Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T12:40:01Z No. of bitstreams: 2 joao_gabriel_guimares_luz.pdf: 5854830 bytes, checksum: 3b384576c220fecec7431b8c107b9ea1 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T12:40:28Z (GMT) No. of bitstreams: 2 joao_gabriel_guimares_luz.pdf: 5854830 bytes, checksum: 3b384576c220fecec7431b8c107b9ea1 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T12:40:47Z (GMT) No. of bitstreams: 2 joao_gabriel_guimares_luz.pdf: 5854830 bytes, checksum: 3b384576c220fecec7431b8c107b9ea1 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2015-01-07T12:40:47Z (GMT). No. of bitstreams: 2 joao_gabriel_guimares_luz.pdf: 5854830 bytes, checksum: 3b384576c220fecec7431b8c107b9ea1 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2014 / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / O objetivo desse trabalho foi desenvolver um imunosensor fundamentado em espectroscopia de resson?ncia de pl?smons de superf?cie (SPR) para a detec??o de anticorpos anti-Trypanosoma cruzi por meio da imobiliza??o de ant?genos brutos do parasito em monocamadas auto-organizadas mistas (SAMs) formadas por ?cido 11-mercaptoundecan?ico (11-MUA) e ?cido 3-mercaptopropi?nico (3-MPA). Na sua constru??o, as concentra??es de cada alcanotiol, bem como a propor??o de cada um deles na composi??o das SAMs mistas foram definidas atrav?s de c?lculos de cobertura de superf?cie Posteriormente, o ant?geno em diferentes concentra??es foi imobilizado sobre as SAMs mistas ativadas constru?das em disco de ouro e, a varia??o do ?ngulo de SPR (??SPR) decorrente dessa etapa foi monitorada em tempo real. O imunosensor desenvolvido foi ent?o eletroquimicamente caracterizado por voltametria c?clica (VC) e espectroscopia de imped?ncia eletroqu?mica (EIE) e as respostas do mesmo frente a um pool de soros de indiv?duos infectados e n?o infectados pelo T. cruzi foram avaliadas. Em seguida, foram determinados par?metros para otimiza??o do dispositivo, incluindo tempo de imobiliza??o e concentra??o do ant?geno, bloqueio e regenera??o da superf?cie sensora e tempo envolvido nas an?lises do imunoensaio. Por fim, a dilui??o de trabalho do soro e o ponto de corte do imunosensaio foram escolhidos e os ?ndices de desempenho do mesmo como t?cnica para o imunodiagn?stico da DC foram calculados empregando 171 soros, sendo 99 de indiv?duos infectados pelo T. cruzi, 30 de n?o infectados e 42 portadores de outras infec??es. Os maiores valores de foram encontrados para a concentra??o 1.0 mmol L-1 de 11-MUA e 3-MPA e para as SAMs mistas formadas na propor??o de 1:10. A imobiliza??o dos ant?genos foi conduzida com ?xito, de modo que houve linearidade entre a concentra??o utilizada e o ??SPR. Por VC e EIE, foi demonstrado que a liga??o covalente das prote?nas aumentou a resist?ncia ? transfer?ncia eletr?nica quando comparado com o comportamento da sonda eletroqu?mica sobre as SAMs mistas. Ademais, o imunosensor se mostrou, semelhante ao ELISA, capaz de detectar anticorpos anti-T. cruzi em amostras de soros dilu?das at? 1280 vezes. J? em rela??o aos soros negativos, o dispositivo detectou ??SPR significativas apenas at? a dilui??o 1:160, o que demonstrou razo?vel capacidade do m?todo em distinguir indiv?duos infectados de n?o infectados. Dentre os par?metros anal?ticos avaliados ficou definido o tempo de imobiliza??o e a concentra??o do ant?geno em 20 minutos e 30 ?g mL-1, o bloqueio da superf?cie por 10 minutos com solu??o de BSA 1.0%, a regenera??o pela inje??o de SDS 1.0% e o tempo de avalia??o da resposta do dispositivo em 20 minutos. A dilui??o do soro em 1:320 e o ponto de corte a 17.2 m? foram os crit?rios metodol?gicos definidos para o imunosensaio. Os ?ndices de desempenho calculados determinaram um excelente potencial do imunosensor no diagn?stico