Biosensors monitor physiological activities for diagnosis and treatment of disease. Molecularly imprinted polymers (MIPs) are a viable synthetic approach for molecular recognition in biosensing. For biosensing purposes, the most important properties in MIP optimization are sensitivity and selectivity towards a desired analyte. This study aims to optimize MIP sensitivity and selectivity by varying the amount and type of cross-linker used in the synthesis of cortisol and melatonin. The four cross-linkers tested were trimethylpropane trimethacrylate (TRIM), ethyleneglycodimethacrylate (EGDMA), divinylbenzene (DVB), and pentaerythritol triacrylate (PETRA). Based on literature, the following ratios were used for the template molecule to functional monomer to cross-linker in MIP synthesis: for EGDMA cross-linked polymers, 1:6:30; for TRIM and PETRA cross-linked polymers, 1:8:8, 1:6:3, and 1:8:35; for DVB cross-linked polymers, 1:6:30, 1:4:16, and 4:1:60. The polymers were ground and washed, then suspended in a polyvinyl matrix which was spin-coated onto an organic electrochemical transducer (OECT). The device performance was evaluated using electrochemical impedance spectroscopy. For each device, the impedance was measured in electrolyte solutions containing target molecules in concentrations ranging from 1 pM to 100 uM. The impedance was plotted against the analyte concentration to give the sensing slope, which is a measurement for the binding affinity of the polymer. For a device to be considered sensitive, its sensing slope should be greater than its non-imprinted counterpart by a factor above the error margin (+/- 1.79). Of the devices tested, CM1835T (highly cross-linked with TRIM) showed sensitivity towards cortisol, but lacks selectivity towards cortisol over its structural analog, estradiol. Of the melatonin selective polymers, MM163T (low cross-linking with TRIM), MM1630D, and MM4160D (both highly cross-linked with DVB) all showed promising results in sensitivity to melatonin. Overall, the results indicate that high degrees of cross-linking in MIPs improve sensitivity for large, rigid, non-aromatic molecules such as cortisol; however there is no correlation between selectivity and the degree of cross-linking. Meanwhile, divinylbenzene as a cross-linker improves sensitivity and selectivity towards aromatic analytes such as melatonin and estradiol. This study could be improved upon by further characterization of imprinted and non-imprinted polymers, investigation of molecular dynamics, and optimization of devices.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24234号 / 工博第5062号 / 新制||工||1790(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 大塚 浩二, 教授 沼田 圭司, 教授 大内 誠 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Orientadores: Susanne Rath, Rogério Custódio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T17:08:24Z (GMT). No. of bitstreams: 1 Barros_LeonardoAugustode_D.pdf: 4314419 bytes, checksum: 93a1b964e0a2949ef6a2379990e2f089 (MD5) Previous issue date: 2014 / Resumo: Esse trabalho teve como objetivo principal otimizar e sintetizar polímeros de impressão molecular (MIP) para serem empregados em processos de extração em fase sólida (SPE), visando a determinação de diuréticos tiazídicos em urina. Cálculos teóricos de modelagem molecular, usando o programa computacional Gaussian 09 e os métodos DFT e PCM, no nível B3LYP e conjunto de base 6-31G(d), foram realizados para selecionar o monômero funcional (MF) e o solvente porogênico mais adequados para serem utilizados na síntese dos MIP. Para o desenvolvimento dos MIP compatíveis com água, foi utilizado hidroclorotiazida, clorotiazida ou hidroflumetiazida como molde, acrilamida como MF, etilenoglicol dimetacrilato como reagente de ligação cruzada (RLC) e tetraidrofurano como solvente porogênico. Foram avaliados alguns parâmetros que afetam a eficiência do polímero de impressão, tais como a quantidade de MF e a natureza do RLC. Foram construídas as isotermas de adsorção para cada um dos polímeros sintetizados e foi avaliada a seletividade dos MIP frente a análogos estruturais dos moldes. Os polímeros foram caracterizados por infravermelho com transformada de Fourier, 13C RMN, microscopia eletrônica de varredura, porosimetria de sorção de nitrogênio e análise termogravimétrica. Os MIP foram empregados como fase estacionária em SPE para a determinação de diuréticos tiazídicos em urina por cromatografia líquida de alta eficiência e os mesmos apresentaram seletividade cruzada em relação aos análogos estruturais / Abstract: This work aimed to optimize and synthesize molecularly imprinted polymers (MIP) to be employed in processes of solid phase extraction (SPE), for the determination of thiazide diuretics in urine. Theoretical calculations of molecular modeling, using the Gaussian 09 software and the density functional theory and PCM methods, at the B3LYP/6-31G(d) level, were performed to select a the most appropriate functional monomer (FM) and porogenic solvent for the synthesis of the molecularly imprinted polymers (MIP). For the development of a water-compatible MIP chlorothiazide, hydrochlorothiazide or hydrfoflumethiazide were used as template, acrylamide as FM, ethyleneglycol dimethacrylate as cross-linker and tetrahydrofuran as porogenic solvent. Parameters that affect thepolymer efficiency, such as the amount of the monomer and nature of the cross-linker were evaluated. The adsorption isotherms for each of the synthesized polymers were constructed and the selectivities of the MIPs in relation to structural analogues of the templates were evaluated. The polymers were characterized by Fourier transform infrared, 13C NMR, scanning electron microscopy, nitrogen sorption porosimetry and thermogravimetric analysis. The MIPs were employed as stationary phase in SPE for the determination of thiazide diuretics in urine by high performance liquid chromatography and they showed cross-selectivity in relation to their structural analogues / Doutorado / Quimica Analitica / Doutor em Ciências

