Sun Yung-Shin, Zhu Xiangdong
Department of Physics, Fu-Jen Catholic University, New Taipei City, Taiwan
Department of Physics, University of California at Davis, CA, USA.
J Lab Autom. 2016 Oct;21(5):625-31. doi: 10.1177/2211068215586977. Epub 2015 May 14.
Microarrays provide a platform for high-throughput characterization of biomolecular interactions. To increase the sensitivity and specificity of microarrays, surface blocking is required to minimize the nonspecific interactions between analytes and unprinted yet functionalized surfaces. To block amine- or epoxy-functionalized substrates, bovine serum albumin (BSA) is one of the most commonly used blocking reagents because it is cheap and easy to use. Based on standard protocols from microarray manufactories, a BSA concentration of 1% (10 mg/mL or 200 μM) and reaction time of at least 30 min are required to efficiently block epoxy-coated slides. In this paper, we used both fluorescent and label-free methods to characterize the BSA blocking efficiency on epoxy-functionalized substrates. The blocking efficiency of BSA was characterized using a fluorescent scanner and a label-free oblique-incidence reflectivity difference (OI-RD) microscope. We found that (1) a BSA concentration of 0.05% (0.5 mg/mL or 10 μM) could give a blocking efficiency of 98%, and (2) the BSA blocking step took only about 5 min to be complete. Also, from real-time and in situ measurements, we were able to calculate the conformational properties (thickness, mass density, and number density) of BSA molecules deposited on the epoxy surface.
微阵列提供了一个用于生物分子相互作用高通量表征的平台。为了提高微阵列的灵敏度和特异性，需要进行表面封闭以最小化分析物与未打印但已功能化表面之间的非特异性相互作用。为了封闭胺基或环氧基功能化的底物，牛血清白蛋白（BSA）是最常用的封闭试剂之一，因为它价格便宜且使用方便。根据微阵列制造商的标准方案，需要1%（10mg/mL或200μM）的BSA浓度和至少30分钟的反应时间才能有效封闭环氧涂层载玻片。在本文中，我们使用荧光和无标记方法来表征BSA在环氧基功能化底物上的封闭效率。使用荧光扫描仪和无标记斜入射反射率差（OI-RD）显微镜来表征BSA的封闭效率。我们发现：（1）0.05%（0.5mg/mL或10μM）的BSA浓度可产生98%的封闭效率；（2）BSA封闭步骤仅需约5分钟即可完成。此外，通过实时和原位测量，我们能够计算沉积在环氧表面的BSA分子的构象性质（厚度、质量密度和数量密度）。