sorol?gico da doen?a de Chagas, com valores de sensibilidade de 100%, especificidade de 97.2%, valor preditivo positivo igual a 98%, valor preditivo negativo de 100% e acur?cia global estimada em 99.6%. O n?mero de rea??es cruzadas em soros de indiv?duos com leishmaniose visceral foi menor no m?todo proposto (1/7) do que no ELISA (6/7). A partir dos resultados ? poss?vel afirmar que foi desenvolvido com ?xito um imunosensor sens?vel, espec?fico, r?pido, simples e de f?cil execu??o para o diagn?stico sorol?gico da doen?a de Chagas. / Disserta??o (Metrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014. / ABSTRACT The aim of the present study was to describe the development of an unpublished SPR-based immunosensor for detection of anti-T. cruzi antibodies in human serum through the covalently immobilized T. cruzi antigen on a mixed SAM of 11-mercaptoundecanoic acid (11-MUA) and 3-mercaptopropionic acid (3-MPA) on a SPR sensor chip. In its construction, the concentrations of each alkanethiol, and the proportion of each in the composition of the mixed SAMs were defined by calculation of surface coverage Subsequently, the antigen at different concentrations was immobilized on the activated mixed SAMs built on gold disc, and the change in the SPR angle (??SPR) resulting from this step was monitored in real time. The imunosensor developed was electrochemically characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and the responses of a pool of sera from individuals infected and not infected with T. cruzi were also evaluated. Then, some parameters for the optimization of the device were defined, including immobilization time and antigen concentration, blocking and regeneration of the sensor surface and time involved in the analysis of the immunoassay. Finally, the working dilution of the serum and the cut-off point were chosen and the performance indices of the technique were calculated using 171 sera - 99 individuals infected with T. cruzi, 30 not infected and 42 with other infections. The highest values ??of ? were found for the 1.0 mmol L-1 concentration of 11-MUA and 3-MPA and mixed SAMs formed at 1:10 proportion. The immobilization of antigens was conducted successfully, so there was linearity between the concentration used and ??SPR. For VC and EIS has been demonstrated that covalent attachment of the protein increased the electron transfer resistance when compared with the behavior of the electrochemical probe on the mixed SAMs. Furthermore, imunosensor showed, similarity to ELISA, capable of detecting anti-T. cruzi in sera diluted up 1280 times. In relation to the negative sera, the device detected significant ??SPR only until dilution 1:160, which demonstrated the ability of the method to distinguish infected from uninfected individuals. The following analytical parameters were defined: time of immobilization and antigen concentration at 20 minutes and 30 mg mL-1, blocking surface for 10 minutes with 1.0% BSA solution, regeneration by injection of 1.0% SDS and time evaluation of the response of the device in 20 minutes. The serum dilution at 1:320 and cut-off point at 17.2 m? were the methodological criteria for immunoassay. The performance indices calculated for the imunosensor demonstrated a great potential in serological diagnosis of Chagas' disease, with a sensitivity of 100%, specificity of 97.2%, positive predictive value of 98%, negative predictive value of 100% and global accuracy estimated in 99.6%. The number of cross-reactivity with sera from individuals with visceral leishmaniasis was lower (1/7) than in ELISA (6/7). Based on the results, it is possible to stay that a sensitive, specific, rapid, simple and easy immunosensor was successfully with potential application at serological diagnosis of Chagas disease.