By using the technology of molecularly imprinted polymer (MIP), we propose to synthesize a protein conformational imprint that also acts as a plastic enzyme, inducing protein structural transitions. The imprint aims at MIP-induced stabilization and / or formation of bound protein secondary structure and the applications associated with analysis and correction of misfolded proteins. The screening of polymeric functional monomers being able to induce the conformational transitions in proteins is investigated in this report. The fluoro-silanes (3-heptafluoroisopropoxy)propalethoxysilane (7F) and 3,3,3-trifluoropropylmethoxysilane (3F) were employed as functional monomers for synthesis of this catalytic protein conformational imprint via sol-gel reactions. 3F was demonstrated superior to 7F for fluoro-modification of tetraethylorthosilicate (TEOS) gel in terms of retaining gel transparency and increasing hydrophobicity while maintaining a uniform distribution of encapsulated protein. Both hydrolyzed 3F and polymerized 3F exhibited strong influences on structure transitions of three template proteins: bovine serum albumin (BSA), beta-lactoglobulin (BLG), and bovine carbonic anhydrase (BCA). The formation of molten globule intermediates that stabilized by increased alpha-helices was induced by the trifluoro-silane in BLG and BCA. Additionally, 3F was effective at a lower concentration than the benchmark fluoro-alcohol 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), validating the application of 3F as a functional monomer for protein conformational imprinting.

<p>An intrinsic mathematical model for simulation of fixed bed chromatography was demonstrated and compared to more simplified models. The former model was shown to describe variations in the physical, kinetic, and operating parameters better than the latter ones. This resulted in a more reliable prediction of the chromatography process as well as a better understanding of the underlying mechanisms responsible for the separation. A procedure based on frontal liquid chromatography and a detailed mathematical model was developed to determine effective diffusion coefficients of proteins in chromatographic gels. The procedure was applied to lysozyme, bovine serum albumin, and immunoglobulin γ in Sepharose™ CL-4B. The effective diffusion coefficients were comparable to those determined by other methods.</p><p>Molecularly imprinted polymers (MIPs) are traditionally prepared as irregular particles by grinding monoliths. In this thesis, a suspension polymerization providing spherical MIP beads is presented. Droplets of pre-polymerization solution were formed in mineral oil with no need of stabilizers by vigorous stirring. The droplets were transformed into solid spherical beads by free-radical polymerization. The method is fast and the performance of the beads comparable to that of irregular particles. Optimizing a MIP formulation requires a large number of experiments since the possible combinations of the components are huge. To facilitate the optimization, chemometrics was applied. The amounts of monomer, cross-linker, and porogen were chosen as the factors in the model. Multivariate data analysis indicated the influence of the factors on the binding and an optimized MIP composition was identified. The combined use of the suspension polymerization method to produce spherical beads with the application of chemometrics was shown in this thesis to drastically reduce the number of experiments and the time needed to design and optimize a new MIP.</p>

An intrinsic mathematical model for simulation of fixed bed chromatography was demonstrated and compared to more simplified models. The former model was shown to describe variations in the physical, kinetic, and operating parameters better than the latter ones. This resulted in a more reliable prediction of the chromatography process as well as a better understanding of the underlying mechanisms responsible for the separation. A procedure based on frontal liquid chromatography and a detailed mathematical model was developed to determine effective diffusion coefficients of proteins in chromatographic gels. The procedure was applied to lysozyme, bovine serum albumin, and immunoglobulin γ in Sepharose™ CL-4B. The effective diffusion coefficients were comparable to those determined by other methods. Molecularly imprinted polymers (MIPs) are traditionally prepared as irregular particles by grinding monoliths. In this thesis, a suspension polymerization providing spherical MIP beads is presented. Droplets of pre-polymerization solution were formed in mineral oil with no need of stabilizers by vigorous stirring. The droplets were transformed into solid spherical beads by free-radical polymerization. The method is fast and the performance of the beads comparable to that of irregular particles. Optimizing a MIP formulation requires a large number of experiments since the possible combinations of the components are huge. To facilitate the optimization, chemometrics was applied. The amounts of monomer, cross-linker, and porogen were chosen as the factors in the model. Multivariate data analysis indicated the influence of the factors on the binding and an optimized MIP composition was identified. The combined use of the suspension polymerization method to produce spherical beads with the application of chemometrics was shown in this thesis to drastically reduce the number of experiments and the time needed to design and optimize a new MIP.

