Multi intercalators FRET enhanced detection of minute amounts of DNA

IF 2.2 4区生物学 Q3 BIOPHYSICS

European Biophysics Journal Pub Date : 2023-05-04 DOI:10.1007/s00249-023-01655-y

Luca Ceresa, Jose Chavez, Magdalena M. Bus, Bruce Budowle, Emma Kitchner, Joseph Kimball, Ignacy Gryczynski, Zygmunt Gryczynski

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引用次数: 0

Abstract

A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays.

Graphical abstract

Abstract Image

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多嵌入物FRET增强了对微量DNA的检测。

提出了一种新的方法，该方法提高了检测液体和固体样品中微量DNA的灵敏度和特异性。从YOYO到溴化乙锭（EtBr）的Förster共振能量转移（FRET）显著增加了来自DNA结合的EtBr的信号，高度增强了DNA检测的灵敏度和特异性。当与DNA结合时，EtBr受体的长荧光寿命允许通过时间门控（MPPTG）检测进行多脉冲泵浦，这大大增加了与DNA结合的EtBr的可检测信号。直接的光谱/图像相减消除了样本背景，并允许整体检测灵敏度的大幅提高。使用FRET和MPPTG检测的组合，可以在没有任何额外的样品纯化/操作或使用扩增技术的情况下检测微升样品中小至10pg的DNA量。这个数量的DNA相当于一到两个人类细胞的DNA含量。这种基于简单光学的检测方法为在现场进行稳健、高灵敏度的DNA检测/成像、对收集的DNA样本进行快速评估/分类（即试验）开辟了潜力，并可以支持各种诊断测定。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

European Biophysics Journal 生物-生物物理

CiteScore

4.30

自引率

0.00%

发文量

审稿时长

6-12 weeks

期刊介绍： The journal publishes papers in the field of biophysics, which is defined as the study of biological phenomena by using physical methods and concepts. Original papers, reviews and Biophysics letters are published. The primary goal of this journal is to advance the understanding of biological structure and function by application of the principles of physical science, and by presenting the work in a biophysical context. Papers employing a distinctively biophysical approach at all levels of biological organisation will be considered, as will both experimental and theoretical studies. The criteria for acceptance are scientific content, originality and relevance to biological systems of current interest and importance. Principal areas of interest include: - Structure and dynamics of biological macromolecules - Membrane biophysics and ion channels - Cell biophysics and organisation - Macromolecular assemblies - Biophysical methods and instrumentation - Advanced microscopics - System dynamics.