Surface immobilization of probe molecules in surface based assays is a key area of research in the continued development of immunoassay microarrays. Interest continues to grow in microarray based immunoassays given their potential as a high throughput technique for immunodiagnostics. Therefore, it is important to thoroughly study and understand the implications of interfacial chemistry and immobilization conditions on the performance of the assay. This thesis presents a body of work that examines the impact of probe density, interfacial chemistry, and enhancement factors for arrays read with surface plasmon resonance (SPR) imaging. An array of structurally similar Salmonella disaccharides was immobilized at varying densities and the interface formed was thoroughly investigated to determine the properties of the interface. The arrays were then used with SPR imaging to evaluate the binding of an antibody specific for one disaccharide of the three stereoisomers on the array. A dilute disaccharide surface was found to provide optimal antibody binding. Higher densities result in steric hindrance of antibody binding by not allowing the disaccharide to insert into the antibody binding pocket. The role of interfacial chemistry in antibody attachment was studied to determine optimum conditions. The study examined physical adsorption, covalent attachment, and affinity capture. It was found that covalent attachment provided the most stable attachment and resulted in the lowest levels of antigen detection. Both the physical adsorption and affinity capture provided larger antigen binding capacity and therefore more sensitive antigen detection. The covalent attachment was chosen to evaluate an enhanced assay with the incorporation of gold nanoparticles. These particles provided detection limits that were an order of magnitude improved over those excluding the nanoparticles. A novel surface chemistry for antibody immobilization in SPR imaging studies was evaluated. This involved the electrochemical driven formation of mono- to multilayers of diazonium benzoic acid films. The studies showed the ability to control the thickness of the films formed and also the ability of the antibody chips to capture antigen from solution.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / As mais importantes lactases, em termos de interesse biotecnolÃgico, sÃo aquelas produzidas por leveduras do gÃnero Kluyveromyces, que sÃo intracelulares e, em sua maioria, sÃo obtidas por fermentaÃÃo em cultura submersa. Esta tÃcnica, assim como a maioria dos processos biotecnolÃgicos, envolve a necessidade de purificaÃÃo de proteÃnas e peptÃdeos a partir de uma variedade de fontes. Neste contexto, uma das tÃcnicas mais notavelmente promissoras à a cromatografia de modo misto, que permite interaÃÃes iÃnicas e hidrofÃbicas simultaneamente entre o adsorvente e o adsorbato. O objetivo do presente trabalho foi estudar a viabilidade da recuperaÃÃo e purificaÃÃo da enzima β-galactosidase, produzida por meio de processo fermentativo e utilizando o micro-organismo Kluyveromyces lactis, por tÃcnica de cromatografia de modo misto. OperaÃÃes unitÃrias de precipitaÃÃo proteica e diÃlise foram tambÃm realizadas com o intuito de concentrar a enzima de interesse e eliminar detritos celulares e outros interferentes advindos do meio de fermentaÃÃo, o que ocasionaria uma diminuiÃÃo do rendimento do processo. A produÃÃo se apresentou satisfatÃria, com uma mÃdia de valores de concentraÃÃo de enzimas totais de 0,45 mg/mL, atividade enzimÃtica de 67 U/mL, atividade especÃfica de 167,9 U/mg. O Fator de PurificaÃÃo obtido foi de 1,17. Uma precipitaÃÃo seguida de diÃlise foi realizada e a posterior corrida cromatogrÃfica em leito fixo com esse material rendeu valores de recuperaÃÃo de 41,0 e 48,2% de proteÃna total e atividade total, respectivamente. A anÃlise de eletroforese