Currently, around 28 million people globally suffer from the consequences of corneal blindness and most of them are part of a long waiting list; availability of donor tissue is highly limited. Furthermore, even those who are treated are in risk of developing post- surgery complications, mainly due to microbial infections. Hence, cell-free biomaterials with enhanced properties to prevent corneal associated infections would provide a safe alternative. We evaluated the efficacy of different peptides for the functionalization of collagen-based hydrogels through the in situ synthesis of silver nanoparticles (AgNPs). The produced biomaterials were characterized and evaluated in vitro for biocompatibility and potential antimicrobial activity. From the diverse strategies evaluated, the localized formation of AgNPs onto the periphery of cornea-shaped collagen hydrogels may represent a more promising option.

Owing to their increasing application and potential toxicity, engineered nanoparticles (ENPs) have been considered as a potential agricultural contaminant that may pose unknown risk to human beings. However, many techniques require invasive and complicated sample preparation procedures to detect and characterize engineered nanomaterials in complex matrices. In the first part of this thesis, we present a non-destructive and label-free approach based on surface enhanced Raman spectroscopic (SERS) mapping technique to qualitatively detect and characterize gold nanoparticles (AuNPs), on and in spinach leaves in situ. We were able to detect the clearly enhanced signals from AuNPs at 15 to 125 nm on and in spinach leaves. Peak characterizations revealed the aggregation status of Au NPs and their interactions with plant biomolecules, such as chlorophylls and carotenoids. This developed approach will open a new analytical platform for various researches on studying ENPs' adhesion and accumulation. The second part focuses on investigating the interaction between AgNPs and plant leaves using surface enhanced Raman spectroscopy. AgNPs of different surface coating (citrate, CIT and polyvinylpyrrolidone, PVP) and size (40 and 100 nm), were deposited onto spinach leaves. SERS signals produced from all kinds of AgNPs exhibited a unique C-S stretching peak at 650-680 cm-1. In vitro study indicates this peak may originate from the interaction between AgNPs and cysteine-like compounds based on the peak pattern recognition. The interaction between AgNPs and the cysteine-like compounds happened as soon as 0.5 h after AgNPs exposure. The in situ replacement of the CIT with the cysteine-like compounds on the AgNP surfaces was faster compared to that of the PVP. Based on the mapping of the highest C-S peak, we observed the CIT-AgNPs penetrated faster in spinach leaves than the PVP-AgNPs, although the penetration profile for both of them is similar after 48 h (P ˂ 0.05). The 40 nm CIT-AgNPs was able to penetrate deeper (to the depth of 183 ± 38 µm) than the 100 nm CIT-AgNPs (to the depth of 90 ± 51 µm) after 48 h. The results obtained here demonstrate the size of AgNPs is the main factor that affects the penetration depth, and the surface coating mainly affects the initial speed of interaction and penetration. This study helps us to better understand the distribution and biotransformation of AgNPs in plants. In the third part, the removal efficiency of postharvest washing on AgNPs that had accumulated on fresh produce was evaluated. Ten µL commercially available 40 nm citrate coated AgNPs (0.4 mg L-1) were dropped to a (1×1 cm2) spot on spinach leaves, followed by washing with deionized water (DI water), Tsunami® 100 (80 mg L-1) or Clorox® bleach (200 mg L-1). Then, AgNPs removal efficiency of the three treatments was evaluated by surface enhanced Raman spectroscopy (SERS), scanning electron microscopy (SEM)-energy dispersive spectrometer (EDS), and inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS results showed that deionized water removed statistically insignificant amounts of total Ag, whereas Tsunami® 100 and Clorox® bleach yielded 21% and 10% decreases in total Ag, respectively (P < 0.05). The increased removal efficiency resulted from Ag NPs dissolution and Ag+ release upon contact with the oxidizing agents in Tsunami® 100 (peroxyacetic acid, hydrogen peroxide) and Clorox® bleach (sodium hypochlorite). According to the SERS results, the deionized water and Tsunami® 100 treatments removed nonsignificant amounts of AgNPs. Clorox® bleach decreased Ag NPs by more than 90% (P < 0.05), however, SEM-EDS images revealed the formation of large silver chloride (AgCl) crystals (162 ± 51 nm) on the leaf, which explained low total Ag removal from ICP-MS. This study indicates current factory washing methods for fresh produce may not be effective in reducing AgNPs (by water and Tsunami® 100) and total Ag (by all three means). This highlights the necessity to develop an efficient washing method for NP removal from food surfaces in the future.

No description available.

