Diagnóstico diferencial da proteína ns1 de Zika virus e Dengue vírus através do reconhecimento label-free com polímeros impressos molecularmente

Abstract

The similarity of symptoms developed after Zika virus and Dengue virus infections makes clinical diagnosis difficult. Furthermore, the structural similarity between these viruses makes it difficult to develop a differential and accurate diagnostic method. Nonstructural protein 1 (NS1) is a glycoprotein common to all flaviviruses, secreted in the serum of patients infected with Dengue virus at concentrations from 1x10-2 to 50 mg L-1. The artificial recognition of proteins by electropolymerized sensors has recently gained space in clinical diagnosis for providing fast, sensitive, selective, robust, non-destructive chemosensors, with a low attributed cost, while being portable and easy to handle. The present work proposed the development of chemosensors capable of detecting and differentiating the NS1 proteins of Zika virus (ZIKV) and Dengue virus type I (DENV-1). Based on structural information of linearity, flexibility, accessibility and immunogenicity, six epitopes were selected to be used as a template molecule for the production of molecularly printed polymers, two for DENV-1 and four for ZIKV. Films of phenol, aminophenyl boronic acid and 3- and 4-aminophenol were tested and studies were carried out to determine ideal conditions for polymerization. A molecularly printed film was polymerized on the surface of commercial carbon printed board electrodes using the cyclic voltammetry technique. Removal of the template molecule was performed by cleavage with proteinase K and cyclic voltammetry. Three sensor models were tested, two for NS1 from DENV-1 and one for NS1 from ZIKV. Furthermore, the influence of a previous temperature denaturation step to convert NS1 to its monomeric form before recognition was evaluated. Device performance was evaluated in terms of precision, sensitivity, selectivity and specificity. Sensors synthesized from 3-aminophenol were able to detect NS1 in diluted human serum samples (1:1000) at concentrations from 50 to 200 μg L-1. One of the tested models was able to distinguish NS1 from Dengue virus and Zika virus. This same prototype was able to detect the denatured protein in the range of 50 to 200 μg L-1 with a DPR below 5.04%. The calculated LOD and LOQ values were 29.3 and 88.7 μg L-1, respectively.