SDS-PAGE confirmou a evoluÃÃo do processo de purificaÃÃo no decorrer das operaÃÃes unitÃrias, atestando a viabilidade do emprego das tÃcnicas utilizadas para obtenÃÃo de enzimas com considerÃvel grau de pureza com alto valor comercial agregado. / The most important lactases, as far as biotechnological interest is concerned, are those produced by Kluyveromyces yeasts, which are intracellular and currently obtained mostly by submerged-state fermentation. This technique, just as the mainstream biotechnological processes, involves a need for protein and peptide purification from a variety of sources. In this context, one of the most promising notable techniques that can be highlighted is Mixed Mode Chromatography, which allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. Thus, the aim of this work was to assess the feasibility of recovery and purification of a Kluyveromyces lactis β-galactosidase, produced via fermentation process, by employing Mixed Mode Chromatography. Unit operations, such as protein precipitation and dialysis were also performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium, something that would result in a decrease in the process yield. The production showed satisfactory results, with mean values for total enzyme concentration of 0.45 mg/mL, enzymatic activity of 77 U/mL and specific activity of 167,9 U/mg. The Purification Factor obtained was 1.17. A precipitation step, followed by a dialysis process, was performed and the later chromatographic run carried out in fixed bed with this material yielded recovery values of 41.0 and 48.2% of total protein and activity, respectively. SDS-PAGE Electrophoresis confirmed the purification evolution throughout the unit operations employed, confirming the viability of the employment of the techniques used to obtain an enzyme of considerable degree of purity and possessing high-added value.

Nitrones are alternatives to azides in rapid strain-promoted 1,3-dipolar cycloadditions with cyclooctynes. To evaluate the differences between nitrones and azides we have performed kinetic studies of strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions of biarylazacyclooctynone (BARAC) with various acyclic and cyclic nitrones. The reactions were conducted under pseudo first-order reaction conditions using UV-visible spectroscopy. The reactivity of the acyclic nitrones was evaluated by varying the stereoelectronic and steric character of substituents at both the α-aryl and nitrogen positions. Cyclic nitrone reactivity was assessed according to the size of the ring and additional steric and strain effects. The obtained second-order rate constants for reactions of BARAC with cyclic nitrones were found to be greater than those for acyclic nitrones. However, all nitrones employed in the kinetic studies herein displayed significantly greater reactivity than azides in the analogous cycloadditions with BARAC. It is of particular note that the five-membered cyclic nitrones showed exceptional reactivity and, if used as rapid alternatives to azides in reactions with BARAC, can increase the reaction rates by up to 50 fold. An attempt to synthesize an allylated BARAC analogue is also described; the rearrangement reaction leading to the unexpected products is reported. The reaction rate for the novel rearrangement under both neutral and acidic conditions was obtained and plausible mechanisms for formation of products are proposed. Based on the results reported herein we anticipate that development of a labelling probe based on BARAC and a five-membered cyclic nitrone would allow for significant decrease of the concentrations of labelling reagents, thereby minimizing reaction time and reagent usage in life sciences applications. Nevertheless, a possible labelling decrease due to side reactions should be given consideration for prolonged labelling.

