Bacterial Diagnostic Chip By the Detection of Fluorescence from Legionella pneumophila in a Microbeads Suspension

  Sensing of bacteria is attracting a great deal of interest to avoid an outbreak of bacterial infection [1, 2]. The sensors used for this purpose should integrate at least two functions in a small volume: trapping bacteria and detecting them such as by a photo detector on a chip, as schematically illustrated in Fig. 1. We successfully devised a microfluidic chip with Si-pillar structure for trapping Legionella pneumophila (L. pneumophila) that is widely known as one of a pathogenic bacterium and confirmed its effectiveness by observing fluorescence produced by the trapped L. pneumophila [1].  In the chip, Si pillars work as a kind of sieve in which the introduced L. pneumophila cells are captured. During the course of this study, we further found that L. pneumophila might emit fluorescence just when they are squeezed in a narrow space such as one surrounded by the Si pillars, although its detailed mechanism is still unclear.

  The Si-pillar structure was indeed effective for trapping L. pneumophila, however, the amount of L. pneumophila cells trapped in the Si pillars was not necessarily sufficient to observe fluorescence because the introduced L. pneumophila cells were stuffed at the inlet side of Si pillars as shown schematically in Fig. 2(a). To solve this problem, we put L. pneumophila cells in a narrow space surrounded by glass microbeads instead of Si pillars as shown in Fig. 2(b).  Figure 3 illustrates how L. pneumophila cells are squeezed among the beads in a microfluidic chip: We previously make a mixed suspension of L. pneumophila cells and the beads, and then inject them into a microfluidic chip having protrusions which work as stoppers both for the cells and the beads. This method enables us to expect the observation of fluorescence from almost all the L. pneumophila cells injected into the chip.

  The microfluidic bacterial trap chip was fabricated by using poly(dimethylsiloxane), (PDMS), in which the depth was 30 um, the channel between stoppers was 10 um (Fig. 4). We made a mixed suspension consisting of 1x10⁶ cells of L. pneumophila, 5×10⁷ microbeads with the diameter of 7 um, and 1 mL of pure water. After injecting the 1 mL of mixed suspension into the chip and making the cells densely packed among the beads, the fluorescence was observed by a fluorescence microscope. Spectroscopic analyses of the fluorescence showed that the shape of the spectrum for the mixture of the cells and beads are different from that of the beads and PDMS as shown in Fig. 5,  indicating that the L. pneumophila cells packed densely among microbeads emit fluorescence.

  In summary, making narrow space by microbeads instead of Si pillars is effective way for detecting the fluorescence from L. pneumophila. Applying this simple microbeads approach paves the way for making L. pneumophila sensor by the combination with a photo detector.

 References

[1] K. Katsube, et al., Abstract ac12000224, APCOT 2012, Nanjing, China (2012).

[2] M. F. Bolanos, et al., Proc. SSDM2013, p. 860, JSAP, Fukuoka, Japan (2013).