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0Localization of dye molecules in zero mode waveguides
A. Gritchenko*, M. Markov, A. Kalmykov, V. Balykin, P. Melentiev
Institute of Spectroscopy RAS, 108840, Fizicheskaya st. 5, Troitsk, Moscow, Russia
* gritchenkoant@gmail.com
Detection, sensing and spectroscopy at the single-molecule level is a rapidly developing field of science at the interface of nanophotonics, chemistry and biochemistry. One of the fundamental problems in this field is the localization and long-term tracking of single molecules in space with nanometer precision. To date, there are several approaches to the localization of single molecules: embedding molecules in a polymer matrix [1], optical tweezers [2], immobilization of molecules by chemical and biochemical reactions on surfaces [3], electrophoretic methods [4].
Recently, approaches have been developed in the field of single-molecule detection and spectroscopy that involve the use of zero-mode waveguides (ZMWs). ZMWs are nanoholes with diameter of 50-200 nm in an approximately 100 nm thick metal film on the glass surface. The use of ZMWs provides many advantages: an increase in the signal-to-noise ratio during detection, the ability to work with physiological analyte concentrations, the possibility of parallel detection and spectroscopy with ZMW arrays. Detection and spectroscopy methods with ZMWs are actively used in single-molecule sequencing of DNA molecules [5].
In our work, we propose an approach to localize single fluorescent molecules in ZMW by using hydrogen bonds that can be effectively formed between the molecules and the quartz bottom of the ZMW (Figure 1a). We demonstrate, through measurements, the possibility to localize dye molecules in ZMWs on a time scale ranging from 10 ms to 10 seconds. This process can be finely controlled by the local density of free charges in the buffer solution used. By using different salts at appropriate concentrations, we can deterministically switch the localization process on and off (see Figure 1b). We discuss the use of the single molecule localization approach in ZMW in various applications: (i) detection and study of single molecules by fluorescence; (ii) sensing at the single molecule level; (iii) sequencing of single DNA molecules. The study was supported by a grant Russian Science Foundation No. 23-42-00049, https://rscf.ru/project/23-42-00049/.
Figure 1. Localization of a dye molecule in ZMW: (a) schematic representation of the trajectory of the AF555 dye molecule in ZMW, (b) fluorescence image of a single ZMW with localized AF555 molecule (the molecule is negatively charged, four upper images in a row) and mito-TMRE molecule (the molecule is positively charged, four lower images in a row, note that the images are amplified by a factor of 300 compared to the upper images) at different KCl concentrations in the buffer solution used (the concentration level is shown schematically above).
[1] W.E. Moerner and M. Orrit, Illuminating single molecules in condensed matter, Science (1979). American Association for the Advancement of Science, 1999. Vol. 283, № 5408. P. 1670-1676.
[2] G. Sirinakis, et al, Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy, Review of Scientific Instruments, AIP Publishing, 2012. Vol. 83, № 9.
[3] J.L. Zimmermann, et al, Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments, Nature Protocols, 2010 5:6. Nature Publishing Group, 2010. Vol. 5, № 6. P. 975-985.
[4] A.E. Cohen and W.E. Moemer, Method for trapping and manipulating nanoscale objects in solution, Appl Phys Lett. AIP Publishing, 2005. Vol. 86, № 9. P. 1-3.
[5] J. Eid, et al, Real-time DNA sequencing from single polymerase molecules, Science (1979), American Association for the Advancement of Science, 2009. Vol. 323, № 5910. P. 133-138.
