[NEDO] World’s first technology to detect and separate microorganisms inside droplets has been developed
NEDO Press release: September 26, 2024 We have developed the world’s first technology that can detect and separate microorganisms inside droplets. Accelerating the search for microbial resources that support biomanufacturing In the
NEDO-entrusted project, “Development of bio-based product production technology to accelerate the realization of carbon recycling (Bio-manufacturing project)” (hereinafter referred to as this project), NEDO is working with the National Institute of Advanced Industrial Science and Technology (hereinafter referred to as INPEX). Research Institute) and OnChip Biotechnologies Co., Ltd. are collaborating to dye microbial membrane components with fluorescence to improve water-in-oil production. We have developed the world’s first technology (hereinafter referred to as this technology) that can detect and separate microorganisms inside droplets (hereinafter referred to as droplets). The detection reagent used in this technology will be commercialized by OnChip Biotechnologies and will be on sale from October 1st. This technology is the basic technology for million screening, which uses droplets to screen a wide variety of microorganisms that exist in the environment at the level of 1 million samples. Through demonstration tests using E. coli, we have confirmed that droplets containing microorganisms can be clearly identified and separated within a microchannel. In the future, in addition to developing examples of the use of this technology, expanding its scope of application, and developing useful applications, we will work on further strengthening and improving the technology. In addition, we aim to provide solutions to the requests for microbial screening using this technology from companies aiming at bio-manufacturing. This will accelerate bio-based manufacturing and contribute to the realization of a bioeconomy society. Some of the results of this technology will be exhibited at the NEDO booth at “BioJapan2024” to be held at Pacifico Yokohama from October 9th to 11th, 2024.
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expectations for the creation of highly optimized cells called “smart cells” that can be applied to manufacturing production processes, such as microorganisms. However, for the development of more advanced industrial smart cells, there are not enough host cells and genetic resources (microbial resources) to form the parts, and diversification and expansion are desired. In order to meet this demand, million screening using droplets, which can speed up the search for microbial resources, is attracting attention. Million screening requires the development of technology to detect and separate microbial growth and target products in droplets. Against this background, NEDO has been working on the development and demonstration of basic technology for million screening in this project*1 since fiscal 2020. As part of this effort, we are collaborating with AIST and OnChip Biotechnologies to develop technology to detect and isolate microbial growth inside droplets. 2. This result (1) Development of technology to detect microbial growth in droplets using membrane staining reagents In Million Screening, microorganisms are cultured in liquid in droplets with a diameter of several tens of micrometers, and then positive droplets in which microorganisms have grown inside are selectively separated based on detection of fluorescence, scattered light, etc. To date, the growth of microorganisms within droplets has been detected by detecting cell autofluorescence or by using reagents that react with extracellular secretions. The problem with autofluorescence detection is low sensitivity, and methods that use reagents that react with extracellular secretions have had problems with false positives and increased background due to extracellular substances mixed in from the environment. Therefore, we developed a method for detecting microbial growth using a fluorescent reagent that specifically stains the membrane of microorganisms, as a detection method that keeps cells alive, has high sensitivity, and suppresses background increase. By coexisting membrane-staining dyes that increase fluorescence intensity when localized on the membrane with microorganisms in droplets, the droplets emit strong fluorescence as microorganisms grow, making it possible to specifically detect them. (Figure 2)
https://prcdn.freetls.fastly.net/release_image/135644/115/135644-115-e11a0843b52afaa350ed4ccbcb8f6fed-1453×522.png Figure 2 Microscopic image of a droplet (30 µm) encapsulated with membrane staining reagent (green) and E. coli and cultured overnight (Left: dark field microscope image, right: fluorescence microscope image) Scale bar: 100 µm (2) Demonstration on the Million Screening Platform In order to verify the usefulness of the microorganism detection technology developed this time, we used Escherichia coli to detect microorganisms in a microchannel, which is a platform for million screening, and a droplet sorter that sorts positive droplets in a microchannel. I tried preparative separation. By staining each droplet with a membrane staining reagent and detecting and evaluating the fluorescence intensity of each droplet within the microchannel, it is possible to clearly distinguish between droplets that do not contain microorganisms and droplets that have grown microorganisms inside. (Figure 3). Furthermore, from the situation where
approximately 90% of droplets are empty and approximately 10% of droplets contain microorganisms, by using this technology, the percentage of droplets that contain microorganisms can be increased to 100% at maximum. We succeeded in such preparative separation. The Million Screening platform has been shown to be able to detect and selectively separate droplets with growing microorganisms. As a result, for the first time in the world, we have developed a fluorescence detection and preparative separation method based on the amount of microbial film (number of microorganisms) present within a droplet *2.
https://prcdn.freetls.fastly.net/release_image/135644/115/135644-115-b3e95e1e668833fd9ec696da9fa87481-727×353.png Figure 3 Sorting and fractionation of droplets (120 µm) encapsulated with membrane staining reagent (red) and E. coli and cultured overnight Scale bar: 100 µm 3. Future plans The detection reagent used in this technology will be commercialized by OnChip Biotechnologies and will be on sale from October 1st. In the future, we will work on further strengthening and improving the technology, in addition to developing examples of the use of this technology, expanding the range of applications, and developing useful applications. In addition, we aim to solve problems by supporting companies aiming for
biomanufacturing and requesting screening using this technology. This will accelerate bio-based manufacturing and contribute to the realization of a bioeconomy society. Some of the results of this technology will be exhibited at the NEDO booth at “BioJapan2024” to be held at Pacifico Yokohama from October 9th to 11th, 2024. [Note] *1 This project Business name: Development of bio-based product production technology to accelerate the realization of carbon recycling Project period: FY2020-FY2026 Business overview: Development of bio-based product production technology that accelerates carbon recycling https://www.nedo.go.jp/activities/ZZJP_100170.html *2 Intellectual property information JP2024-018130 “Method for detecting microbial growth, method for obtaining microorganisms, kit for detecting microbial growth, kit for obtaining microorganisms, and use of dye as microbial growth reporter” (Publication date: February 8, 2024)