(128d) A Microfluidic Approach to Detect Heterogenous Alkaline Phosphatase Activity in Single Chlamydomonas Reinhardtii Cells

The unicellular algal species Chlamydomonas reinhardtii require phosphorous (P) for growth and typically utilize dissolved orthophosphate in order to satisfy cellular P requirements. Inconsistencies in the availability of nutrients, due to water stratification or fertilizer influx, can decrease or enhance algal growth. One of these inconsistencies is a sudden influx of P into a body of fresh or marine water resulting in an undesired overgrowth of algal referred to as a harmful algal bloom (HAB). Since P content has been connected to HAB formation and persistence, it is important to be able to measure available P levels in the water. Traditional methods such as ELISA or mass spectrometry average P levels over a large area consisting of a substantial population of algae. However, quantifying the free P levels in the water fails to demonstrate how much P is being fixed by the algae. A more accurate metric is to measure the activity of the enzyme responsible for fixing extracellular P, alkaline phosphatase (AP), on the surface of algal cells. Measuring AP activity will demonstrate how the algal cells are fixing the available P which can be correlated to the size and density of the bloom. AP functions by cleaving the P monoesters from organic P so that it can be utilized and consumed by the algae cells. Traditional methods used to quantify AP activity are time consuming, require multiple reagents, and a large sample size. Moreover, this approach suffers from a relatively small number of cells to analyze at the end due to substantial cell death during the multiple wash and centrifuge steps needed to incorporate all components of the assay.

In this study, a microfluidic device was designed and fabricated to trap single algae cells and quantify AP activity using a single fluorescence stain in response to different spiked P concentrations. Fluid flow in the device was simulated by COMSOL to ensure accurate trapping in the device along the flow lines in the middle of the single fluidic channel. In-line closed-end traps allowed for facile isolation of single algal cells coupled with the ability to incorporate staining and wash steps without loss in cell number or a decrease in cell viability. The final design of the devices consisted of a 110-member trapping array with ~90% trapping efficiency. In order to mimic the real-time sudden influx of P in water, C. reinhardtii cells were subjected to a 24-h starvation period with no P followed by a 24-h-spiked P incubation period at concentrations ranging from 0.1 – 41 mM. Additionally, two populations of cells were interrogated: one population that was allowed to accumulate to basal P levels (1 mM) for several weeks while the second population was incubated with enhanced P levels (21 mM) to evaluate AP activity in single cells from normal and bloom-like conditions. Cells were injected into the microfluidic device after the 24-h P spike period and stained on-chip with the commercially available green fluorescent AP stain. Cells from each of the two culture P concentrations (1 and 21 mM) showed different degrees of heterogeneity in their response to different spike concentrations. Interestingly, cells with a basal culture level of 1 mM P when exposed to a spike concentration of 1 mM had a relatively homogenous response compared to the other spike concentrations. Also interestingly, the 1 mM acclimated cells showed greater heterogeneity in AP at the extreme spiked concentrations of 0.1 and 41 mM P suggesting that dramatic shifts in exogeneous P can result in several distinct subpopulations of cells with respect to AP activity. Cells acclimated to 21 mM P showed homogenous response to the 11 mM spike but not the 21 mM suggesting that cells under bloom-like conditions adapt differently to dramatic changes in P levels than those growing under basal levels. The findings of distinct subpopulations of cells should be taken into account by research sampling P levels and measuring AP levels in algae as looking at only a small population of cells or a small number of samples could inadvertently mask these distinct subpopulations, especially those with high AP activity. Additionally, these results further show the advantage of quantifying and analyzing AP activity at the single cell level compared to bulk measurement of P levels or traditional methods of AP activity quantification as they all fail to demonstrate the heterogenous nature of the cells response to extreme conditions.