fin de proponer nuevos sistemas de liberación controlada por vía oral. La primera sección de esta tesis muestra el efecto de la encapsulación de los compuestos de aceites esenciales (EOCs, por sus siglas en inglés) en soportes mesoporosos de sílice. Por un lado, seevalúa la eficiencia de los EOCs libres y encapsulados para reducir la viabilidad en líneas celulares de cáncer de colon. Además, se evalúa la selectividad de los EOCs frente a células de colon normales (líneas no tumorales). Por otro lado, seestudia la capacidad de enmascaramiento de olor de los soportes. Los resultados obtenidos, evidencian en primer lugar, que los EOCs encapsulados mejoran su actividad frente a células de cáncer,en comparacióncon la respuesta de los compuestos sin encapsular. La encapsulación hace que el efecto de los EOCs sea sostenido en el tiempo, y muestra índices de especificidad prometedores, cuandose evalua el efecto toxico de los EOCsfrente a células de cáncer de colon y células normales. Los resultados de esta primera sección, indican que los soportes basados en partículas de sílice mesoporosa (MSPs, por sus siglas en inglés) protegen y liberan eficientemente los compuestos, sino que, a la vez que la funcionalización de la superficie de las MSPs permite enmascarar el olor de los compuestos de mayor volatilidad, y con mayores inconvenientes a nivel sensorial (p.e. compuestos derivados del ajo). Por lo tanto, el sistema de encapsulación se plantea como una excelente alternativa para (i) promover la liberación controlada de EOCs, (ii) aprovechar y mejorar el efecto de sus propiedades bioactivas en células de cáncer de colón y (iii) controlar las desventajas técnicas relacionadas con la volatilidad y limitaciones organolepticas. Por último, se ha comprobado que los soportes empleados en la encapsulación de los compuestos derivados de ajo, mantienen su funcionalidad luego der ser inmovilizados en nanofribras de nylon.Con esto, se busca desarrollar un nuevo sistema de 'composite';un material híbrido y homogéneo, fácil de manejar, que libera controladamente los compuestos encapsulados desde soportes tipo fibras (composites).Estoexpande el abanico de aplicaciones de los EOCs en laindustria alimentaria y farmacológica. La segunda sección de esta tesis, evalúa la toxicidad de los soportes de sílice mesoporosa (MSPs) mediante ensayos in vitro e in vivo. En primer lugar, la viabilidad celular permite identificar el impacto citotóxico de los MSPs sobre líneas celulares de colón. En particular, se evalúa los soportes mesoporosos de sílice, tipo MCM41, en función de (i) las dosis empleadas, (ii) la diferencia de tamaño (micro y nanopartículas) y (iii) el efecto que la funcionalización de la superficie genera en la viabilidad celular. Por otro lado, empleando el modelo Caednorhabditis elegans, yadministrando por vía oral las MSPs,se evalua la influencia de las características de laspartículas (MSPs) en función de la esperanza de vida (lifespan) y la calidad con la que viven y envejecen (healthspan) los nematodos. Los resultados de este estudio,muestran que el tamaño y la estructura de la superficie de las partículas, son parámetros determinantesal momento de diseñar soportes de bajoriesgo toxicológico. En resumen, la presente tesis ha evaluado las características de la sílice mesoporosa, micro y nanoparticulada, como soporte de encapsulación para mejorar la actividad y las aplicaciones de los compuestos de aceites esenciales, al mismo tiempo
