(229ap) Quantification of Alkaline Phosphatase Activity in Single Algal Cells Using a Microfluidic Device

Authors: 
Melvin, A., Louisiana State University
Dugas, T., Louisiana State University
Roberts, B. S., Louisiana State University
Harmful algal blooms (HABs) are the result of the overabundance of naturally occurring algae due, in part, to an excess of nutrients such as nitrogen and phosphorus. While measuring nitrogen and phosphorus content in the water column can provide information about their respective availability, this analytical metric does not account for how much of each nutrient is fixed by the algae. Conversely, measuring alkaline phosphatase (AP) activity provides a more direct metric for how much phosphorus is being fixed by algae in the bloom. AP is expressed on the surface of algae and is responsible for converting the dissolved organic phosphorus to inorganic phosphorus required by algae. One limitation to using AP as a metric for dissolved phosphorus in a bloom is that it requires several washing and centrifugation steps confining the assay to a lab and resulting in a 1-2 week lag time between sample acquisition and analysis. Microfluidics devices however can be used to drastically decrease the time required to run such biochemical assays while using a fraction of the reagents coupled with their ability to serve a portable readouts of cellular behavior. The goal of this study was to develop a microfluidic device capable of single cell capture and isolation followed by directly measuring AP activity in the trapped algal cells. The device consisted of an array of 15 μm semi-circular cups capable of trapping individual Chlamydomonas reinhardtii cells followed by detection of AP activity using a commercially available fluorescent biomolecule with fluorescent microscopy. The device eliminated the need for the washing and centrifugation steps as the introduction and removal of all reagents was achieved by changing the inlets to the device. To induce AP activity, C. reinhardtii cells were starved in phosphorus free 1X TA buffer followed by resuspension in phosphorus-rich 1X TAP prior to initiation of the AP assay. A range of increasing phosphorus concentrations were tested which correlated to increasing levels of AP activity, all performed in a fraction of the time in the device. In order to demonstrate the utility of this device, similar experiments were performed examining AP activity in the small centric diatom Thalassiosira, with similar results of increasing AP activity correlating to increasing dissolved organic phosphorus levels. Thus, the microfluidic device allows for facile single cell analysis and the ability to incorporate all the steps in the biochemical assay in a single device. This technology exhibits the potential for portability needed to measure AP activity in HAB samples collected during cruises without the need for long-term storage and analysis in a lab.