A manipulação de materiais em escala nanométrica representa uma das fronteiras em nanociência e nanotecnologia, devido à possibilidade de controle de propriedades específicas do material. No caso de materiais biológicos, em particular, a manipulação e imobilização na forma de filmes ou camadas ultrafinas é crucial para seu emprego em dispositivos biotecnológicos. Neste trabalho, objetivou-se o estudo de detecção de diferentes hormônios tireoidianos (HTs) e análogos a partir da imobilização da região LBD do receptor de hormônio tireoidiano humano TRTRβ1 em um eletrodo interdigitado, para o desenvolvimento de um biossensor capacitivo. Este sistema consiste em um arranjo estrutural na forma de filme fino capaz de distinguir a interação específica receptor-ligante de outras interações possivelmente interferentes, visando a quantificação dos níveis de HTs. Para isto, a técnica de SAMs (Self-Assembled Monolayers) foi empregada, por permitir um alto controle da espessura e ordenamento molecular dos filmes, assim como a preservação das atividades das biomoléculas. Análises espectroscópicas e morfológicas foram realizadas para o estudo de adsorção das biomoléculas no filme. As interações específicas receptor-ligante foram avaliadas por meio de respostas elétricas (impedância) do biossensor contendo o TRβ1-LBD imobilizado em um filme orgânico ultrafino, e também por SPR (Surface Plasmon Resonance). Os resultados mostraram a capacidade dos eletrodos contendo TRTRβ1-LBD de detectar e diferenciar entre diferentes HTs em concentrações da ordem de nanomolar, compatível com níveis fisiológicos, evidenciando o grande potencial de aplicação para este sistema no diagnóstico e tratamento de disfunções tireoidianas. / Manipulation of materials at the nanoscale represents one of the frontiers in nanoscience and nanotechnology, mainly due to the possibility of specific controlling, improved properties, not observed if conventional bulk processing is applied. For biomolecules, in particular, processing via immobilization in the form of nanostructured films has allowed their use in biotechnological applications and devices. In this master dissertation, we aimed at investigating the immobilization of the LBD domain of human thyroid hormone receptor TRTRβ1 on interdigitated electrodes, to be used as capacitive biosensors for thyroid hormones (THs) and analogues detection. The nuclear receptors were immobilized via SAMs (Self-Assembled Monolayers), since this technique allows a high control of molecular order and thickness of the films, as well as the preservation of biological activities. Spectroscopic and morphological analyses were performed to investigate the adsorption of biomolecules on the nanostructured film. The interactions between receptor - ligand were also evaluated by means of electrical response (impedance) and SPR (Surface Plasmon Resonance). The bioelectrodes containing immobilized TRTRβ1 were capable of detecting and distinguishing among different HTs, including T3, T4, TRIAC and GC-1 at concentrations down to nanomolar, compatible with physiological levels. The latter results point to the possibility of applications of the bioelectrodes in the diagnosis and treatment of thyroid dysfunctions.

A manipulação de materiais em escala nanométrica representa uma das fronteiras em nanociência e nanotecnologia, devido à possibilidade de controle de propriedades específicas do material. No caso de materiais biológicos, em particular, a manipulação e imobilização na forma de filmes ou camadas ultrafinas é crucial para seu emprego em dispositivos biotecnológicos. Neste trabalho, objetivou-se o estudo de detecção de diferentes hormônios tireoidianos (HTs) e análogos a partir da imobilização da região LBD do receptor de hormônio tireoidiano humano TRTRβ1 em um eletrodo interdigitado, para o desenvolvimento de um biossensor capacitivo. Este sistema consiste em um arranjo estrutural na forma de filme fino capaz de distinguir a interação específica receptor-ligante de outras interações possivelmente interferentes, visando a quantificação dos níveis de HTs. Para isto, a técnica de SAMs (Self-Assembled Monolayers) foi empregada, por permitir um alto controle da espessura e ordenamento molecular dos filmes, assim como a preservação das atividades das biomoléculas. Análises espectroscópicas e morfológicas foram realizadas para o estudo de adsorção das biomoléculas no filme. As interações específicas receptor-ligante foram avaliadas por meio de respostas elétricas (impedância) do biossensor contendo o TRβ1-LBD imobilizado em um filme orgânico ultrafino, e também por SPR (Surface Plasmon Resonance). Os resultados mostraram a capacidade dos eletrodos contendo TRTRβ1-LBD de detectar e diferenciar entre diferentes HTs em concentrações da ordem de nanomolar, compatível com níveis fisiológicos, evidenciando o grande potencial de aplicação para este sistema no diagnóstico e tratamento de disfunções tireoidianas. / Manipulation of materials at the nanoscale represents one of the frontiers in nanoscience and nanotechnology, mainly due to the possibility of specific controlling, improved properties, not observed if conventional bulk processing is applied. For biomolecules, in particular, processing via immobilization in the form of nanostructured films has allowed their use in biotechnological applications and devices. In this master dissertation, we aimed at investigating the immobilization of the LBD domain of human thyroid hormone receptor TRTRβ1 on interdigitated electrodes, to be used as capacitive biosensors for thyroid hormones (THs) and analogues detection. The nuclear receptors were immobilized via SAMs (Self-Assembled Monolayers), since this technique allows a high control of molecular order and thickness of the films, as well as the preservation of biological activities. Spectroscopic and morphological analyses were performed to investigate the adsorption of biomolecules on the nanostructured film. The interactions between receptor - ligand were also evaluated by means of electrical response (impedance) and SPR (Surface Plasmon Resonance). The bioelectrodes containing immobilized TRTRβ1 were capable of detecting and distinguishing among different HTs, including T3, T4, TRIAC and GC-1 at concentrations down to nanomolar, compatible with physiological levels. The latter results point to the possibility of applications of the bioelectrodes in the diagnosis and treatment of thyroid dysfunctions.

