(Invited) Observation of Stress Responses of Bacteria Confined in a MEMS Microfluidic Chip
Authors: Nishimura Yusuke , Ishida Makoto , Sawada Kazuaki , Ishii Hiromu , Machida Katsuyuki , Masu Kazuya , Wang Changle , Iida Kenichiro , Saito Mitsumasa , Yoshida Shinichi 
Published: February 2020

This work describes the new and simple method for observing bacterial behavior of stress response such as to light irradiation in a polydimethylsiloxane (PDMS) -based MEMS microfluidic chip. The fluorescence was observed from Legionella cells confined in small spaces among microbeads. Ultraviolet (UV) light irradiation was found to be a trigger for producing fluorescent material for Legionella.Confining bacteria in the small spaces made by the MEMS technique is an effective way for investigating their stress response.

Sensing bacteria is attracting a great deal of interest to avoid an outbreak of bacterial infection. We have been developing sensors for bacteria, Legionella pneumophila (L. pneumophila), which is widely known as one of the pathogenic bacteria [1]. The sensors used for this purpose have small cavity structures for trapping the bacteria. During the course of the study for trapping bacteria in the small cavities, we found that the cells emit fluorescence by the UV excitation just when they are trapped and restricted their motion in the cavities or narrow spaces [2]. This fact implies that such bacteria might show previously-unknown stress responses in a small space. In this work, applying MEMS technique, we made the cells of L. pneumophila and Legionella dumoffii (L. dumoffii) confined in small spaces and investigated their stress responses to UV light irradiation.

To confine Legionella cells in small spaces, we used a glass microbeads suspension and a PDMS-made microfluidic chip. We previously made a mixed suspension of Legionella cells and the beads. Then we introduced them into a PDMS-made MEMS microfluidic chip that has protrusions which work as stoppers both for the cells and the beads. Thus Legionella cells are trapped in small spaces among the microbeads.

The blue fluorescence from the trapped L. pneumophila cells was observed, indicating that the cells were restricted their motion among the beads. Spectroscopic analyses then compared the fluorescence with and without the beads. Both L. pneumophila and L. dumoffii showed their fluorescence for the case with the beads, meaning that they emit fluorescence when they are restricted their motion and not when they swim freely in water. The UV light irradiation time dependence of the fluorescence spectra for L. pneumophila and L. dumoffii in the case with the beads was further investigated. The spectra for L. pneumophila had a main and a shoulder peaks at around 450 nm and 550 nm respectively at the initial stages of the irradiation. Then the main peak grew with the time, showing the photo irradiation triggers to produce fluorescent materials. The spectra for L. dumoffii, on the other hand, drastically varied depending on the irradiation time. The spectral analysis showed that the fluorescent material which L. dumoffii originally had in their cells as reported in ref. 3 is photodissociated and then the production of the different fluorescent materials begins to occur. Thus our proposed method successfully revealed the previously-unknown characteristics of Legionella confined in the small spaces.

In summary, the fluorescent characteristics of Legionella were investigated by using the PDMS-made MEMS microfluidic chip with the combination of microbeads suspension. Our developed MEMS-based method for observing bacterial behavior paves the way for obtaining new knowledge about the stress response of bacteria by controlling their surrounded conditions in a MEMS microfluidic chip.

References

 [1] H. Ishii et al., “Bio-MEMS Chip for Bacteria Detection -A Challenge of Si Technology to Biomedical Field-”, ECS Trans., Vol. 58, No. 9, pp. 125-133, 2013.

 [2] Y. Nishimura et al., “PDMS-Based Microfluidic Chip for Detection of Legionella pneumophila by Mixing with Microbeads”, Abs. P1-32, APCOT 2014, Daegu, Korea (2014).

 [3] J. Amemura-Maekawa et al., “Legioliulin, a new isocoumarin compound responsible for blue-white autofluorescence in Legionella (Fluoribacter) dumoffii under long-wavelength UV light”, Biochem. Biophys. Res. Commun., Vol. 323, No. 9, pp. 954-959, 2004.