Researchers at Delft University of Technology (TU Delft) have developed drums made out of ultrathin bilayers of graphene that can detect the tiny movements, or vibrations, made by a single, live bacterium, and which could pave the way to development of a high-throughput, rapid screening system for testing antibiotic resistance. Led by Farbod Alijani, PhD, the TU Delft team carried out experiments using the suspended graphene drum technology to investigate the source of the nanometer-scale oscillations made by individual E. coli bacteria, and to evaluate use of the system to assess antibiotic resistance. In their report in Nature Nanotechnology, they concluded, “With the significant reduction in size and increase in sensitivity presented in this work, nanomotion detection potentially can evolve into an important non-invasive monitoring tool in cell biology and provide new routes for rapid screening tests in personalized medicine and drug development…This opens new routes towards faster, label-free detection of antimicrobial resistance at the single-cell level with potential applications in drug screening and rapid diagnostics.” The team’s paper is titled “Probing the nanomotion of single bacteria with graphene drums.”

Have you ever wondered if bacteria make distinctive sounds? If they do, and we could listen to them, we could detect whether the bacteria were alive or not. And if the bacteria were killed using a particular antibiotic, those sounds would stop—unless the bacteria were resistant to the antibiotic, in which case the sounds would continue.

In fact, motion is a key characteristic of every form of life, the researchers noted. “Living cells exhibit nanomechanical vibrations as a result of the biological processes that govern their growth, function and reproduction.” And while there are many hypotheses explaining the underlying driving mechanisms, such as the movement of organelles, ion pumps, or redistribution of the cell membrane, a consensus hasn’t been reached, partly because investigating cellular biomechanics noninvasively is hugely challenging. Newer technologies, such as mechanical cantilevers, have been used to detect vibrations of bacteria in liquid, and have found that nanomotion of these bacterial populations does rapidly decrease in the presence of antibiotics, “which holds great promise for the development of rapid antibiotic susceptibility testing technologies,” the team noted. However, scientists still need to understand the origins of these cellular nanomotions.

Farbod Alijani’s team was originally looking into the fundamentals of the mechanics of graphene, but they also wondered what would happen if this extremely sensitive material came into contact with a single biological object. “Graphene is a form of carbon consisting of a single layer of atoms and is also known as the wonder material,” says Alijani. “It’s very strong with nice electrical and mechanical properties, and it’s also extremely sensitive to external forces.”

To investigate further, the researchers initiated a collaboration with the nano biology group of Cees Dekker, PhD, and the nanomechanics group of Peter Steeneken, PhD, together with PhD student Irek Roslon and postdoc Aleksandre Japaridze, PhD. The graphene drums were made of an ultrathin (less than 1 nm) bilayer of graphene that was used to cover circular cavities of diameter 8 micrometers, and a depth of 285 nm, etched into silicon dioxide. A silicon chip containing an array of thousands of these graphene-covered cavities was used to detect the oscillations of individual E. coli bacteria in growth medium, attached to the graphene surface. The nanomotion of a bacterium effectively caused deflection of the suspended graphene membrane, and this could be measured by laser interferometry.

Artist’s impression of a graphene drum detecting nanomotion of a single bacterium [Irek Roslon, TU Delft]

Describing the results of tests with drums containing a single live bacterium, Dekker said, “What we saw was striking! When a single bacterium adheres to the surface of a graphene drum, it generates random oscillations with amplitudes as low as a few nanometers that we could detect. We could hear the sound of a single bacterium!”

The extremely small oscillations, they found, are a result of the biological processes of the bacteria, with the main contribution from the bacterial flagella, which are the tails on the cell surface that propel the bacteria. This was confirmed through experiments using strains of E. coli that were genetically engineered to exhibit varying levels of motility, dependent on the number, or activity of their flagella. “To understand how tiny these flagellar beats on graphene are, it’s worth saying that they are at least 10 billion times smaller than a boxer’s punch when reaching a punch bag,” Alijani said. “Yet, these nanoscale beats can be converted to sound tracks and listened to—and how cool is that.”

The research could feasibly have potential applications in the development of detection systems for antibiotic resistance, so the team carried out tests in which they monitored nanomotion of the graphene drums resulting from the vibrations of single E. coli bacteria that were either susceptible, or resistant to an antibiotic. The experimental results unequivocally showed that if the bacteria were resistant to the antibiotic, the oscillations just continued at the same level. When the bacteria were susceptible to the drug, vibrations decreased until one or two hours later, and then they were completely gone. Thanks to the high sensitivity of graphene drums, this phenomenon could be detected using just a single cell. “This experiment demonstrates the potential of graphene devices as an indicator of bacterial physiology, and opens new routes for determining the temporal response of bacteria to antibiotics at single-cell level,” the authors stated.

Alijani commented, “For the future, we aim at optimizing our single-cell graphene antibiotic sensitivity platform and validate it against a variety of pathogenic samples. So that eventually it can be used as an effective diagnostic toolkit for fast detection of antibiotic resistance in clinical practice.” Steeneken further stated, “This would be an invaluable tool in the fight against antibiotic resistance, an ever- increasing threat to human health around the world.”

The ability to generate data at the single cell level is particularly valuable, the authors noted, as it is a label-free approach that can be used directly on clinical samples for antibiotic resistance screening. Generating data at the single cell level could allow for the identification and study of “persister cells” that are related to the emergence of antibiotic resistance within a population. “Our antibiotic susceptibility experiments demonstrated that the graphene drum sensing platform can trace the effect of antibiotics on bacterial nanomotion in real-time,” the team stated. “This opens the way to fast, label-free susceptibility testing down to the single bacterial level.” In comparison with other techniques for detecting antibiotic susceptibility, the newly reported method stands out with respect to both sensitivity and speed, the investigators added. “The small size of the graphene drums enables high-throughput sensing, allowing, in principle, millions of cells to be monitored in parallel in the presence of antibiotics. Similar benefits might apply in the field of personalized medicine, where the right antibiotic can be rapidly selected on the basis of the nanomotion response.”