Current high-throughput screening (HTS) tools (i.e., single-use 96-well filter plate) are limited to the few membrane types that are sold commercially, restricting the ability to screen membrane materials for targeted applications. In this thesis, a multi-well device capable of screening any flat-sheet membrane was designed, where multiple devices can be stacked for extensive HTS (>32 experiments). Confocal imaging of a Natrix Q cross-section – a membrane type not sold in a commercial filter plate – was carried out after 24 h in contact with green fluorescent protein to visually confirm protein-membrane interactions. The static binding capacity (SBC) of bovine serum albumin (BSA) and Herring testes DNA was found for specific parameters: membrane type (Mustang Q, Sartobind Q, Natrix Q, Durapore), salt concentration (0, 50, 100 mM NaCl), and contact time (1 min, 4 h, 8 h, 24 h). Considering solution conditions, the highest BSA SBC was observed with Natrix Q at 0 M NaCl with a contact time of 24 h. The DNA and BSA SBC values for Natrix Q were the highest among the membrane types evaluated, demonstrating consistency with literature trends. These findings suggest that SBC experiments can predict promising membrane materials for scaled-up applications. Finally, the chromatography process was replicated in this multi-well device (Natrix Q), showing 50% BSA elution from the membrane. The results of this thesis confirmed this ability to accommodate any membrane adsorber, simultaneously compare different membrane materials, and extract the membrane for post-experimental analysis. This work’s significance was emphasized in its future potential to aid with membrane material selection, particularly with exploring the properties of next-generation membrane materials (e.g., 3D-printed membranes). Three future areas for optimization with this multi-well device were highlighted: biotherapeutic purification, sequencing of membrane materials within a process, and applying it as a tool to understand ion selectivity. / Thesis / Master of Applied Science (MASc) / Membranes are used in many industries, such as water treatment, environmental remediation, and biopharmaceuticals. In the biopharmaceutical industry, high-throughput screening (HTS) tools (e.g., filter plates), which allow for miniaturized experiments, are used to perform extensive experimental analysis to determine optimal solution conditions (e.g., pH) for biomolecule binding. Unfortunately, commercial filter plates are limited in customizability for HTS of membrane materials. To address these limitations, this thesis focuses on designing and validating a multi-well device capable of incorporating any membrane adsorber. Different biomolecules (proteins, DNA), solution conditions, and membrane materials were evaluated. The results of this thesis confirmed this ability to accommodate any membrane adsorber, simultaneously compare different membrane materials, and extract the membrane for post-experimental analysis. This work also discussed using this device for future rapid membrane material selection in multiple industries (e.g., biotherapeutics, ion extraction).

No description available.

No description available.

2024 / This thesis uses single molecule conductance spectroscopy to probe the binding mechanisms and conductance characteristics of various biologically relevant molecules with gold at the atomic level. Firstly, we identify imidazole as a pH-activated linker for forming stable single gold molecule junctions, which present several distinct configurations and reproducible electrical characteristics. We then examine the resulting conductance signatures and identify corresponding binding geometries, which involve up to four imidazole molecules binding in the junction in parallel. In addition, we discover the distinct conductance signatures that indicate the in-situ formation of molecule-metal-molecule chains within the molecular junction. Building on this foundation, the investigation continues into the origin of the conductance enhancement observed in benzimidazole dimers compared to imidazole dimers. Density Functional Theory (DFT) calculations reveal that the parallel stacking of two benzimidazoles, due to the large π system between electrodes, represents the most energetically favorable configuration, leading to dimer conductance enhancement. The smaller size and greater conformational freedom of imidazole enable it to access a variety of stacking angles. Having understood the underlying mechanism, we use substituents to promote the cooperative in situ assembly of imidazole derivatives into a parallel binding configuration, subsequently enhancing conductance. Next, we shift our focus to adenine, one of the most important biological building blocks of deoxyribonucleic acid (DNA). By using structurally similar molecules, we can assign different conductance signals to various binding configurations of adenine. This approach also enables the differentiation between adenine and its biological derivatives, 2’deoxyadenosine and 6-methyladenine. Using single molecule conductance signals, we demonstrate the potential of single molecule conductance spectroscopy as a biosensing platform. Finally, we present a detailed study of the pH-activated intramolecular conductance features of histamine. By employing histamine and its derivatives, we associate different conductance features with specific binding sites. DFT calculation is used to simulate the different ethylamine configurations of histamine in molecular junctions, and flicker noise analysis is applied to identify and assign one of the conductance features to a hydrogen bond-assisted binding configuration. These results and insights collectively establish single molecule conductance spectroscopy as a robust platform for studying complex gold-biomolecule interactions.

