Análises em dispositivos de papel: síntese de carbon dot aplicada à determinação de compostos de interesse clínico e paper-spray ionization para detecção direta de doping esportivo
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Data
2020-02-14
Autores
Rossini, Eduardo Luiz [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A primeira parte deste trabalho foi desenvolvida no laboratório Fritz Feigl-UNESP/CAr, na qual foi descrito um novo carbon dot (CD) sintetizado a partir de ácido cítrico e tiramina usando uma rota em única etapa por meio de aquecimento por micro-ondas doméstico. O CD sintetizado apresenta emissão no azul quando irradiado com luz UV, que foi suprimida na presença de H2O2 e peroxidase. O CD foi utilizado para desenvolver metodologias para quantificar glicose em amostras biológicas utilizando plataforma de papel. O método desenvolvido foi aplicado em amostras de soro e urina certificadas, e não há diferenças estatisticamente significativas entre os resultados obtidos usando o dispositivo de papel com adição de padrão e as amostras certificadas. Também foram desenvolvidas metodologias utilizando 3D-µPAD com o reagente fluorescente e enzimas oxidase para quantificação de glicose e lactato em amostras de saliva. O preparo de amostra foi simples e rápido e o método foi validada utilizando adição de padrão e recuperação, além de ser aplicado em amostras de soro certificadas, não houve diferenças estatisticamente significativas entre os resultados obtidos e os valores de referência. A segunda parte deste trabalho foi desenvolvida no Badu’s research group na The Ohio State University durante o período sanduiche com bolsa da FAPESP, na qual foi desenvolvida a quantificação direta de agentes dopantes em amostras de urina sem tratamento da amostra por PS-MS. Paper-spray mass spectrometry é um método de ionização em condições ambientes que combina as vantagens das plataformas de papel com a confiabilidade das análises de espectrometria de massas, reduzindo o tempo de análise e o consumo de reagente e de solventes orgânicos. O uso de substâncias ilícitas para aumentar a desempenho de atletas em esportes é proibido e considerado doping. A superfície do papel foi tratada com triclorometilsilano utilizando reação em fase gasosa para aumentar a ionização dos compostos alvo. Essa abordagem foi aplicada para análise de dois agentes anabólicos (trembolona e clembuterol) e dois diuréticos (furosemida e hidroclorotiazida) em amostras de urina. Após a otimização das condições experimentais, os métodos desenvolvidos apresentaram repetibilidade satisfatória e boa correlação linear entre concentração e intensidade absoluta. Foram alcançados limites de detecção na faixa de sub-ng mL-1, abaixo dos níveis de desempenho mínimos requeridos pela WADA, utilizando o método de PS-MS com papel hidrofóbico sem nenhuma etapa de preparo ou clean-up da amostra. Os métodos propostos foram aplicados em amostras de urina fortificadas com os agentes dopantes e se mostraram rápidos, simples, sensíveis, exatos e precisos.
The first part of this work was developed on the Fritz Feigl-UNESP/CAr laboratory, in which a novel carbon dot synthesized from citric acid and tyramine was described using a one-step microwave heating route. Under UV irradiation, the CD displayed blue emission that was quenched in the presence of H2O2 and peroxidase. This property was used to develop a method to quantify glucose in biological samples using a paper platform. The method developed was applied in the analysis of certified serum and urine samples, and there were no statistically significant differences between the certified concentrations and the results obtained using the paper device with standard additions. In addition, it was developed method using 3D-µPAD, the CD fluorescence reagent and oxidase enzymes to quantify glucose and lactate in saliva samples. The sample preparation used was simple and fast. The method was validated in saliva samples utilizing standard addition and recovery, and applied in the analysis of certified serum samples, and there were no statistically significant differences between the certified concentrations and the results obtained using the 3D-µPAD. The second part of this work was developed at the Badu’s research group at The Ohio State University during the internship period funded by FAPESP, in which a direct quantification of doping agents in urine samples with no sample preparation by PS-MS was developed. Paper-spray mass spectrometry (PS-MS) is an ambient ionization method that combines the advantages of the paper platform with the reliability of MS analysis, reducing the consumption of time, reagent, and organic solvent. The use of illicit substances to improve enhance athletic performance in sport is forbidden and considered doping. The paper surface was treated with trichloromethylsilane (TCMS) using a gas-phase reaction to increase the target compounds ionization. This approach was applied for the analysis of two anabolic agents (trenbolone and clenbuterol) and two diuretics (furosemide and hydrochlorothiazide) in urine samples. Under optimized conditions, the developed methods presented satisfactory repeatability and a good correlation between concentration and absolute intensity. Highly sensitive detections as low as sub-ng mL 1 which is below the minimum required performance levels (MRPL) proposed by the WADA has been reached using the hydrophobic PS-MS without any pre-concentration and clean-up step. The proposed methods were applied in fortified urine samples with doping agents and showed to be fast, simple, sensitive, accurate and precise.
The first part of this work was developed on the Fritz Feigl-UNESP/CAr laboratory, in which a novel carbon dot synthesized from citric acid and tyramine was described using a one-step microwave heating route. Under UV irradiation, the CD displayed blue emission that was quenched in the presence of H2O2 and peroxidase. This property was used to develop a method to quantify glucose in biological samples using a paper platform. The method developed was applied in the analysis of certified serum and urine samples, and there were no statistically significant differences between the certified concentrations and the results obtained using the paper device with standard additions. In addition, it was developed method using 3D-µPAD, the CD fluorescence reagent and oxidase enzymes to quantify glucose and lactate in saliva samples. The sample preparation used was simple and fast. The method was validated in saliva samples utilizing standard addition and recovery, and applied in the analysis of certified serum samples, and there were no statistically significant differences between the certified concentrations and the results obtained using the 3D-µPAD. The second part of this work was developed at the Badu’s research group at The Ohio State University during the internship period funded by FAPESP, in which a direct quantification of doping agents in urine samples with no sample preparation by PS-MS was developed. Paper-spray mass spectrometry (PS-MS) is an ambient ionization method that combines the advantages of the paper platform with the reliability of MS analysis, reducing the consumption of time, reagent, and organic solvent. The use of illicit substances to improve enhance athletic performance in sport is forbidden and considered doping. The paper surface was treated with trichloromethylsilane (TCMS) using a gas-phase reaction to increase the target compounds ionization. This approach was applied for the analysis of two anabolic agents (trenbolone and clenbuterol) and two diuretics (furosemide and hydrochlorothiazide) in urine samples. Under optimized conditions, the developed methods presented satisfactory repeatability and a good correlation between concentration and absolute intensity. Highly sensitive detections as low as sub-ng mL 1 which is below the minimum required performance levels (MRPL) proposed by the WADA has been reached using the hydrophobic PS-MS without any pre-concentration and clean-up step. The proposed methods were applied in fortified urine samples with doping agents and showed to be fast, simple, sensitive, accurate and precise.
Descrição
Palavras-chave
Microfluídica, Fluorimetria, Nanopartículas, Espectrometria de massa, Química clínica, Carbon dot, Ionização ambiente, Agentes anabolizantes, Diuréticos, Glicose, Lactato, Dispositivos de papel, Imagem digital, Bionálises, Paper spray, Doping