Palermo Andrea, Thelen Richard, Weber Laura K, Foertsch Tobias, Rentschler Simone, Hackert Verena, Syurik Julia, Nesterov-Mueller Alexander
Institute of Microstructure Technology, Karlsruhe Institute of Technology (KIT), 76131 Baden-Württemberg, Germany.
High Throughput. 2019 Mar 27;8(2):7. doi: 10.3390/ht8020007.
Peptide microarrays are a fast-developing field enabling the mapping of linear epitopes in the immune response to vaccinations or diseases and high throughput studying of protein-protein interactions. In this respect, a rapid label-free measurement of protein layer topographies in the array format is of great interest but is also a great challenge due to the extremely low aspect ratios of the peptide spots. We have demonstrated the potential of vertical scanning interferometry (VSI) for a detailed morphological analysis of peptide arrays and binding antibodies. The VSI technique is shown to scan an array area of 5.1 square millimeters within 3⁻4 min at a resolution of 1.4 μm lateral and 0.1 nm vertical in the full automation mode. Topographies obtained by VSI do match the one obtained by AFM measurements, demonstrating the accuracy of the technique. A detailed topology of peptide-antibody layers on single spots was measured. Two different measurement regions are distinguished according to the antibody concentration. In the case of weakly diluted serum, the thickness of the antibody layer is independent of the serum dilution and corresponds to the physical thickness of the accumulated antibody layer. In strongly diluted serum, the thickness measured via VSI is linearly proportional to the serum dilution.
肽微阵列是一个快速发展的领域，能够绘制针对疫苗接种或疾病的免疫反应中的线性表位图谱，并对蛋白质-蛋白质相互作用进行高通量研究。在这方面，以阵列形式对蛋白质层拓扑结构进行快速无标记测量非常有意义，但由于肽点的纵横比极低，这也是一个巨大的挑战。我们已经证明了垂直扫描干涉术（VSI）在肽阵列和结合抗体的详细形态分析方面的潜力。VSI技术在全自动模式下，能在3至4分钟内扫描5.1平方毫米的阵列区域，横向分辨率为1.4μm，纵向分辨率为0.1nm。VSI获得的拓扑结构与原子力显微镜（AFM）测量获得的拓扑结构相匹配，证明了该技术的准确性。测量了单个斑点上肽-抗体层的详细拓扑结构。根据抗体浓度区分出两个不同的测量区域。在血清稀释度较低的情况下，抗体层的厚度与血清稀释度无关，相当于积累的抗体层的物理厚度。在血清高度稀释的情况下，通过VSI测量的厚度与血清稀释度呈线性比例关系。
