Eletross?ntese, caracteriza??o e aplica??o de filmes polim?ricos de poli(?cido 2-hidroxicin?mico) em imunossensores impedim?tricos para diagn?stico de leishmaniose visceral em amostras de soros caninos

• Main Author: Xavier, Marcelo de Sousa
• Other Authors: Ferreira, Lucas Franco
• Language: Portuguese
• Published: UFVJM 2017
• Subjects: Leishmaniose visceral; Imunossensor impedim?trico; ?cido 2-hidroxicin?mico; Filmes polim?ricos; Eletropolimeriza??o; Visceral leishmaniasis; Impedimetric immunosensor; 2-hydroxycinnamic acid; Polymeric films; Electropolymerization
• Online Access: http://acervo.ufvjm.edu.br/jspui/handle/1/1479

Submitted by Jos? Henrique Henrique ([e-mail]) on 2017-09-22T18:10:08Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_sousa_xavier.pdf: 3096603 bytes, checksum: dae91257943ac86316978d8deb31ff3e (MD5) === Approved for entry into archive by Rodrigo Martins Cruz ([e-mail]) on 2017-10-09T13:42:33Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_sousa_xavier.pdf: 3096603 bytes, checksum: dae91257943ac86316978d8deb31ff3e (MD5) === Made available in DSpace on 2017-10-09T13:42:33Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_sousa_xavier.pdf: 3096603 bytes, checksum: dae91257943ac86316978d8deb31ff3e (MD5) Previous issue date: 2017 === A Leishmaniose Visceral Canina (LVC) ? considerada um problema de sa?de p?blica causada por um protozo?rio do g?nero Leishmania que acomete c?es, o qual no ciclo urbano de transmiss?o ? o principal reservat?rio. Neste trabalho, investigou-se o desenvolvimento de um imunossensor impedim?trico sobre eletrodos de grafite (EG) e eletrodos impressos de ?xido de grafeno (EIG) modificados com filmes polim?ricos para detec??o de Leishmaniose infantum em amostras de soros caninos. Os EG e o EIG foram modificados pela eletrodeposi??o do ?cido 2-hidroxicin?mico (2-HCA). A eletropolimeriza??o do 2-HCA foi realizada por voltametria c?clica (VC), utilizando-se solu??o monom?rica 2,50 mM preparada em H2SO4 0,50 M contendo 1,0 mL de etanol. Para o EG utilizou-se 100 ciclos de potencial na faixa de +0,20 a +1,20 V a 50 mV/s e para o EIG 15 ciclos consecutivos de potencial de 0,00 a +1,00V a 100 mV/s. Para ambos os eletrodos modificados o poli(2-HCA) apresentou caracter?sticas adsortivas e eletroativas. Estudos de Espectroscopia de Imped?ncia Eletroqu?mica (EIE) foram realizados para avaliar as propriedades el?tricas dos eletrodos modificados. Para o EG/2-HCA utilizou-se o circuito equivalente descrito como (Rs(RQp)(Qdl[RtcW]) onde obteve-se o valor de resist?ncia a transfer?ncia de carga (Rtc) de 13,2 k?, enquanto que para EIG/2-HCA o circuito equivalente proposto foi o de Randles, no qual o valor de Rtc foi de 4,2 k?. Visando a miniaturiza??o do sistema, optou-se pelo desenvolvimento do biossensor sobre os EIG. Desta forma, foram colocados sobre a superf?cie dos EIG/2-HCA, utilizando a t?cnica de adsor??o, 35 ?L de diferentes concentra??es do ant?geno L. infantum (Ag) (15, 30, 50, 70 e 100 ?g/mL), sendo realizados testes de intera??o com amostras de soros caninos contendo os anticorpos positivos (Ac+) de L. infantum em diferentes propor??es 1:80, 1:160, 1:320, 1:640 e 1:1280 dilu?dos em tamp?o HBS-EP pH 7,40 com 20 minutos de intera??o a 37 ?C. Os melhores resultados foram obtidos utilizando-se o ant?geno em concentra??o de 50 ?g/mL durante 20 min a 37 ?C e a dilui??o do soro de 1:320. Posteriormente, avaliou-se o tempo de imobiliza??o do Ag e de imunorrea??o Ag-Ac em 20, 40 e 60 minutos. Obteve-se 20 min de como tempo