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...

The colonization of immersed surfaces by a myriad of marine organisms is a complex, multi-stage, species-specific process giving rise to economic and environmental costs. This unwanted accumulation of organisms in the marine environment, called biofouling, has been attacked from different fronts, going from the ‘problem-elimination-as-problem-solving’ strategy (essentially through the use of biocides) to more elaborated and environmentally-friendly options based on the principle of ‘non-stick’ or ‘easy foul-release’ surfaces, which do not jeopardize marine life viability. Several marine organisms rely on proteinaceous adhesives to secure a holdfast to surfaces. Proteolytic enzymes have been demonstrated to be effective agents against settlement and settlement consolidation onto surfaces of marine bacteria, algae, and invertebrates, their proposed mode-of-action being the enzymatic degradation of the proteinaceous components of the adhesives. So far, however, the evidence remains inconclusive since most of the published investigations refer to commercial preparations where the enzyme is mixed with other components, like additives, which obviously act as additional experimental variables. This work aims at providing clear, conclusive evidence about the potential of serine proteases to target the adhesives produced by a group of model marine biofoulers. The strategy towards the goal consisted in the preparation and characterization of maleic anhydride copolymer nanocoatings modified by a surface-bound enzyme, Subtilisin A, the active constituent of the commercial preparations reported as effective against biofouling. The enzyme-containing maleic anhydride copolymer films were characterized (enzyme surface concentration, activity, stability, roughness and wettability) and thereafter tested in biological assays with three major biofoulers: spores of the green alga Ulva linza, cells of the pennate diatom Navicula perminuta, and cyprid larvae of the barnacle Balanus amphitrite. The purpose of the biological assays was to elucidate the efficacy of the immobilized catalyst to discourage settlement and/or to facilitate removal of these organisms from the bioactive layers. Results confirmed the initial hypotheses related to the enzymatic degradation of the biological adhesives: the immobilized protease was effective at reducing the adhesion strength of Ulva spores and Navicula diatoms in a manner that correlated with the enzyme activity and surface concentration, and deterred settlement of Balanus amphitrite barnacle cyprids even at the lowest surface activity tested. By facilitating the removal of biofilm-forming diatoms and of spores of the troublesome alga Ulva linza, as well as by interfering with the consolidation of adhesion of the calcareous Balanus amphitrite macrofouler, the enzyme-containing coatings here disclosed are considered to constitute an appealing and promising alternative to control marine biofouling without jeopardizing marine life.

El presente trabajo de investigación busca Identificar de qué manera afecta el congestionamiento causado por la demora en la disposición de mercancías en Abandono y/o inmovilizadas a los Depósitos Temporales autorizados por la SUNAT en la Intendencia Aduana Marítima del Callao, en el 2019; para ese efecto nuestro estudio se divide en cuatro (4) capítulos, los cuales son detallados a continuación: El primer capítulo se centra en el plan de investigación, por medio del cual planteamos la situación problemática, los problemas, los objetivos principales y específicos; y la formulación de la hipótesis de trabajo de la investigación. El segundo capítulo da a conocer los antecedentes de la investigación nacionales e internacionales, el marco conceptual y el análisis del sector a investigar. El tercer capítulo se centra en la metodología de la investigación, donde detallamos el tipo, propósito, categorías, delimitaciones, diseño de la investigación y tamaño de muestra. Asimismo, en este capítulo desarrollamos las entrevistas de profundidad, las cuales serán de utilidad para el recojo de información. Finalmente, el cuarto capítulo se enfoca en el análisis y discusión de resultados de nuestra investigación para así encontrar los hallazgos y realizar tanto las conclusiones como las recomendaciones finales por medio del análisis de documentos y de la información obtenida mediante las entrevistas de profundidad desarrolladas en nuestro estudio. / This research work seeks to identify how the congestion caused by the delay in the disposal of goods in Abandonment and/or immobilized in the Temporary Deposits authorized by SUNAT in the Callao Maritime Customs Administration, until 2019; therefore, our study is divided into four (4) chapters, which are detailed below. The first chapter focuses on the research plan, through which we pose the question, the problem and the objectives, both main and specific, and the formulation of the initial research hypothesis. The second chapter discloses the research background, both national and international, and the conceptual framework, and the analysis of the sector to be investigated. The third chapter focuses on the research methodology, where we detail the type, purpose, categories, delimitation, design and sample size of the research. Likewise, in this chapter we develop in-depth interviews, which will be useful for gathering information. Finally, the fourth chapter focuses on the analysis and discussion of the results of our research in order to find the findings and make both the conclusions and the final recommendations through the analysis of documents and the information obtained through the in-depth interviews carried out in our study. / Tesis

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.