Sample preparation has a critical role as a first step in analytical processes, especially in bioanalysis and environmental analysis. A good sample preparation technique should be robust and stable, regardless of the sample matrix. The aim of this thesis is to design and synthesize molecularly imprinted polymers that can be used in various sample preparation techniques, such as on-line MEPS, on-line SPE and on-line monolithic pre-columns used for the extraction of drugs, hormones, and cancer biomarkers from human plasma and urine samples. Additional aim was to provide full automation, on-line coupling, short sample preparation time and high-throughput. In this thesis MIP in MEPS was used on-line with liquid chromatography-tandem mass spectrometry (LC/MS/MS) for the determination of sarcosine in human urine and plasma samples. The method was fully automated and the packed sorbent could be used for about hundred extractions. In additional work a coated needle with MIP-Sol-Gel as thin layer was prepared and used for the microextraction of bilirubin from human plasma and urine. Small sample volumes could be handled and the validation of the method showed that the method was robust and selective. In a further work MIP-SPE on-line with HPLC was used for the extraction and determination of dextromethorphan in human plasma samples. MIP-SPE showed a good selectivity and high recovery (87% - 92%). On-line MIP monolithic pre-column was prepared and used in a coupled system for the extraction of tramadol in human plasma and urine samples. The MIP monolithic pre-column showed good selectivity and high extraction recovery was obtained (91-96%). The extraction and analysis of human insulin in plasma and pharmaceutical formulation solutions were carried out using MIP-SPE on-line with HPLC. The validation of the method showed that the method was accurate and robust. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Submitted.</p>

Orientador: Lauro Tatsuo Kubota / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T02:29:01Z (GMT). No. of bitstreams: 1 Santos_WilneydeJesusRodrigues_D.pdf: 6401846 bytes, checksum: 72d1c8253a6ec5d1d17792b0bd34ca39 (MD5) Previous issue date: 2009 / Resumo: O presente trabalho descreve as aplicações de nanoreatores biomiméticos à peroxidase baseados em MIP ("Molecularly Imprinted Polymers") como uma ferramenta promissora para determinação de substâncias de grande interesse biológico e ambiental, tais como os compostos fenólicos (4-aminofenol e serotonina). Neste sentido, a síntese dos MIPs foi baseada na polimerização convencional em "bulk". Cada polímero foi sintetizado a partir do ácido metacrílico (monômero funcional), etileno glicoldimetilacrilato (reagente de ligação cruzada), 2¿2-azo-bis-isobutironitrila (iniciador radicalar), em presença de Fe(III)protoporfrina(IX) (hemina) como centro catalítico, o qual é responsável pela mimetização do sítio ativo da peroxidase, criando portanto, um polímero com impressão molecular cataliticamente ativo para o reconhecimento do 4-aminofenol e serotonina (moléculas molde). Além disso, a fim de avaliar a seletividade do material, foram preparados, paralelamente, polímeros sem a impressão molecular (NIP Non Imprinted Polymers) e também na ausência de hemina. Os MIPs foram caracterizados pelas técnicas de espectroscopia no infravermelho, área superficial específica, volume específico dos poros, análise termogravimétrica, microscopia eletrônica de varredura. Parâmetros cinéticos, incluindo valores de velocidade máxima, Vmax e constante aparente de Michaelis¿Menten, Km foram obtidas pelo gráfico de Lineweaver-Burk. Para aplicação analítica, em amostras de água e soro sanguíneo, sistemas amperométricos foram otimizados através de análise multivariada / Abstract: The present work describes the applications of biomimetic nanoreactor to the based peroxidase in molecularly imprinted polymers (MIP) as a promising tool for determination of substances of high biological and environmental interest, such as phenolic compounds (4-aminophenol and serotonin). In this sense, the synthesis of MIPs was based on the conventional polymerization in bulk. Each polymer was synthesized from methacrylic acid (functional monomer), ethylene glycol dimethacrylate (cross-linking reagent), 2,2'-azobis-isobutyronitrile (initiator), in the presence of Fe(III)protoporphyrin(IX) (hemin) as a catalytic center, which is responsible for the mimic of the active site of peroxidase, creating therefore, a molecularly imprinted polymer active catalytically for the recognition of the 4-aminophenol and serotonin (template molecules). Furthermore, in order to evaluate the selectivity of the material, were prepared, parallel, polymers without the molecular impression (NIP - Non imprinted polymers) and also in the hemin absence. The MIPs were characterized by the techniques of infrared spectroscopy, specific surface area, specific pore volume, thermogravimetric analysis, scanning electron microscopy. Kinetic parameters, including values for maximum rate, Vmax and Michaelis-Menten apparent constant, Km were obtained from Lineweaver-Burk plots. For analytical application, in samples of water and blood serum, amperometric systems were optimized through multivariate analysis / Doutorado / Quimica Analitica / Doutor em Ciências

Orientador: Ivo Milton Raimundo Júnior / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T16:51:10Z (GMT). No. of bitstreams: 1 Santos_KleciaMoraisdos_D.pdf: 2306751 bytes, checksum: bf7642f623ca7798b881b810cd6dd821 (MD5) Previous issue date: 2012 / Resumo: Neste trabalho diferentes fases sensoras foram desenvolvidas para implementar novas estratégias para a determinação de íons cobre em águas naturais, empregando Espectroscopia Óptica de Emissão em Plasma Induzido por Laser (LIBS) e Espectroscopia de Luminescência. Nas determinações por LIBS, membranas de PVC plastificadas foram empregadas para extração e pré-concentração de íons cobre em meio aquoso, fornecendo curvas analíticas na faixa de 10 mg L - 100mg L (R > 0,90). Esses estudos mostraram que a técnica LIBS é capaz de detectar íons cobre em concentrações da ordem de mg L, indicando que membranas extratoras podem ser aplicadas como uma eficiente estratégia de pré-concentração. Dessa forma, LIBS pode ser considerada como uma potencial e viável ferramenta para determinação de íons Cu(II) em águas. Determinações baseadas em Espectroscopia de Luminescência foram realizadas usando o bis(1,10-fenantrolina)1-(4-hidroxi 3-(1H-imidazol [4,5-f][1,10] rutênio(II) bis hexafluorofosfato ([Ru(phen)2HAIP](PF6)2), que foi imobilizado em membranas de metacrilato, de PVC e iônicas, sol-gel e nanopartículas (NP) de sílica. O reagente luminescente apresenta alta seletividade para íons Cu (II) em solução, enquanto somente as nanopartículas mostraram sensibilidade adequada para esse íon. As nanopartículas de sílica foram sintetizadas através do método Stöber, sendo que o reagente luminescente foi adicionado em diferentes estágios da síntese (0, 3, 6 e 8 horas), a qual foi realizada em um total de 10 horas. A microscopia de transição eletrônica (TEM) mostrou nanopartículas com diâmetros em torno de 10-20 nm. As nanopartículas sintetizadas pela adição do reagente após 8h de síntese apresentaram uma faixa linear entre 2,0-8,0 mmol L, um limite de detecção de 0,30 mmol L, um limite de quantificação de 1,0 mmol L e um tempo de resposta (t90%) < 30 s. A nanopartícula apresentou alta seletividade para íons cobre, não mostrando interferências dos íons Zn(II), Cd(II), Ni(II), Hg(II), Pb(II) e Fe(III). As nanopartículas foram aplicadas para determinação de íons Cu(II) em amostras de água mineral e de torneira que foram enriquecidas com 5,0 e 7,0 mmol L do analito. Recuperações na faixa de 88,1-109,6% foram obtidas, indicando a potencialidade das NPs fluorescentes para determinação de Cu(II) em águas. Além disso, os íons Cu (II) podem ser liberados na NP depois de tratamento ácido, sendo regenerado para um novo conjunto de medidas, tornando o reagente reutilizável, fato que está de acordo com os princípios da Química Verde / Abstract: In this work, different sensing phases were developed to implement new strategies for determination of copper ions in natural waters, employing Laser Induced Breakdown Spectroscopy (LIBS) and Luminescence Spectroscopy. In the determinations by LIBS, plasticized PVC membranes were employed for extraction and pre-concentration of copper ions in aqueous medium, providing analytical curves in the range from 10 mg L to 100mg L (R > 0.90). These studies have shown that LIBS is able to