This licentiate thesis is focused on two methods for protein immobilization to biosensor surfaces for future applications in protein microarray formats. The common denominator is a surface chemistry based on a gold substrate with a self-assembled monolayer (SAM) of functionalized alkanethiolates. Both methods involve photochemistry, in the first case for direct immobilization of proteins to the surface, in the other for grafting a hydrogel, which is then used for protein immobilization. Paper I describes the development and characterization of Chelation Assisted Photoimmobilization (CAP), a three-component surface chemistry that allows for covalent attachment and controlled orientation of the immobilized recognition molecule (ligand) and thereby provides a robust sensor surface for detection of analyte in solution. The concept was demonstrated using His-tagged IgG-Fc as the ligand and protein A as the analyte. Surprisingly, as concluded from IR spectroscopy and surface plasmon resonance (SPR) analysis, the binding ability of this bivalent ligand was found to be more than two times higher with random orientation obtained by amine coupling than with homogeneous orientation obtained by CAP. It is suggested that a multivalent ligand is less sensitive to orientation effects than a monovalent ligand and that island formation of the alkanethiolates used for CAP results in a locally high ligand density and steric hindrance. Paper II describes the development of nanoscale hydrogel structures. These were photografted on a SAM pattern obtained by dip-pen nanolithography (DPN) and subsequent backfilling. The hydrogel grew fast on the hydrophilic patterns and slower on the hydrophobic background, which contained a buried oligo(ethylene glycol) (OEG) chain. Using IR spectroscopy, it was found that the OEG part was degraded during UV light irradiation and acted as a sacrificial layer. In this process other OEG residues were exposed and acted as new starting points for the self-initiated photografting and photopolymerization (SIPGP). A biotin derivative was immobilized to the hydrogel density pattern and interaction with streptavidin was demonstrated by epifluorescence microscopy.

No description available.

The scope of this work is to fathom different possibilities to create functional coatings with polymer brushes. The immobilization of nanoparticles and enzymes is investigated, as well as the affection of their properties by the stimuli-responsiveness of the brushes. Another aspect is the coating of 3D-nanostructures by polymer brushes and the investigation of the resulting functional properties of the hybrid material. The polymer brush coatings are characterized by a variety of microscopic and spectroscopic techniques, with a special emphasis on the establishment of the combinatorial quartz crystal microbalance/spectroscopic ellipsometry technique as a tool to characterize the functional properties of the polymer brush systems insitu. The pH-responsive swelling of the polyelectrolyte brushes poly(acrylic acid) and poly(2-vinylpyridine), as well as the thermoresponsive swelling of poly(N-isopropylacryl amide) is studied in detail by this technique. Poly(2-vinylpyridine) and binary poly(N-isopropylacryl amide)-poly (2-vinylpyridine) brushes are used as templates for the insitu-synthesis of palladium and platinum nanoparticles with catalytic activity. As an example for the use of polymer brushes to immobilize enzymes, the model enzyme glucose oxidase is physically adsorbed to poly (2-vinylpyridine) and poly (acrylic acid) brushes and also covalently bound to poly (acrylic acid) brushes. In the last part of this thesis, sculptured thin films are coated with poly (acrylic acid) and poly (N-isopropylacryl amide) brushes and the swelling characteristics as well as the adsorption behavior of the model protein bovine serum albumin are investigated.

