Study of dendrimer immobilization in the construction of a SPR-based immunosensor for detecting troponin T in early infarct diagnoses

Author(s)

Juliana de Fátima Giarola

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2020-03-26

Examining board members

Lauro Tatsuo Kubota; Maria Valnice Boldrin; Lucio Angnes; Denize Cristina Favaro; William Reis de Araujo

• Advisor: Lauro Tatsuo Kubota

Abstract

Cardiovascular diseases are the number one cause of death in the world, 85% of them due to strokes and acute myocardial infarctions (AMI). AMI affects people of all ages, and accurate diagnosis and appropriate medical treatments are critical to increasing the survival rate. This doctoral work involved the study of the immobilization of different generations of PAMAM dendrimers (G3, G4, and G5) in the construction of an immunosensor based on surface plasmon resonance (SPR) aiming at the diagnosis of early AMI using troponin T biomarker. The best performance of the immunosensor was obtained using the 0.1mmol L¡1 PAMAM(G4) dendrimers, combined with the self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA - 1.0 mmol L¡1 ). The capture antibodies (20 ¹g mL¡1 ) were immobilized over dendrimers, and the non-specific sites in immunosensor were blocked with BSA (30 ¹g mL¡1 ) and ethanolamine (1.0 mol L¡1 pH 8.5) for 5 minutes. The value of KD estimated (8.3 x 10¡7 mol L¡1 ) suggests a high affinity between the capture antibody and the biomarker, favoring higher detectability. Repeatability studies indicated a good precision of the immunosensor developed with a relative standard deviation of less than 10%. The regeneration of the capture antibody binding sites was performed using 0.01 mol L¡1 pH 4.8 sodium acetate buffer with 0.1% Tween 20, allowing the reuse of the immunosensor for one more time. The most promising results for the proposed immunosensor were obtained using the "sandwich"assay, employing two types of secondary antibodies: monoclonal, with a response range of 5.0 ¡ 300 ng mL¡1 and LOD of 3.6 ng mL¡1 and polyclonal, with a response range of 3.0 ¡ 30 ng mL¡1 and LOD of 0.98 ng mL¡1 , both with a response time of approximately 30 minutes. These LOD values are very close to the values found in the bloodstream of patients with initial AMI, and the immunosensor can be used for this diagnosis. The immunosensor application was performed in human serum samples from healthy patients, by recovering troponin T added to the matrix (88% to 104%), indicating reliable results at this determination. Another application was carried out on a sample of patients with an initial AMI condition, in which the recovered value by the immunosensor agrees with the electrochemiluminescence immunoassay method (ECLIA). Thus, the results obtained in this thesis work are substantial for the contribution in the rapid diagnosis of early AMI, as well as in the development of new methodologies sensitive to troponin T with reduced costs (AU)

• FAPESP's process: 16/08166-3 - Development of an electrochemical immunosensor based on lateral flow for cardiac markers detection
• Grantee: Juliana de Fátima Giarola
• Support Opportunities: Scholarships in Brazil - Doctorate