|The following chapters introduce and describe novel droplet microfluidic techniques to assist with limits of detection of analytes with low signal-to-noise ratios. The approach is proven feasible with absorbance measurements over short path lengths and with on-chip protein assays using fluorescence detection. This is accomplished by applying a lock-in detection method to alternating sample/reference droplet formation, which allows for the recovery of low signal in the presence of overwhelming background noise. Also described are methods of droplet formation control through pressure operated variable fluidic resistors in channel. These resistors use thin membranes that deflect into the channels to alter fluidic resistance for adjusting droplet volume and frequency.
The absorbance measurements take place within a 3-channel device consisting of one oil phase channel input and two aqueous channels. The two aqueous channels contain sample and reference solutions. Absorbance measurements are used to obtain a limit of detection (LOD) of 500 nM bromophenol blue (BPB) in a phosphate buffered saline (PBS) solution, as well as to determine the extinction coefficient at varying pH.
On-chip protein assays were performed using a similar device, with the exception of having two reservoirs per aqueous channel and incorporating mixing and incubation regions on the chip after droplet formation. A fluorescence calibration curve was established showing a LOD of 10 pM fluorescein. The proximity FRET (pFRET) assay was used for on-chip hormone
detection of thrombin and insulin, resulting in a decrease in fluorescence signal when analyte is present. A calibration curve was created for various concentrations of insulin. Finally, living murine pancreatic islets were loaded into a sample reservoir, then stimulated with 11 mM
glucose. Secreted insulin was then mixed with probe and connector and allowed to incubate on-chip for approximately 2 minutes. Fluidic lock-in detection was used to show a decrease in signal as more insulin was released.
Finally, a variable fluidic resistor was developed and used to control droplet size and frequency on-chip. By applying pressure to thin channel ceilings, we were able to increase the fluidic resistance in the channels. Various fabrication approaches were tested, with the two most effective methods involving magnetic pins and using multiple masks to create two separate layers (multilayer photolithography) that could be plasma oxidized then aligned and bonded. The magnetic pin method was capable of fabricating devices that could control droplet size over a few nL, while the multilayer lithography approach gave devices capable of generating wide ranges of droplet sizes and frequencies on the same chip, even in the same channel.
Overall, the work outlined in this dissertation provides proof-of-concept that absorbance and fluorescent detection can be applied to droplet microfluidic devices at low signal intensity with the aid of a lock-in detection method and at high temporal resolution. In conjunction with frequency and volume control over the droplets through the use of variable resistors, this should allow further advances in on-chip, stimulated hormone secretion analysis.