Highly sensitive virus detection is essential for controlling infectious disease and for monitoring viruses in medical, food, and environmental settings. Polymerase chain reaction (PCR) can detect very small amounts of viral nucleic acid, but it also detects genetic material from inactivated viruses. PCR therefore does not directly measure whether a virus remains capable of infection.
An international research team has now developed a fluid material that enables highly sensitive quantitative measurement of viral infectivity. The material was inspired by liquid-liquid phase-separated microdroplets inside cells, which act as microreactors by concentrating biomolecules and accelerating biochemical reactions.
The new material consists of dextran-rich microdroplets dispersed in a polyethylene glycol-rich aqueous phase. Conventional dextran droplets rapidly fuse and undergo macroscopic phase separation, limiting their use as stable three-dimensional reaction compartments. The researchers overcame this limitation by adding nanofibers formed by an azobenzene-containing self-assembling peptide, AzSAP. The nanofiber network suppresses droplet coalescence for extended periods while maintaining the macroscopic fluidity needed for flow cytometry.
When M13 bacteriophages and their host cells, E. coli, were localized within the stabilized droplets, newly produced phages were largely prevented from spreading to additional droplets. This confinement preserved the relationship between the initial phage concentration and the number of fluorescent infection-positive droplets. The researchers used a fluorogenic substrate, DDAO galactoside, which produces far-red DDAO fluorescence after cleavage by beta-galactosidase generated in infected E. coli cells. Counting fluorescent droplets by confocal microscopy or flow cytometry therefore provided a quantitative readout of viral infectivity.
The resulting method, termed the microflow assay, achieved more than a tenfold improvement in sensitivity compared with conventional plaque assays and substantially shortened the measurement workflow.
Figure 1. (a) Molecular structure of AzSAP and (b,c) schematic comparison of conventional dextran-rich droplets with AzSAP-stabilized droplets. (b) Without AzSAP, droplets rapidly coalesce and undergo macroscopic phase separation. (c) In the presence of AzSAP nanofibers, the droplets remain stable and fluid and can be manipulated in space and time. (Figure adapted from Nature Communications)
Figure 2. Structural characterization of AzSAP nanofibers. (a) Circular dichroism spectroscopy and (b) transmission electron microscopy show beta-sheet-rich nanofiber formation. (c) Cryogenic electron microscopy reveals (d) two protofilaments and (e) a 14-molecule, butterfly-shaped cross-section with azobenzene-rich hydrophobic cores. (Figure adapted from Nature Communications)
Figure 3. Stability and photo-control of dextran-rich droplets. (a,c) Conventional droplets fuse within minutes, (b,d) whereas AzSAP-stabilized droplets remain dispersed for more than 24 hours. (e–i) Ultraviolet irradiation disrupts the nanofiber network and induces rapid fusion in the illuminated region. Scale bar: 100 μm. (Figure adapted from Nature Communications)
Figure 4. Quantitative detection of M13 bacteriophage infection. (a) M13 phages and E. coli are confined within AzSAP-stabilized dextran-rich droplets. DDAO galactoside is converted to fluorescent DDAO by beta-galactosidase produced in infected cells, allowing infection-positive droplets to be quantified by (b–e) confocal microscopy or (f) flow cytometry. (Figure adapted from Nature Communications)
Papers
Journal: Nature Communications
Title: Supramolecular nanofiber stabilization of microdroplets enables ultra-sensitive virus infection analysis
Authors: Noriyuki Uchida, Atsuya Yaguchi, Shiori Kondo, Kazuyoshi Muranishi, Haruka Kawabata, Haruka Umezawa, Naito Ishimoto, Michiko Tajiri, Satoko Akashi, Takuya Seki, Go Watanabe, Ayano Yoshida, Genki Higuchi, Jakuei Fu, Takanobu Takenouchi, Daisuke Yoshino, Hirotsugu Hiramatsu, Masaki Okumura, Tomohide Saio, Hiroyuki Noji, Young-Ho Lee, Sam-Yong Park, Takahiro Muraoka
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