Dificuldades no processo de construção de imunossensor para tipagem sanguínea ABO
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Data
2021-05-31
Autores
Silva, Pâmela Leite da
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Universidade Estadual Paulista (Unesp)
Resumo
Os antígenos ABO são carboidratos adicionados à superfície da membrana das hemácias, que conferem o tipo A, B, AB ou O aos indivíduos. Pelo fato do organismo humano produzir naturalmente anticorpos regulares contra antígenos desse sistema, ele se torna o mais importante na prática transfusional. Entre os testes pré- transfusionais, destaca-se a fenotipagem do sistema ABO/Rh, (fase direta e reversa da tipagem sanguínea). Tal técnica baseia-se na hemaglutinação, com leitura subjetiva e sem interfaceamento digital. Com os avanços das metodologias de Point of Care e Lab on a Chip, biossensores são desenvolvidos para modernizar e otimizar técnicas manuais como essas. Dentre estes, destaca-se neste trabalho o imunossensor, que se baseia na ligação antígeno- anticorpo, detectada por um transdutor e convertido em informação digital. O objetivo deste trabalho é a construção de um imunossensor para tipagem sanguínea ABO. Foram utilizados anticorpos anti- A e - B monoclonais produzidos home made e policlonais purificados de plasma de bolsa de descarte, na concentração de 0,05mg/mL, que compuseram a molécula biorreceptora do imunossensor, fixados à superfície do eletrodo de carbono pelo polímero quitosana. A espectrofotometria UV-visível foi utilizada para monitoramento de construção do filme. As amostras analisadas foram de doadores de sangue do Hemocentro de Botucatu, que foram previamente fenotipadas conforme protocolo de doação em A, B e O. A técnica de construção do filme polímero + anticorpos foi a de automontagem por Layer-by-Layer, (LbL) que permite obter filmes finos compostos de anticorpos e polímeros, por adsorção física. O tempo de exposição da amostra sobre o imunossensor inicialmente foi de 5, 15 e 30 minutos e submetidas à medidas eletroquímicas e comparadas com sistemas controle positivo e negativo. Dadas as análises, os tempos de incubações passaram a ser de 1, 2,3,4 e 5minutos. A técnica de voltametria cíclica foi utilizada para caracterizar os filmes, monitoramento de cada camada depositada e para detecção de diferentes amostras de sangue. A absorção de luz em espectrofotometria na região UV-vis demonstrou que o número ideal de bicamadas para a composição do filme é de 2 bicamadas para o anti-B. Para o anti-A os resultados não foram conclusivos, embora a adsorção de luz fosse maior com 3 bicamadas, não houve pico de adsorção. Foi adotado então o número de duas bicamadas pra ambos os filmes dos dois anticorpos, para uso de anticorpos monoclonais. Os anticorpos policlonais foram funcionalizados com nanopartículas de ferro, à qual propiciaram o uso de uma única bicamada do filme. As leituras em Voltametria cíclica com o eletrodo Quitosana + Anti-A monoclonal demonstrou um alto potencial de especificidade com a hemácia A, quando incubados com a hemácia A e B por 5 minutos. Acima desse período observou a inespecificidade do imunossensor. Com o sensor contento Quitosana + Anti-B monoclonal os resultados não foram conclusivos, pois as análises de comportamento das curvas de voltamograma e as áreas dos mesmos não demonstraram diferenças entres as análises com hemácias A e B em todos os tempos analisados. Os eletrodos Quitosana + Anti-A e Anti-B policlonais funcionalizados com nanopartículas de ferro, tanto nos testes de especificidade, quando no de reprodutibilidade não demonstraram diferenças em amostras especificas e inespecíficas. Um dos principais problemas em potencial são os resquícios de albumina na purificação dos anticorpos que possam influenciar nas leituras e ser depositados de forma inespecífica sobre o filme. Além disso, a hipótese de que a hemácia é atraída quimicamente pela quitosana pode ser um dos fatores que influenciaram os resultados. Entretanto, pode-se afirmar que as nanopartículas permitiram uma melhor fixação de anticorpos da classe IgM em eletrodos impressos de carbono, o que era um grande desafio para construção desse biossensor. Diante disso, constata-se o grande potencial da produção de imunossensores para identificação do grupo sanguíneo ABO, sendo necessários ajustes para que o sistema se torne específico e reprodutivo.
