Bacterial Diagnostic Chip By the Detection of Fluorescence from Legionella pneumophila in a Microbeads Suspension
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.
 K. Katsube, et al., Abstract ac12000224, APCOT 2012, Nanjing, China (2012).