A simple, sensitive and reduced cost paper-based device with low quantity of chemicals for the early diagnosis of Plasmodium falciparum malaria using an enzyme-based colorimetric assay

摘要

In this study, we report the development of a paper-based platform with low amount of reagents for the detection of the histidine-rich protein 2 by an enzyme-based colorimetric assay aiming for the early diagnosis of Plasmodium falciparum malaria. A CO_2 laser cutter was used for the fabrication of the device in a simple layout suitable to perform the lateral flow assay, which consisted in a region for sample addition, a detection zone and an absorbent pad, all of which were linked by microfluidic channels. Investigations related to the performance of the sandwich immunoassay in the nitrocellulose membrane shown that the blocking and washing steps are essential to promote efficient antigen-antibody recognition and to generate reliable results, avoiding misinterpretations. Several parameters were also evaluated and the experimental conditions were optimized: concentration and incubation time with the capture antibody (50 µg mL~(-1) and 5min, respectively); BSA solution at 1.5% (w/v) containing 0.1% (v/v) Tween 20 for blocking the nitrocellulose and 10min as the incubation time; 10mmol L~(-1) Tris-HCl buffer solution at pH 7.4 containing 0.15 mol L~(-1) NaCl and 0.1% (v/v) Tween 20 for washing the device before adding the ready to use TMB substrate at the detection zone. The system demonstrated a good sensitivity and detectability reaching a visual and calculated limit of detection of 5.0 ng mL~(-1) (65 parasites µL~(-1)) and 4.5 ng mL~(-1) (59 parasites µL~(-1)) respectively, a clinically relevant concentration that should be sufficient to identify the disease in the appearance of the first symptoms and with the advantage of not needing to use nanostructures or other strategy for signal amplification beyond the enzyme.