Departamento de Química Analítica, Universidad Nacional de Rosario, Argentina.
Anal Biochem. 2011 Jan 1;408(1):86-94. doi: 10.1016/j.ab.2010.09.002. Epub 2010 Sep 7.
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.
临床免疫分析通常具有适当的灵敏度，但特异性不足或反之亦然。作为特异性提高和灵敏度降低之间的权衡，生物传感器被设计为使用定制的嵌合受体进行间接免疫分析，以进行安培检测，以与分析物（特异性抗克氏锥虫免疫球蛋白 G (IgG)）反应。设计重组嵌合体以有利于其定向共价附着。这允许嵌合体适当地暴露其表位，有效地捕获分析物，并承受苛刻的化学处理以重复使用生物传感器。通过进一步结合次级抗体，辣根过氧化物酶标记的抗人 IgG，在可溶性介质和酶底物的存在下，测量与分析物浓度成正比的电流。使用嵌合结构的生物传感器对样品具有 100%的特异性，而使用其他生物受体时，这些样品会出现假阳性结果。一种带有聚-Lys 链和硫氧还蛋白作为导向元件的蛋白质显示出最高的信噪比（P<0.05）。检测限为 62ngml⁻¹，比目前使用的商业克氏锥虫酶联免疫吸附测定（ELISA）试剂盒低 8 倍。评估了生物传感器的可重复使用性。进行了 10 次连续测定后，信号约为原始信号的 80%。