Made available in DSpace on 2017-07-24T19:38:09Z (GMT). No. of bitstreams: 1 CRISTIANE A ERDMANN.pdf: 1781032 bytes, checksum: 803e9255fa555b2e79d98e8d126018f8 (MD5) Previous issue date: 2013-04-10 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / In this work prepared an imunosensor for Chagas disease. This sensor was onstructed by immobilizing the antigen (AG) of Chagas disease under an LbL film between the polyvinyl sulfonate (PVS) and silver nanoparticles incorporated in an inorganic matrix, 3-n-propilpiridíneo silsesquioxane chloride(Ag-SiPy+Cl-) using ITO as the electrochemical probe. The silver nanoparticles were synthesized through chemical reduction using NaBH4 and AgNO3. DLS measurements showed that the suspension of Ag-SiPy+Clconsisted of particles on the order of 20 nm. UV-Vis spectra confirmed the growth of bilayers through the LbL technique using PVS and the suspension Ag-SiPy+Cl- by means of bands at 200 and 260 nm which is referring to the SiPy+Cl- and a band near 400 nm which is referring to the silver nanoparticles. This growth was also demonstrated by AFM technique, which showed that the increase in the number of bilayers leads to an increase in surface roughness and film thickness. The modified films with AG were submitted to the technique electrochemical impedance spectroscopy and showed a pronounced change in impedance in the presence of the antibody (AB) of Chagas disease, indicating that this type of film can be used in a device for the diagnosis of Chagas. Were made tests with different dilutions the samples contaminated with antibody (AB) of Chagas disease and the imunosensor proposed was more sensitive compared to the laboratory test for the qualitative and semi-quantitative of Chagas disease (Gold analyzes Diagnostic Ltd.) / Neste presente trabalho preparou-se um imunosensor para doença de Chagas.Este sensor foi construído por meio da imobilização do Antígeno (AG) da doença de chagas sob um filme LbL entre o sulfonato de polivinil (PVS) e nanopartículas de prata incorporadas na matriz inorgânica cloreto de 3-npropilpiridíneo silsesquioxano (Ag-SiPy+Cl-) usando como transdutor eletroquímico o ITO. As nanopartículas de prata foram sintetizadas via redução química, com o uso de NaBH4 e AgNO3. Medidas de DLS mostraram que a suspensão de Ag-SiPy+Cl- era composta por partículas na ordem de 20 nm. Espectros de UV-Vis comprovaram o crescimento das bicamadas por meio da técnica LbL como uso do PVS e da suspensão Ag-SiPy+Cl-, por meio das bandas em 200 e 260 nm referentes ao SiPy+Cl- e uma banda próxima a 400 nm referente a nanopartículas de prata. Este crescimento também foi comprovado por meio da técnica de AFM, que mostrou que o aumento no número de bicamadas leva ao aumento na rugosidade e espessura do filme.Os filmes modificados com O AG forma submetidos à técnica de impedância eletroquímica e mostraram uma mudança de impedância pronunciada quando na presença do anticorpo (AB) da doença de Chagas, indicando que este tipo de filme pode ser utilizado num dispositivo para o diagnóstico da doença de Chagas. Foram feitos testes com diferentes diluições das amostras contaminadas com o AB da doença de Chagas e o imunosensor proposto mostrou-se mais sensível em relação ao teste laboratorial para determinação qualitativa e semi-quantitativa da doença de Chagas (Gold Analisa Diagnóstica Ltda).

