Abstract: Techniques for single fluorescent molecules detection have garnered a great amount of interests from science community in recent years largely due to their ability to reveal the individual molecular properties and behaviors which were often obscured by techniques based on ensemble  average. In order to overcome diffraction-limit of optical observation, fluorescent molecules must be diluted to a very low concentration-way below normal physiological concentration. However, this may adversely impact the kinetics for the chemical or biochemical reaction under study. Zero-mode waveguides（ZMWs）, as a new kind of single fluorescent molecule detection device, effectively reduce the observation volume to the level of zeptoliter (10-21L), which is much lower than optical diffraction-limited volume, by utilizing of a nanoaperture structure. The zeptoliter-observation volume provides an environment where a single fluorescent molecule can be observed at normal physiological concentration. The ZMWs have been extensively used in the field of single fluorescent molecule detection due to its outstanding performance than diffraction-limited optics.Therefore we provided an overview of ZMWs, including discussions on its principle, fabrication techniques, and applications in DNA sequencing, biomembrane structure elucidation,  biomacromolecule interaction studies and kinetics study of single-molecule reaction.

Key words: zero-mode waveguides, single molecule, nano microstructure devices

摘要： 单分子荧光检测作为一种能够表征分子个体性质及行为的分析方法，有助于揭示利用传统荧光检测方法无法得到的信息，在近年来受到人们的广泛关注。利用传统光学检测设备进行单分子荧光检测时，由于受到衍射极限的限制，同时为了保证在观测体积内只有单个荧光分子，仅能采用无限稀释溶液的方法实现单分子荧光检测。虽然这种方法可以满足单分子检测的要求，但是由于大部分酶分子正常工作时底物的生理浓度都非常高，底物浓度的大幅度降低会对酶分子的反应机制等方面造成影响。零模波导作为一种新型的单分子检测器件，通过纳米微孔结构突破了光学衍射极限的限制将观测体积降至仄升量级（10-21L），使得在生理浓度范围内检测单分子荧光成为可能，在单分子荧光检测领域得到了广泛应用。因此，就零模波导的原理、制备工艺及其在单分子DNA测序、生物膜、生物大分子之间的相互作用及单分子反应动力学方面的具体应用进行综述。

关键词: 零模波导, 单分子, 纳米微结构器件