National ESCA and Surface Analysis Center for Biomedical Problems and Department of Chemical Engineering, University of Washington, Seattle, Washington 98195-1750, USA.
Biointerphases. 2007 Jun;2(2):64-72. doi: 10.1116/1.2738854.
The interactions of fibronectin (Fn) with group B streptococci (GBS) were investigated using the atomic force microscope (AFM) and surface plasmon resonance (SPR) biosensing. Submonolayer amounts of Fn were immobilized onto the AFM tip by two different methods, using either a sulfosuccinimidyl-4-(N-maleimidomethyl) cycholhexane-1-carboxylate (SMCC) linker or a pyridyldithio poly(ethylene glycol) succinimidylpropionate (NHS-PEG-PDP) linker. Each step of both immobilization methods was characterized using x-ray photoelectron spectroscopy. Time-of-flight secondary ion mass spectrometry experiments indicated both methods produced Fn immobilized in a similar conformation. AFM force-distance curves from live GBS plated onto polystyrene exhibited several types of interactions between the Fn functionalized AFM tip and the surface of capsule-deficient GBS (no interactions, interactions with the cell wall, Fn unfolding, large specific unbinding events, and small specific unbinding events). From analysis of the force-distance curves that exhibited only a single specific unbinding event, the work of adhesion and rupture force for the SMCC immobilized Fn tips (11,131 pN nm and 213 pN) were larger than the corresponding values for the NHS-PEG-PDP immobilized Fn tips (8115 pN nm and 189 pN). The unbinding event occurred at distances approximately 100 nm further from the surface with the NHS-PEG-PDP immobilized Fn tip compared to SMCC immobilized Fn tip. The SPR experiments of soluble Fn with adsorbed serine protease C5a peptidase (Scp), the surface protein on GBS that binds Fn, showed that both low (millimolar) and high binding (nanomolar) affinity interactions were present. However, the low binding affinity interactions dominated the adsorption process and, with increasing Fn solution concentration, the amount of Scp bound to Fn via the high binding affinity interaction decreased. These data confirm that Scp binds only to adsorbed Fn at the Fn concentrations typically present in blood plasma.
使用原子力显微镜（AFM）和表面等离子体共振（SPR）生物传感技术研究了纤连蛋白（Fn）与 B 组链球菌（GBS）的相互作用。通过两种不同的方法将亚单层量的 Fn 固定在 AFM 尖端上，一种方法使用磺基琥珀酰亚胺-4-（N-马来酰亚胺基甲基）环己烷-1-羧酸盐（SMCC）接头，另一种方法使用吡啶基二硫代聚乙二醇琥珀酰亚胺基丙酸酯（NHS-PEG-PDP）接头。两种固定化方法的每一步都使用 X 射线光电子能谱进行了表征。飞行时间二次离子质谱实验表明，这两种方法都使 Fn 以相似的构象固定。将活 GBS 接种到聚苯乙烯上的 AFM 力-距离曲线显示，Fn 功能化 AFM 尖端与无荚膜 GBS 表面之间存在几种类型的相互作用（无相互作用、与细胞壁相互作用、Fn 展开、大特异性非结合事件和小特异性非结合事件）。从仅显示单个特异性非结合事件的力-距离曲线分析中，SMCC 固定 Fn 尖端的粘附功和断裂力（11131 pNnm 和 213 pN）大于 NHS-PEG-PDP 固定 Fn 尖端的相应值（8115 pNnm 和 189 pN）。与 SMCC 固定 Fn 尖端相比，NHS-PEG-PDP 固定 Fn 尖端的非结合事件发生在距离表面约 100nm 处。与吸附在 GBS 表面蛋白丝氨酸蛋白酶 C5a 肽酶（Scp）上的可溶性 Fn 的 SPR 实验表明，存在低（毫摩尔）和高（纳摩尔）亲和力相互作用。然而，低结合亲和力相互作用主导了吸附过程，并且随着 Fn 溶液浓度的增加，通过高结合亲和力相互作用与 Fn 结合的 Scp 量减少。这些数据证实 Scp 仅在通常存在于血浆中的 Fn 浓度下与吸附的 Fn 结合。