De nombreuses techniques de lithographie sont proposées aujourd'hui pour structurer des surfaces à l'échelle micrométrique et nanométrique. Parmi elles, la lithographie colloïdale est intéressante en raison notamment du faible coût du procédé. Dans cette thèse, nous avons développé une nouvelle technique appelée « lithographie électro-colloïdale » qui est basée sur l'utilisation de particules colloïdales soumises à des champs électriques continus et alternatifs. Avec des temps de préparation courts et une instrumentation peu coûteuse, des surfaces structurées polymériques et métalliques ont été produites puis testées pour différentes applications (immobilisation de protéines, réseaux de microélectrodes, surfaces superhydrophobes). / Many lithography techniques have been developed to structure surfaces at the micrometer and sub-micrometer ranges. Among them, colloidal lithography is interesting because the process is inexpensive and does not require the use complex instruments. In this thesis, we have developed a new technique called « electro-colloidal lithography » which is based on the use of colloidal particles organized using alternating and direct electric fields. With short preparation times and inexpensive instruments, polymeric and metallic structured surfaces have been prepared and tested for different applications (protein immobilization, microelectrode arrays, superhydrophobic surfaces)

No description available.

Title: Nano-structured multicomponent plasma polymers for controlled immobilization of biomolecules Author: Iurii Melnichuk Department / Institute: Department of Macromolecular Physics/Charles University Supervisor of the doctoral thesis: Doc. Ing. Andrey Shukurov, Ph.D. Abstract: The aim of this thesis is to highlight the feasibility of tailored nano- structures in functionalizing surfaces for biointerfacial interactions. Development of new techniques for the production of nanoscaled biomaterials can be of use in a variety of medical and biological applications, e.g. biosensors, microarrays, drug sensors, implants, blood-contacting devices. This thesis first examines the early stages of nano-structured thin film growth fabricated by vapor phase deposition of poly(ethylene). We discuss island growth within a framework of rate equation theory, dynamic scaling theory and capture zone distribution. In a second stage, we test dielectric barrier discharge to activate PE nano-pattern for covalent immobilization of proteins. Finally, we assess cell behavior on surfaces in dependence on morphology and the presence of cell adhesive protein tropoelastin. We employ plasma polymerization to produce ultrathin hydrocarbon layer capable of protein anchoring. The thesis findings for the first time manifest the critical...

