Nanobiosensorics Group, Institute for Technical Physics and Materials Science, Centre for Energy Research, Hungarian Academy of Sciences, Budapest, Hungary; Department of Biological Physics, Eötvös University, Budapest, Hungary.
Department of Hydrodynamic Systems, Budapest University of Technology and Economics, Budapest, Hungary.
J Colloid Interface Sci. 2019 Nov 1;555:245-253. doi: 10.1016/j.jcis.2019.07.102. Epub 2019 Jul 31.
Characterization of the binding of functionalized microparticles to surfaces with a specific chemistry sheds light on molecular scale interactions. Polymer or protein adsorption are often monitored by colloid particle deposition. Force measurements on microbeads by atomic force microscopy (AFM) or optical tweezers are standard methods in molecular biophysics, but typically have low throughput. Washing and centrifuge assays with (bio)chemically decorated microbeads provide better statistics, but only qualitative results without a calibrated binding force or energy value. In the present work we demonstrate that a computer controlled micropipette (CCMP) is a straightforward and high-throughput alternative to quantify the surface adhesion of functionalized microparticles. However, being an indirect force measurement technique, its in-depth comparison with a direct force measurement is a prerequisite of applications requiring calibrated adhesion force values. To this end, we attached polystyrene microbeads to a solid support by the avidin-biotin linkage. We measured the adhesion strength of the microbeads with both a specialized robotic fluid force microscope (FluidFM BOT) and CCMP. Furthermore, the bead-support contact zone was directly characterized on an optical waveguide biosensor to determine the density of avidin molecules. Distribution of the detachment force recorded on ∼50 individual beads by FluidFM BOT was compared to the adhesion distribution obtained from CCMP measurements on hundreds of individual beads. We found that both methods provide unimodal histograms. We conclude that FluidFM BOT can directly measure the detachment force curve of 50 microbeads in 150 min. CCMP can provide calibrated binding/adhesion force values of 120 microbeads in an hour.
对具有特定化学性质的功能化微粒与表面的结合进行表征，可以深入了解分子尺度的相互作用。聚合物或蛋白质的吸附通常通过胶体粒子沉积来监测。原子力显微镜 (AFM) 或光镊对微球的力测量是分子生物物理学中的标准方法，但通常通量较低。用（生物）化学修饰的微球进行洗涤和离心分析可以提供更好的统计数据，但没有校准的结合力或能量值，只能得到定性结果。在本工作中，我们证明计算机控制的微吸管 (CCMP) 是一种简单、高通量的替代方法，可以定量测量功能化微球的表面附着力。然而，作为一种间接的力测量技术，它与直接力测量的深入比较是应用于需要校准粘附力值的前提。为此，我们通过亲和素-生物素键将聚苯乙烯微球附着到固体载体上。我们使用专门的机器人流体力显微镜 (FluidFM BOT) 和 CCMP 测量微球的粘附强度。此外，我们还在光波导生物传感器上直接对微球支撑接触区进行了表征，以确定亲和素分子的密度。通过 FluidFM BOT 记录的约 50 个微球的分离力分布与通过 CCMP 测量获得的数百个微球的粘附分布进行了比较。我们发现这两种方法都提供了单峰直方图。我们得出的结论是，FluidFM BOT 可以直接测量 50 个微球的分离力曲线，用时 150 分钟。CCMP 可以在一小时内提供 120 个微球的校准结合/粘附力值。