La present tesi titulada: "Components d'olis essencials encapsulats en suports mesoporosos de sílica: una avaluació de les seves propietats bioactives i un enfocament toxicològic" se centra en estudis de protecció i alliberament controlat d'agents naturals bioactius, derivats de components d'olis essencials, encapsulats en materials mesoporosos de sílica. Els components d'olis essencials encapsulats milloren les seves propietats funcionals i redueixen els problemes sensorials per aplicacions futures, garantint, al mateix temps, la baixa toxicitat dels suports desenvolupats. La primera secció de la tesi mostra l'efecte d'encapsulació dels components d'olis essencials (EOCs, per les seves sigles en anglès) en suports mesoporosos de sílica sobre la millora de les seues propietats bioactives i el camuflament de problemes sensorials. Este estudi avalua l'eficiència dels EOCs lliures i encapsulats per a reduir la viabilitat en línies cel¿lulars de càncer còlon. A més, la selectivitat dels EOCs es va provar enfront de cèl¿lules de còlon normals (no canceroses). Els resultats han demostrat que l'efecte dels EOCs pot ser millorat i sostingut en el temps quan els EOCs estan encapsulats. Encara més, l'encapsulació dels EOCs mostra índexs d'especificitat prometedors, arribant a duplicar la toxicitat en l'efecte en les cèl¿lules de càncer de còlon amb comparacio en les cèl¿lules normals. Els resultats també mostren que els suports basats en partícules de sílice mesoporoses (MSPs, per les seves sigles en anglès) no sols protegixen i alliberen EOCs eficientment, sinó que, a més, la funcionlització en superfície de les MSPs permet emmascarar l'olor dels EOCs d'alta volatilitat, que té una aplicació limitada a causa dels seus problemes sensorials(p.e. compostos derivats de l'all). Per tant, el sistema de subministrament proposat resulta una excel¿lent alternativa per a (i) promoure l'alliberament controlat de EOCs, (ii) avançant en les seues propietats bioactives en cel¿lulas de càncer còlon i (iii) controlant els desavantatges tècnics relacionats amb la volatilitat i la disseminació desagradable de les olors. Finalmet, les mostres utilitzades per encapsulació de compostos d'all es van immobilitzar en nanofibres per a proporcionar un sistema híbrid homogeni i fàcil de manejar amb administració controlada i característiques bioactives, per aplicacions potencials en l'àrea d'alimentació, farmacologia, medicina o enginyeria. La segona secció avalua la toxicitat del suports de sílice mesoporosa per mitjà d'avaluacions in vitro e in vivo. La viabilitat cel¿lular permet identificar l'impacte citotòxic basat en el tipus de suport base de sílice i les seues característiques (rang de dosi, grandària i canvis en l'estructura superficial).A més, utilitzant el model in vivo Caednorhabditis elegants, s'ha estudiat la influència de les característiques de la sílice mesoporosa, administrant micro i nanopartícules de base sílice, no sols en l'esperança de vida, sinó també en el comportament dels nematodes durant el seu envelliment. Aquest estudi ha demostrat que la grandària i l'estructura superficial, són decisius per a reduir el risc de toxicitat dels suports de sílice mesoporosa i obrir la possibilitat d'utilitzar estos materials en aplicacions d'ingesta oral. En resum, la present tesi ha avaluat les característiques de les partícules de sílice mesoporosa, com a suports d'encapsulació per a millorar l'activitat i les aplicacions dels EOCs, alhora que es va avaluar el seu principal risc tòxicologic. En conseqüència, els resultats obrin una opció adequada i de seguretat per als dispositius d'administració oral.
