适体是越来越多地用于开发生物传感策略的识别元素,特别是在蛋白质或小分子靶标的检测中。溶菌酶,它被认为是各种疾病的重要生物标志物,也是蛋白中发现的主要过敏原蛋白,是基于适体的生物传感器的主要分析目标之一。然而,由于基于适体的策略可能容易出现伪影和数据误解,需要对适体-靶标相互作用进行多方面表征的严格策略。在这项工作中,已经设计了一种多技术方法来进一步了解文献中常用的抗溶菌酶DNA适体的结合性能。为了研究溶菌酶与不同抗溶菌酶DNA适配体之间的分子相互作用,基于磁电化学apta分析的测量,圆二色光谱,荧光光谱法,并进行了不对称流场-流动分馏。通过研究文献报道的SELEX选择的RNA适体,证明了该方法的可靠性和多功能性。作为一个积极的控制。结果证实,在所研究的结合缓冲液中存在低微摩尔范围的相互作用,并且该结合与蛋白质或DNA适体的构象变化无关。与随机序列和多聚胸腺嘧啶相比,抗溶菌酶DNA适体的行为相似,用作阴性对照,显示非序列特异性相互作用。这项研究表明,对SELEX选择产生的适体进行严格测试是在分析领域将这些生物识别元件推向可靠和可重复结果的独特方式。
Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To
study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This
study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.