Proof of concept

Overview

The aim of our experiments was to design a more efficient and effective serological detection system for Dengue virus (DENV). The design was based on Ammit, a chimeric protein with multiple DENV epitopes that would be recognized by antibodies present in an infected patient´s serum and a biochip that can recognize this interaction. Our biochip was based on electrochemical biosensors that can detect antigen-antibody binding. To prove that our system is viable, we compared our biochip detection to the enzyme-linked immunosorbent assay (ELISA), which is a conventional method for serological diagnosis.

Ammit and detection

Ammit refers to the chimeric protein created by the team, made of specific DENV epitopes and will be used as a probe antigen in serological diagnostic tests for this disease. Thus, the detection systems that we are proposing, will be responsible for reporting the binding of anti-dengue antibodies present in a patient's serum to Ammit.

ELISA

The immunoenzymatic ELISA assay is a method that permits antibody binding to an antigen bound to a solid surface. Upon binding the antibody is fixed and it can be detected directly or indirectly by the use of labeled primary or secondary antibodies, respectively. The label consists commonly of enzymes that can be detected easily either colorimetrically or by fluorescence, such as peroxidase and alkaline phosphatase. In this project we used the indirect assay in which the secondary antibody was labeled with horseradish peroxidase. The detection was based on the color change of a chromogenic peroxidase substrate , o-Phenylenediamine(OPD). Given this, to perform the indirect ELISA technique we proposed the use of our protein AMMIT as an input, immobilized on a plate, which can capture the immunoglobulins IgM or IgG present in the serum of patients, and a secondary antibody bound to HRP.

We performed an Elisa test with DME-C and DME-BR proteins to assess their interaction with Dengue and Zika antibodies. Therefore, different protein dilutions were used. For this purpose, the primary antibodies Anti-Dengue Protein E or anti-Zika NS1 at a 1:1000 dilution were used. The secondary antibody was Anti-Mouse monoclonal antibody at a 1:10,000 dilution. According to the results DME-C and DME-BR had interaction with both antibodies, both with anti-Dengue and anti-Zika. However, a possible explanation for this is that since it is anti-Zika NS1, the protein could probably contain NS1 epitopes that helped the antibody interaction, or that the anti-Zika NS1 antibody is not specific for Zika, but also for Dengue.

Biochip application

Ammit's idealization proves to be viable for the construction of electrochemical biosensors, which can be integrated into a service device, with the premise of being more sensitive and specific than the lateral flow serological tests. We manufactured printed carbon electrodes (SPCE) modified with gold nanoparticles, which could serve as a surface for immobilizing our multi-epitope protein. Then an indirect ELISA test could be carried out using peroxidase as a label in secondary antibodies. The peroxidase would catalyze an oxidation-reduction reaction of hydrogen peroxide and thus produce an electrochemical signal. This in turn could be read by cyclic voltammetry and interpreted by a reader, who will indicate the presence or absence of the target. (For full protocol saber, go to Experiments)

Cyclic Voltammetry Experiment

To check the feasibility of the biochip for establishing the detection system, the electrode was tested using the potential scanning method based on cyclic voltammetry. This electrochemical technique enables the measurement of high selectivity and sensitivity, through the signal alteration caused by analytes, during the oxidation-reduction process of chemical species on the electrode surface. It is based on the dynamic voltage/current relationship over time that arises in a cell with three electrodes: working electrode, reference electrode, and auxiliary electrode which are ideal for the system developed in the biochip. While nanoparticles increase the electroactive area of the electrodes and favor oxidation-reduction reactions, exhibiting a greater potential during cyclic voltammetry. Our protein, on the other hand, causes a reduction in the electroactive area, disfavoring oxidation-reduction reactions. As a result, the working electrode emits a reduced potential when compared to the pure carbon electrode.

Conclusion