Quantification of Alkaline Phosphatase Activity in Single Algal Cells Utilizing a Microfluidic Device

Travis Dugas, Kelly Yates, David Englehardt, B. Seth Roberts, Sibel Bargu, and Adam T. Melvin

Microfluidic devices are commonly used to decrease the incubation required and reagent volume needed to perform certain biochemical assays. The goal of this project is to develop a microfluidic device to trap single algae cells and quantify the activity of the enzyme alkaline phosphatase (AP) in the green algal species Chlamydomonas reinhardtii in the presence and absence of organic phosphorous. This assay is capable of assessing the activity enzyme alkaline phosphatase (AP), which plays an important role in informing researchers of the available levels of organic phosphorous available in the water column. In fact, measuring AP activity provides a more accurate metric of phosphorous levels in the water that can specifically impact the initiation and propagation of harmful algal blooms (HABs). As part of this study three microfluidic devices were fabricated. The first device fabricated was capable of isolating single algae cells in a trapping array consisting of 230 semicircular wells. AP activity was assessed in cells using a commercially available stain (AP Live Stain). AP activity was quantified across a population of, ~70 cells per experiment at increasing concentrations of organic phosphorous. These results confirmed that AP activity is directly proportional to the phosphorous concentration in the cellular environment. This device was also capable of finding variations in AP activity across an entire population. However, one significant challenge encountered during experimentation was a low trapping efficiency in the 230 trap array. In an effort to increase single cell trapping efficiency, numerical simulations were performed using COMSOL to obtain a more comprehensive understanding of the fluid flow dynamics within the microfluidic device. From these computational results, two new microfluidic devices were designed consisting of 120 and 60 cell trappers. The hallmark of this device was the introduction of two parallel streams at different velocities to create a sheath flow and increase trapping efficiency. Preliminary experimentation determined a trapping efficiency of ~70-80% in these new designs. Ultimately, this work demonstrates the utility of incorporating an enzymatic assay into a microfluidic device, modeled through COMSOL, to not only decrease the reagent cost and incubation time, but also to increase the overall trapping efficiency of single cells to be evaluated in a much shorter time.