Acosta Romero, C. (2017). Essential oil components encapsulated in mesoporous silica supports: a bioactive properties evaluation and toxicological approach [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90653
TESIS

Les matériaux poreux représentent une solution idéale en ingénierie tissulaire car leur structure peut offrir un environnement tridimensionnel aux cellules similaire à leur matrice extracellulaire tout en maintenant de bonnes propriétés mécaniques. Une première partie de cette thèse consiste à développer des matériaux poreux en polyuréthane (PU), dont l’architecture est contrôlée pour favoriser au mieux la survie et la croissance des cellules. Ces matériaux sont combinés à des traitements de surface (revêtement de polydopamine (PDA) et traitement plasma) pour augmenter notamment l’adhésion des cellules. Nous avons pu démontrer que le diamètre des interconnexions (i.e. l’ouverture connectant deux pores adjacents) impacte profondément la survie et l’organisation des cellules à long terme dans le matériau. Le revêtement de PDA s’est révélé efficace pour des cellules de type fibroblaste, alors que le traitement plasma favorise la colonisation des cellules souches mésenchymateuses (MSCs). Par ailleurs, nous avons étudié l’influence de la formulation du PU sur les capacités d’adhésion des cellules au matériau. Nous avons démontré que pour un ratio donné entre les réactifs, l’adhésion des cellules peut être exclue ou permise. Finalement, nous avons mis un gel de peptides auto-assemblés dans les pores du matériau pour fournir aux cellules un environnement similaire à leur matrice extracellulaire. Nous avons pu montrer que le gel permet d’augmenter la prolifération des MSCs
Porous materials are an ideal solution in tissue engineering since they can provide a three-dimensional environment to the cells that is close to their extracellular matrix while keeping suitable mechanical properties. In the first part of this Thesis we develop porous materials made from polyurethane (PU) whose architecture is controlled to allow cells colonisation and growth. These materials are subsequently surface-treated (polydopamine (PDA) coating and plasma treatment) to enhance the adhesion of the cells. We were able to show that the interconnection diameter (i.e. the aperture connecting two adjacent pores) has an important impact on the long-term cell survival and organization in the material. Polydopamine coating was shown to be efficient for fibroblasts, whereas plasma treatment promoted mesenchymal stem cells (MSCs) colonisation. Besides, we also studied the influence of the PU formulation on the adhesion capacity of the cells. We demonstrated that at a given ratio between the reactants, cell adhesion could be allowed or prevented. Finally, we put a hydrogel of self-assembled peptides inside the pores of the material to provide an environment close to the extracellular matrix for the cells. We could show that the gel increases the proliferation ability of MSCs. In summary, this Thesis puts forward the important interplay between material properties and morphology of porous scaffolds

Cette thèse se focalise sur le design, la synthèse et la caractérisation photophysiques de complexes de platine(II) neutres contenant un ligand chromophore tridenté et un ligand auxiliaire monodenté avec des groupes fonctionnels différents. De tels complexes montrent des changements drastiques de leurs propriétés de photoluminescence à cause de leur auto-assemblage, déclenché par l’établissement de faibles liaisons non covalents tels que l’empilement p-p et des interactions métallophiliques. Il a été démontré que ces complexes de platine(II) peuvent être attaché de manière covalente à des surfaces de silice et d’or mais aussi encapsulé dans des particules de polymère. Les propriétés d’autoassemblage de complexes de platine(II) amphiphiles ont également été étudiées en détails. Comprendre le comportement d’un complexe de platine dans un mélange de solvant nous a permis d’en apprendre plus sur le co-assemblage supramoléculaire de deux complexes de platine(II) différents. Cette approche conduit à la formation de fibres solvatochromiques avec des propriétés photophysiques spécifiques et réversibles. Les composés étudiés et leur auto-assemblage sonti ntéressant pour le développement de nouveaux matériaux fonctionnels aux applications sensoriels et réactives à certains stimuli
This thesis focuses on the design, synthesis and the photophysical characterisation of luminescent neutral platinum(II) compounds containing a tridentate chromophoric ligand and a monodentate ancillary moiety with different functionalities. Such complexes exhibited drastic changes to their photoluminescence properties upon self-assembly, triggered by the establishment of weak noncovalent p-p stacking and metallophilic interactions. It was demonstrated that these platinum(II) complexes can be covalently attached to silicon and gold substrates but also encapsulated into polymer particles. The self-assembly properties of amphiphilic platinum(II) complexes have also been intensively studied. Understanding the behaviour of one complex in a solvent mixture allowed further investigation into the supramolecular co-assembly of two different platinum(II) complexes. This approach leads to the formation of solvatochromic fibers with specific and reversible phototophysical properties. The investigated compounds and their assemblies were useful for the development of novel functional materials for sensing or stimuli-responsiveness applications

