1. Active Nanostructures as Probes in Biophysics

2. Resolving Subdiffraction Limit Contacts in Live Cell High-Speed Nanoparticle Tracking

3. Photonic Plasmonic Crystals with Multiscale Field Enhancement

4. Higher Functionalities through Rational Combination of Photonic and Plasmonic Resonators

5. Investigating the Influence of Receptor Clustering on Signaling Activity using Plasmon Coupling Microscopy

1. Active Nanostructures as Probes in Biophysics: Plasmon Rulers

We have developed DNA and RNA programmed self-assembly procedures and Agarose gel purification strategies that yield bulk quantities of dimers of polymer tethered gold and silver nanoparticles, so called plasmon rulers. Since the optical response of plasmon rulers depends on the interparticle separation, plasmon rulers can act as dynamic molecular rulers. Noble metal nanoparticles have advantageous photophysical properties. They are extremely bright and they do not blink or bleach. Plasmon rulers enable distance measurements beyond the spatial and temporal barriers of alternative fluorescence based molecular rulers such as fluorescence resonance energy transfer (FRET). If the distance dependent polarization anisotropy of coupled nanoparticles is utilized as well, plasmon rulers allow simultaneous distance and orientation measurements. Due to their large optical cross-sections, plasmon rulers can be used in massively parallel single molecule assays with high temporal resolution.

We are currently using employing plasmon rulers to investigate nuclease activity on RNA and RNA-protein complexes. We have demonstrated that due to the single RNA molecule sensitivity and the high temporal resolution plasmon rulers give rise to details in the cleavage kinetics and dynamic structures of nucleid acids that are difficult to access with conventional techniques.


Plasmon ruler RNase A cleavage assay. (A) The RNA plasmon rulers are bound to the surface of a glass flow chamber using a BSA (bovin serum albumin)-Biotin-NeutrAvidin surface chemistry. Upon addition of RNase A, the RNA tether is cleaved, and the dimer converted into a monomer. (B) Single RNA plasmon ruler cleavage trajectory (recorded at 96 Hz). (I) The plasmon ruler is first incubated in buffer containing spermidine at defined concentrations (0 -5 mM), (II) the buffer is exchanged with a 1 nM RNase A solution, causing (III) a strong drop in intensity upon RNA cleavage. Inset: Number of cleavage events for flushing with/without enzyme. ∆tcl is defined as the time between enzyme addition and cleavage.

Relevant Publications:

“Spermidine Modulated Ribonuclease Activity Probed by RNA Plasmon Rulers” L. R. Skewis, B. M. Reinhard, Nano Lett. 2008, 8, 214.
“Correlated Optical Spectroscopy and Transmission Electron Microscopy of Individual Hollow Nanoparticles and their Dimers”, L. Yang, Bo Yan, B. M. Reinhard, J. Phys. Chem. C 2008, 112, 15989 .
“Monitoring Simultaneous Distance and Orientation Changes in Discrete Pairs of DNA Linked Gold Nanoparticles”, H. Wang, B. M. Reinhard, J. Phys. Chem. C 2009, 113, 11215.


2. Resolving Subdiffraction Limit Contacts in Live Cell High-Speed Nanoparticle Tracking: Plasmon Coupling Microscopy

Biological complexity required for instance for controlling and regulating cell signaling often involves dynamic interactions between several cell receptor molecules. To unravel the underlying molecular mechanisms governing fundamental cell signaling processes, it is necessary to quantify the interactions between individual cell surface bound receptors. For the latter, optical tools are required that can resolve interactions between individual molecules on deeply subdiffraction limit distances with high temporal resolution.

In response to this need we are developing plasmon coupling based microscopies that detect direct interactions between individual nanoparticle labeled receptors through plasmon coupling between the labels. Plasmon coupling microscopy detects direct interactions between individual membrane confined and laterally diffusing nanoparticles as a spectral shift in their plasmon resonance. The large optical cross-sections enable precise localization of individual nanoparticles with high spatial resolution. As nanoparticles to not blink or bleach and they have clear advantages in tracking applications especially when long observation times are required.

If two nanoparticles approach each other below the diffraction limit, the individual particles can no longer be distinguished in optical microscopy. However, in plasmon coupling microscopy, further information about the real separation between the particles is available by decoding the spectral information encoded in the nanoparticle plasmons.

Polarization resolved plasmon coupling microscopy can be used to simultaneously monitor the orientation of individual nanoparticles labeled receptors and their separation on deeply sub-diffraction limit distances.


Gold nanoparticle labeled surface receptors (left) and spectral signature (right) as function of interparticle distance. (a) For interparticle separations ∆ larger than the particle diameter D, the near-field interactions between the particles is small and the resonance wavelength λres is that of an individual particle. (b) For interparticle separations ∆ < D the plasmons in the individual particles couple and the resonance wavelength λres red-shifts with decreasing separation. This spectral shift is observable as an increase in the intensity ratio R = I580nm/I530nm.

Relevant Publications:

“Resolving Sub-Diffraction Limit Encounters in Nanoparticle Tracking Using Live Cell Plasmon Coupling Microscopy”, G. Rong, H. Wang, L. R. Skewis, B. M. Reinhard, Nano Lett. 2008, 8, 3386.
“Insights from a Nanoparticle Minuet: Two-Dimensional Membrane Profiling through Silver Plasmon Ruler Tracking”, G. Rong, H. Wang, B. M. Reinhard, Nano Lett., 10, 230 (2010).