detect copper ions in concentrations as low as mg L, indicating that extraction membranes can be applied as an efficient strategy of pre-concentration. In this way, LIBS can be considered as a potential and feasible tool for Cu(II) determination in waters. Determinations based on luminescence spectroscopy were performed by using the bis(1,10-phenanthroline)1-(4-hydroxy 3-(1H-imidazo [4,5-f][1,10] ruthenium(II) bis(hexafluorophosphate) reagent ([Ru(phen)2HAIP](PF6)2), which was immobilized in methacrylate, PVC and ionic membranes, sol-gel silica and silica nanoparticles (NP). The luminescent reagent presented high selectivity to Cu(II) ions in solution, while only the silica nanoparticles showed adequate sensitivity to this ion. The nanoparticles were synthesized by the Stöber method, being the luminescent reagent added at different stages of the synthesis (0, 3, 6 and 8 hours), which was a performed in a total interval of 10 hours. The transmission electron microscopy (TEM) showed nanoparticles with diameters around 10-20 nm. The nanoparticles synthesized by adding the reagent after 8 hours provided a linear response range from 2.0 to 8.0 mmol L, a detection limit of a 0.30 mmol L, a quantification limit of 1.0 mmol L and response time (t90%) < 30 s. The nanoparticle presented high selectivity for copper ions, showing no interference of Zn(II), Cd(II), Ni(II), Hg(II), Pb(II) and Fe(III) ions. The nanoparticles were applied to determination of Cu(II) ions in mineral and tap water samples, which were spiked with 5.0 and 7.0 mmol L of the analyte. Recovery within 88.1-109.6% were attained, indicating the capability of the fluorescent NPs for Cu(II) determination in waters. In addition, the Cu(II) ions can be released from the NP by acidic treatment, being regenerated for another set of measurements, making the reagent reusable, fact that is in agreement with the principles of Green Chemistry / Doutorado / Quimica Analitica / Doutor em Ciências

Recently, laser scribed graphene (LSG) technology has shown great potential for the development of a plethora of sensing platforms due to its high sensitivity, 3D porous structure, and flexibility. Molecularly imprinted polymers (MIPs) have shown high potential as recognition elements for many applications such as biosensing. Hence, we report in this thesis a novel biosensing platform that utilizes nanostructured gold to enhance the performance of LSG sensors coupled with a biomimetic MIP biosensor. To the best of our knowledge, this is the first report of a nanostructured gold modified MIP based LSG biosensor to detect HER-2, which is an important breast cancer biomarker. HER-2 positive breast cancer is more aggressive and does not respond to the same treatment as standard breast cancer. As such, a simple and accurate sensing approach is highly needed for early detection of this type of cancer biomarkers. The LSG sensor platform was fabricated by irradiation of polyimide substrates using a CO2 laser under optimized conditions. Nanostructured gold was electrodeposited onto LSG to enhance its sensitivity and active surface area. Deposition parameters such as deposition voltage, deposition time, and gold chloride (HAuCl4) concentration were optimized to yield complete nanostructured gold coverage and enhanced electrical conductivity of LSG-Au electrodes. A deposition voltage of -0.9 V at 50 mM HAuCl4 for 4 minutes proved to be the optimal condition for gold deposition to yield a 150% peak current enhancement. To fabricate our MIP biosensor, 3,4- ethylenedioxythiophene (EDOT) was chosen from several functional monomers to form PEDOT due to its high conductivity and synergy with nanostructured gold. Electropolymerization of EDOT is performed after adsorbing 0.4mg/mL of HER-2 on the LSG-Au electrode for 20 min. The efficiency of LSG-Au-MIP was optimized by choosing an appropriate extraction agent and HER-2 concentration to be adsorbed on gold. The developed sensing strategy could differentiate between three rebinding concentrations of 10 ng/mL, 100ng/mL, and 200 ng/mL, which is sufficient to determine the HER-2 status of breast cancer since the clinical cut-off is 30.5ng/mL. The developed sensing strategy showed a high degree of novelty and could be useful for the non-invasive detection of cancer biomarkers.