La formation de biofilms bactériens pathogènes est un problème important, particulièrement dans les secteurs médicaux et agro-alimentaires. La formation contrôlée de biofilms de la bactérie probiotique Lactobacillus rhamnosus GG (LGG) est sélectionnée ici comme méthode potentielle pour prévenir la contamination de surfaces par des bactéries pathogènes. Nous avons étudié le développement de biofilms de LGG ainsi que leur possible contrôle en combinant des approches physico-chimiques et de fonctionnalisation de surface. L’impact des conditions environnementales sur la cinétique de croissance des biofilms et sur leur composition biochimique a été analysé par des mesures in situ et en temps réel par spectroscopie infrarouge à transformée de Fourier en réflexion totale atténuée (ATR-FTIR) sous conditions de flux. Ces données ont été complétées par des images de microscopie en épifluorescence permettant d’obtenir des informations sur la distribution et la forme des cellules bactériennes sur la surface à des étapes clé du développement du biofilm. Compatible avec les mesures ATR-FTIR, un cristal de séléniure de zinc a été choisi comme substrat, nu ou fonctionnalisé avec des monocouches auto-assemblées d’alcane-thiols (SAMs). Différents groupes fonctionnels ont été étudiés : méthyl (-CH3), hydroxyle (-OH) ou amine (-NH2) pour obtenir respectivement des substrats hydrophobe, hydrophile ou chargé positivement. La cinétique d’auto-assemblage des SAMs, leur organisation et l’énergie de surface ont été étudiées en combinant ATR-FTIR, spectroscopie de rétrodiffusion de Rutherford à hautes énergies et mesures d’angles de contact. L’analyse des spectres ATR-FTIR des biofilms de LGG enregistrés in situ et en temps réel pendant 24 heures a montré un rôle important du milieu nutritif sur la composition biochimique et le métabolisme bactériens. Les propriétés du substrat ont un impact faible sur la composition biochimique des biofilms, mais ont un rôle crucial sur leur force d’attachement à la surface. Ce travail pluridisciplinaire a fourni des informations sur l’influence de l’environnement, et particulièrement des caractéristiques du support, sur les propriétés des biofilms aux échelles moléculaire et cellulaire. La méthodologie développée dans ce travail peut notamment être utilisée dans la recherche des conditions les plus favorables à la croissance des biofilms de bactéries probiotiques
Biofilm formation by pathogenic bacteria brings concerns, particularly in food and medical sectors, and is associated with high sanitary risks and economic losses. Biofilms of probiotic bacteria can potentially be used to prevent the surface contamination by pathogenic species. This work was focused on the investigation of the development of biofilms of probiotic Lactobacillus rhamnosus GG (LGG) and the possible control of their formation by combining surface functionalisation and physico-chemical approaches. The effect of different environmental conditions on the kinetics of the biofilm growth and on its biochemical composition was analysed by in situ and real time measurements with infrared spectroscopy in attenuated total reflection mode (ATR-FTIR) under flow conditions. These data were complemented by epifluorescence images providing information on the surface distribution and the shape of the bacterial cells at specific stages of the biofilm development. Compatible with ATR-FTIR measurements, a zinc selenide (ZnSe) crystal was chosen as a substrate, bare or functionalised with self-assembled monolayers (SAMs). SAMs were formed from alkanethiols terminated by methyl (-CH3), hydroxyl (-OH) or amine (-NH2) groups to obtain hydrophobic, hydrophilic and positively charged substrates, respectively. The kinetics of self-assembly of the alkanethiols onto ZnSe, the organisation of the molecules, their areal density and the surface energy of thus obtained surfaces were studied preliminarily to the biofilm cultivation by means of ATR-FTIR spectroscopy, high energy Rutherford backscattering spectrometry, and contact angle measurements. The analysis of the ATR-FTIR spectra of LGG biofilms recorded in situ and in real time during 24 hours revealed an important role of the nutritive medium in the biosynthesis of nucleic acids, phospholipids, polysaccharides and lactic acid. Substrate properties had low impact on the biochemical composition of LGG biofilms, but had a critical role in the strength of attachment of cultivated biofilms. The findings of this multidisciplinary work provide a fundamental understanding of how the direct environment, including a support surface, influences the properties of bacterial biofilms at the molecular and cellular scales, based on which favourable conditions for the enhancement of probiotic biofilm growth and its mechanical stability can be chosen

