A biosensor made of fluorescent proteins that are embedded in the shell of microscopic marine algae called diatoms could help detect chemicals in water samples. The device could help produce nanomaterials that can solve sensing, catalysis and environmental remediation problems. The fluorescent biosensor was developed by researchers at the Department of Energy’s Pacific Northwest National Laboratory, who were inspired by previous research showing that it is possible to insert proteins in diatom shells through genetic engineering. Diatoms make up the bulk of phytoplankton, the plant base of the marine food chain. Using that work as a starting point, PNNL Laboratory Fellow Guri Roesijadi and molecular biologist Kate Marshall and their colleagues had the goal of using fluorescent proteins to turn diatoms into a biosensor. They specifically wanted to create a reagentless biosensor — one that detects a target substance on its own without depending on another chemical or substance. PNNL researchers genetically engineered this microscopic marine diatom to become a biosensor for the sugar ribose. From left to right: The engineered diatom without fluorescence; the same diatom exhibiting blue fluorescence; and when no ribose is present, the diatom generates a bright-yellow fluorescence via fluorescence resonance energy transfer. Amnis Corp. of Seattle used its ImageStream imaging flow cytometer to take these images. (Image: Pacific Northwest National Laboratory) The team inserted genes for its biosensor into Thalassiosira pseudonana, a marine diatom whose shell resembles a hatbox. The new genes allowed the diatoms to produce a protein that is the biosensor. At the heart of the biosensor is ribose-binding protein. Each ribose-binding protein is flanked by two other proteins, one that glows blue and one that glows yellow. This three-protein complex attaches to the silica shell while the diatom grows. In the absence of ribose, the two fluorescent proteins sit close enough to one another that the energy in the blue protein's fluorescence is easily handed off, or transferred, to the neighboring yellow protein. This process, called fluorescence resonance energy transfer, is akin to the blue protein’s shining a flashlight at the yellow protein, which then glows yellow. When ribose binds to the diatom, however, the ribose-binding protein changes its shape. In the process, the blue and yellow fluorescent proteins are moved apart, and the amount of light energy that the blue protein shines on the yellow protein decreases, causing the biosensor to display more blue light. A side and overhead view of the microscopic marine diatom Thalassiosira pseudonana. PNNL scientists used this species to develop a fluorescent biosensor that changes its glow in the presence of the sugar ribose. (Image: Nils Kröger, Universität Regensburg) The biosensor will always emit a blue or yellow glow when it's exposed to energy under a microscope, regardless of whether ribose is bound to the diatom's biosensor. The key difference, however, is how much of each kind of light is displayed. The team distinguished between the light from the two proteins using a fluorescence microscope equipped with a photon sensor. The sensor allowed the investigators to measure the intensities of the unique wavelengths of light given off by each fluorescent protein. They calculated the ratio of the two wavelengths to determine whether the diatom biosensor was exposed to ribose, and how much ribose was present. The team also succeeded in making the biosensor work with the shell alone, after it was removed from the living diatom. Removing the living diatom provides researchers greater flexibility in how and where the silica biosensor can be used. The Office of Naval Research, which funded the research, believes biosensors based on modifying a diatom's silica shell may prove useful for detecting threats such as explosives in the marine environment. “Like tiny glass sculptures, the diverse silica shells of diatoms have long intrigued scientists,” Marshall said. “With this research, we’ve made our important first steps to show it’s possible to genetically engineer organisms such as diatoms to create advanced materials for numerous applications.” The biosensor was described in PLoS One. For more information, visit: www.pnnl.gov