Department of Chemistry, National Core Research Center for Systems Bio-Dynamics, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea.
Anal Chem. 2010 Jun 15;82(12):5189-94. doi: 10.1021/ac100476b.
The mapping capability of atomic force microscopy (AFM) enabled us to see captured prostate-specific antigens (PSAs) on a spot microarrayed with the corresponding antibody and count the number of the antigens in a submicrometer area. To enhance the reliability and the reproducibility of the approach, a third-generation dendron was employed for the surface treatment. The specific force between the captured PSA and the detection antibody (5A6) was measured after cross-linking, and the mean unbinding force was 56 +/- 2 pN. At 100 fM, there were 12 captured antigens in 4.32 x 10(4) nm(2), and the number was dependent upon the concentration. A larger hydrodynamic distance (8 +/- 2 nm) of the immunocomplex resulted in a cluster of pixels corresponding to the single complex in a map recorded over a selected area with a positional interval of 3 nm, and this feature helped to discriminate between pixels of the specific interaction and the nonspecific ones. The results indicate that the approach can be applicable to the quantitative analysis of the antigen in a sample and imply that it can be extended to a sample of very low copy numbers as long as the size of the microarrayed spot is reduced.
原子力显微镜（AFM）的成像能力使我们能够在带有相应抗体的点微阵列上看到捕获的前列腺特异性抗原（PSA），并计算亚微米区域内的抗原数量。为了提高方法的可靠性和重现性，使用第三代树枝状大分子进行表面处理。交联后测量捕获的 PSA 和检测抗体（5A6）之间的特定力，平均解缚力为 56 ± 2 pN。在 100 fM 时，在 4.32 x 10(4) nm(2)中捕获了 12 个抗原，并且数量取决于浓度。免疫复合物的较大流体动力距离（8 ± 2 nm）导致在记录所选区域的映射中，对应于单个复合物的像素簇，其位置间隔为 3 nm，该特征有助于区分特异性相互作用和非特异性像素之间的区别。结果表明，该方法可适用于样品中抗原的定量分析，并表明只要微阵列点的尺寸减小，它就可以扩展到拷贝数非常低的样品。
