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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >Nano-Affi: a solution-phase, label-free, colorimetric aptamer affinity assay based on binding-inhibited aggregation of gold nanoparticles
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Nano-Affi: a solution-phase, label-free, colorimetric aptamer affinity assay based on binding-inhibited aggregation of gold nanoparticles

机译：纳米AFFI：基于结合抑制的金纳米粒子聚集的溶液相，无标记，比色体互相分析

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摘要

The ideal way to assess aptamer affinity is when both aptamer and target are in a native state, without the unpredictable interference associated with labelling and surface immobilization. However, most current aptamer affinity assays need aptamer (or target) immobilization on surface and/or labelling. Ideally, such a solution-phase assay should also be high-throughput, in order to accelerate aptamer identification, binding site study, and engineering for various downstream applications. So far, only isothermal titration calorimetry (ITC) enables label-free solution-phase affinity measurements, but with low-throughput and the need of large amount of samples. Here, we report a solution-phase, label-free, colorimetric gold nanoparticle (AuNP)-based affinity assay (Nano-Affi) that addresses this need. Nano-Affi is based on kinetically- favoured, adsorbate charge-tuned aggregation of AuNPs, wherein positively-charged or nearneutral proteins induce instantaneous aggregation of negatively-charged AuNPs at the pH below or near the isoelectric point of the target protein. In contrast, protein-aptamer complexes possess a greater negative charge than free targets, and thus induce little or no aggregation of AuNPs due to electrostatic repulsion. The higher an aptamer's affinity for the protein, the less AuNP aggregation occurs. We demonstrate here that Nano-Affi enables the reliable aptamer screening and dissociation constant determination for diverse protein targets, as well as binding site identification, with readouts based on colour observation or absorbance or dynamic light scattering size measurements. Nano-Affi possesses sub-nanomolar sensitivity and can be performed with nanogram amounts of protein in less than half an hour with minimal training and minimal instrument requirements.
机译：评估Aptamer亲和力的理想方式是当适体和靶标处于原生状态时，而没有与标记和表面固定相关的不可预测的干扰。然而，大多数电流适体亲和力测定需要在表面和/或标记上进行适体（或靶）固定化。理想地，这种溶液相测定也应该是高通量，以便加速适体鉴定，结合位点研究和工程，以获得各种下游应用。到目前为止，只有等温滴定量热法（ITC）使无标记的溶液相位亲和力测量，但具有低通量和大量样品的需要。在这里，我们报告了解决了这种需求的解决方案，无标记的比色金纳米粒子（AUNP）的基础和纳米AFFI）。纳米Affi基于动力学，吸附的AUNPS充气聚集，其中带正电荷或接近的蛋白质诱导靶蛋白的等电点在pH下或附近的pH下的带负电荷的aUnps的瞬时聚集。相反，蛋白质 - 适体络合物比游离靶具有更大的负电荷，因此由于静电排斥而诱导肛周的少量或没有聚集。适于对蛋白质的Aptamer的亲和力越高，发生较少的AUNP聚集。我们在此证明，纳米AFFI使得可靠的适体筛选和解离常数确定不同蛋白质靶标，以及基于颜色观察或吸光度或动态光散射尺寸测量的读出的结合位点识别。纳米Affi具有亚纳摩尔敏感性，并且可以在不到半小时的培训和最小仪器要求中以不到半小时的百分之一的蛋白质进行。

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