Favorably orienting recombinant proteins to develop amperometric biosensors to diagnose Chagas' disease.

Clinical immunoassays often display suitable sensitivity but some lack of specificity or vice versa. As a trade-off between specificity improvement and sensitivity loss, biosensors were designed to perform indirect immunoassays with amperometric detection using tailor-made chimeric receptors to react with the analyte, specific anti-Trypanosoma cruzi immunoglobulin G (IgG). Recombinant chimeras were designed to favor their oriented covalent attachment. This allows the chimeras to properly expose their epitopes, to efficiently capture the analyte, and to withstand severe chemical treatment to reuse the biosensors. By further binding the secondary antibody, horseradish peroxidase-labeled anti-human IgG, in the presence of the soluble mediator and the enzyme substrate, a current that increased with the analyte concentration was measured. Biosensors using the chimeric constructions showed 100% specificity with samples that had revealed false-positive results when using other bioreceptors. A protein bearing a poly-Lys chain and thioredoxin as directing elements displayed the highest signal-to-noise ratio (P<0.05). The limit of detection was 62 ng ml⁻¹, which is eight times lower than that obtained with a currently used commercial Chagas enzyme-linked immunosorbent assay (ELISA) kit. Reusability of the biosensor was assessed. The signal was approximately 80% of the original one after performing 10 consecutive determinations.

Assembling Amperometric Biosensors for Clinical Diagnostics.

Clinical diagnosis and disease prevention routinely require the assessment ofspecies determined by chemical analysis. Biosensor technology offers several benefits overconventional diagnostic analysis. They include simplicity of use, specificity for the targetanalyte, speed to arise to a result, capability for continuous monitoring and multiplexing,together with the potentiality of coupling to low-cost, portable instrumentation. This workfocuses on the basic lines of decisions when designing electron-transfer-based biosensorsfor clinical analysis, with emphasis on the strategies currently used to improve the deviceperformance, the present status of amperometric electrodes for biomedicine, and the trendsand challenges envisaged for the near future.