Sandia National Laboratories, Livermore, California 94551, United States, University of Michigan, Ann Arbor, Michigan 48109, United States, and AM Biotechnologies, LLC, Houston, Texas 77034, United States.
Anal Chem. 2010 Nov 1;82(21):8813-20. doi: 10.1021/ac101106m. Epub 2010 Oct 14.
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-κB using a thio-modified 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.
核酸基亲和试剂（例如，适体）作为生化分析中的特异性识别元件，与抗体相比具有几个潜在优势。除了提供更高的成本效益和稳定性外，适体还非常适合快速电泳分析，因为它们分子量小且带负电荷高。虽然适体已被证明非常适合亲和移位电泳分析，但展示完全集成的基于适体的分析平台是实现低成本即时检测分析的重要成就，特别是在偏远或资源匮乏的环境中，试剂的成本和环境稳定性是关键考虑因素。在这里，我们在集成电泳凝胶迁移分析平台中对两种临床相关靶分析物（使用已知适体的 IgE 和使用硫代修饰适体的 NF-κB）的基于适体的亲和分析进行了评估。（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成功地在基于荧光的凝胶迁移分析步骤之前在芯片上进行了集成。这种方法利用这里优化的用于适体保留和样品预浓缩的排阻膜，能够在 10 分钟或更短的时间内实现痕量分析物的自动化从样品到答案。我们通过添加类似的核酸竞争物来抑制与适体的相互作用，从而解决了来自血清蛋白的显著非特异性干扰问题。证明了纳摩尔灵敏度，并且样品的集成预浓缩提供了进一步提高检测灵敏度的重要手段。在许多方面，适体都优于抗体试剂，尤其是在与靶标特异性结合相关的凝胶迁移的速度和分辨率方面。