ideal de imobiliza??o de Ag e para imunorrea??o Ag-Ac+. Ap?s otimiza??o destes par?metros, avaliou-se a resposta do imunossensor na presen?a de amostras de soros caninos diagnosticados como positivos (Ac+) e negativos (Ac-) para Leishmaniose infantum, onde observou-se valores de Rtc para o sistema Ag-Ac+ 82% maior do que o obtido para o Ag-Ac-, demonstrando margem de seguran?a confi?vel no reconhecimento do analito de interesse. O imunossensor proposto para detec??o de Leishmaniose infantum apresentou boas perspectivas para aplica??o ao diagn?stico da doen?a, podendo-se tornar mais uma alternativa para realiza??o de testes r?pidos e de simples aplica??o para detec??o de LVC. === Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. === Visceral Canine Leishmaniasis (LVC) is considered a public health issue caused by a protozoan of the Leishmania genre that attacks dogs, which is the main reservoir in the urban transmission cycle. In this work, we investigated the development of an impedimetric immunosensor on graphite electrodes (GE) and graphene oxide-modified screen-printed carbon electrode (GSP) both modified with polymeric films to detect Leishmaniose infantum in canine serum samples. The GE and GSP were modified by the electrodeposition of 2-hydroxycinnamic acid (2-HCA). Electropolymerization of 2-HCA was performed by cyclic voltammetry (CV) using a 2.50 mM monomeric solution prepared in 0.50 M H2SO4 containing 1.0 mL of ethanol. For the EG and EIG, 100 cycles of potential between +0.20 and +1.20 V at 50 mV/s and 15 cycles of potential between 0.00 and + 1.00 V at 100 mV/s were used, respectively. Poly(2-HCA) shown adsorptive and electroactive characteristics for both electrodes. Electrochemical Impedance Spectroscopy (EIS) studies were performed to evaluate the electrical properties of the modified electrodes. For the GE/2-HCA was used an equivalent circuit described as (Rs(RpQp)(Qdl[RtcW]), and a resistance of charge transfer (Rct) of 13.2 k? was obtained. For GSP/2-HCA the proposed equivalent circuit was that of Randles in which the value of Rct obtained was 4.2 k?. For system miniaturization it was developed the biosensor over GSP. Thus, 35 ?L of different concentrations of L. infantum antigen (Ag) (15, 30, 50, 70 e 100 ?g/mL) were placed on the GSP/2-HCA surface using the adsorption technique. Interaction tests were carried out with canine serum samples containing the positive antibodies (Ac+) of L. infantum in different proportions of 1:80, 1:160, 1:320, 1:640 e 1:1280, diluted in HBS-EP buffer pH 7.4 with 20 minutes of interaction at 37 ?C. The best results were obtained using the antigen at a concentration of 50 ?g/mL for 20 min at 37 ?C and a serum dilution of 1:320. Subsequently, the Ag immobilization and Ag-Ac immunoreaction time were evaluated in 20, 40 and 60 min. 20 min was obtained as the ideal time for Ag immobilization and Ag-Ac+ immunoreaction. After optimization of these parameters, the immunosensor response was evaluated in the presence of canine serum diagnosed as positive (Ac+) and negative (Ac-) for Leishmaniose infantum, where the Rct values for the Ag-Ac+ system were 82% higher than those obtained for Ag-Ac-, demonstrating a reliable safety margin in the recognition of the analyte of interest. The proposed immunoassay for the detection of Leishmaniasis infantum presented good prospects for application in the diagnosis of the disease, and it could become an alternative for performing rapid tests and simple application for the detection of LVC.