A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces.
A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces.
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DOI：
10.1038/s41598-022-14948-7
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发表时间：
2022-06-29
影响因子：
4.6
通讯作者：
Richter, Ralf P.
中科院分区：
文献类型：
--
作者：
Srimasorn, Sumitra;Souter, Luke;Green, Dixy E.;Djerbal, Lynda;Goodenough, Ashleigh;Duncan, James A.;Roberts, Abigail R. E.;Zhang, Xiaoli;Debarre, Delphine;DeAngelis, Paul L.;Kwok, Jessica C. F.;Richter, Ralf P.
透明质酸（HA）是细胞周围和细胞外基质的主要成分，在细胞粘附、增殖和迁移等许多生物学过程中发挥着重要作用。HA的丰度、大小分布和呈现决定了其生物学效应，并且也是病理学和疾病进展的有用指标。评估自由漂浮HA和其他糖胺聚糖（GAG）分子量的方法已得到充分确立。然而，在许多生物和技术环境中，GAG展示在表面上，并且缺乏获得表面附着的GAG的大小的方法。在这里，我们提出了一种方法来确定HA的大小，HA是端连接到表面。该方法是基于石英晶体微天平与耗散监测（QCM-D），并利用接枝HA的膜的柔软度和厚度的增加与HA的大小。这两个量灵敏地反映在形成HA膜时QCM-D测量的耗散位移（ΔD）和负频移（-Δf）的比率上。使用一系列大小确定的HA制备物，大小范围从~ 2 kDa四糖到~ 1 MDa多糖，我们建立了作为HA大小函数的ΔD/ − Δf比率的单调但非线性的标准曲线，这反映了接枝HA链根据其大小和表面覆盖度所采用的不同构象。我们证明，标准曲线可用于确定HA以及其他糖皮质激素（例如硫酸软骨素和硫酸乙酰肝素）的平均大小，其大小范围为1至500 kDa，分辨率优于10%。对于多分散样品，我们的分析表明，表面接枝的过程中优先选择较小的GAG链，从而降低了与原始溶液样品相比，固定在表面上的GAG的平均尺寸。我们的研究结果建立了一个定量的方法来确定HA和其他接枝在表面上的GAG的大小，也突出了直接在表面上确定GAG大小的重要性。该方法应是有用的GAG为基础的表面涂层的开发和质量控制在广泛的研究领域，从分子相互作用分析，生物材料涂层。
Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (− Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ − Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.
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影响因子：
3.4
作者：
Bano F;Tammi MI;Kang DW;Harris EN;Richter RP
通讯作者：
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4.3
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Jing, W;DeAngelis, PL
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