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Aptamers as Affinity Reagents in an Integrated Electrophoretic Lab-on-a-Chip Platform

机译:在集成的电泳芯片实验室平台中作为亲和试剂的适体。

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Nucleic acid based affinity reagents (e.g., aptamers) offer several possible advantages over antibodies as specific recognition elements in biochemical assays. Besides offering improved cost and stability, aptamers are ideal for rapid electrophoretic analysis due to their low molecular weight and high negative charge. While aptamers have proven well-suited for affinity-shift electrophoretic analysis, demonstrating a fully integrated aptamer-based assay platform represents an important achievement toward low-cost point-of-care analysis, particularly for remote or resource poor settings where cost and ambient stability of reagents is a key consideration. Here we perform and evaluate the suitability of aptamer-based affinity assays for two clinically relevant target analytes (IgE using a known aptamer and NF-(kappa)B using a thiomodified aptamer) in an integrated electrophoretic gel-shift platform. Key steps of (i) mixing sample with aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated on-chip upstream of a fluorescence-based gel-shift analysis step. This approach, utilizing a size-exclusion membrane optimized here for aptamer retention and preconcentration with sample, enables automated sample-to-answer for trace analytes in 10 min or less. We addressed notable nonspecific interference from serum proteins by adding similar nucleic acid competitors to suppress such interactions with the aptamer. Nanomolar sensitivities were demonstrated and integrated preconcentration of sample provides an important means of further improving detection sensitivities. Aptamers proved superior in many respects to antibody reagents, particularly with regard to speed and resolution of gel-shifts associated with specific binding to target.
机译:在生物化学测定中,基于核酸的亲和试剂(例如适体)相对于作为特异性识别元件的抗体具有多个可能的优点。除了提供改进的成本和稳定性之外,由于适体分子量低且负电荷高,因此非常适合用于快速电泳分析。尽管适体已被证明非常适合亲和移位电泳分析,但展示出完全集成的基于适体的测定平台代表了低成本现场护理分析的重要成就,特别是对于成本和环境稳定性较差的偏远或资源匮乏的环境试剂的数量是关键考虑因素。在这里,我们在集成的电泳凝胶移位平台中,对两种临床相关的目标分析物(使用已知的适体的IgE和使用硫代修饰的适体的NF-κB)进行基于适体的亲和力测定并评估其适用性。 (i)将样品与适体混合,(ii)缓冲液交换和(iii)样品预浓缩的关键步骤已成功整合到基于荧光的凝胶位移分析步骤的芯片上游。这种方法利用此处优化的尺寸排阻膜进行适体保留和样品预富集,可以在10分钟或更短的时间内自动完成痕量分析物的样品到样品的应答。我们通过添加相似的核酸竞争物来抑制与适体的这种相互作用,从而解决了血清蛋白引起的非特异性干扰。证明了纳摩尔灵敏度,样品的预浓缩对进一步提高检测灵敏度提供了重要的手段。适体被证明在许多方面优于抗体试剂,特别是在与特异性结合靶标相关的凝胶移位的速度和分辨率方面。

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