Wang B, Lou Z, Park B, Kwon Y, Zhang H, Xu B
Single Molecule Study Laboratory, Faculty of Engineering and Nanoscale Science and Engineering Center, University of Georgia, Athens, GA 30602, USA.
Phys Chem Chem Phys. 2015 Jan 7;17(1):307-14. doi: 10.1039/c4cp03190c.
We used atomic force microscopy (AFM) and surface plasmon resonance (SPR) to study the surface conformations of an anti-ricin aptamer and its specific binding affinity for ricin molecules. The effect of surface modification of the Au(111) substrate on the aptamer affinity was also estimated. The AFM topography images had a resolution high enough to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The aptamer on a Au(111) surface modified with carboxymethylated-dextran (CD) showed both similarities to and differences from the one without CD modification. The influence of CD modification was evaluated using AFM images of various aptamer conformations on the Au(111) surface. The affinity between ricin and the anti-ricin aptamer was estimated using the off-rate values measured using AFM and SPR. The SPR measurements of the ricin sample were conducted in the range from 83.3 pM to 8.33 nM, and the limit of detection was estimated as 25 pM (1.5 ng mL(-1)). The off-rate values of the ricin-aptamer interactions were estimated using both single-molecule dynamic force spectroscopy (DFS) and SPR as (7.3 ± 0.4) × 10(-4) s(-1) and (1.82 ± 0.067) × 10(-2) s(-1), respectively. The results show that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single-molecule measurements, the various conformations of the aptamer immobilized on the gold surface determined the availability of each specific binding site to the ricin molecules. The SPR bulk solution measurements averaged the signals from specific and non-specific interactions. AFM images and DFS measurements provide more specific information on the interactions of individual aptamer and ricin molecules.
我们使用原子力显微镜（AFM）和表面等离子体共振（SPR）来研究抗蓖麻毒素适体的表面构象及其与蓖麻毒素分子的特异性结合亲和力。还评估了Au(111)基底的表面修饰对适体亲和力的影响。AFM形貌图像具有足够高的分辨率，能够区分不同的适体构象。通过AFM识别图像可以清楚地定位适体分子上的特异性结合位点。在经羧甲基化葡聚糖（CD）修饰的Au(111)表面上的适体与未修饰CD的适体既有相似之处，也有不同之处。利用Au(111)表面上各种适体构象的AFM图像评估了CD修饰的影响。利用AFM和SPR测量的解离速率值估算了蓖麻毒素与抗蓖麻毒素适体之间的亲和力。对蓖麻毒素样品的SPR测量在83.3 pM至8.33 nM范围内进行，检测限估计为25 pM（1.5 ng mL(-1)）。分别使用单分子动态力谱（DFS）和SPR估算蓖麻毒素 - 适体相互作用的解离速率值为(7.3 ± 0.4) × 10(-4) s(-1)和(1.82 ± 0.067) × 10(-2) s(-1)。结果表明，单分子测量能够从本体溶液测量中获得不同的反应参数。在AFM单分子测量中，固定在金表面的适体的各种构象决定了每个特异性结合位点与蓖麻毒素分子结合的可用性。SPR本体溶液测量对特异性和非特异性相互作用的信号进行了平均。AFM图像和DFS测量提供了关于单个适体和蓖麻毒素分子相互作用的更具体信息。