In this work, the Collision Induced Dissociation under Energy Control method was extended to study the fragmentation of gas-phase biomolecules adenine (H5C5N5) and porphyrin FeTPPCl (C44H28N4FeCl). The population distribution for each dissociation channel has been experimentally determined as a function of the excitation energy of the parent molecular ions at a well-determined initial charge state. In collisions between Cl+ and adenine (Ade) at 3keV, the fragmentation pattern of Ade2+ is dominated by the loss of H2CN+ and the successive emission of HCN. The energy distribution of the parent dications confirms the successive emission dynamics. A specific decay channel is observed, i.e., the emission of a charged H2CN+ followed by the emission of HC2N2. In Kr8+-FeTPPCl collisions at 80keV, parent ions FeTPPCl1+,2+,3+ are observed, along with the corresponding decay patterns. It is found that in the first step the dominant low-energy-cost decay channel is the emission of Cl0 independent of the initial charge state of FeTPPClr+ (r=1-3). For the resulted dication FeTPP2+, the dominant fragmentation channel is the neutral evaporation; for the trication however, the dominant fragmentation channel is the asymmetrical fission. In the case of H+ and F+ impact at 3keV, due to the different reaction windows opened in the two collision systems, different fragmentation patterns are observed. Furthermore, nH2 loss processes are observed. Additionally, the production yield of the negative ion emerged in F2+-Ade collision at 30keV is measured to be about 1% using a new experimental approach.

Investigation of RNA and DNA function often requires sequence-specific incorporation of various reporter and affinity probes. This can be achieved using AdoMet-dependent methyltransferases (MTases) as they can be active with synthetic AdoMet analogues equipped with transferable chains larger than the methyl group. These chains usually carry reactive groups that can be further chemically appended with required reporters. For this, azide-alkyne 1,3-cycloaddition (AAC), also called “click”, reaction is particularly attractive. This work shows that the HhaI cytosine-5 DNA MTase (variant Q82A/Y254S/N204A) catalyzes efficient sequence-specific transfer of hex-2-ynyl side chains containing terminal alkyne or azide groups from synthetic cofactor analogues to DNA. Both the enzymatic transfer and subsequent “click” coupling of a fluorophore can be performed even in cell lysates. For RNA labeling, the activity of an archaeal RNA 2‘-O-MTase C/D ribonucleoprotein complex (RNP) with synthetic cofactors was investigated. It was shown that synthetically reprogrammed guide RNA sequences can be used to direct the C/D RNP-dependent transfer of a prop-2-ynyl group to predetermined nucleotides in substrate RNAs. Followed by AAC this can be used for programmable sequence-specific labeling of a variety of RNA substrates in vitro. These new possibilities for specific labeling of nucleic acids can be adopted in biochemistry, biomedical, nanotechnology, etc. research. / Tiriant DNR ir RNR, neretai svarbu prijungti įvairius reporterinius ar giminingumo žymenis griežtai apibrėžtose (sekos) vietose – t.y. specifiškai. Tam galima pasitelkti fermentus metiltransferazes (MTazes). Natūraliai jos naudoja kofaktorių AdoMet, tačiau gali būti aktyvios ir su sintetiniais jo analogais, turinčiais ilgesnes nei metil- pernešamas grandines. Jei šios grandinės turi galines funkcines grupes, prie jų vėliau cheminių reakcijų pagalba galima prijungti norimus žymenis. Tam itin patogi azidų-alkinų cikloprijungimo (AAC), dar vadinama „click“, reakcija. Šiame darbe parodyta, kad DNR citozino-5 MTazė HhaI (variantas Q82A/Y254S/N204A) efektyviai katalizuoja sekai specifinę heks-2-inil- grandinių, turinčių