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-02T19:23:04Z No. of bitstreams: 1 RitaCarvalho.pdf: 1268991 bytes, checksum: 9f30de20b2377b94837ba5ffe305debe (MD5) / Made available in DSpace on 2017-06-02T19:23:04Z (GMT). No. of bitstreams: 1 RitaCarvalho.pdf: 1268991 bytes, checksum: 9f30de20b2377b94837ba5ffe305debe (MD5) Previous issue date: 2016-07-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Cardiovascular diseases (CVDs) are considered as an important cause of morbidity and premature mortality worldwide. Rapid diagnosis of CVD is extremely important to ensure the survival of the patient. Myoglobin (Mb), for its low molecular weight is quickly released in the body, resulting in high sensitivity in order to be as valuable biomarker of acute myocardial infarction (AMI). Therefore, this study aims to develop an efficient platform, sensitive and stable for the immobilization of antibodies (Ab) myoglobin applicable to the monitoring of antigens (Ag) myoglobin using surface plasmon resonance (SPR). In this sense, the cyclic voltammetry, differential pulse voltammetry and impedance spectroscopy were used in selfassembled monolayers research (SAMs) simple and mixed acid 11-mercaptoundecanoic (MUA) and 3-mercaptopropionic acid (MPA). The mixed SAM MPA / MUA 3: 1 ratio showed low resistance to charge transfer and high surface coverage. In order to investigate the SAM capacity MPA / MUA opposite the locking Ac-MB were made comparative studies for the evaluation of the interaction of Ac-MB SAM MPA / MUA well as for SAM MPA / MUA modified polyamidoamine dendrimer of generation 5 (G5 PAMAM). Then, after choosing the more sensitive platform made up AC-Mb interaction studies with different concentrations of Ag-Mb (0,001; 0,5; 0,8; 1 and 500 µg.mL-1) through immunoassay in a "sandwich". The three-dimensional structure of the dendrimer PAMAM G5 favored amplification of SPR signal as grants most accessible area for the immobilization of antibody Mb. The sandwich immunoassay amplified the interaction signal Ac / Ag Mb, with significant advantages in sensitivity immunosensor. The results show that it was possible to develop an immunosensor capable of detecting low concentrations (0.00067 μg.mL-1) Ag-Mb. In this sense, the proposed system has great potential alternative to early diagnosis, simple and fast IAM. / As doenças cardiovasculares (DCVs) são consideradas como importante causa de morbidade e mortalidade prematura em todo o mundo. O diagnóstico rápido das DCVs é extremamente importante para garantir a sobrevivência do paciente. A mioglobina (Mb), por seu baixo peso molecular, é rapidamente liberada no organismo, resultando em elevada sensibilidade de forma a se constituir como precioso biomarcador de infarto agudo do miocárdio (IAM). Portanto, este trabalho tem por objetivo o desenvolvimento de uma plataforma eficiente, sensível e estável para a imobilização de anticorpos (Ac) de mioglobina aplicável ao monitoramento de antígenos (Ag) de mioglobina empregando ressonância de plásmons de superfície (SPR). Neste sentido, as técnicas de voltametria cíclica, voltametria de pulso diferencial e impedância eletroquímica foram empregadas na investigação de monocamadas auto-organizadas (SAMs) simples e mistas de ácido 11-mercaptoundecanóico (MUA) e ácido 3-mercaptopropiônico (MPA). A SAM mista MPA/MUA de proporção 3:1 apresentou baixa resistência à transferência de carga e elevada cobertura de superfície. Com o propósito de investigar a capacidade da SAM de MPA/MUA frente à imobilização de Ac-Mb foram feitos estudos comparativos para a avaliação da interação da do Ac-Mb com a SAM de MPA/MUA bem como para a SAM de MPA/MUA modificada com dendrímero de poliamidoamina de geração 5 (PAMAM G5). Em seguida, após a escolha da plataforma mais sensível, fez-se os estudos da interação do Ac-Mb com diferentes concentrações do Ag-Mb (0,001; 0,5; 0,8; 1 e 500 µg mL-1) através de imunoensaio em formato “sanduíche”. A estrutura tridimensional do dendrímero PAMAM G5 favoreceu a amplificação do sinal de SPR, pois concede uma maior área acessível para a imobilização do anticorpo da Mb. O imunoensaio em sanduíche amplificou o sinal de interação Ac/Ag da Mb, apresentando vantagens significativas na sensibilidade do imunossensor. Os resultados mostram que foi possível desenvolver um imunossensor capaz de detectar baixas concentrações (0,00067 µg.mL-1) do Ag-Mb. Neste sentido, o sistema proposto apresenta grande potencial alternativo de diagnóstico precoce, simples e rápido do IAM.

