Favorably orienting recombinant proteins to develop amperometric biosensors to diagnose Chagas' disease
| Autor: | María Soledad Belluzo, María E. Ribone, Cecilia María Camussone, Iván Sergio Marcipar, Claudia M. Lagier |
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| Rok vydání: | 2010 |
| Předmět: |
Analyte
Trypanosoma cruzi Molecular Sequence Data Biophysics Antibodies Protozoan Antigens Protozoan Biosensing Techniques Biochemistry Immunoglobulin G Epitope law.invention law Humans Chagas Disease Amino Acid Sequence Molecular Biology Electrodes Immunoassay biology Cell Biology Electrochemical Techniques Primary and secondary antibodies Fusion protein Molecular biology Recombinant Proteins biology.protein Recombinant DNA Thioredoxin Biosensor |
| Zdroj: | Analytical biochemistry. 408(1) |
| ISSN: | 1096-0309 |
| Popis: | 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 (P0.05). 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. |
| Databáze: | OpenAIRE |
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