We are developing a portable small sensor that can detect pathogenic bacteria, Legionella without their cultivation. During the course of the study, we found that the cells emit fluorescence by the ultraviolet (UV) light irradiation just when they are restricted in their motion in microscopic spaces . To confine Legionella cells in such small spaces, we used a glass microbeads suspension and a PDMS-made MEMS microfluidic chip .
The fluorescence was observed for L. pneumophila and L. dumoffii confined in the MEMS microfluidic chip using a fluorescence microscope . The fluorescent spectra obtained for both bacteria varied with the UV-photoirradiation time. The spectra of L. pneumophila with beads showed a main and a shoulder peaks at around 450 nm and 520 nm respectively in the initial stages of the UV-photoirradiation with a wavelength of 365 nm. Then the main peak grew with the irradiation time, showing the UV light triggers to produce fluorescent materials. The spectra obtained from L. dumoffii also varied drastically depending on the UV-photoirradiation time. In the initial stages of the photoirradiation, the spectra with multiple peaks appeared and fell off. Subsequently, the spectra with a broad peak gradually grew. This shows that the fluorescent materials which L. dumoffii originally has in their cells as reported in Ref. 4, are photodissociated. After the initial fluorescent materials disappear, the production of the different fluorescent materials begins to occur. Thus, the bacteria might show their own fluorescent characteristics in the motion-restricted situation such as in microfluidic chips.
The fluorescent spectral characteristics of Legionella shown above were acquired by the combination of a bulky fluorescence microscope and a spectrometer. However, the integration with the optical sensor on a chip is necessary for easy portable use or in-field use. The successful detection of fluorescence from L. dumoffii is finally described using the microfluidic chip and a fluorescence sensor of Si photodetector with a poly-Si photogate . In the fluorescence detection, the transparent PDMS-made microfluidic chip was set above the detector without any optical filters because the poly-Si photogate prevents the excitation light from entering the sensor by absorbing UV light. This helps the bacterial sensor to be made small. The photocurrent generated by the fluorescence from L. dumoffii cells confined with the beads in the microfluidic chip was able to be observed just after the UV photoirradiation with a wavelength of 365 nm. The time course of the photocurrent intensity was similar to that of fluorescent spectral intensity obtained by the fluorescence microscope mentioned earlier. This indicates the combination of the PDMS-made MEMS microfluidic chip and the Si photodetector enables to make an integrated on-chip bacterial sensor.
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