Šioje daktaro disertacijoje buvo nagrinėjami paviršiaus plazmonų rezonanso (PPR) ir elektrocheminiai imuniniai jutikliai žmogaus augimo hormonui (ŽAH) ir antikūnams prieš žmogaus augimo hormoną (anti-ŽAH) nustatyti bei pritaikyti juos realių mėginių analizei. Šiame darbe buvo nagrinėjami nekryptingas ir kryptingas antikūnų imobilizavimo būdai PPR jutiklio lusto paviršiuje ir tirtas antikūno-antigeno komplekso formavimasis. Buvo nustatyta, kad tinkamiausias iš tirtų anti-ŽAH imobilizavimo PPR jutiklio lusto paviršiuje būdų yra kryptingas anti-ŽAH fragmentų gautų redukuojant anti-ŽAH 2-merkatoetilaminu (2-MEA) imobilizavimas. Atominės jėgos mikroskopijos pagalba buvo stebėti įvairiais būdais modifikuotų PPR jutiklio lusto paviršių skirtumai, kurie parodė, ar fragmentai gauti redukuojant anti-ŽAH buvo tinkamai prijungti prie auksinio PPR jutiklio lusto paviršiaus, o imuninis kompleksas susiformavo dėl specifinės antikūno-antigeno sąveikos. PPR ŽAH imuninis jutiklis pasižymi maža aptikimo riba (3,4 nmol/L), stabilumu, atrankamu. Aktyvus PPR jutiklio lusto paviršius visiškai regeneruojamas, o tai leidžia imuninio jutiklio daugkartinį panaudojimą. Tiesioginio ir netiesioginio tipo PPR anti-ŽAH imuniniai jutikliai buvo tiriami kombinuojant du analizinius metodus – PPR ir elektrocheminius (impulsinė amperometrija (PA) ir ciklinė voltamperometrija (CV)). Anti-ŽAH imuniniai jutikliai pasižymi maža aptikimo riba (0,056, 0,051 ir 0,027 nmol/L, atitinkamai CV, PPR ir PA), stabilumu... [toliau žr. visą tekstą] / In this doctoral thesis the development of surface plasmon resonance (SPR) and electrochemical (EL) immunosensors has been examined and used for the detection of human growth hormone (HGH) and antibodies against human growth hormone (anti-HGH). The designed immunosensors were applied for the real sample analysis. In this research work random and oriented techniques of anti-HGH immobilization on the SPR sensor chip were compared and the interaction between immobilized anti-HGH and HGH was evaluated. Fragments of anti-HGH immobilization via their native thiol-groups directly coupled to the SPR chip is the most suitable technique for the development of an SPR immunosensor for the HGH detection. Fragments of antibody were obtained by chemical reduction of 2-mercaptoethylamine (2-MEA). In our study atomic force microscopy has been successfully applied for the imaging of the the differently modified surfaces. The achieved results indicate how much adequately the fragments of anti-HGH molecules bound onto the gold substrate and how immune complexes were formed. SPR HGH immunosensor is suitable for HGH detection with a limit of detection (3.4 nmol/L), good stability and selectivity. Furthermore, active SPR-chip surface is fully regenerated that allows multiply usage of designed immunosensor. Direct and indirect anti-HGH immunosensor was studied by combining two analytical methods – SPR and EL (pulsedamperometry (PA) and cyclic voltammetry (CV)). The limit of detection values of... [to full text]