Current strategies for designing biomaterials involve creating materials and interfaces that interact with biomolecules, cells and tissues.  This thesis aims to investigate several bioactive surfaces, such as nanocrystalline diamond (NCD), hydroxyapatite (HA) and single crystalline titanium dioxide, in terms of material synthesis, surface functionalization and characterization. Although cochlear implants (CIs) have been proven to be clinically successful, the efficiency of these implants still needs to be improved. A CI typically only has 12-20 electrodes while the ear has approximately 3400 inner hair cells. A type of micro-textured NCD surface that consists of micrometre-sized nail-head-shaped pillars was fabricated. Auditory neurons showed a strong affinity for the surface of the NCD pillars, and the technique could be used for neural guidance and to increase the number of stimulation points, leading to CIs with improved performance. Typical transparent ceramics are fabricated using pressure-assisted sintering techniques. However, the development of a simple energy-efficient production method remains a challenge. A simple approach to fabricating translucent nano-ceramics was developed by controlling the morphology of the starting ceramic particles. Translucent nano-ceramics, including HA and strontium substituted HA, could be produced via a simple filtration process followed by pressure-less sintering. Furthermore, the application of such materials as a window material was investigated. The results show that MC3T3 cells could be observed through the translucent HA ceramic for up to 7 days. The living fluorescent staining confirmed that the MC3T3 cells were visible throughout the culture period. Single crystalline rutile possesses in vitro bioactivity, and the crystalline direction affects HA formation. The HA growth on (001), (100) and (110) faces was investigated in a simulated body fluid in the presence of fibronectin (FN) via two different processes. The HA layers on each face were analysed using different characterization techniques, revealing that the interfacial energies could be altered by the pre-adsorbed FN, which influenced HA formation. In summary, micro textured NCD, and translucent HA and FN functionalized single crystalline rutile, and their interactions with cells and biomimetic HA were studied. The results showed that controlled surface properties are important for enhancing a material’s biological performance.

This work describes the successful preparation and characterisation of peptide functionalised gold surfaces, with a focus towards developing enzyme response applications. The serine protease, elastase, was the primary focus of this work; however, all of the systems described herein have the utility to be modified for alternative proteolytic enzymes. 1.0 provides background and context to the work presented in this thesis and additionally describes the layout of the work to be presented. 2.0 reviews the literature on enzyme responsive nanomaterials (ERNMs), providing a brief overview of the design considerations, routes to functionalisation and characterisation techniques which must be taken into account when developing such materials. 3.0 describes the development of a method for the step-wise functionalisation of planar gold surfaces with short peptide derivatives, which contained elastase cleavable moieties. (±)-α-lipoic acid was utilised to provide an anchor to the gold surface and conventional Fmoc chemistry used to prepare the peptide sequences. The fluorenyl functional group provided a convenient marker for surface analysis, enabling the verification of each synthetic step by ToF-SIMS, XPS and LDI-ToF MS. Step-wise solid-phase synthesis on the gold surface is a facile means of preparing peptide-conjugates, providing a method whereby no additional purification or final “grafting-on” steps are required. The use of LDI-ToF MS and ToF-SIMS in tandem ensured confidence in the stepwise synthesis method. These techniques provided complimentary data to one another through the analysis of both the fully desorbed peptide derivative and molecular fragments respectively. 4.0 presents investigations into the use of SERS as a technique to follow the elastase responsive system developed in 3.0. The nanostructure surface of Klarite, a commercially available SERS substrate, was functionalised using the same methodology and proof-of-concept work towards a SERS-active system was detailed. 5.0 discusses the development of the first elastase detection system found on gold nanoparticles, progressing on from the work on flat surfaces presented in 3.0 and 4.0. The remainder of the work presented in the chapter focuses on the development of a potentially more robust system, with several target molecules successfully synthesised; however, none of these molecules provided an elastase responsive system. 6.0 then provides conclusions of the work carried out, followed by a description of potential future investigations that could be carried out. Finally, 7.0 provides a detailed description of the experimental methodology followed, providing evidence for the findings and conclusions presented in 3.0-6.0.