galines alkinil- arba azido- grupes, pernašą nuo sintetinių kofaktorių ant DNR. Naudojant šią MTazės-kofaktorių sistemą bei AAC, visą specifinio DNR žymėjimo procesą galima atlikti netgi ląstelių lizate. RNR žymėjimui ištirtas archėjų RNR 2‘-O-MTazės C/D ribonukleoproteininio komplekso aktyvumas su sintetiniais kofaktoriais. Parodyta galimybė sintetiškai keičiant kreipiančiąją RNR, prop-2-inilgrupės pernašą nukreipti į norimas įvairių substratinių RNR sekos vietas ir po to AAC reakcijos pagalba prijungti fluoroforą. Taigi, sukurtas naujas molekulinis įrankis, leidžiantis be suvaržymų pasirinkti norimą pažymėti RNR seką. Šios naujos specifinio nukleorūgščių žymėjimo galimybės gali būti pritaikytos biochemijos, biomedicinos, nanotechnologijų ir kitose tyrimų srityse... [toliau žr. visą tekstą]

Tiriant DNR ir RNR, neretai svarbu prijungti įvairius reporterinius ar giminingumo žymenis griežtai apibrėžtose (sekos) vietose – t.y. specifiškai. Tam galima pasitelkti fermentus metiltransferazes (MTazes). Natūraliai jos naudoja kofaktorių AdoMet, tačiau gali būti aktyvios ir su sintetiniais jo analogais, turinčiais ilgesnes nei metil- pernešamas grandines. Jei šios grandinės turi galines funkcines grupes, prie jų vėliau cheminių reakcijų pagalba galima prijungti norimus žymenis. Tam itin patogi azidų-alkinų cikloprijungimo (AAC), dar vadinama „click“, reakcija. Šiame darbe parodyta, kad DNR citozino-5 MTazė HhaI (variantas Q82A/Y254S/N204A) efektyviai katalizuoja sekai specifinę heks-2-inil- grandinių, turinčių galines alkinil- arba azido- grupes, pernašą nuo sintetinių kofaktorių ant DNR. Naudojant šią MTazės-kofaktorių sistemą bei AAC, visą specifinio DNR žymėjimo procesą galima atlikti netgi ląstelių lizate. RNR žymėjimui ištirtas archėjų RNR 2‘-O-MTazės C/D ribonukleoproteininio komplekso aktyvumas su sintetiniais kofaktoriais. Parodyta galimybė sintetiškai keičiant kreipiančiąją RNR, prop-2-inilgrupės pernašą nukreipti į norimas įvairių substratinių RNR sekos vietas ir po to AAC reakcijos pagalba prijungti fluoroforą. Taigi, sukurtas naujas molekulinis įrankis, leidžiantis be suvaržymų pasirinkti norimą pažymėti RNR seką. Šios naujos specifinio nukleorūgščių žymėjimo galimybės gali būti pritaikytos biochemijos, biomedicinos, nanotechnologijų ir kitose tyrimų srityse... [toliau žr. visą tekstą] / Investigation of RNA and DNA function often requires sequence-specific incorporation of various reporter and affinity probes. This can be achieved using AdoMet-dependent methyltransferases (MTases) as they can be active with synthetic AdoMet analogues equipped with transferable chains larger than the methyl group. These chains usually carry reactive groups that can be further chemically appended with required reporters. For this, azide-alkyne 1,3-cycloaddition (AAC), also called “click”, reaction is particularly attractive. This work shows that the HhaI cytosine-5 DNA MTase (variant Q82A/Y254S/N204A) catalyzes efficient sequence-specific transfer of hex-2-ynyl side chains containing terminal alkyne or azide groups from synthetic cofactor analogues to DNA. Both the enzymatic transfer and subsequent “click” coupling of a fluorophore can be performed even in cell lysates. For RNA labeling, the activity of an archaeal RNA 2‘-O-MTase C/D ribonucleoprotein complex (RNP) with synthetic cofactors was investigated. It was shown that synthetically reprogrammed guide RNA sequences can be used to direct the C/D RNP-dependent transfer of a prop-2-ynyl group to predetermined nucleotides in substrate RNAs. Followed by AAC this can be used for programmable sequence-specific labeling of a variety of RNA substrates in vitro. These new possibilities for specific labeling of nucleic acids can be adopted in biochemistry, biomedical, nanotechnology, etc. research.