Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Fluorogenic "on-off" nanosensor based on dual-quenching effect for imaging intracellular metabolite of various microalgae.

Sensing intracellular compounds such as ATP in living microalgal cells is of great importance in diverse fields. To achieve this, nanosensing platform composed of graphene oxide (GO) and ATP aptamer (APT) was applied to diverse microalgal cells (Chlamydomonas reinhardtii, Chlorella vulgaris, Anabaena flos-aquae, and Ochromonas danica). The nanosized GO was characterized with atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The nanosensing platform (GO-APT) was prepared by attaching fluorophore-labeled APT to GO. GO-APT was applicable to only cell wall-deficient species (O. danica and mutant strains of C. reinhardtii) and the existence of flagella did not affect the uptake of the GO-APT by the cells. These results indicate that the cell wall is the primary barrier of GO-APT internalization for sensing application. To reduce the background fluorescence signal elicited by nonspecific displacement of the fluorophore-labeled probe, APT was modified as molecular beacon (MB) type (APTMB). Owing to the double quenching effect (by GO and quencher-labeled complementary sequence), the background signal significantly reduced. Cytotoxicity of GO-APTMB on the microalgal species was also tested. The application of GO-APTMB had no effect on the growth of microalgae. Given that diverse aptamer sequences had been screened, the sensing platform is not limited for detecting ATP only, but also can be applied to other metabolite imaging by simply changing the aptamer sequences. Our research will contribute to broadening the application of GO and aptamer beacon complex for intracellular metabolite imaging and detecting.

A strategic approach to apply bacterial substances for increasing metabolite productions of Euglena gracilis in the bioreactor.

Bacterial extracellular polymeric substances (EPS) are promising materials that have a role in enhancing growth, metabolite production, and harvesting efficiency. However, the validity of the EPS effectiveness in scale-up cultivation of microalgae is still unknown. Therefore, in order to verify whether the bacterial metabolites work in the scale-up fermentation of microalgae, we conducted a bioreactor fermentation following the addition of bacterial EPS derived from the marine bacterium, Pseudoalteromonas sp., to Euglena gracilis. Various culture strategies (i.e., batch, glucose fed-batch, and glucose and EPS fed-batch) were conducted to maximize metabolite production of E. gracilis in scale-up cultivation. Consequently, biomass and paramylon concentrations in the continuous glucose and EPS-treated culture were enhanced by 3.0-fold and 4.2-fold (36.1 ± 1.4 g L-1 and 25.6 ± 0.1 g L-1), respectively, compared to the non-treated control (12.0 ± 0.3 g L-1 and 6.1 ± 0.1 g L-1). Also, the supplementation led to the enhanced concentrations of α-tocopherols and total fatty acids by 3.7-fold and 2.8-fold, respectively. The harvesting efficiency was enhanced in EPS-supplemented cultivation due to the flocculation of E. gracilis. To the best of our knowledge, this is the first study that verifies the effect of bacterial EPS in scale-up cultivation of microalgae. Also, our results showed the highest paramylon productivity than any other previous reports. The results obtained in this study showed that the scale-up cultivation of E. gracilis using bacterial EPS has the potential to be used as a platform to guide further increases in scale and in the industrial environment. KEY POINTS: Effect of EPS on Euglena gracilis fermentation was tested in bioreactor scale. EPS supplement was effective for the paramylon, α-tocopherol, and lipid production. EPS supplement induced the flocculation of E. gracilis.

In Vivo Monitoring of Intracellular Metabolite in a Microalgal Cell Using an Aptamer/Graphene Oxide Nanosheet Complex.

Real-time sensing and imaging of intracellular metabolites in living cells are crucial tools for the characterization of complex biological processes, including the dynamic fluctuation of metabolites. Therefore, additional efforts are required to develop in vivo detection strategies for the visualization and quantification of specific target metabolites, particularly in microalgae. In this study, we developed a strategy to monitor a specific microalgal metabolite in living cells using an aptamer/graphene oxide nanosheet (GOnS) complex. As a proof-of-concept, ß-carotene, an antioxidant pigment that accumulates in most microalgal species, was chosen as a target metabolite. To achieve this, a ß-carotene-specific aptamer was selected through graphene oxide-assisted systematic evolution of ligands by exponential enrichment (GO-SELEX) and characterized thereafter. The aptamer could sensitively sense the changes in the concentration of ß-carotene (i.e., the target metabolite) and more specifically bind to ß-carotene than to nontargets. The selected aptamer was labeled with a fluorophore (fluorescein; FAM) and allowed to form an aptamer/GOnS complex that protected the aptamer from nucleic cleavages. The aptamer/GOnS complex was delivered into the cells via electroporation, thus enabling the sensitive monitoring of ß-carotene in the cell by quantifying the aptamer fluorescence intensity. The results suggest that our biocompatible strategy could be employed to visualize and semiquantify intracellular microalgae metabolites in vivo, which holds a great potential in diverse fields such as metabolite analysis and mutant screening.