Nanometrology: Plasmon rulers: The optical angstrometers

Sept. 8, 2014
Evolving nanofabrication techniques suggest that plasmon rulers can potentially serve as easy-to-use optical "angstrometers".

RYAN T. HILL

Plasmon rulers are a fascinating class of metrology devices because of their exquisite, atomic-bond-length sensitivity to distance and their transduction of signal as a simple spectroscopic measurement. They are composed of plasmon resonant nanoparticles (NPs), and hence report distance information from a very localized, nanoscale environment—be it a biologically relevant, aqueous sample reporting structural dynamics of molecules or a molecularly thin surface coating. While their development into robust metrology tools is not without challenges, evolving nanofabrication techniques suggest that plasmon rulers can function as optical "angstrometers".

The optical signal from a plasmon ruler arises from a localized surface plasmon resonance (LSPR) from metallic NPs. This LSPR is produced when broadband illumination of plasmon resonant NPs with sub-wavelength dimensions causes the free electrons on the NP surfaces to oscillate in response to the oscillating electric field of the incident light.

Excitation of a LSPR results in absorbance and elastic scattering of the incident illumination at the resonant wavelength, both of which are easily detectable using optical imaging and spectroscopy. The LSPR of a NP occurs at a very specific wavelength, depending on the NP size, shape, and composition, and also on the dielectric properties of the surrounding medium.1

The use of NP LSPRs as optical reporters has several attractive features that contribute to the unique properties of plasmon rulers. First, the scattering from a NP LSPR is bright enough that single NPs can be identified using optical microscopes and dark-field illumination, even though the physical dimension of the NP is smaller than the resolution limit of optical microscopes. This bright signal from such a small structure enables studies on the single-molecule level. Also, scattering from ensembles of NPs can be seen by the naked eye, promoting their use in biosensing diagnostic devices that utilize colorimetric detection (such as a lateral flow assay as a pregnancy test). A second advantage of NP LSPRs is that their optical properties are stable over time even with prolonged excitation, unlike fluorophores that are susceptible to photobleaching.1 A third advantage is that NP LSPRs are completely tunable by varying the size, shape, and composition of the chemically synthesized NPs.

Plasmonic coupling

As mentioned above, NP LSPRs are sensitive to their surroundings and as such, are sensitive to the proximity of other nearby plasmon resonant NPs. When two NPs are separated by a distance less than their diameters, they become plasmonically coupled and produce a dramatically red-shifted, coupled LSPR (see Fig. 1). If the coupled NPs are then pushed apart, the coupled LSPR blue-shifts in a distance-dependent manner. This color shift in the LSPR of coupled plasmon resonant NPs with separation distance is the basic underlying principle by which plasmon rulers function.

Plasmonic coupling used in the context of molecular rulers was first established as a technique to rival Förster Resonance Energy Transfer (FRET) molecular rulers, which probe dynamic molecular interactions.2 The plasmon ruler effect was demonstrated by linking two NPs together using single-stranded DNA to produce a red-shifted coupled LSPR. It was then observed that the coupled LSPR of the NP dimer blue-shifted as the DNA linking the NPs hybridized to a complimentary DNA molecule in solution, which increased the separation distance between the NPs.

Since this initial demonstration, the plasmon ruler has been well characterized by experimental and theoretical studies.3 A unique feature of the plasmon ruler is that the coupled LSPR shift in wavelength scales nonlinearly with NP separation distance in a way that makes the plasmon ruler extremely sensitive when the coupled NPs are very close together.

In comparison to FRET molecular rulers, plasmon rulers have the advantage of using non-degradable LSPR scattering as a transduction mechanism, which allows continuous monitoring of dynamically changing molecular events without photodamage concerns. Furthermore, plasmon rulers are sensitive over longer distance ranges than FRET rulers, thus increasing their utility.

Plasmon rulers have been used for various sensing schemes, such as detection of DNA and protein.4,5 The NP coupling mechanism has also been developed into a special form of microscopy called plasmon coupling microscopy, which has been used to study protein expression on cell membranes.6

Implementing plasmonic rulers

Perhaps the largest challenge in using plasmon rulers is the high degree of difficulty involved in reproducibly creating NP dimers with precisely defined separation distances. In many instances NP dimers are fabricated such that they become immobilized onto substrates. However, the efficiency in the chemical linking procedures used to create dimers in this way is notoriously low, which often results in a small population of active plasmon rulers embedded within a large population of unusable light scatterers (usually single NPs or large NP aggregates) that contribute to spurious background signal. Thus, the functional plasmon ruler NP dimers must be selected and characterized one-by-one using dark-field microscopy, which increases the complexity and reduces the throughput capabilities of the sensing platform.

A variant of the NP dimer plasmon ruler developed recently called the NP-film plasmon ruler addresses the concerns of the high complexity and low throughput of NP dimer plasmon rulers.7-10 NP-film plasmon rulers can be created with approximately 100% yield by simply immobilizing plasmon resonant NPs onto a metallic surface such as gold (see Fig. 2). In doing so, the NPs become plasmonically coupled to the gold film and display dramatically red-shifted LSPRs when near the gold film. As the NPs are separated from the film, for example by using increasingly thick molecular spacer layers deposited onto the film prior to NP deposition, the NP LSPRs blue-shift similarly to the NP dimer plasmon rulers. These optical properties of the film-coupled NPs are understood conceptually as the formation of a virtual NP dimer via an induced, mirror-image dipole within the film.
The key advantage of NP-film plasmon rulers is that each NP immobilized on the film becomes a plasmon ruler (see Fig. 3). It is straightforward to create billions of identical NP-film plasmon rulers over large surface areas (coverage of a full 75 mm × 25 mm microscope slide is easily done) using a self-assembly fabrication strategy. First, the gold film is coated with a uniform molecular spacer layer, and then NPs are attached to the molecular layer so that a high NP surface coverage is achieved.

