The synthesis and biological sensing applications of novel water soluble poly(para-phenyleneethynylene)s (PPEs) are presented. The ease of synthesis, synthetic variability, and dramatic chromicity of PPEs makes them well suited for biological and sensing applications. Molecular recognition and signal transduction can be achieved by using PPEs as sensory materials. By incorporating biological functional groups (e.g. sugars), PPEs can efficiently detect the presence of toxic heavy metals, proteins, and bacteria through either fluorescence quenching or enhancement. Rapid, precise, and convenient sensory arrays for the detection of biological analytes are possible through the formation of gold nanoparticle-PPE constructs.

Recent advances in multilayer self-assembly have enabled the precise construction of nanocomposite ultrathin films on a variety of substrates, from large-area planar surfaces to nanoparticles. As a result, a wide range of physico-chemical properties may be represented by selecting from an array of surface preparations, molecules, assembly conditions, and post-assembly treatments. Such multilayer nanofilm assemblies are particularly attractive for use as specialized membranes for selective transport, which have many applications for separations, sensors, and drug delivery systems. In this work, nanocomposite ultrathin films built with layer-by-layer (LbL) self-assembly methods have been applied to surface modification to control interfacial behavior, including diffusion, anti-fouling, and biomimetic membranes. Transport and interfacial properties of nanocomposite membranes constructed using LbL self-assembly with synthetic and/or bio-polymers were characterized, and permeability values of clinically relevant small molecules through the nanofilms were determined. Correlations between permeability and film properties were also examined. Nanofilm coatings around 100nm thickness decreased diffusion coefficients of glucose up to five orders of magnitude, and were found to greatly affect enzymatic glucose sensor responses. Surface modification on top of the nanofilms with poly(ethylene glycol) provided anti-fouling effects. However, weak-weak polyelectrolyte multilayers (PEMs) should not be used to control transport due to their susceptibility under normal physiological conditions. Natural/biological polymers also provided multilayer film structures at the specific conditions, but their transport-limiting properties were not significant compared to synthetic PEMs. Even when covalently crosslinked, biological PEMs did not reduce the permeability of a small molecule. Finally, the predicting model of projecting analyte permeation through multi-phase nanocomposite films comprised with known diffusion coefficients was theoretically and experimentally evaluated. The modeling was matched reasonably well to experimental data. The outcomes will be the key knowledge or engineering principles to support future efforts in research and development. It is anticipated that the system developed for determining transport properties will provide a general platform for assessing new candidate materials. The theory developed will be useful in estimating transport properties of novel nanocomposite materials that may be interesting in a broad array of chemical and biological systems, from analytical separations to implantable biomedical applications, and will provide useful design rules for materials and fabrication process selection.

This thesis focuses on surface science and bioengineering investigations, first for the development of an improved biomaterial for orthopaedic implant applications, and second, for the development of a biosensor device for biomedical diagnostics. A key component considered in this thesis was the covalent linkage of proteins to the material’s surface for retaining the protein’s immunological and biological activities and for generating a functional interface. Part 1 of this thesis investigated surface modification procedures for improving the bioactivity of titanium substrates. Titanium is first coated with a bioactive silica film grown by plasma enhanced chemical vapour deposition (PECVD), referred to as PECVD-Si-Ti. In previous studies, the bone-implant integration process was enhanced 1.6-fold for titanium implants coated with PECVD-Si films compared to uncoated titanium implants in vivo. However, in vitro studies carried out in this thesis showed that the growth of MG63 osteoblast-like cells was 7-fold higher on uncoated titanium compared to PECVD-Si coated titanium. Therefore, to improve cell growth on the surface and, by inference, the integration of PECVD-Si-Ti implants into bone tissue, the implant’s surface was functionalised with a mitogenic factor, insulin-like growth factor-1 (IGF-1). This was accomplished by modifying the PECVD-Si-Ti surface with an alkoxysilane, 3-isocyanatopropyl triethoxysilane (IPTES), and then by covalent bioconjugation of IGF-1 through isocyanate-amino chemistry. After 72 h of in vitro cell culture in serum-free medium, the growth of MG63 cells was enhanced 1.9-fold on IPTES functionalised PECVD-Si-Ti, which was loaded with covalently immobilised IGF-1 compared to IPTES functionalised PECVD-Si-Ti without IGF-1 (isocyanate reactive groups were quenched with ethanolamine hydrochloride). The attachment and adhesion of MG63 cells were also enhanced on PECVD-Si-Ti by the covalently immobilised IGF-1 in serum-free cell culture conditions. Therefore, the bioactivity of PECVD-Si-Ti was improved by covalently linking IGF-1 to the substrate surface through isocyanate-amino chemistry. Part 2 of this thesis involved the development of a new optical interferometric biosensor. The biosensor platform was constructed from electrochemically-prepared thin films of porous silicon that acted as a sensing matrix and transducer element. By reflective interferometry using white light, an enzyme-catalysed reaction was discovered (horseradish peroxidase (HRP) mediated oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB)), which led to an acceleration in the rate of porous silicon corrosion and represented the biosensor’s readout signal. We discovered that another substrate, which is also oxidised by HRP, OPD, produces an even more pronounced readout signal. The HRP-OPD system was used in an immunoassay for detecting human IgG from an Intragam solution. An important part in the design of the biosensor was the surface functionalisation approach where anti-human IgG, referred to as the capture antibody, is immobilised on the porous silicon surface. The readout signal (produced from the capture of human IgG) was enhanced 4-fold on the porous silicon biosensing platform functionalised with covalently linked anti-human IgG through isocyanate-amino chemistry compared to the porous silicon biosensing platform functionalised with adsorbed anti-human IgG. The optimised biosensor was used to detect IgG from a total human protein concentration of Intragam to a sensitivity of 100 ng/ml. In summary, isocyanate-amino bioconjugate chemistry was used to covalently link either IGF-1 to PECVD-Si-Ti for improving the biological activity of the orthopaedic implant and to covalently link IgG to porous silicon for developing a sensitive biosensor for the detection of proteins. This surface chemistry approach is very useful for biomaterial and biosensing applications.

