Roach Paul, Shirtcliffe Neil J, Farrar David, Perry Carole C
Interdisciplinary Biomedical Research Centre, School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton, Nottingham NG11 8NS, UK.
J Phys Chem B. 2006 Oct 19;110(41):20572-9. doi: 10.1021/jp0621575.
Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.
蛋白质吸附是一个备受广泛关注的重要领域，它对于从界面生物过程的基础理解到新材料的开发都具有重要意义。许多技术通常用于研究蛋白质粘附，但直接定量的技术却很少。在此，我们描述了使用荧光分析法Nano Orange来定量评估牛纤维蛋白原和白蛋白在亲水性（羟基封端）和疏水性（甲基封端）模型表面上的吸附情况。使用该方法获得的结果能够对先前通过石英晶体微天平测量和氨基黑蛋白质分析法在相同表面上获得的无法量化的数据进行校准。结果发现，两种蛋白质在疏水性表面上的吸附亲和力更高，但饱和水平更低。尽管荧光分析是唯一能够给出表面结合蛋白绝对量的方法，但在一系列蛋白质浓度范围内，所有三种分析技术在结合强度和吸附相对量方面均显示出相似的趋势。通过分析蛋白质在多孔超疏水和超亲水表面上的吸附情况，还探究了荧光分析法的通用性。结合这些表面使用该分析法获得的结果取决于表面化学性质。水渗入超亲水涂层为蛋白质吸附提供了更大的表面积，尽管蛋白质表面密度低于在类似的平坦亲水表面上发现的密度。超疏水表面可防止蛋白质溶液渗透。本文展示了荧光分析法在蛋白质吸附过程后用作其他技术的外部校准或作为研究蛋白质吸附的补充方法的潜力。突出了蛋白质在亲水与超亲水以及疏水与超疏水表面上吸附的差异。
