Toward single enzyme analysis in a droplet-based micro and nanofluidic system

In this thesis, we have demonstrated the application of micro- and nanofluidic devices to generate an array of aqueous droplets in oil phase for single-enzyme encapsulation and activity measurement. We chose droplet-based microfluidics for this purpose of monitoring single-enzyme reactions since the generated droplets can be modulated by changing the fluidic flows as well as the geometry of the micro- and nanofluidic channels. This dropletbased microfluidics was discussed regarding the aspects of the generation and manipulation of droplets, their applications, and the recent work which has been performed by many researchers. The enzymatic reaction of beta-glucosidase to produce fluorescein was chosen as the model reaction for this assay. Glass material was used to fabricate the devices to avoid a high background noise for the optical measurements. In addition, a femtolitre compartmentalization of the aqueous system was achieved by using devices with both nanochannels and microchannels to diminish the background signal from solvent molecules and substrate. Since the glass-based micro- and nanofluidic device is inherently hydrophilic, it requires to be hydrophobized for facilitating the formation of an water-in-oil emulsion. Conventional hydrophobization methods were performed on plain substrates and then multisubstrates were integrated by several integration techniques. We propose an integration technique operated at room temperature at the chip level by using an UV adhesive. However, our integration method puts a limit to the geometry of the fluidic channels which should be above 10 µm wide and 15 µm high and therefore is not suitable to implement on devices containing nanofluidic channels. Subsequently therefore, a new in-channel hydrophobization method was developed by using a silicone oil and UV light. After the thermal bonding of two patterned glass substrates, the glass-based micro- and nanofluidic device can be hydrophobized by our method at the chip level. Finally, the hydrophobized chip can be used to generate water-in-oil emulsions. For the single-enzyme activity measurements, we demonstrated the successful encapsulation of single enzymes into femtolitre carriers. The histograms of the enhancing fluorescence intensity express the periodic increase in the fluorescence intensity, and hence the turnover number of the enzymatic reaction. The occupancies of the enzyme per droplet were determined by fitting to the Poisson distribution and were found to be in agreement with the calculated values. The obtained kinetic activity was somewhat lower than the value observed from the experiment in bulk probably due to effects of the confined volume.