Department of Physics and Astronomy, Texas Christian University, 2800 S. University Dr. Fort Worth, Texas 76129, USA.
Analyst. 2018 Jun 11;143(12):2819-2827. doi: 10.1039/c8an00136g.
Fluorescence signal enhancement induced by the binding of intercalators to DNA has been broadly utilized in various DNA detection methods. In most instances the increase in fluorescence intensity is associated with a concomitant increase of fluorescence lifetime. This increase of the fluorescence lifetime presents an additional opportunity to increase detection sensitivity. In this paper, we present a new approach to significantly enhance the sensitivity in detecting minute DNA concentrations. The approach is based on simultaneous use of time-gated detection and multi-pulse pumping. By using a calibrated burst of short pulses we greatly enhance the contribution of long-lived fluorescence species, thus enabling easy time-gated detection. Using a classic DNA intercalator - Ethidium Bromide (EtBr) - as an example with our novel multi-pulse pumping and time-gated detection technique, we were able to increase detection sensitivity over 70-fold with only 3 pulse excitation. This approach is generic and can be used with any analytical probe (exhibiting about 10 times change in lifetime) that shows an increase in fluorescence signal and fluorescence lifetime upon binding to a target.
荧光信号增强诱导的嵌入剂与 DNA 的结合已广泛应用于各种 DNA 检测方法。在大多数情况下，荧光强度的增加与荧光寿命的同时增加有关。这种荧光寿命的增加提供了另一个增加检测灵敏度的机会。在本文中，我们提出了一种新的方法，可显著提高检测微量 DNA 浓度的灵敏度。该方法基于同时使用时间门控检测和多脉冲泵浦。通过使用校准的短脉冲爆发，我们大大增强了长寿命荧光物质的贡献，从而实现了简单的时间门控检测。使用经典的 DNA 嵌入剂 - 溴化乙锭（EtBr） - 作为我们的新型多脉冲泵浦和时间门控检测技术的示例，我们仅用 3 个脉冲激发就能将检测灵敏度提高 70 多倍。这种方法是通用的，可以与任何分析探针（其在与靶标结合时显示荧光信号和荧光寿命增加约 10 倍）一起使用。