This thesis is concerned with understanding and directing the functionalisation of solid surfaces with materials: molecules, nanoparticles and crystals. Both conducting (electrode) and insulating surfaces are of interest. For molecular functionalisation, a sweep potential procedure has been developed to assist the formation of self assembled monolayers (SAMs) of a ruthenium thiolated complex. Electrochemical investigations were employed to characterised the SAM formed on a platinum electrode. Nanoparticles formation explored two distinct routes. First Pd nanoparticles were successfully formed within ultra-thin Nafion films via impregnation and a chemical reduction method. Morphological investigations utilised atomic force microscopy. The electrocatalytic properties of the nanocomposite material were elucidated for the hydrogen oxidation reaction. The methodology used for the preparation of this nanocomposite material shows promise for applications in sensors and fuel cells. Second, the potential-assisted deposition of pre–formed perthiolated-ß-cyclodextrin-capped Pt nanoparticles method is described. Pt nanoparticles (5 nm diameter) were deposited in a controlled fashion on indium tin oxide and highly oriented pyrolytic graphite electrodes. The Pt nanoparticles formed in this way were electrocatalytically active towards hydrogen generation and oxidation. This new approach for the deposition of metal nanoparticles with controlled surface density provides a new tool for the investigation of electrocatalytic processes. A major focus of the second part of the thesis has been the development of methods to study crystal deposition at extreme supersaturation. For this purpose a delivery system for calcium carbonate at high-supersaturation ion has been coupled with a quartz crystal microbalance and in–situ optical microscopy. The dynamics and quantitative evaluation of calcium carbonate deposition onto foreign solid substrates, and the effect of various additives, are described. Ex– situ studies, scanning electron microscopy and microRaman spectroscopy, allowed the morphological characterisation of the phases deposited. The transformation of ACC to calcite has been explored in details. In the study of additives, a significant finding was that citrate concentration shows a nonmonotonic behaviour on the amount of scale deposited. Fast screening of different additives (polymeric and molecular) and a quantitative ranking of their inhibitory properties on calcium carbonate deposition on a gold surface is described. Molecular and polymeric additives showed different inhibitory mechanisms on the scaling process and the technique employed gave a better insight into their mode of action.

Plasmachemical functionalisation offers a versatile route to surface functionalisation, allowing a wide variety of functional groups to be deposited in a substrate independent process. This thesis presents the application of pulsed plasmachemical functionalisation in two areas. Firstly, the construction of covalently tethered DNA or protein arrays is examined, and novel routes are presented based on the introduction of aldehyde and bromine functions onto substrates such as glass, silicon, polystyrene beads and carbon nanotubes. Secondly, the use of plasmachemical layers for the tethering of metal centres is also presented, with aldehyde and vinylpyridine functional surfaces presented, and their use in the metallization of substrates such as carbon nanotubes demonstrated. The functionalisation and reaction of plasmachemical surfaces is monitored by a variety of surface sensitive methods including XPS, FT-IR, contact angles, and reflectometry.

Ce travail de thèse a consisté en la conception et la réalisation d'une nano-sonde hybride luminescente visant à permettre la détection précoce du cancer de la prostate. La première partie de ce projet a été consacré à la synthèse, par une voie de chimie click, d'une bibliothèque d'acides 4-triazolyl dipicoliniques substitués en position 4 du triazole par une large gamme de substituants. Ces diacides ont permis d'obtenir les complexes d'europium(III) et de terbium(III) correspondant, qui ont montré d'excellentes propriétés optiques, avec des rendements quantiques de luminescence sous excitation UV pouvant atteindre 60% et 36%, pour les complexes d'europium(III) et deterbium(III) respectivement. D'autre-part, ces fluorophores ont pu être excités efficacement en régime biphotonique, à la fois au travers des transitions S0 ®S1 et S0 ®T1. Sur la base de ces résultats, certains de ces chélates ont été sélectionnés afin de les incorporer dans des nanoparticules de silice. Le procédé d'élaboration par microémulsion inverse s'est révélé efficace pour l'incorporation des complexes électriquement neutres, mais n'a pas permis celle de nanohybrides incorporant des complexes chargés négativement. Ces nanohybrides présentent des propriétés optiques caractéristiques des lanthanides, avec des rendements quantiques allant jusqu'à 30%. La surface de ces nano-objets a ensuite été fonctionnalisée par des groupements amino, qui ont permis le greffage de bras espaceur et d'un vecteur ciblant la PSMA, l'un des signaux du cancer de la prostate, nous donnant ainsi accès à un modèle de nano-sonde luminescente. Un autre volet de ce travail a été dédié à l'étude de nouveaux analogues du NAAG, substrat naturel de la PSMA. Bien que la synthèse des deux composés cibles, sélectionnés parmi une vingtaine de structures par modélisation moléculaire, n'ait pu aboutir, elle a été largement avancée. Enfin, la dernière partie de ce travail décrit les premiers résultats obtenus in vitro et in vivo avec les nanosondes. Ces études ont porté sur l'évaluation de la cytotoxicité des nanoparticules ainsi que sur leur biodistribution chez la souris saine et chez la souris porteuse d'une tumeur prostatique. Cette étude a révélé une élimination rapide des nanoparticules par l'organisme, mais n'a malheureusement pas pu mettre en évidence un marquage des zones tumorales par les nanosondes.

