Practical Plasmonic Crystal Biosensors

In the realm of myths, legends, and the occult, crystals have always been believed to have extraordinary powers, from protection and healing with crystal talismans to foretelling the future with crystal balls. Scientific discoveries about crystals may be less dramatic, but they are no less amazing. In a recent example, researchers at the University of Illinois, Urbana-Champaign (UIUC) and Argonne National Laboratory developed a small, low-cost crystal array that makes a highly sensitive biosensor, and used computational modeling to explain how it works.

What the researchers achieved was a breakthrough in a common but, up till now, expensive technique for measuring binding interactions, such as those between DNA and proteins, based on changes in the refractive index near a metal surface. The technique, called surface plasmon resonance or SPR, is used in diagnostic bioassays and in research ranging from drug discovery to immunology, virology, and other fields.

Just as a pebble tossed into a pond produces waves on the surface of the water, a beam of light shining on a plasmonic crystal produces electron waves on the crystal’s surface. “SPR is simply light causing a collective excitation of electrons near the surface of a metal,” explained Stephen Gray, a chemist at Argonne National Laboratory, who created the simulations that analyzed the experimental results.

 “Those collective excitations of electrons are like waves on the metal’s surface,” Gray continued, “and light shining around different objects above the metal creates different waves. When we use plasmonic crystals as sensors, small changes in the material specimen produce changes in the refraction index which can be measured with a spectrophotometer. From those responses, you can infer what the material is and how it has changed.”

While the scientific understanding of SPR may be modern, its application has a long history. “The ruby red color in some medieval stained-glass windows is the result of surface plasmon resonance,” Gray pointed out. “Gold nanoparticles that were added to the glass scatter and absorb light in a way that produces a pleasing color.”

As a sensing technology, SPR has the advantage of not requiring that fluorescent labels be added to samples, as in fluorescence microscopy. But SPR to date has had a variety of limitations. The first SPR systems used prisms, but those systems were too bulky to be portable. More recent systems employ plasmonic crystals in the form of nanostructured films or nanoparticles; these systems are more portable but less sensitive, and fabricating large and uniform arrays of plasmonic crystals has been prohibitively expensive.

But all that may be changing. In an experiment reported in the Proceedings of the National Academy of Sciences, [1] Ralph Nuzzo, John Rogers and co-workers at UIUC’s Frederick Seitz Materials Research Laboratory developed a low-cost crystal array to make a highly sensitive sensor. Using soft nanoimprint lithography, a technique that uses a soft polymeric mold to stamp and create structures on a substrate, the researchers created a plasmonic crystal consisting of a regular array of cylindrical wells in gold film on a polyurethane substrate (Figure 1). The SPR effects were produced on the nanoscale holes in the gold film and on the separate gold disks at the bottoms of the wells. SPR waves can be modeled mathematically using Maxwell’s equations, so Gray was able to do a detailed computational analysis of the optical properties of the new crystals and the complex electromagnetic field distributions around the multilevel nanostructured features (Figure 2).

Figure 1. Images and schematic illustrations of a quasi-3D plasmonic crystal. (A) Scanning electron micrograph (SEM) of a crystal. (Upper Inset) A low-resolution optical image illustrating the diffraction colors produced by these structures. (Lower Inset) A high-magnification SEM that shows the upper and lower levels of gold. (B) Schematic illustration of the normal incidence transmission mode geometry used to probe these devices. The intensity of the undiffracted, transmitted light is monitored across the UV, visible, and near-infrared regions of the spectrum. (Inset) A close-up schematic illustration of the crystal. (Click image for larger view)		Figure 2. Correlation of transmission spectral features with hole/disk plasmonic excitations. (A) Normal incidence transmission spectrum of a quasi-3D plasmonic crystal (blue), and rigorous electrodynamics modeling of the spectrum for an ideal crystal (green) and one that includes subtle isolated nanoscale grains of gold near the edges of the gold disks (red). (B) Computed electromagnetic field distribution associated with the resonance at 883 nm (labeled B in A). The intensity is concentrated at the edges of the nanoholes in the upper level of the crystal. (C) Field distribution associated with the resonance at 1,138 nm (labeled C in A), showing strong coupling between the upper and lower levels of the crystal. (Click image for larger view)

Interestingly, Gray’s initial idealized crystal model produced spectral features (Figure 2A, green line) that did not quite match the experimental results (blue line); but when he added small defects in the form of isolated grains of gold on the sides of the wells near the bottom, the match was close to perfect (red line). Scanning electron micrographs confirmed that there were indeed grains of gold at the edges of the recessed gold disks (Figure 3).

Figure 3. Tilted SEM image of an individual nanohole showing grains of gold at the edges of the recessed gold disk. (Click image for larger view)

“This showed how, at the nanoscale, very small defects can have important effects,” Gray said. It also shows how computational modeling could be used to figure out how to fine-tune the performance of the system.

The unusual geometry and uniformity of these crystals gives them high sensitivity to multiple wavelengths over large sample areas with micrometer spatial resolution. The research team used a well studied ligand–receptor pair, biotin and avidin, as a model system to illustrate the functionality of these crystals in a quantitative analytical bioassay, and they were able to detect molecular binding in a single layer.

Because these plasmonic crystal arrays are smaller than typical sensing or imaging systems, and in view of their high sensitivity, low-cost fabrication, and simple readout apparatus, this technology could be used in developing the next generation of portable diagnostic sensors. They could easily be integrated into microfluidic lab-on-a-chip instrumentation.

[1] Matthew E. Stewart, Nathan H. Mack, Viktor Malyarchuk, Julio A. N. T. Soares, Tae-Woo Lee, Stephen K. Gray, Ralph G. Nuzzo, and John A. Rogers, “Quantitative multispectral biosensing and 1D imaging using quasi-3D plasmonic crystals,” PNAS 103, 17143 (2006).