Šioje daktaro disertacijoje buvo nagrinėjami paviršiaus plazmonų rezonanso (PPR) ir elektrocheminiai imuniniai jutikliai žmogaus augimo hormonui (ŽAH) ir antikūnams prieš žmogaus augimo hormoną (anti-ŽAH) nustatyti bei pritaikyti juos realių mėginių analizei. Šiame darbe buvo nagrinėjami nekryptingas ir kryptingas antikūnų imobilizavimo būdai PPR jutiklio lusto paviršiuje ir tirtas antikūno-antigeno komplekso formavimasis. Buvo nustatyta, kad tinkamiausias iš tirtų anti-ŽAH imobilizavimo PPR jutiklio lusto paviršiuje būdų yra kryptingas anti-ŽAH fragmentų gautų redukuojant anti-ŽAH 2-merkatoetilaminu (2-MEA) imobilizavimas. Atominės jėgos mikroskopijos pagalba buvo stebėti įvairiais būdais modifikuotų PPR jutiklio lusto paviršių skirtumai, kurie parodė, ar fragmentai gauti redukuojant anti-ŽAH buvo tinkamai prijungti prie auksinio PPR jutiklio lusto paviršiaus, o imuninis kompleksas susiformavo dėl specifinės antikūno-antigeno sąveikos. PPR ŽAH imuninis jutiklis pasižymi maža aptikimo riba (3,4 nmol/L), stabilumu, atrankamu. Aktyvus PPR jutiklio lusto paviršius visiškai regeneruojamas, o tai leidžia imuninio jutiklio daugkartinį panaudojimą. Tiesioginio ir netiesioginio tipo PPR anti-ŽAH imuniniai jutikliai buvo tiriami kombinuojant du analizinius metodus – PPR ir elektrocheminius (impulsinė amperometrija (PA) ir ciklinė voltamperometrija (CV)). Anti-ŽAH imuniniai jutikliai pasižymi maža aptikimo riba (0,056, 0,051 ir 0,027 nmol/L, atitinkamai CV, PPR ir PA), stabilumu... [toliau žr. visą tekstą] / In this doctoral thesis the development of surface plasmon resonance (SPR) and electrochemical (EL) immunosensors has been examined and used for the detection of human growth hormone (HGH) and antibodies against human growth hormone (anti-HGH). The designed immunosensors were applied for the real sample analysis. In this research work random and oriented techniques of anti-HGH immobilization on the SPR sensor chip were compared and the interaction between immobilized anti-HGH and HGH was evaluated. Fragments of anti-HGH immobilization via their native thiol-groups directly coupled to the SPR chip is the most suitable technique for the development of an SPR immunosensor for the HGH detection. Fragments of antibody were obtained by chemical reduction of 2-mercaptoethylamine (2-MEA). In our study atomic force microscopy has been successfully applied for the imaging of the the differently modified surfaces. The achieved results indicate how much adequately the fragments of anti-HGH molecules bound onto the gold substrate and how immune complexes were formed. SPR HGH immunosensor is suitable for HGH detection with a limit of detection (3.4 nmol/L), good stability and selectivity. Furthermore, active SPR-chip surface is fully regenerated that allows multiply usage of designed immunosensor. Direct and indirect anti-HGH immunosensor was studied by combining two analytical methods – SPR and EL (pulsedamperometry (PA) and cyclic voltammetry (CV)). The limit of detection values of... [to full text]

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-19T13:03:29Z No. of bitstreams: 5 denio.pdf: 1745055 bytes, checksum: 531bb76e60dd34c17e7c27620ce5b5c0 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-20T10:55:25Z (GMT) No. of bitstreams: 5 denio.pdf: 1745055 bytes, checksum: 531bb76e60dd34c17e7c27620ce5b5c0 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Made available in DSpace on 2015-02-20T10:55:25Z (GMT). No. of bitstreams: 5 denio.pdf: 1745055 bytes, checksum: 531bb76e60dd34c17e7c27620ce5b5c0 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) Previous issue date: 2013-02-21 / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Neste trabalho, foi realizado o desenvolvimento de um imunosensor para detec??o de anticorpos anti-Leishmania infantum. O princ?pio de transdu??o foi a Resson?ncia de Pl?smons de Superf?cie (SPR) e o sensor foi produzido atrav?s da imobiliza??o covalente de ant?genos sol?veis de Leishmania infantum sobre Monocamada Auto-Organizada (SAM) do ?cido 