L'objectif de ce travail de thèse est d'étudier la fonctionnalisation de textiles par des particules contenant un principe actif cosmétique. L'approche choisie repose sur la fixation sur le textile de particules de charges opposées. Dans ce travail, nous avons étudié dans un premier temps l'état de surface des textiles synthétiques et naturels. Les résultats ont montré que les textiles présentent une surface chargée négativement. Dans un second temps, l'adsorption de particules cationiques “modèles” de PMMA sur un textile synthétique à base de polyamide a été étudiée. Les résultats ont montré une bonne adsorption des particules sur le textile ainsi qu'une bonne résistance à la désorption lors des étapes de lavage. A la suite de ces résultats, des particules cationiques formulées par émulsion-diffusion à partir de différents polymères ont été réalisées. Ce criblage des polymères a permis de sélectionner un polymère biocompatible et biodégradable capable de fonctionnaliser durablement le textile. La dernière partie de ce travail a consisté en l'étude du rendement d'encapsulation et du profil de libération de deux principes actifs : rafraichissant et blanchissant
The aims of this work is to study textiles functionalization with particles containing an cosmetics active ingredient. The approach selected is based on textiles functionalization by opposite charged particles. At first, we studied synthetic and natural textiles surface and results showed that textiles exhibit negative charged surface. In a second step, adsorption of cationic PMMA particles onto synthetic polyamide textile was studied. The results showed proper particles adsorption on textile and a good resistance to desorption during washing steps. Then, cationic particles formulated by emulsion-diffusion from different polymer were made. This screening polymers was used to select a biocompatible and biodegradable polymer able to functionalized textiles. The last part of this work consisted in studying the encapsulation efficiency and release profile of two active ingredient : refreshing and whitening

The research undertaken in this thesis is concerned with the attachment of fluorophores to both optical fibres and nanodiamonds to provide sensing devices with application to biological sensing. Fluorescent sensors are commonly used to detect biological analytes through a change in fluorescence, a concept introduced in Chapter 1. However, the successful application of fluorescent sensors to biological systems is inhibited by a number of challenges, including delivery of the fluorophore to the measurement site, photobleaching, and fluorophore cell permeability. Chapter 1 introduces optical fibres and nanodiamonds as solid supports for attachment of fluorophores to overcome these challenges. Chapter 2 presents a new fibre functionalisation architecture for dual imaging and sensing within a single optical fibre probe. The fluorescent pH sensor, 5(6)carboxy-SNARF2, was reacted with the N-terminus of a silk-binding peptide to form an amide bond and provide the peptide SBP-SNARF. A fluorescence polarisation assay showed this peptide to bind with a Kd of 36 μM when added to aqueous silk fibroin protein. Fibre probes were prepared by dip-coating the fibre tip into the silk and peptide mixture, which provided a uniform silk coating (determined by scanning electron microscopy) that was stable to repeated washing in water and did not affect the imaging light emitted. This allows concurrent optical coherence tomography (OCT) imaging and pH sensing, which was demonstrated in an in vitro fertilisation (IVF) setting. Specifically, the probe detected a change in pH of 0.04 in cumulus-oocyte complexes after metabolic stimulation with CoCl2 to produce lactic acid, with the distance between the probe tip and the oocyte monitored by simultaneous OCT acquisitions. Notably, OCT imaging of an ovary revealed the presence/absence of an oocyte within an ovarian follicle, an important first step toward improving IVF patient outcomes by limiting the number of follicle punctures required to collect oocytes. Collectively, these results demonstrate the utility