Biochar application as a soil additive is gaining global acceptance. In this era of climate change, biochar use for improved soil productivity is not just a sustainable eco-friendly substitute to synthetic fertilizer, but a noble contributor to the fight against climate change. Although biochar has been accredited with some environmental and agricultural benefits, most studies concentrated on agricultural and biowaste products as feedstocks. This research was designed to explore P-enriched modified-Douglas fir biochar potential as a soil additive. Using corn as a test crop, greenhouse studies were performed on acidic sandy soil, comparing phosphorus enriched modified-Douglas fir biochar efficacy to a commercial synthetic triple superphosphate fertilizer and a control treatment. Incubation studies were also performed to evaluate the liming and heavy metal immobilization efficacies. Firstly, P-enriched modified-Douglas fir biochar’s ability to release plant soluble P was investigated. At various P enrichment concentration, soil plant availability P from P enriched modified-Douglas fir biochar treatments differed insignificantly from superphosphate fertilizer treatment. The direct correlations between both K and Mg recoveries with available soil P, suggested P enriched modified-Douglas fir biochar potential to supply multiple plant nutrients. Secondly, the influence of P uptake on plant growth and P use efficiency was examined. The greater agronomic P use efficiency obtained in P enriched modified-Douglas fir biochar (~32 kg kg-1) than the triple supper phosphate fertilizer (~17 kg kg-1) treatment confirmed P enriched modified-Douglas fir biochar potential as a multiple nutrient released soil additive. Thirdly, biochar-supported phosphate (BP) effectively reduced Pb2+ mobility in simulated contaminated soil. Pb2+reacted with phosphate from Ca10(PO4)6(OH)2 embedded in the biochar supported phosphate at pHPb10(PO4)6(OH)2. Finally, the amendment of acidic soils with modified P-enriched modified-biochar improved soil buffering capacity because of its enhanced ash contents, alkalinity, and surface functional groups. Spectroscopic methods were used to analyze biochar, soil, and plant materials extracts. Several other analytical methods including BET and thermogravimetric analyses were used to characterized biochar. These findings suggest that the use of phosphorus enriched modified-Douglas fir biochar in agricultural soil is a feasible relatively low-cost, effective, and environmentally sustainable soil management and P recycling strategy

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.

Microcantilevers have been investigated as high sensitivity, label free biosensors for approximately 15 years. In nearly all cases, a thin gold film deposited on the microcantilevers is used as an intermediate attachment layer because of the convenience of thiol-gold chemistry. Unfortunately, this attachment chemistry can be unstable when used with complex sample media such as blood plasma. The Nordin group at BYU has recently developed an all-silicon in-plane photonic microcantilever (PMCL) technology to serve as a platform for label-free biosensing. It has the advantage of being readily scalable to simultaneous readout of many PMCLs in array format, and allows integration with polymer microfluidics to facilitate the introduction of biological samples and reagents. An essential processing step for the transformation of the PMCL into a practical biosensor is the ability to effectively immobilize active biological receptors directly on silicon PMCL surfaces such that ligand binding generates sufficient surface stress to cause measureable PMCL deflection. This dissertation presents the development of a method to functionalize the sensor surface of all-silicon in-plane photonic microcantilever (PMCL) arrays. This method employs a materials inkjet printer for non-contact jetting and a fluid that is custom designed for ink-jetting and biological applications with approximately 1 pL droplet size. The method facilitates the application of different receptors on select PMCLs with drop placement accuracy in the +/- 7.5 μm range. The functionalization fluid facilitates further processing using humidity control to achieve full coverage of only the PMCL's top surface and removal of dissolved salts to improve uniformity of receptor coverage and to prevent fouling of the sensor surface. Once a functionalization method was successfully developed, a series of experiments were performed to investigate the amount of surface stress that can be generated when receptors are immobilized directly to the silicon surface. In one series of experiments, a 4.8 μM streptavidin solution was used with biotin immobilized on multiple PMCLs to demonstrate adsorption-induced surface stress and concomitant deflection of the PMCL. The group observed ~ 15 nm PMCL deflection on average, with a corresponding surface stress of approximately 4 mN/m. These experiments yield the sensor response in real-time and employ a combination of multiple PMCLs functionalized as either sensors or unfunctionalized to serve as references. Investigation of various attachment chemistries is included, as well as a comparison with and without passivation of non-sensor surfaces. Investigated passivation strategies prevented ligand binding from generating a differential surface stress. Failure modes and physical mechanisms for adsorption-induced surface stress are discussed. Immobilization and passivation strategies for antibody-based biosensing are demonstrated with fluorescence microscopy and a corresponding PMCL sensing experiment using rabbit anti-goat F(ab') fragments as the receptors and Alex Fluor 488 labeled goat anti-rabbit IgGs as the ligand. While the results of these experiments remain inconclusive, suggestions for future research involving the PMCL sensor array are recommended.