11-Mercaptoundecan?ico (11-MUA) ativado pela mistura contendo 100,0 mmol.L-1 de 1-etil-3-(3-dimetilaminopropil) carbodiimida (EDC) e 150 mmol.L-1 de N-hidroxisuccinimida (NHS). Atrav?s das t?cnicas de Voltametria C?clica e Redu??o Dessortiva por Voltametria de Pulso Diferencial o processo de forma??o da SAM foi otimizado. Sob condi??es otimizadas, 1,0 mmol.L-1 da solu??o etan?lica de 11-MUA e 24 horas foram suficientes para a forma??o de uma SAM est?vel sobre a superf?cie de ouro. Para obten??o de informa??es complementares da SAM, foram realizados c?lculos da Cobertura de Superf?cie, Constante Diel?trica e Espessura do tiol adsorvido no substrato de ouro. Ap?s esta etapa, a adi??o de ant?genos sol?veis de L. infantum sobre a SAM foi acompanhada por SPR. As t?cnicas de Voltametria C?clica e Espectroscopia de Imped?ncia Eletroqu?mica foram usadas para caracterizar a imobiliza??o do ant?geno. Ap?s constru??o do imunosensor, foram adicionados em sua superf?cie soros caninos positivos para Leishmaniose Visceral, evidenciando varia??o significativa no ?ngulo de SPR, mostrando excelente sensibilidade da t?cnica para detec??o da intera??o ant?geno-anticorpo. Por outro lado, a adi??o dos soros negativos n?o foi acompanhada com resposta significativa, concluindo-se tamb?m que o imunosensor apresentou boa especificidade contra anticorpos anti-L. infatum. Portanto, neste trabalho foi desenvolvido com sucesso um sensor empregando SPR para detec??o em tempo real de anticorpos anti-L. infatum, apresentando grande perspectiva como sistema de sensoriamento da Leishmaniose Visceral em ?reas end?micas. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2012. / ABSTRACT In this work, the development of an immunosensor for detection of anti-Leishmania infantum antibodies was performed. The principle of transduction used was the Surface Plasmon Resonance (SPR) and the sensor was constructed by covalent immobilization of soluble antigens of Leishmania infantum on self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (11-MUA) which was activated by the mixture 100.0 mmol.L-1 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) and 150 mmol.L-1 N-hidroxisuccinimide (NHS). Cyclic Voltammetry and Dessortive Reduction by Differential Pulse Voltammetry techniques were employed in monitoring of SAM formation. Under optimized conditions, 1.0 mmol.L-1 of the 11-MUA in ethanolic solution and 24 hours were sufficient for the formation of a stable SAM on the gold surface. To obtain further information about the SAM, the Surface Coverage, Dielectric Constant and Thickness of the thiol film adsorbed on gold substrate were performed. After this step, the addition of soluble antigens?of L.?infantum?on the?SAM?was monitored?by SPR. Cyclic Voltammetry and Electrochemical Impedance Spectroscopy techniques were employed in characterization of antigen immobilization. After construction of immunosensor, positive canine serum for Visceral Leishmaniasis were added on its surface showing significant variation in the SPR angle, indicating excellent sensitivity of the technique for?antigen-antibody interaction detection. On the other hand, the addition of negative serum was not accompanied with a significant response, demonstrating that the immunosensor shows excellent specificity against anti-L infantum antibodies. Therefore, this work shows a successfully development of a SPR sensor for anti-L. infatum antibodies detection in real time, showing a great perspective as a sensing system of Visceral Leishmaniasis in endemic regions.