We’ve recently witnessed the stunning images of distant galaxies revealed by the James Webb telescope, previously only visible as blurry spots. Researchers at the University of Washington in St. Louis have developed a new method for visualizing the proteins secreted by cells with stunning resolution, making it the James Webb version for visualizing the secretion of single-cell protein.
The researchers, led by Srikanth Singamaneni, the Lilyan & E. Lisle Hughes Professor of Mechanical Engineering & Materials Science at the McKelvey School of Engineering, and Anushree Seth, a former postdoctoral scientist in Singamaneni’s lab, developed the FluoroDOT assay, which they introduced in an Aug. 5 paper in the journal Cell Reports Methods. The highly sensitive assay can see and measure proteins secreted by a single cell in about 30 minutes.
Working with researchers from Washington University School of Medicine and other universities, they found that the FluoroDOT test is versatile, inexpensive, adaptable to any lab setting and has the potential to provide a more comprehensive look at these proteins than the commonly used ones. existing tests. Biomedical researchers look to these secreted proteins for information about cell-to-cell communication, cell signaling, activation and inflammation, among other actions, but existing methods are limited in sensitivity and can take up to 24 hours to process.
What makes the FluoroDOT test different from existing tests is that it uses a plasmonic-fluorine, a plasmon-enhanced nanolabel developed in Singamaneni’s lab that is 16,000 times brighter than conventional fluorescent labels and has a signal-to-noise ratio nearly 30 times higher. is.
Plasmonic fluorine is composed of metallic nanoparticles that act as an antenna to pull the light in and amplify the fluorescence emission of molecular fluorophores, making it an ultra-bright nanoparticle.”
Srikanth Singamaneni, the Lilyan & E. Lisle Hughes Professor of Mechanical Engineering & Materials Science, McKelvey School of Engineering
This ultra-bright emission of plasmonic fluorine allows the user to see extremely small amounts of secreted protein, which they cannot do in existing tests, and digitally measure the high-resolution signals using the number of particles, or dot pattern, per cluster, or spot, using a custom algorithm. In addition, it does not require any special equipment. Singamaneni and his collaborators first published their work with the plasmonic fluorine in Nature Biomedical Engineering in 2020.
The patent-pending plasmonic fluorine technology is licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC.
“Using a simple fluorescence microscope, we can simultaneously image a cell along with the spatial distribution of the proteins secreted around it,” said Seth, who had worked in Singamaneni’s lab and is now a lead scientist in cellular applications for Auragent. bioscience. “We saw interesting secretion patterns for different cell types. This assay also allows for simultaneous visualization of two types of proteins from individual cells. When the multiple cells are subjected to the same stimuli, we can distinguish the cells that secrete two proteins at the same time. Over time from those that only secrete one protein or not secrete at all.”
To validate the technology, the team used proteins secreted by both human and mouse cells, including immune cells infected with Mycobacterium tuberculosis.
One of the contributors and co-authors, Jennifer A. Philips, MD, PhD, the Theodore and Bertha Bryan Professor in the Departments of Medicine and Molecular Microbiology and co-director of the Department of Infectious Diseases at the School of Medicine, has used the FluoroDOT test in her lab.
“When Mycobacterium tuberculosis infects immune cells, those cells respond by secreting important immune proteins called cytokines,” Philips said. “But not all cells respond to infection in the same way. With the FluoroDOT assay, we were able to see how individual cells in a population respond to infection — to see which cells secrete and in which direction. This was not possible with the older technology.” .”
Washington University in St. Louis
Seth, A., et al. (2022) High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay. Cell reporting methods. doi.org/10.1016/j.crmeth.2022.100267.