This research proposes to develop single-molecule spectroscopy (SMS) on plasmonic antennas to achieve 1nm spatial resolution, 1?s temporal resolution, and 1mM working concentration, representing improvement by 3 orders of magnitude over state-of-the-art SMS. The invention of SMS has made it possible to follow the biochemical reactions of an individual enzyme molecule in real time, and sequence a single DNA molecule with long reads (10-15 kb). However, two key barriers have hampered the expanded use of SMS: 1). Concentration barrier. To detect a single molecule requires that, on average, no more than one molecule be within the detection volume at a time. Therefore, it is difficult to carry out single-molecule detection at concentrations above 1micromolar, out of the range of physiologically relevant concentrations of high micro- to millimolar range. 2). Resolution barrier. The spatiotemporal resolution of current single-molecule F?rster resonance energy transfer spectroscopy (SM-FRET) is about 4nm and 1ms, respectively, whereas the dynamics of most protein enzymes is on a sub-nanometer length scale and nano- to microsecond time scale, too small and too fast for SMS to capture. This project proposes to break the concentration and the resolution barriers by harnessing the extreme light manipulation power from recent breakthroughs in plasmonic antennas. Plasmonics is a flourishing field of science and technology that exploits the surface plasmon of metallic nanostructures to confine, route, and manipulate light at the nanometer length scale. In the past decade, thanks to better electromagnetic wave simulation algorithms, massive computational resources, and increasingly precise nanofabrication techniques, plasmonic antennas have been demonstrated to 1) coherently magnify fluorescent signals, 2) suppress photo-bleaching and blinking of single fluorescent molecules, and 3) reduce background noise. As a result, the antennas have been shown to enhance the fluorescence signal-to-noise ratio by up to 3 orders of magnitude, offering a means to break the concentration and resolution barriers. To develop this "lab-on-antennas" platform, this project will: 1) use computer simulation, single-molecule super-resolution microscopy, and atomic-force microscopy (AFM) assisted fabrication to rationally design and fabricate plasmonic antennas for high-resolution SMS;2) fabricate plasmonic antennas on a large scale with novel polymer-assisted methods;and 3) use DNA molecular ruler, polypeptides, and adenylate kinase as models to establish the protocols of capturing transient dynamics of a single enzyme. By leveraging the advance of plasmonics, "lab-on-antennas" will be a general and powerful tool to visualize enzymes working at the single-molecule level with spatial and temporal resolution that has been previously unattainable.

Public Health Relevance

This research proposes to develop plasmonic antennas to enhance the resolution and sensitivity of single-molecule microscopy by 1,000 fold, which will assist in capturing transient states of proteins for drug targets and reduce the error rate of single-molecule DNA sequencing.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
NIH Director’s New Innovator Awards (DP2)
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Special Emphasis Panel (ZRG1-MOSS-C (56))
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Conroy, Richard
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Salk Institute for Biological Studies
La Jolla
United States
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Beuzer, Paolo; Axelrod, Joshua; Trzoss, Lynnie et al. (2016) Single dish gradient screening of small molecule localization. Org Biomol Chem 14:8241-5
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