Nanopartículas de prata (AgNPs) vêm ganhando grande importância na prevenção contra infecções, principalmente pela sua incorporação em dispositivos médicos, tecidos entre outros produtos relacionados. Estudos prévios indicaram que partículas de menores tamanhos têm ação biocida mais efetiva. Nesse contexto, a separação granulométrica pode ser explorada para se obter partículas com as dimensões desejadas. A centrifugação foi escolhida, entre outros métodos de separação granulométrica, por sua possibilidade de ressuspensão das AgNPs após a separação, pela facilidade do aumento de escala e baixa complexidade. Um modelo do perfil de concentração (em uma cela centrífuga) de AgNPs em função do tempo e intensidade de centrifugação foi proposto a partir da Equação de Lamm, considerando a difusividade Browniana e o coeficiente de Svedberg calculado a partir do balanço de forças. A equação foi resolvida com a solução semi-analítica proposta inicialmente por Fujita (1962) e melhorada pelos alemães Joachim Behlke e Otto Ristau (2001). O desempenho da centrifugação de AgNPs sintetizadas pelo Método Turkevich foi avaliado analisando-se as partículas sobrenadantes por DLS, UV-Vis e microscopia eletrônica e comparando com o modelo proposto. O modelo previu razoavelmente a concentração total de partículas e a sua distribuição de tamanhos em diferentes tempos de centrifugação para forças de campo baixas (força centrífuga relativa até 1480). Para maiores forças de campo, houve uma sedimentação mais acelerada que a prevista pelo modelo. / Silver nanoparticles (AgNPs) have gained great importance in preventing infections, especially by their incorporation into medical devices, tissues and related products. Previous studies indicated that particles of smaller sizes have more effective biocidal action. In this context, granulometric separation may be explored to deliver nanoparticles of desired size. Centrifugation was chosen, among other particle size separation methods, for their ability to resuspend the AgNPs after separation, for the ease of scale-up and low complexity. A profile concentration model (in the centrifuge cell) of AgNPs as a function of time and intensity of centrifugation has been proposed from the Lamm equation, considering the Svedberg and Brownian diffusivity coefficients calculated from a force balance. The equation was solved with the semi-analytical solution proposed initially by Fujita (1962) and improved by the Germans Joachim Behlke and Otto Ristau (2001). The centrifugation performance of AgNPs synthesized by the Turkevich method was evaluated by analyzing the supernatant particles by DLS, UV-Vis and electron microscopy and comparing to the proposed model. The model reasonably predicted the total concentration of particles and their size distribution at different centrifugation times at low field strengths (1480 RCF). For higher field strengths, the sedimentation was faster than expected by the model.

Nanopartículas de prata (AgNPs) vêm ganhando grande importância na prevenção contra infecções, principalmente pela sua incorporação em dispositivos médicos, tecidos entre outros produtos relacionados. Estudos prévios indicaram que partículas de menores tamanhos têm ação biocida mais efetiva. Nesse contexto, a separação granulométrica pode ser explorada para se obter partículas com as dimensões desejadas. A centrifugação foi escolhida, entre outros métodos de separação granulométrica, por sua possibilidade de ressuspensão das AgNPs após a separação, pela facilidade do aumento de escala e baixa complexidade. Um modelo do perfil de concentração (em uma cela centrífuga) de AgNPs em função do tempo e intensidade de centrifugação foi proposto a partir da Equação de Lamm, considerando a difusividade Browniana e o coeficiente de Svedberg calculado a partir do balanço de forças. A equação foi resolvida com a solução semi-analítica proposta inicialmente por Fujita (1962) e melhorada pelos alemães Joachim Behlke e Otto Ristau (2001). O desempenho da centrifugação de AgNPs sintetizadas pelo Método Turkevich foi avaliado analisando-se as partículas sobrenadantes por DLS, UV-Vis e microscopia eletrônica e comparando com o modelo proposto. O modelo previu razoavelmente a concentração total de partículas e a sua distribuição de tamanhos em diferentes tempos de centrifugação para forças de campo baixas (força centrífuga relativa até 1480). Para maiores forças de campo, houve uma sedimentação mais acelerada que a prevista pelo modelo. / Silver nanoparticles (AgNPs) have gained great importance in preventing infections, especially by their incorporation into medical devices, tissues and related products. Previous studies indicated that particles of smaller sizes have more effective biocidal action. In this context, granulometric separation may be explored to deliver nanoparticles of desired size. Centrifugation was chosen, among other particle size separation methods, for their ability to resuspend the AgNPs after separation, for the ease of scale-up and low complexity. A profile concentration model (in the centrifuge cell) of AgNPs as a function of time and intensity of centrifugation has been proposed from the Lamm equation, considering the Svedberg and Brownian diffusivity coefficients calculated from a force balance. The equation was solved with the semi-analytical solution proposed initially by Fujita (1962) and improved by the Germans Joachim Behlke and Otto Ristau (2001). The centrifugation performance of AgNPs synthesized by the Turkevich method was evaluated by analyzing the supernatant particles by DLS, UV-Vis and electron microscopy and comparing to the proposed model. The model reasonably predicted the total concentration of particles and their size distribution at different centrifugation times at low field strengths (1480 RCF). For higher field strengths, the sedimentation was faster than expected by the model.