3. Photonic Plasmonic Crystals with Multiscale Field Enhancement: Nanoparticle Cluster Arrays and their Application for Sensitive SERS Applications

Surface Enhanced Raman Scattering (SERS) Spectroscopy is a label free vibrational spectroscopy that provides direct molecular information. SERS is enabled by strong field enhancements in the vicinity of nanostructured metallic surfaces, which can effectively localize and focus incident electromagnetic fields. Due to the strong distance dependence of the SERS effect, it is a useful method to selectively obtain information about the surface of pathogenic bacteria cells placed directly on the SERS substrate. The surface of a bacterium is essential for its interaction with the environment and is thus supposed to be highly specific to the bacterium species and possible even bacterium strain. The chemically characteristic bacterial surface properties in combination with the surface specificity of SERS could potentially pave the way to a rapid spectral pathogen identification.

The applicability of SERS for sensitive sensing applications is, however, challenged by the difficulty to fabricate SERS substrates that generate high and reproducible field enhancements. To increase the reliability and applicability of SERS in biological sensing, it is therefore necessary to develop novel SERS substrates with improved performance characteristics in terms of reproducibility and field enhancement.

Our approach to overcome this problem is based on fabrication technologies that enable an engineering of electromagnetic hot-spots – small volumes with giant field enhancement – with nanometer precision. We combined top-down (electron beam lithography) and chemical bottom-up fabrication (nanoparticle self assembly) to create clusters of nanoparticles with defined size at defined locations. Using this method, we generated two-dimensional nanoparticle cluster arrays with defined geometry. Control of the size and separation of the nanoparticle clusters gives control over the nanoparticle generated field enhancement. Since the field in arrays of nanoparticle clusters is enhanced through intra- and inter-cluster nanoparticle interactions, they rise to a multiscale field enhancement. This synergistic effect lead to a field enhancement that is larger than that of the individual clusters (which is higher than that of the individual particles).

We are currently investigating the fundamental electromagnetic interactions in these photonic plasmonic crystals and apply them for sensitive biosensing applications, such as a rapid and reliable identification of bacterial pathogens.


SEM images from extracts of nanoparticle cluster arrays with varying diameters of e-beam defined binding size D = 50 nm (a), 80 nm (b), 100 nm (c), 130 nm (d), 200 nm (e). The SEM images confirm that through control of the diameter of the e-beam fabricated binding site the cluster size can be continuously varied. The enlargement of an individual cluster in (f) shows junctions and crevices between nearly touching particles constituting a high degree of roughness on the nanoscale.

Relevant Publications:

“Engineered SERS Substrates with Multiscale Signal Enhancement: Nanoparticle Cluster Arrays”, B. Yan, A. Thubagere, R. Premasiri, L. Ziegler, L. Dal Negro, B. M. Reinhard, ACS Nano 2009, 3, 1190.
“Photonic-Plasmonic Scattering Resonances in Deterministic Aperiodic Structures” A. Gopinath, S. Boriskina, N.-N. Feng, B. M. Reinhard, L. Dal Negro, Nano Lett. 2008, 8, 2423.


4. Higher Functionalities through Rational Combination of Photonic and Plasmonic Resonators : On-Chip Integrated Optoplasmonic Atoms and Molecules

Optical microcavity (OM) resonators with high-Q whispering gallery modes (WGM) generate greatly enhanced local fields which makes the attractive for many critical sensing applications. The WGMs are, however, strongly confined to the interior resonator volume, which is not accessible to potential analytes. The fields associated with localized surface plasmon (LSP) resonances of metal nanoparticles penetrate the ambient medium more efficiently, but the optical properties of metal nanoparticles suffer from high dissipative losses. Rationally designed photonic-plasmonic nanostructure offer a potential route to overcoming the intrinsic limitation of both conventional OM resonators and noble metal nanoparticles. Coupling of photonic and plasmonic modes in optoplasmonic nanostructures results in a redistribution of the optical power between OM and plasmonic nanostructures. This redistribution cumulates in a field amplification in a narrow frequency ranges located on the plasmonic components where it is accessible to external analytes.
We are currently exploring the electromagnetic coupling mechanisms of photonic and plasmonic components in complex optoplasmonic structures with multiple photonic and plasmonic building blocks and develop precise fabrication approaches to realize these exciting new electromagnetic materials.


SEM images of optoplasmonic structures that contain one (a, b), two (c, d), or three (e, f) 2.048 μm diameter PS microspheres. The edge-to-edge distance between the pillars of two neighboring binding cavities is Dp = 330 nm, and the height of the pillars is h = 870 nm. The tilted SEM images (g, h) show that spherical Au NPs with a diameter of d = 148 nm are located in the equatorial plane of the PS microspheres trapped in the pillar cavities (scale bars = 1 μm).

Relevant Publications:

“Photonic-Plasmonic Mode Coupling in On-Chip Integrated Optoplasmonic molecules”, W. Ahn, S. V. Boriskina, Y. Hong, B.M. Reinhard, ACS Nano 2012,,6951.

5. Investigating the Influence of Receptor Clustering on Signaling Activity using Plasmon Coupling Microscopy