Det uppskattas att 15% av alla par världen över lider avinfertilitet. Ungefär hälften beror på manlig infertilitet och 40% av dessa fall kan ännu inte förklaras. Därmed är nuvarande metoder för att diagnostisera manlig infertilitet otillräckliga och ytterligare tekniker behövs. En lyckad befruktning kräver att spermierna uttrycker membranproteinet Izumo1 som måste känna igen dess receptorprotein Juno, belägen vid ytan av äggmembranet. Bindningen mellan Juno och Izumo1 är essentiell för befruktning hos däggdjur då den bidrar till att gameternabinder och skapar en ny distinkt organism. Juno är ett relativt nyupptäckt protein och mekanismen med Izumo1 är fortfarande okänd. Ett nystartat företag vid namn Spermosens vill mäta interaktionen mellan Juno ochIzumo1 i ett nytt diagnostikverktyg som ska diagnostisera manlig infertilitet. Tanken är att Juno ska immobiliseras på guld-nanopartiklar och användas för att mätainteraktionen med spermaprover. Det nya verktyget ska hjälpa par att fastställa felet i befruktningen, vilket som en följd skulle hjälpa paret att välja lämplig assisteradreproduktionsmetod. I utvecklingen av det nya diagnostikverktyget behöver det konfirmeras att den Juno som används i enheten kan binda korrekt till mänskligt Izumo1. Därför måste interaktionerna mellan de mänskliga, rekombinanta proteinerna Juno och Izumo1 mätas och karakteriseras. Syftet med detta projekt var att utveckla en metod för att immobilisera Juno på guldnanopartiklar och sedan mäta interaktionerna med Izumo1 genom UV-Visspektroskopi. Detta är teoretiskt möjligt eftersom guld-nanopartiklarna framkallar ett fenomen som kallas lokaliserad ytplasmonresonans som varierar beroende påstorleken på guld-nanopartikelkomplexet. Immobiliseringsmetoden var en process som involverade flera steg som designades, polerades och förbättrades underarbetets gång. Dithiobis(C2-NTA) konjugerades till guldytan och koboltjonerkonjugerades till NTA. Det sista steget som innebar konjugering av Juno till kobolt genom en His-tag lyckades inte, och interaktionerna kunde därför inte mätas genom denna metod. Istället mättes protein-protein-interaktionen genom SPR-mätningar med Biacore, ett instrument som också är baserat på ytplasmonresonans. Interaktioner mellan Izumo1 och Juno kunde uppmätas både vid användning av Juno producerad från E. coli ochfrån däggdjursceller. Dissociationskonstanten (Kd) beräknades till 7-33 nM (för Junooch Izumo1 producerade i däggdjursceller) vilket kan jämföras med ett experimentfrån 2016 där 48 nM beräknades. Ett mer exakt Kd kunde inte fastställas och entrolig anledning till detta var att regenereringen av sensorytan som utfördes med NaOH varierade i effektivitet, vilket ledde till en osäkerhet då ytförhållandena kan ha varierat mellan mätningarna. De två Juno-proteinerna, som är producerade i olika organismer, visade två skilda affinitetsprofiler med Izumo1 vilket tyder på att glykosyleringen påverkar bindningsmekanismen mellan Juno och Izumo1. / It is estimated that 15% of all couples worldwide suffer from infertility. Roughly half is male-factor infertility and 40% of these cases cannot be explained. Thus, current methods for diagnosing male infertility are not enough and further techniques are needed. To have a successful fertilisation event, it is required that the sperm expresses membrane surface-protein Izumo1 which must recognise its counterpart protein Juno, located at the surface of the egg membrane. The recognition step between Juno and Izumo1 is essential in mammalian fertilisation for the gametes to bind and start the creation of a new distinct organism, but the molecular mechanism is still unknown.A start-up company named Spermosens want to measure the Juno-Izumo1 interaction in a new diagnostic device designed to diagnose male infertility. The idea is to have Juno immobilised on gold nanoparticles and measure the interaction between Juno and various semen samples. The new device is supposed to help couples pin-point the procreation issue which would help in the selection of suitable assisted reproductive technology. In the development of the new device, it had to be established that the Juno used in the device will bind correctly to human Izumo1. Therefore, the interactions between the human recombinant proteins Juno and Izumo1 had to be measured and characterized.The objectives of this project were to develop a method to immobilise Juno on gold nanoparticles and then measure the interactions with Izumo1 using UV-vis spectroscopy. This is theoretically possible since the gold nanoparticles exhibit a phenomenon called localized surface plasmon resonance that vary depending on the size of the gold nanoparticle-complex. The immobilisation procedure was a process involving several steps that were designed, polished and improved along the way. Dithiobis(C2-NTA) was conjugated to the gold surface and a cobalt ion was conjugated to the NTA. The last step involving conjugation of Juno to the cobalt through a His-tag was not succeeded, and the interactions could therefore not be measured this way.Instead, the protein-protein interaction was measured through SPR-measurements using Biacore, an instrument that is based on surface plasmon resonance as well. Interactions between Izumo1 and Juno could be detected using Juno produced in E. coli and in mammalian cells. The dissociation constant (Kd) could be calculated to 7-33 nM which can be compared to a previously published Kd of 48 nM. A more precise Kd could not be established, possibly due to that the regeneration of the sensor surface with NaOH varied in efficiency, leading to changing surface conditions during the measurements. The two Juno proteins, that were produced in different hosts, showed two different affinity profiles with Izumo1, which contributes to the suggestion that the glycosylation plays a role in the binding mechanism between Juno and Izumo1.

The scope of this work is to fathom different possibilities to create functional coatings with polymer brushes. The immobilization of nanoparticles and enzymes is investigated, as well as the affection of their properties by the stimuli-responsiveness of the brushes. Another aspect is the coating of 3D-nanostructures by polymer brushes and the investigation of the resulting functional properties of the hybrid material. The polymer brush coatings are characterized by a variety of microscopic and spectroscopic techniques, with a special emphasis on the establishment of the combinatorial quartz crystal microbalance/spectroscopic ellipsometry technique as a tool to characterize the functional properties of the polymer brush systems insitu. The pH-responsive swelling of the polyelectrolyte brushes poly(acrylic acid) and poly(2-vinylpyridine), as well as the thermoresponsive swelling of poly(N-isopropylacryl amide) is studied in detail by this technique. Poly(2-vinylpyridine) and binary poly(N-isopropylacryl amide)-poly (2-vinylpyridine) brushes are used as templates for the insitu-synthesis of palladium and platinum nanoparticles with catalytic activity. As an example for the use of polymer brushes to immobilize enzymes, the model enzyme glucose oxidase is physically adsorbed to poly (2-vinylpyridine) and poly (acrylic acid) brushes and also covalently bound to poly (acrylic acid) brushes. In the last part of this thesis, sculptured thin films are coated with poly (acrylic acid) and poly (N-isopropylacryl amide) brushes and the swelling characteristics as well as the adsorption behavior of the model protein bovine serum albumin are investigated.