No description available.

Aluminium (Al) and silver (Ag), through human activities, are present in the environment at concentrations sufficient to cause toxicity. The aim of this study was to administer Al and Ag to the short lived model organism Drosophila melanogaster, so that developmental toxicity and potential ameliorative interventions could be examined over a compressed timescale relative to mammalian models. Aluminium was administered to Drosophila in food as either the chloride salt or citrate complex at concentrations of 1, 10 and 100 mM and various developmental parameters were assessed. The lowest concentration to delay pupation relative to the control was 10 mM but this depended upon the food in which it was administered. Higher whole body tissue levels of Al were seen following Al citrate administration compared to AlCl3, but Al citrate was less toxic as this did not did not impair larval viability at 100 mM; 100 mM AlCl3 resulted in 100% mortality. Eclosion success was significantly impaired with either form of Al at 10 mM, but no difference was seen between the forms of Al. When Drosophila were fed AlCl3 over their entire lifespan, a small but significant reduction in the lifespan of male flies was seen. No behavioural toxicity could be demonstrated. Existing studies have demonstrated significant tissue Al concentrations and toxicity whereas these have been minimal in this study. It is suggested that these differences may have a genetic component, with food composition exerting an influence also. Silver, either as AgNO3 or Ag nanoparticles (AgNPs) was administered in concentrations up to 500 micromolar and 10 mM, respectively. Either form of Ag, at 50 micromolar was sufficient to significantly retard pupation rate, although pupation or eclosion success was not impaired until 100 micromolar. The concentration-response relationship for AgNO3 was steep with pupation success dropping to nearly zero by 300 micromolar; Drosophila in this study were far more sensitive to AgNO3 than those in other reports. Animals exposed to AgNPs were still able to pupate at 500 micromolar, but these pupae were almost all non-viable when exposed to 400 micromolar AgNPs. At 1 mM and above, AgNPs, however, showed reduced toxicity compared to lower concentrations. The reasons for this are unclear. Both forms of Ag caused de-pigmentation in adults after larval exposure that may be explainable by inhibition of polyphenol oxidase enzymes by Ag (I) ions. The de-pigmentation was preventable by pre-loading larvae with Cu. Ascorbate prevented the de-pigmentation caused by AgNPs but not AgNO3 suggesting that AgNP toxicity is due to Ag (I) ion release. Oxidation of AgNPs was found to be greatly accelerated by Fe (III) and Cu (II) ions in the presence of Cl- ions. Although some of the results here conflict with the literature, developmental toxicity has been observed here, for both Al and Ag, and the variability across studies may provide an opportunity for dissecting the mechanisms behind Al and Ag toxicity through identification of the traits that confer sensitivity or resistance.