of the new fibre coating system to enable simultaneous OCT imaging and sensing, which provides significant insight into complex biological systems. Chapter 3 reports the detection of AlexaFluor-532 tagged streptavidin through its binding to D-biotin, which was reacted with the N-terminus of a silk-binding peptide to form the complex SBP-Biotin. This complex then adheres to a silk-coated fibre tip to provide an optical fibre probe for streptavidin, which is stable to repeated washing and long-term exposure to water. The probes were prepared by two methods that either distribute the SBP-Biotin throughout the silk fibroin matrix, or isolate the SBP-Biotin to the exterior of the silk layer. Only probes with surface bound SBP-Biotin successfully detected streptavidin, with a fluorescence-based detection limit of 15 mg/mL. Atomic absorption spectroscopy revealed that silk coating formation was inhibited by a lithium concentration of 200 ppm, however reduction to less than 20 ppm by dialysis re-enabled fibre coating. Together, Chapters 2 and 3 demonstrate that judicious preparation of optical fibre probes provides an opportunity for a wide array of fibre-based sensors using the silk fibroin and silk-binding peptide-based fibre functionalisation architecture described herein. Chapter 4 explores functionalisation of carboxylic acid laden nanodiamonds (NDs) through amide bond formation. This was first probed using an analytical high pressure liquid chromatography system to quantify the amount of 4-fluorobenzylamine attached to NDs. However, it was found that 4-fluorobenzylamine did not bind to NDs. Next, 1H nuclear magnetic resonance spectroscopy was employed to determine that the amount of diamino-PEG molecules attached to the NDs varied between 0 and 0.2 mmol/g. This indicated an inconsistent yield from the amidation reactions, which was investigated in more detail across ten separate amidation reactions between 4-cyanobenzylamine and NDs. However, none of these reactions resulted in an observable loading of 4-cyanobenzylamine, and it was concluded that amide bond formation is not an effective strategy for ND functionalisation. Chapter 5 presents four carbon-binding peptides (1-DLC, 2-CN, 3-DF and 4-GF) for ND functionalisation. Retention of each peptide on NDs was assessed by colourimetric assay and their presence confirmed through infrared spectroscopy and thermogravimetric analysis. 1-DLC was found to be the most well-retained peptide, at 87% and 35% on detonation and high-pressure high-temperature NDs respectively. This interaction was determined to be predominantly electrostatic, while 2-CN bound through non-polar interactions. Both 1-DLC and 2-CN coatings improved the colloidal stability of detonation NDs in aqueous solution, however neither peptide affected the colloidal stability of high-pressure, high-temperature NDs. This study provides a new, highly adaptable approach to functionalise NDs using carbon-binding peptides. Chapter 6 presents the synthesis of 4-aminobutanenitrile, an important synthetic intermediate for neurological disorder therapeutics, including Parkinson’s and Alzheimer’s diseases. Preparation of 4-aminobutanenitrile by Co(II) catalysed reduction, or a one-pot Staudinger reduction, of 4-azidobutanenitrile was low yielding. The reported Staudinger reaction was investigated through 1H-NMR analysis to reveal formation of the iminophosphorane intermediate after 22 h at rt, and increasing the temperature to 40 °C promoted hydrolysis of this intermediate to the desired amine. The Staudinger reduction was performed using pyridine solvent in place of THF, with water added 3 h after reaction initiation. These conditions gave rise to 4-aminobutanenitrile in 69% yield and 94% purity (calculated by qHNMR) without chromatography. However, 4-aminobutanenitrile was found to be unstable at rt, and cyclised to 2-aminopyrroline over several days. This was circumvented by preparation of the hydrochloride salt, which was shown to be stable at rt. Hence, 4-aminobutanitrile is best stored as the corresponding hydrochloride salt.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2021