Fu Yi, Zhang Jian, Lakowicz Joseph R
Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Baltimore, USA.
Photochem Photobiol. 2009 May-Jun;85(3):646-51. doi: 10.1111/j.1751-1097.2008.00500.x. Epub 2009 Jan 13.
Single molecule detection (SMD) is usually performed on surface-immobilized molecules with diffraction-limited observation volumes, typically with confocal optics to suppress background from the sample and instrument. In this paper we consider the more difficult task of detecting single fluorophores in the presence of a substantial fluorescence background. We determined that for a readily accessible macroscopic observation volume of 1 pL that the background from undiluted blood serum was approximately equal to 2700 Cy5 molecules, and the background from whole blood equal to about 14 000 Cy5 molecules in whole blood. These high backgrounds appear to preclude the possibility of SMD of Cy5 molecules. However, we show that the signal-to-noise ratio (SNR) in high background samples can be increased dramatically by reduction of the observed volume. We were able to detect single surface-bound Cy5-labeled DNA (Cy5-DNA) oligomers in diluted blood serum with an SNR near 40. We also examined freely diffusing Cy5-DNA in blood serum. The data showed that single Cy5-DNA molecules could be detected even in the undiluted serum. We further investigated the SNR on silver island films. We found that the fluorescence signal was greatly enhanced in the presence of metallic nanostructures showing a larger SNR in the application tested. These results suggest the possibility of clinical assays based on SMD in blood serum and possibly whole blood. Increased SNR near metallic nanostructure could probably overcome the need for diffraction-limited volumes and enhance our ability to do in situ SMD.
单分子检测（SMD）通常是在表面固定的分子上进行，其观察体积受衍射限制，通常使用共聚焦光学器件来抑制来自样品和仪器的背景。在本文中，我们考虑在存在大量荧光背景的情况下检测单个荧光团这一更具挑战性的任务。我们确定，对于易于获得的1皮升宏观观察体积，未稀释血清的背景约等于2700个Cy5分子，全血的背景约等于全血中14000个Cy5分子。这些高背景似乎排除了对Cy5分子进行单分子检测的可能性。然而，我们表明，通过减小观察体积，高背景样品中的信噪比（SNR）可以显著提高。我们能够在稀释血清中检测到单个表面结合的Cy5标记DNA（Cy5-DNA）寡聚物，信噪比接近40。我们还研究了血清中自由扩散的Cy5-DNA。数据表明，即使在未稀释的血清中也能检测到单个Cy5-DNA分子。我们进一步研究了银岛膜上的信噪比。我们发现，在存在金属纳米结构的情况下，荧光信号大大增强，在所测试的应用中显示出更大的信噪比。这些结果表明了基于血清甚至可能是全血中的单分子检测进行临床检测的可能性。金属纳米结构附近信噪比的提高可能克服对衍射限制体积的需求，并增强我们进行原位单分子检测的能力。
