Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements.
Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements.
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DOI：
10.1021/acsami.2c16647
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发表时间：
2022-10-28
影响因子：
9.5
通讯作者：
Hudson AJ
中科院分区：
文献类型：
--
作者：
Bueno-Alejo CJ;Santana Vega M;Chaplin AK;Farrow C;Axer A;Burley GA;Dominguez C;Kara H;Paschalis V;Tubasum S;Eperon IC;Clark AW;Hudson AJ
单分子成像对于研究生物分子的异质行为和相互作用是非常宝贵的。然而，单分子精确采样的障碍是组分不可逆地吸附到盖玻片表面上。这导致从分配样品的那一刻起溶解或悬浮在水相中的不同分子的浓度连续变化，这将随着时间的推移改变单体和多聚体组分之间的化学平衡的位置。干涉散射显微镜（iSCAT）是单分子工具包中的一种技术，能够检测吸附在玻璃表面上和从玻璃表面解吸的未标记蛋白质和蛋白质复合物。在这里，我们通过分析单分子水平上蛋白质的吸附和解吸来研究许多不同蛋白质与玻璃之间可逆和不可逆的相互作用。此外，我们提出了一种表面钝化方法，该方法几乎消除了不可逆吸附，同时仍确保分子在表面上的停留时间足以通过iSCAT检测吸附。通过在盖玻片表面上接枝高密度全氟烷烃刷，我们观察到在测量表面上蛋白质的吸附和解吸事件的数量大致相等（±1%）。氟-水界面还防止蛋白质复合物的动力学捕获，并有助于在单体和多聚体组分之间建立热力学平衡。这种表面钝化方法对于使用iSCAT显微镜的体外单分子实验是有价值的，因为它允许连续监测蛋白质的吸附和解吸，而不会由于蛋白质与表面的不可逆结合而导致检测事件下降或样品组成变化。
Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous–aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces.
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影响因子：
48
作者：
Hua, Boyang;Han, Kyu Young;Zhou, Ruobo;Kim, Hajin;Shi, Xinghua;Abeysirigunawardena, Sanjaya C.;Jain, Ankur;Singh, Digvijay;Aggarwal, Vasudha;Woodson, Sarah A.;Ha, Taekjip
通讯作者：
Ha, Taekjip
DOI：
10.1039/c7cc00288b
发表时间：
2017-03-09
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Flynn GE;Withers JM;Macias G;Sperling JR;Henry SL;Cooper JM;Burley GA;Clark AW
通讯作者：
Clark AW
DOI：
10.1073/pnas.1105555108
发表时间：
2011-09-06
影响因子：
11.1
作者：
Knowles, Tuomas P. J.;White, Duncan A.;Weitz, David A.
通讯作者：
Weitz, David A.
DOI：
10.1007/s00249-007-0140-8
发表时间：
2007-07-01
影响因子：
2
作者：
Arouri, Ahmad;Garidel, Patrick;Blume, Alfred
通讯作者：
Blume, Alfred
影响因子：
8.4
作者：
Chang, Jianguo;Gao, Nailong;Li, Lu
通讯作者：
Li, Lu