Στην παρούσα διπλωματική εργασία γίνεται χρήση τριών διαφορετικών ειδών λεπτών υμενίων διοξειδίου του τιτανίου (TiO2) ως στερεό υπόστρωμα για την ακινητοποίηση πρωτεϊνών με σκοπό την ανάπτυξη και σύγκριση αμπερομετρικών βιοαισθητήρων με ευαισθησία στο υπεροξείδιο του υδρογόνου (H2O2). Το πρώτο είδος των λεπτών υμενίων TiO2 είναι από την εμπορική πάστα της Dyesol η οποία παρασκευάζεται με τη μέθοδο sol-gel, το δεύτερο είδος είναι από μία πάστα δικής μας παραγωγής με τη μέθοδο sol-gel και το τρίτο είδος είναι από μία πάστα που παρασκευάσαμε χρησιμοποιώντας την έτοιμη νανοδομημένη σκόνη TiO2, Degussa P – 25. Τα υμένια TiO2 που έχουν παρασκευαστεί από την εμπορική πάστα της Dyesol χρησιμοποιήθηκαν και για τη δημιουργία υβριδικού υποστρώματος με νανοσωματίδια Αργύρου το οποίο μπορεί να βρει εφαρμογή τόσο στη φωτοκατάλυση όσο και στην ανάπτυξη πιο ευαίσθητων αμπερομετρικών βιοαισθητήρων. Αρχικά περιγράφεται η λειτουργία των βιοαισθητήρων καθώς και οι σημαντικότεροι τύποι βιοαισθητήρων που έχουν κατασκευαστεί μέχρι σήμερα. Σημαντικό ρόλο στην επιτυχή κατασκευή ενός βιοαισθητήρα παίζει η επιλογή του υλικού που θα χρησιμοποιηθεί ως υπόστρωμα / ηλεκτρόδιο (στη συγκεκριμένη περίπτωση, υμένια TiO2) καθώς και ο τρόπος που ακινητοποιείται το βιομόριο πάνω σε αυτό, γι’ αυτό και έχει δοθεί έμφαση στην ανάλυση των παραπάνω πληροφοριών. Στη συνέχεια περιγράφεται η δομή και η φυσική λειτουργία της πρωτεΐνης, (κυτόχρωμα c), που χρησιμοποιήθηκε ως το βιομόριο επιλογής για την ανάπτυξη του βιοαισθητήρα. Αναλύονται οι κρυσταλλικές δομές του διοξειδίου του τιτανίου και οι βασικές φυσικοχημικές τους ιδιότητες. Επίσης γίνεται περιγραφή του φαινομένου της φωτοκατάλυσης, ενώ αναφέρονται τα πλεονεκτήματα και τα μειονεκτήματα του TiO2 για την εφαρμογή αυτή. Στο τέλος του πρώτου κεφαλαίου γίνεται αναφορά και στα νανοσωματίδια αργύρου καθώς και στους λόγους που βοηθούν στην αύξηση της φωτοκαταλυτικής απόδοσης του TiO2. Στη συνέχεια περιγράφονται οι πειραματικές διατάξεις που χρησιμοποιήθηκαν τόσο για τον χαρακτηρισμό των υμενίων TiO2 όσο και για την αναλυτική μελέτη της ακινητοποίησης του κυτοχρώματος c και των νανοσωματιδίων αργύρου πάνω σε αυτά. Περιγράφεται επίσης, η ηλεκτροχημική κυψελίδα 3 ηλεκτροδίων και οι τεχνικές της κυκλικής βολταμμετρίας και της φασματοηλεκτροχημείας που επιλέχθηκαν για τη μελέτη των ηλεκτροχημικών ιδιοτήτων των υμενίων TiO2 με ή χωρίς ακινητοποιημένη πρωτεΐνη, των υμενίων TiO2 με ακινητοποιημένα νανοσωματίδια αργύρου καθώς και για την ανάπτυξη των αμπερομετρικών βιοαισθητήρων με ευαισθησία στο H2O2. Τέλος, περιγράφεται η σύνθεση των τριών διαφορετικών παστών TiO2, η πειραματική διαδικασία εναπόθεσης των υμενίων του TiO2 σε υποστρώματα αγώγιμου υάλου και κατόπιν, ακολουθεί η ανάλυση των πειραματικών αποτελεσμάτων. / In this report 3 different types of TiO2 films were used as solid substrates for the immobilization of proteins in order to be used for the development and evaluation of amperometric biosensors for hydrogen peroxide (H2O2). The first type of thin TiO2 films were made using a Dyesol sol gel commercial TiO2 paste, the second type was produced from a TiO2 paste produced in our lab following a standard sol-gel procedure and the third type of films were produced from a paste prepared using Degusa P25 TiO2 powder. In addition, the thin TiO2 films produced from the Dyesol paste were modified with Ag nanoparticles in order to examine their electrochemical behavior which could lead to enhanced photcatalytic and/or biosensing performance. In the first chapter, a general decription of the different types of biosensors developed so far is presented and emphasis is given to their function and applications. In order to develop a successful biosensor, the choice of the material to be used as the solid substrate is very important as well as the type of the biomolecule used as the recognition element. The sensitivity and response of the biosensor is greatly enhanced by the method used to immobilize the biomolecule on the solid support in a stable and functional way. Therefore in this work both the material, TiO2 films, and the biomolecule of choice, Cytochrome-c, are presented in detail and in particular their physicochemical properties, their functions and applications. Moreover the different methods that have been used for the successful immobilization of biomolecules on solid surfaces are well documented. Furthermore, the photocatalytic properties of the TiO2 films are discussed and how they are enhanced by the deposition of silver nanoparticles on their surfaces that could also lead to the development of more sensitive and accurate amperometric biosensors. In the second chapter, the experimental techniques and procedures used for the characterization of the resulting TiO2 films and for the adsorption process of protein and/or silver nanoparticles on their surfaces are well presented. Furthermore, details are given of the electrochemical techniques (cyclic voltammetry and spectroelectrochemistry) used to evaluate the electrochemical performance of the resulting films with or without protein or silver nanoparticles. A detailed description of the 3 electrode electrochemical used to perform these experiments is also presented. Finally emphasis is given to the procedures used for the development of the electrochemical biosensors for H2O2. Finally, a description of the procedures used for the synthesis of the 3 different TiO2 pastes and of the method used for the production of thin TiO2 films on conducting glass is given followed by the presentation, analysis and discussion of the data collected.