Universiẗat, Diss., 2003--Tübingen.

Universiẗat, Diss., 2004--Münster (Westfalen).

Universiẗat, Diss., 2003--Stuttgart.

Orientador: Valber de Albuquerque Pedrosa / Resumo: O desenvolvimento de novos dispositivos para monitorar o metabolismo celular e o diagnóstico de doenças expandiu as pesquisas com biossensores, que aliados à nanotecnologia possibilitaram a criação de novos elementos com alta sensibilidade de detecção, especificidade e capacidade de multiplexação, mostrando grande potencial para sua aplicabilidade no diagnóstico clínico. O trabalho foi desenvolvido em duas etapas. A primeira, referiu-se no desenvolvimento de uma metodologia para acoplar o aptâmero conjugado com as nanopartículas de ouro sobre o sensor da Ressonância Plasmônica de Superfície (SPR). Foi utilizado MUA para formação das monocamadas auto-organizadas; ativação dos grupos carboxílicos utilizando solução de EDC/NHS e a imobilização do aptâmero conjugado. Após este processo, foram realizadas as injeções de Mucina Epitelial Polimórfica tipo 1 (MUC1). A segunda etapa, consistiu na mesma metodologia de acoplamento do aptâmero, porém substituindo a MUC1 por sobrenadante da linhagem celular LNCaP (células prostáticas tumorais). Desse modo, foi desenvolvida uma metodologia analítica utilizando aptâmeros e biomarcadores para diagnosticar o Câncer de Próstata (PCa) através da SPR. / Mestre

Les réseaux bidimensionnels de nanofils (NFs) d’oxyde de zinc (ZnO) aléatoirement orientés, ou nanonets (pour « nanowire networks »), constituent des nanostructures innovantes et prometteuses pour de nombreuses applications. L’objectif de cette thèse est de développer des nanonets de ZnO en vue d’applications à la détection de molécules biologiques ou gazeuses, en particulier de l’ADN, ceci selon une procédure bas coût et industrialisable. Dans ce but, il est essentiel de bien maitriser les différentes étapes d’élaboration qui sont : (i) le dépôt de couches minces de germination de ZnO sur des substrats de silicium par voie sol-gel, (ii) la croissance de NFs de ZnO sur ces couches de germination par synthèse hydrothermale, et (iii) l’assemblage par filtration sous vide de ces NFs en nanonets de ZnO. Des études approfondies de chacun de ces procédés ont donc été menées. Ces travaux ont permis d’élaborer des couches minces, des NFs et des nanonets de ZnO reproductibles et homogènes dont les propriétés morphologiques sont précisément contrôlées sur une large gamme. Deux protocoles de biofonctionnalisation des nanonets avec de l’ADN ont ensuite été développés et ont abouti à des résultats encourageants mais restant à optimiser. Les nanonets ont également été intégrés au sein de dispositifs fonctionnels et les premières caractérisations électriques ont fourni des résultats prometteurs. A terme, ce travail ouvre la voie à l’intégration collective de NFs de ZnO qui permettrait la réalisation d’une nouvelle génération de capteurs (de biomolécules, de gaz…) à la fois portables, rapides et très sensibles. / Two-dimensional randomly oriented zinc oxide (ZnO) nanowire (NW) networks, or nanonets, represent innovative and promising nanostructures for numerous applications. The objective of this thesis is to develop ZnO nanonets for the detection of biological or gaseous molecules, in particular DNA, by using a low cost and scalable procedure. To this end, it is essential to control the different elaboration steps which are: (i) the deposition of ZnO seed layer films on silicon substrates by sol-gel approach, (ii) the growth of ZnO NWs on these seed layer films by hydrothermal synthesis, and (iii) the assembly of these NWs into ZnO nanonets by vacuum filtration. In-depth studies of each of these processes were thus carried out. This work enabled to elaborate reproducible and homogenous ZnO thin films, NWs and nanonets whose morphological properties are precisely controlled over a wide range. Two DNA biofunctionnalization protocols were then developed for the nanonets and led to encouraging results which need however to be further optimized. The nanonets were also integrated into functional devices and the first electrical characterizations provided promising results. In the longer term, this work opens the way to the collective integration of ZnO NWs which would enable the development of a new generation of portable, fast and ultra-sensitive (bio- or gas-) sensors.

