Mund-Lagowski Department of Chemistry and Biochemistry, Bradley University, 1501 West Bradley Avenue, Peoria, Illinois 61625, USA.
Appl Spectrosc. 2013 Jun;67(6):692-8. doi: 10.1366/12-06848.
The preparation of glass cell surfaces that are chemically functionalized with poly(ethylene glycol) (PEG) chains to reduce sample adsorption and their use in fluorescence correlation spectroscopy (FCS) is described. Optical glass coverslips were acid etched and reacted with either 750 Mr PEG (PEG-750) or 5000 Mr PEG (PEG-5000) to produce adsorption-resistant optical surfaces. FCS data for Nile red-loaded Triton X-100 micelles (NR-TX-100) and Alexa Fluor 555-labeled proteins, bovine serum albumin (BSA-A555), lipidized BSA (lipid-BSA-A555), and three low molecular weight dyes deposited on PEGylated coverslips were evaluated. Measurement artifacts due to sample adsorption on the PEG-5000 functionalized coverslips were reduced significantly for the majority of test materials. Calculations of translational diffusion coefficients and Stokes radii confirmed the effectiveness of this approach. PEG-5000 functionalized coverslips were demonstrated as more effective in inhibiting adsorption than PEG-750 functionalized coverslips. Neither of the functionalized coverslips inhibited the adsorption of one test compound, rhodamine B, a dye that adsorbs strongly on glass surfaces. The use of longer PEG chains in conjunction with chemical cross-linking is proposed for producing a denser, less porous PEG layer for the prevention of strongly glass-adsorbing fluorophores that do not interact with the PEG layer.
描述了一种用聚乙二醇（PEG）链化学功能化玻璃细胞表面以减少样品吸附的方法，并将其用于荧光相关光谱（FCS）。对光学玻璃盖玻片进行酸蚀刻，并与 750 Mr PEG（PEG-750）或 5000 Mr PEG（PEG-5000）反应，以产生抗吸附的光学表面。评估了尼罗红负载的 Triton X-100 胶束（NR-TX-100）和 Alexa Fluor 555 标记的蛋白质、牛血清白蛋白（BSA-A555）、脂质化 BSA（lipid-BSA-A555）以及三种沉积在 PEG 化盖玻片上的低分子量染料的 FCS 数据。对于大多数测试材料，由于样品在 PEG-5000 功能化盖玻片上的吸附而导致的测量伪影显著减少。扩散系数和斯托克斯半径的计算证实了这种方法的有效性。PEG-5000 功能化盖玻片在抑制吸附方面比 PEG-750 功能化盖玻片更有效。两种功能化盖玻片都不能抑制一种测试化合物罗丹明 B 的吸附，罗丹明 B 是一种在玻璃表面强烈吸附的染料。建议使用更长的 PEG 链结合化学交联，以生成更致密、更不易渗透的 PEG 层，以防止与 PEG 层不相互作用但强烈吸附玻璃的荧光染料。
