Biosensing with fluorescent emulsions
The Swager Group combines principles of organic chemistry and materials science to produce innovative solutions for detecting foodborne pathogens
COURTESY OF THE SWAGER GROUP
A deadly foodborne bacteria, Listeria is one of the leading causes of food poisoning deaths via its namesake disease, listeriosis. Able to live through high levels of stress, Listeria is one of the pathogens frequently screened for in foods. However, current methods for screening are costly and require at least one day to grow cultures, a timeline that is often impractical for public safety. With such limitations, a new method for detecting Listeria is needed, a problem that the Swager Group at MIT Chemistry has tackled using a combination of materials science and traditional biosensing.
In order to detect pathogens, the Swager Group utilized a microparticle known as Janus droplets. Aptly named after the two-faced god in Roman mythology, Janus droplets are emulsions with two sides composed of different materials. Just as oil and water don’t mix, Janus droplets are made of organic hydrocarbons on one side and fluorocarbons on the other, held together by careful adjustments of the surface tension of the liquids. The droplets are dispersed in water containing surfactants and naturally orient themselves by density. Fluorocarbons are similar in structure to hydrocarbons, except with many of the hydrogens replaced with the heavier fluorine, so the lower side of the droplets naturally consists of fluorocarbons.
For the actual biosensing aspect, researchers in the Swager lab coat one side of the droplets — the hydrocarbon face — with antibodies specific to Listeria. To do so, they use a burgeoning field in chemistry known as “click chemistry,” which involves functionalizing two chemicals with complementary groups and allowing them to react. In this case, the Janus droplets are modified with trans-cyclooctene (TCO) groups, which react efficiently with tetrazines added to the antibodies. Coated with antibodies, these Janus droplets are now able to bind to Listeria. As multiple Janus droplets begin to surround and attach to the bacterium, a process known as agglutination, the droplets are dragged around. Since only one side of the droplet is covered by antibodies, this dragging motion causes the Janus particles to rotate, resulting in a detectable difference from above; previously only the hydrocarbon phase was visible, but the rotation exposes the fluorocarbon.
Innovative as this approach is, the technical details are challenging. In order to make the rotation of the droplets detectable, the group employs two dyes: a blocker dye to absorb incoming light and a fluorophore that absorbs and re-emits light. Kosuke Yoshinaga, a fifth-year graduate student in the Swager lab, focuses on developing the dyes for this project. He describes the two primary considerations for these dyes: their spectra (what wavelengths they absorb and emit at) and their solubility. “Very limited examples of dyes can be soluble in the fluorocarbon phase,” Yoshinaga says. He adds, “You want to make something that you can manipulate with organic chemistry, but also maintain the fluorocarbon solubility, which is kind of contradictory because it’s organic but kind of fluorous.” In addition to solubility considerations, the two dyes must have compatible spectra; the blocking dye must not only block most incoming radiation but also absorb the emissions of the fluorophore in order to establish maximum contrast. Walking this tightrope, the group settled on subphthalocyanines for the blocking dye and a perylene bisimide for the emissive dye, which they amicably call the “Kosuke dye.”
So how does this technique stack up compared to others? Jie Li, a third-year postdoctoral student in the lab, seems enthusiastic about its potential. Not only does it achieve a high level of sensitivity (100 CFU/mL), but this technique covers its predecessors’ weaknesses. As Li explains, “I think our advantage in comparison with other detection methods of Listeria is that our whole setup is very cheap, and our detection is quick.” Each sample requires only 10–20 microliters of solvent for screening, making the setup significantly more cost-friendly and scalable; meanwhile, the speed and ease of detection also is greatly improved, requiring only two hours and a one-step mixing for detection.
Despite the progress, both Yoshinaga and Li are excited for the further improvements that can be made. For the dyes, Yoshinaga is still searching for more emissive structures to enhance the detection limits. In addition, they’re looking for ways to increase the stability of the droplets, such as by converting the system into solid form, which could increase the shelf life to years. Moreover, while the published work on this Janus detection system was for Listeria, this method is hardly limited to Listeria. By switching out the antibody from one to another, this system can be applied to practically any pathogen, whether bacterial or viral. With such a versatile and novel system, it’s easy to sense the potential the Swager Group has in biosensing.
The Swager Group’s work on the biosensing of Listeria can be found in their paper published in PNAS.