The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The betterperforming Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula ؉OD/cutoff value (where ؉OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

Clinical diagnosis and disease prevention routinely require the assessment of species determined by chemical analysis. Biosensor technology offers several benefits over conventional diagnostic analysis. They include simplicity of use, specificity for the target analyte, speed to arise to a result, capability for continuous monitoring and multiplexing, together with the potentiality of coupling to low-cost, portable instrumentation. This work focuses on the basic lines of decisions when designing electron-transfer-based biosensors for clinical analysis, with emphasis on the strategies currently used to improve the device performance, the present status of amperometric electrodes for biomedicine, and the trends and challenges envisaged for the near future.

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