Shine a light through a stained-glass window and the colors will change depending on where you stand. Metallic nanoplasmonic structures are behind this trick of the light. While artists have been working with these microscopic materials for centuries, scientists have recently become interested, too, largely because of the unique way nanoplasmonic structures interact with light. This is due to a phenomenon called localized surface plasmon resonance, in which metal nanostructures with special dielectric properties allow light to trigger oscillation of free electrons on the metal surface.

Nanoplasmonic materials are currently being investigated for uses ranging from solar cells and light generation to memory storage—and some research teams are interested in using them as biosensors, as well. Examples of applications as biosensors include testing food for contamination, diagnosing disease, monitoring molecular interactions, and counting dividing cells in real time. Now a nanoplasmonic material composed of silicon dioxide stems with gold mushroom-like caps brings researchers one step closer to realizing those goals.

“On the fabrication side, I feel like we’ve opened a door to potentially combine this with industrial protocols and standards,” says Amy Shen, a professor in the Micro/Bio/Nanofluidics Unit at the Okinawa Institute of Science and Technology Graduate University in Japan. “On the application side, we’re exploring [the] nanophotonic substrate for biological applications, including cell proliferation, differentiation, detection, and identification of infections.”   

While Shen and her colleagues’ nanoplasmonic biosensor is not the first to be developed, previous versions tended to detrimentally impact cells, oftentimes killing them or interfering with their ability to thrive. In contrast, as Shen and her colleagues described in a recent issue of Advanced Biosystems, fibroblast cells cultured on their new material survived for over seven days. “Our substrate is completely biocompatible, meaning we don’t kill microorganisms,” Shen says. “That’s quite remarkable.”

In addition, the material possesses excellent sensing properties, which allows real-time counting of cells while they proliferate. “This work demonstrates that nanoplasmonics can be used to monitor cell behavior in real time without interfering with normal cellular processes,” says Nikhil Bhalla, a postdoctoral researcher in Shen’s laboratory.

Nanoplasmonic biosensors have also tended to be difficult to produce in a uniform way without involving a complex, time-consuming, and expensive process. Shen believes this new version can eventually be sold for as little as $1 when manufactured in mass production. As she and her colleagues described in a protocol-focused paper published in ACS Applied Materials and Interfaces in December 2017, fabrication began with evaporating a 4-nm-thick layer of gold onto a glass substrate. Silver, titanium or other metals could also be used, Shen adds, and the thickness of the metallic layer can be altered to change the eventual size of the mushroom-like structures.

Next, they heated the slide to 560°C to anneal the gold and then allowed it to solidify, leaving them with loosely dispersed gold nano-islands. The final step involved exposing the substrate to reactive ion etching inside a standard SF₆ plasma chamber for 3-5 min. This caused the gold nano-islands to transform into mushroom-like structures. The researchers are still teasing out the physics behind this process, but they believe that differences in the etching rate of gold and glass, and differences in the materials’ interactions with the gas, cause the less volatile gold to fill empty spaces left by the glass.

What is left behind is a 2.5 cm × 7.5 cm slide containing a highly homogenous layer of around one million pillar-like silicon dioxide structures topped by 15-20-nm-wide gold mushroom-like caps, spaced at intervals of 10 nm or less. This is a very desirable structure, Shen says, because it provides a higher aspect ratio compared to spherical nano-islands, making the final product more sensitive to reflective light index variations.

Like other nanoplasmonic materials, the new biosensor absorbs and scatters light in the range of visible and infrared spectra that passes through it, allowing the researchers to measure peak wavelength intensity with a spectrometer. If a solution containing antibodies is poured onto the biosensor, the microcontact-printed proteins will cause those pathogen-fighting proteins to attach to the gold nanocaps and change the signal detected by the spectrometer.

By adding certain antibodies to the biosensors, the researchers believe they can create tests for different diseases. When antibody-primed biosensors are exposed to blood, saliva, or urine containing biomarkers for disease, those molecules will bind to the antibodies and cause yet another wavelength shift. “Such shifts could identify specific disease presence,” says Shivani Sathish, a doctoral candidate in Shen’s laboratory, who is currently developing bioassays for the nanomushroom substrate.

“We’re working on developing a small, simple detection unit that can be coupled with a smart phone readout for use in remote areas and developing countries,” Shen says. “In theory, it could be used for detecting any disease.”

Finally, the new biosensor could also be used for more basic medical and biological research, including monitoring of bacterial biofilm growth in real time. In subsequent work, not yet published, the researchers were able to monitor kinetics of bacterial biofilm formation in the presence and absence of antibiotic drugs.

According to Jules Hammond, a biotechnologist at the Université Grenoble Alpes who was not involved in the research, Shen and her colleagues’ sensor is particularly useful because it does away with the need for traditional labelling techniques used for monitoring cells, including dyes and fluorescent molecules, which can add time and cost.

“Furthermore, the printing process used by the group offers an attractive route to scaling up fabrication of the material for use in other applications such as biosensing, environmental monitoring, and biodefense,” he says. “This label-free nanoplasmonic platform therefore offers a promising prospect for low-cost and portable cell assay tools.”

Read the abstract in Advanced Biosystems.