The high density of uniform NP-film plasmon rulers enables high throughput characterization where millions of plasmon rulers can be interrogated with a single optical spectroscopy measurement, eliminating the need to identify and characterize the plasmon rulers one-by-one using an expensive optical microscope with spectroscopic imaging.

Ensemble NP-film plasmon ruler measurements increase the throughput, precision, and sensitivity of the plasmon ruler platform, and as a result the plasmon ruler has achieved angstrom-scale distance resolution on the order of atomic-bond-length distance sensitivity (see Fig. 4). This was accomplished by using the plasmon ruler in the "extreme sensitivity" regime where the NP-film separation distance was extremely small (<2 nm). The NPs were separated from the film by ultrathin molecular spacer layers, with the thinnest layer being only 5 angstroms. In this extreme sensitivity regime, the plasmon ruler produced a spectral shift of 5 nm for every 1 angstrom change in distance.

In a head-to-head comparison with ellipsometry as a metrology tool, the plasmon ruler outperformed ellipsometry for the ultrathin molecular spacer layers. The ellipsometer systematically underestimated the thickness of the ultrathin layers and also was unable to detect the thinnest spacer layer.

The NP-film plasmon ruler format rivals ellipsometry not only in sensitivity, but also in convenience. Once the NP-film ruler is calibrated by characterizing the NP-film plasmon ruler response with increasing separation distance (a study that can easily be done in the NP-film plasmon ruler format using increasingly thick molecular spacer layers), the only thing required for a distance readout is a simple spectroscopic measurement that can instantly be converted to distance via the established calibration curve.

Ellipsometry boasts convenient data collection, but the final metrology measurement produced by ellipsometry is only as good as the model used to fit the acquired data. The models are often generalized, especially for organic materials, so the accuracy of the thickness data can be questionable. It is interesting to note here that NP-film plasmon ruler measurements can actually be acquired using a spectroscopic ellipsometer operating in spectroscopic reflectivity mode.

Plasmon ruler challenges

The NP dimer and the NP-film plasmon ruler platforms each have unique advantages. NP dimer plasmon rulers behave similarly to FRET rulers, acting as nanoprobes that can be deployed in various ways to produce highly localized and dynamic distance information. NP-film plasmon rulers can be used as high-throughput, surface-based metrology devices that rival ellipsometry, especially when used in the "extreme sensitivity" regime to characterize ultrathin surface layers. They are easily used as static rulers in the dry state and also have been demonstrated as dynamic rulers in aqueous solutions. Current and future research efforts are focused on improving their compatibility with aqueous studies so as to be more useful as chip-based biosensors.

There are still significant challenges that limit widespread use of plasmon rulers. The sensitivity of a plasmon ruler diminishes toward larger separation distances due to the nonlinear LSPR coupling trend with NP separation distance. Thus, plasmon rulers are not useful in measuring large separation distances (distances sufficiently greater than the diameter of the NPs used). Also, the NP LSPRs are highly sensitive to the dielectric properties of their surroundings, so any significant changes to the material properties between the plasmonic reporters (between the NP dimers or NP film) or their surroundings can affect plasmon ruler measurements.

In comparison to FRET rulers, plasmon rulers use relatively large reporters—NPs with diameters typically from around 20 to 100 nm—that can adversely affect the dynamics of small molecules being interrogated by the plasmon rulers. Finally, the design of a plasmon ruler experiment must accommodate attachment of plasmon resonant NPs to the material of interest, and so, unlike ellipsometry, the plasmon ruler is not a contact-free measurement system.

Despite these challenges in using plasmon rulers, enthusiasm about them remains high because of their exquisite distance sensitivity, requiring only a simple optical readout that needs essentially no post-processing once the ruler is calibrated. Plasmon rulers also exhibit improved sensitivity over FRET rulers, a non-bleachable signal transduction mechanism, and the ability to probe highly localized, nanoscale environments where the properties of single molecules are observed. With further development, plasmon rulers can likely become more widely used as optical "angstrometers" in various types of sensing devices.

References

1. J. Anker et al., Nat. Mater. 7, 6, 442–453 (2008).

2. C. Sönnichsen et al., Nat. Biotechnol. 23, 6, 741–745 (2005).

3. P.K. Jain et al., Nano Lett. 7, 7, 2080–2088 (2007).

4. L. Guo et al., Small 9, 2, 234–240 (2012).

5. J. I. L. Chen et al., Small 7, 14, 1993–1997 (2011).

6. L. Wu et al., Chem. Soc. Rev. 43, 11, 3884–3897 (2014).

7. R. T. Hill et al., ACS Nano 6, 10, 9237–9246 (2012).

8. J. J. Mock et al., Nano Lett. 12, 4, 1757–1764 (2012).

9. R. T. Hill et al., Nano Lett. 10, 10, 4150–4154 (2010).

10. J. J. Mock et al., Nano Lett. 8, 8, 2245–2252 (2008).

Ryan T. Hill is a senior research scientist in the Department of Biomedical Engineering, Box 90271, Duke University, Durham, NC 27708; e-mail: [email protected]; http://people.duke.edu/~rth5/.

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