Nanobiophysics (NBP), MESA + Institute for Nanotechnology and Technical Medical Centre, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands.
Wetsus, European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911MA, Leeuwarden, The Netherlands.
Methods Appl Fluoresc. 2021 Jan 22;9(2):025001. doi: 10.1088/2050-6120/abd8e4.
In health and environmental research, it is often necessary to quantify the concentrations of single (bio) nanoparticles present at very low concentrations. Suitable quantification approaches that rely on counting and tracking of single fluorescently labelled (bio) nanoparticles are often challenging since fluorophore self-quenching limits the maximum particle brightness. Here we study how the number of labels per nanoparticle influences the total brightness of fluorescently labelled cowpea chlorotic mottle virus (CCMV). We analyze in detail the photophysical interplay between the fluorophores on the virus particles. We deduce that the formation of dark aggregates and energy transfer towards these aggregates limits the total particle brightness that can be achieved. We show that by carefully selecting the number of fluorescent labels per CCMV, and thus minimizing the negative effects on particle brightness, it is possible to quantify fluorescently labelled CCMV concentrations down to fM concentrations in single particle counting experiments.
在健康和环境研究中，通常需要定量分析非常低浓度下存在的单个（生物）纳米颗粒的浓度。由于荧光团自猝灭限制了最大颗粒亮度，因此依赖于对单个荧光标记的（生物）纳米颗粒进行计数和跟踪的合适定量方法通常具有挑战性。在这里，我们研究了每个纳米颗粒上的标记数量如何影响荧光标记豇豆花叶病毒（CCMV）的总亮度。我们详细分析了病毒颗粒上荧光团之间的光物理相互作用。我们推断，暗聚集体的形成和能量转移到这些聚集体限制了可以实现的总颗粒亮度。我们表明，通过仔细选择每个 CCMV 的荧光标记数量，从而最小化对颗粒亮度的负面影响，有可能在单个颗粒计数实验中定量检测到低至 fM 浓度的荧光标记 CCMV 浓度。
