High-Content Screening of Plankton Alkaline Phosphatase Activity in Microfluidics

Abstract

One way for phytoplankton to survive orthophosphate depletion is to utilize dissolved organic phosphorus by expressing alkaline phosphatase. The actual methods to assay alkaline phosphate activity-either in bulk or as a presence/absence of enzyme activity-fail to provide information on individual living cells. In this context, we develop a new microfluidic method to compartmentalize cells in 0.5 nL water-in-oil droplets and measure alkaline phosphatase activity at the single-cell level. We use enzyme-labeled fluorescence (ELF), which is based on the hydrolysis of ELF-P substrate, to monitor in real time and at the single-cell level both qualitative and quantitative information on cell physiology (i.e., localization and number of active enzyme sites and alkaline phosphatase kinetics). We assay the alkaline phosphatase activity of Tetraselmis sp. as a function of the dissolved inorganic phosphorus concentration and show that the time scale of the kinetics spans 1 order of magnitude. The advantages of subnanoliter-scale compartmentalization in droplet-based microfluidics provide a precise characterization of a population with single-cell resolution. Our results highlight the key role of cell physiology to efficiently access dissolved organic phosphorus.

Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates

“…Although the repression of APA can take 3 days for some dinoflagellate species, fast repression of APA has also been observed in on-a-field experiments and in cultures growing under P-deplete conditions [58][59][60]. The fast repression of AP synthesis observed in this study tends to confirm previous analyses showing a decrease in APA after 4-5 days of incubation, concomitant with the presence of APA in the dissolved fraction [34,56,58]. To explain such a quick repression of the AP synthesis while dissolved inorganic phosphate concentrations are still under the detection limit in the culture medium, we previously suggested that free-AP enzyme released from living cells or cell lysates in the dissolved fraction of the culture allowed new P-availability for the cell and consequently repressed intracellular AP synthesis [34].…”

“…The surfaces of the microfluidic channels were treated using fluorosilane (Aquapel) in order to increase the hydrophobic properties of the chip. The microfluidic chip was placed on a microfluidic platform dedicated to APA measurement [34]. In this study, we added a controlled X/Y motorised platform, a microfluidic module to sort droplets containing single cells.…”

“…The fluorescence signal was due to the hydrolysis of ELF-P and subsequent precipitation of phenol form of ELF alcohol into a fluorescent water-insoluble form ELF-A [41]. The measurement of the fluorescent product was made according to the method described previously [34]. Blank experiments were conducted to test the APA response of strains cultivated under P-replete conditions.…”

“…Moreover, the presence of free AP, which is released by cell lyses, can overestimate APA in the sample. To limit these potential biases, microfluidics and image processing algorithms have been recently combined in order to measure APA at single phytoplankton cell levels [34].…”

Microfluidic technology for plankton research

“…Among the enzymes activated during orthophosphate stress, the alkaline phosphatase is reported to be a good indicator of the physiological state and hence the mode of nutrition of the cell. To identify the mode of nutrition and evaluate the phosphate stress, alkaline phosphatase assays at the single cell level was developed in microfluidic device ( Figure 8 ) [ 73 •• ]. The results indicated that cell cultures have a similar timing in the activation of alkaline phosphatase (e.g.…”