The ABO antigens system is composed by carbohydrates added to the red blood cells’ membrane, which includes four blood types known as A, B, O and AB, in humans. Due to the human organism natural production of regular antibodies against this system, it became the most important transfusion practice. Between pre-transfusion tests, it can be highlighted both ABO and Rh phenotyping (direct and reverse phase of blood typing). This technique is based on hemagglutination, with subjective reading and without digital interfacing. With advances on Point of Care and Lab on a Chip methodologies, biosensors have been developed in order to modernize and optimize manual techniques, such as those mentioned. Among this new technology, the immunosensors stand out, which is based on the bond and affinity between an antigen and its respective antibody, that can be detected through a transductor and the information is converted in digital data. The main goal of this research is to construct an immunosensor that recognizes and identifies which blood type group from ABO system. Here are going to be used monoclonal antibodies anti-A and anti-B, home-made produced, and purified polyclonal antibodies from disposal bag’s plasma, in 0,05mg/mL concentration, which both composes the immunosensor’s bioreceptor molecule, fixed against the carbon electrode’s surface by the polymer chitosan. In order to monitor the film construction, the UV-visible spectrophotometry was used. The analyzed samples were from blood donors from Botucatu Hemocenter, which were all previously phenotyped according to donation protocol in A, B and O blood types. For the film made of polymer and antibodies construction, the technique used was self-assembly Layer-by-Layer (LbL), which allows the achievement of fine films with the required composition, through physic adsorption. The time of samples’ exposure over the immunosensors was initially of 5, 15 and 30 minutes, and they were subject to electrochemical measures and compared to both positive and negative control systems. Given the analyses made, the incubation times became 1, 2, 3, 4 and 5 minutes. The cyclic voltammetry technique was applied to distinguish the films, while monitoring each deposited layer, and also to detect different blood type samples. The light absorption in UV-vis spectrophotometry showed that the ideal number of bilayers for the film construction was 2 bilayers of anti-B. The results about anti-A film construction were inconclusive, even though the light adsorption was higher with 3 bilayers, there were no adsorption peak. Accordingly, it was used the number of 2 bilayers for both antibodies film construction. Polyclonal antibodies were functionalized with Fe nanoparticles, which propitiated the use of only one film bilayer. The reading obtained with cyclic voltammetry and chitosan-monoclonal Anti-A electrode showed a high specificity potential with the red blood cell A, when both cell A and B were incubated for five minutes. Above this period of time, it was seen an immunosensor nonspecificity. About the sensor containing chitosan and monoclonal Anti-B the results were not conclusive, since the analyses’ behavior of voltogram curves and the respective graphic area did not show differences between the tests with red blood cell A and B during all used times. Both electrodes with chitosan- polyclonal Anti-A and polyclonal Anti-B functionalized with iron nanoparticles did not show differences in specific and nonspecific samples, during specificity and reproducibility tests. One of the most important potential errors is the albumin remnants during the antibodies purification, that are able to disturb the readings and end up settling nonspecifically above the film. Other than that, the hypothesis that the red blood cell is chemically attracted to the chitosan can be a factor that might influence the results. Nevertheless, it can be assured that the nanoparticles allowed a better fixation the IgM antibodies on printed carbon electrodes, which was a huge challenge during this biosensor construction. In spite of that, it was found the immunosensors production’s great potential for identification of ABO blood types system, even though some adjusts are necessary in order to turn the system specific and reproducible.
The ABO antigens system is composed by carbohydrates added to the red blood cells’ membrane, which includes four blood types known as A, B, O and AB, in humans. Due to the human organism natural production of regular antibodies against this system, it became the most important transfusion practice. Between pre-transfusion tests, it can be highlighted both ABO and Rh phenotyping (direct and reverse phase of blood typing). This technique is based on hemagglutination, with subjective reading and without digital interfacing. With advances on Point of Care and Lab on a Chip methodologies, biosensors have been developed in order to modernize and optimize manual techniques, such as those mentioned. Among this new technology, the immunosensors stand out, which is based on the bond and affinity between an antigen and its respective antibody, that can be detected through a transductor and the information is converted in digital data. The main goal of this research is to construct an immunosensor that recognizes and identifies which blood type group from ABO system. Here are going to be used monoclonal antibodies anti-A and anti-B, home-made produced, and purified polyclonal antibodies from disposal bag’s plasma, in 0,05mg/mL concentration, which both composes the immunosensor’s bioreceptor molecule, fixed against the carbon electrode’s surface by the polymer chitosan. In order to monitor the film construction, the UV-visible spectrophotometry was used. The analyzed samples were from blood donors from Botucatu Hemocenter, which were all previously phenotyped according to donation protocol in A, B and O blood types. For the film made of polymer and antibodies construction, the technique used was self-assembly Layer-by-Layer (LbL), which allows the achievement of fine films with the required composition, through physic adsorption. The time of samples’ exposure over the immunosensors was initially of 5, 15 and 30 minutes, and they were subject to electrochemical measures and compared to both positive and negative control systems. Given the analyses made, the incubation times became 1, 2, 3, 4 and 5 minutes. The cyclic voltammetry technique was applied to distinguish the films, while monitoring each deposited layer, and also to detect different blood type samples. The light absorption in UV-vis spectrophotometry showed that the ideal number of bilayers for the film construction was 2 bilayers of anti-B. The results about anti-A film construction were inconclusive, even though the light adsorption was higher with 3 bilayers, there were no adsorption peak. Accordingly, it was used the number of 2 bilayers for both antibodies film construction. Polyclonal antibodies were functionalized with Fe nanoparticles, which propitiated the use of only one film bilayer. The reading obtained with cyclic voltammetry and chitosan-monoclonal Anti-A electrode showed a high specificity potential with the red blood cell A, when both cell A and B were incubated for five minutes. Above this period of time, it was seen an immunosensor nonspecificity. About the sensor containing chitosan and monoclonal Anti-B the results were not conclusive, since the analyses’ behavior of voltogram curves and the respective graphic area did not show differences between the tests with red blood cell A and B during all used times. Both electrodes with chitosan- polyclonal Anti-A and polyclonal Anti-B functionalized with iron nanoparticles did not show differences in specific and nonspecific samples, during specificity and reproducibility tests. One of the most important potential errors is the albumin remnants during the antibodies purification, that are able to disturb the readings and end up settling nonspecifically above the film. Other than that, the hypothesis that the red blood cell is chemically attracted to the chitosan can be a factor that might influence the results. Nevertheless, it can be assured that the nanoparticles allowed a better fixation the IgM antibodies on printed carbon electrodes, which was a huge challenge during this biosensor construction. In spite of that, it was found the immunosensors production’s great potential for identification of ABO blood types system, even though some adjusts are necessary in order to turn the system specific and reproducible.
Descrição
Palavras-chave
Imonossensor, Tipagem sanguínea, Point of care, Lab on a chip, Immunosensor, Blood typing