Construction and application of paper-based analytical devices in the detection of biomarkers for clinical diagnosis

Author(s)

Glauco Pilon dos Santos

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2017-07-26

Examining board members

Lauro Tatsuo Kubota; João Marcos Madurro; Hideko Yamanaka; Celso Aparecido Bertran; Cátia Ornelas

• Advisor: Scott W Phillips; Lauro Tatsuo Kubota

Abstract

The growing demand for rapid, highly sensitive and cost effective diagnostic devices has stimulated considerable efforts towards the generation of simplified analytical systems capable of being applied in the field. This scientific work involved different approaches for the qualitative and quantitative detection of biomarkers for clinical diagnosis on paper-based analytical platforms. The first strategy consisted of understanding the principles behind the lateral flow immunoassay, in order to evaluate the critical steps and consequently to provide the most adequate experimental conditions for the early diagnosis of Plasmodium falciparum malaria by the identification of the HRP2 biomarker. The best performance of the device was achieved by using 0.05 µg (1 µL / 50 µg mL-1) of capture antibody and incubating for 5 minutes in order for it to adsorb; blocking the nitrocellulose with 1.5% (w/v) BSA containing 0.1% (v/v) surfactant Tween 20 by immersion and incubation for 10 minutes; 0.04 µg (20 µL/ 2 µg mL-1) of detection antibody labeled with the peroxidase enzyme; washing with 0.01 mol L-1 Tris-HCl buffer solution pH 7.4 containing 0.15 mol L-1 NaCl and 0.1% (v/v) Tween 20; addition of 5 µL of the chromogenic substrate TMB followed by reading in the range of 5-20 minutes after this addition. The colorimetric detection system presented a visual limit of detection of 5 ng mL-1 (135 pmol L-1), a value that should be enough for identifying the contamination by malaria in the first day of the appearance of the symptoms. The developed platform was then applied to blood samples from patients infected by the disease, demonstrating efficient performance at qualitatively discriminating a positive and a negative result, thus producing reliable results. In another study, aiming to provide a second generation of the device, a 3D paper-based detection system capable of being coupled to a lateral flow immunoassay platform was constructed. This system enables the obtainment of a quantitative and automated diagnostic assay with signal amplification by employing a new polymeric material from the class of poly(benzyl ethers) that selectively responds to hydrogen peroxide, besides requiring only the color visualization and a timer to obtain the data for subsequent analysis of the results. The delimitation of the hydrophobic barriers on the paper was carried out by wax printing and the orientation and type of paper to be employed in the layer containing the polymer were evaluated. The process of depositing the material on the surface of the paper demonstrated great repeatability when performed automatically by using a liquid handling robot. The highest sensitivity condition for the 3D detection system was achieved by employing 4.0 mg mL-1 of polymer, which allowed obtaining a limit of detection of 0.02 mmol L-1 of hydrogen peroxide. This system was then coupled to a lateral flow platform and employed in initial studies for the detection of creatine kinase MB isoenzyme, one of the biomarkers indicated for the diagnosis of acute myocardial infarction (AU)

• FAPESP's process: 13/09171-2 - Construction and application of a paper-based analytical device for the diagnosis of malaria caused by Plasmodium falciparum
• Grantee: Glauco Pilon dos Santos
• Support Opportunities: Scholarships in Brazil - Doctorate