abstract: In the search for chemical biosensors designed for patient-based physiological applications, non-invasive diagnostic approaches continue to have value. The work described in this thesis builds upon previous breath analysis studies. In particular, it seeks to assess the adsorptive mechanisms active in both acetone and ethanol biosensors designed for breath analysis. The thermoelectric biosensors under investigation were constructed using a thermopile for transduction and four different materials for biorecognition. The analytes, acetone and ethanol, were evaluated under dry-air and humidified-air conditions. The biosensor response to acetone concentration was found to be both repeatable and linear, while the sensor response to ethanol presence was also found to be repeatable. The different biorecognition materials produced discernible thermoelectric responses that were characteristic for each analyte. The sensor output data is presented in this report. Additionally, the results were evaluated against a mathematical model for further analysis. Ultimately, a thermoelectric biosensor based upon adsorption chemistry was developed and characterized. Additional work is needed to characterize the physicochemical action mechanism. / Dissertation/Thesis / M.S. Bioengineering 2011

Neste trabalho relata-se pela primeira vez a determinação de adenosina por um biossensor baseado em tirosinase. O biossensor foi desenvolvido mediante a modificação de um eletrodo de carbono impresso (SPE) com nanopartículas de ouro (AuNPs), tirosinase (Tyr) e Nafion, denominado biossensor Nafion/Tyr/AuNPs/SPE. As AuNPs sintetizadas possuem diâmetro médio de 15,0 ± 1,1 nm e sua função é melhorar a via de condução de elétrons entre a enzima e o eletrodo. Utilizou-se o aprisionamento com filme Nafion® para evitar a lixiviação enzimática da superfície do eletrodo. A tirosinase imobilizada apresentou boa atividade frente ao substrato catecol. Verificou-se que a adenosina atua como um inibidor do tipo não-competitivo. O biossensor é estável durante pelo menos 45 dias. Além disso, foi realizada a eletro-oxidação da adenosina para sua determinação. O biossensor apresenta sensibilidade superior em comparação com SPE, AuNPs/SPE e Nafion/AuNPs/SPE. As curvas de calibração revelaram duas faixas lineares para as concentrações de adenosina, de 1,0 × 10-5 mol L-1 até 5,0 × 10-5 mol L-1 e entre 6,0 × 10-5 mol L-1 e 1,2 × 10-4 mol L -1. O limite de detecção (3 × (desvio padrão + média dos brancos)/coeficiente angular da curva) foi de 7,0 × 10-7 mol L-1. / In this work we report for the first time the determination of adenosine by a biosensor based on tyrosinase. The biosensor was developed by modifying a screen-printed carbon electrode (SPE) with gold nanoparticles (AuNPs), tyrosinase (Tyr) and Nafion, denoted as Nafion/Tyr/AuNPs/SPE biosensor. The synthesized AuNPs have a mean diameter of 15.0 ± 1.1 nm and their function is to improve the electron conduction pathway between the enzyme and the electrode. The entrapment with Nafion® film was selected to prevent the enzyme lixiviation from the electrode surface. Immobilized tyrosinase showed good activity with the catechol substrate. It was found that adenosine acts as a non-competitive type inhibitor. The biosensor is stable for at least 45 days. In addition, the electro-oxidation of adenosine was performed for its determination. The biosensor has superior sensitivity compared to SPE, AuNPs/SPE and Nafion/AuNPs/SPE. Calibration curves revealed two linear ranges for adenosine concentrations of 1,010-5 mol L-1 up to 5,010-5 mol L-1 and from 6,010-5 mol L-1 to 1,210-4 mol L-1. The detection limit (3 × (standard deviation + mean of blanks)/slope of the curve) was 7,010-7 mol L-1.