Den nära förestående bristen på fossila resurser i kombination med deras associerade miljöfarlighet betonar behovet av utveckling av alternativa, mer hållbara kemikalier. I denna studie utvecklades en enzymatisk kaskad för förädling av 5-(hydroxymetyl)furfural (HMF) till 5-(aminometyl)-2-furfuraldehyd (AMFA). Kaskaden omfattar transaminering av HMF till 5-(hydroxymetyl)furfurylamin (HMFA) följt av oxidation av HMFA till AMFA. Transaminas från Silicibacter pomeroyi (SpATA) immobiliserades via his6-taggar på EziG-proteinbärare från EnginZyme AB. Proteinbärarna placerades i spin-kolonner under transamineringen vilket möjliggjorde omhändertagande av SpATA efter transamineringen av HMF. För oxidationen utvärderades alkoholdyhydrogenas från Thermoanaerobacter brockii och hästlever samt galaktosoxidas från Dactylium dendroides (GOase). Omsättning och produktbildning analyserades med HPLC. Resultaten indikerar att SpATA effektivt katalyserar transamineringen av HMF, att alkohol dehydrogenasen inte förmår katalysera oxidationen av HMF till HMFA och att galaktosoxidaset kan oxidera HMFA med hög omsättning vilket leder oss att tro att den föreslagna kaskaden för förädling av HMF till AMFA är möjlig. / The imminent shortage of fossil resources coupled with their associated environmental hazards stresses the need for the development of alternative, more sustainable chemicals. In this study an enzymatic cascade was developed for the valorisation of 5-(hydroxymethyl)furfural (HMF) into 5-(aminomethyl)-2-furfuraldehyde (AMFA). The cascade involves the transamination of HMF into 5-(hydroxymethyl)furfurylamine (HMFA) followed by the oxidation of HMFA into AMFA. Transaminases from Silicibacter pomeroyi (SpATA) was immobilised via his6-tags onto EziG-protein carriers from EnginZyme AB. The protein carriers were placed in spin-columns during the transamination which allowed for salvaging of the SpATA after the transamination of HMF. For the oxidation, alcohol dehydrogenases from Thermoanaerobacter brockii and horse liver as well as galactose oxidase from Dactylium dendroides (GOase) were evaluated. The conversion and product formation were analysed by HPLC. The results indicate that the SpATA efficiently catalyses the transamination of HMF, that the alcohol dehydrogenases are not able to catalyse the oxidation of HMF nor HMFA and that the GOase can oxidize HMFA with high conversion which leads us to believe that the proposed cascade for the valorisation of HMF to AMFA is feasible.

Immobilization of proteins by covalent coupling to polymeric materials offers numerous excellent advantages for various applications, however, it is usually limited by coupling strategies, which are often too expensive or complex. In this study, an electron-beambased process for covalent coupling of the model protein bovine serum albumin (BSA) onto polyvinylidene fluoride (PVDF) flat sheet membranes was investigated. Immobilization can be performed in a clean, fast, and continuous mode of operation without any additional chemicals involved. Using the Design of Experiments (DoE) approach, nine process factors were investigated for their influence on graft yield and homogeneity. The parameters could be reduced to only four highly significant factors: BSA concentration, impregnation method, impregnation time, and electron beam irradiation dose. Subsequently, optimization of the process was performed using the Response Surface Methodology (RSM). A one-step method was developed, resulting in a high BSA grafting yield of 955 mgm−2 and a relative standard deviation of 3.6%. High efficiency was demonstrated by reusing the impregnation solution five times consecutively without reducing the final BSA grafting yield. Comprehensive characterization was conducted by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), and measurements of zeta potential, contact angle and surface free energy, as well as filtration performance. In addition, mechanical properties and morphology were examined using mercury porosimetry, tensile testing, and scanning electron microscopy (SEM).

No description available.