Probing Polarity and pH Sensitivity of Carbon Dots in Escherichia coli through Time-Resolved Fluorescence Analyses

Abstract

Intracellular monitoring of pH and polarity is crucial for understanding cellular processes and functions. This study employed pH- and polarity-sensitive nanomaterials such as carbon dots (CDs) for the intracellular sensing of pH, polarity, and viscosity using integrated time-resolved fluorescence anisotropy (FA) imaging (TR-FAIM) and fluorescence lifetime (FLT) imaging microscopy (FLIM), thereby enabling comprehensive characterization. The functional groups on the surface of CDs exhibit sensitivity to changes in the microenvironment, leading to variations in fluorescence intensity (FI) and FLT according to pH and polarity. The FLT of CDs in aqueous solution changed gradually from 6.38 ± 0.05 ns to 8.03 ± 0.21 ns within a pH range of 2-8. Interestingly, a complex relationship of FI and FLT was observed during measurements of CDs with decreasing polarity. However, the FA and rotational correlation time (θ) increased from 0.062 ± 0.019 to 0.112 ± 0.023 and from 0.49 ± 0.03 ns to 2.01 ± 0.27 ns, respectively. This increase in FA and θ was attributed to the higher viscosity accompanying the decrease in polarity. Furthermore, CDs were found to bind to three locations in Escherichia coli: the cell wall, inner membrane, and cytoplasm, enabling intracellular characterization using FI and FA decay imaging. FLT provided insights into cytoplasmic pH (7.67 ± 0.48), which agreed with previous works, as well as the decrease in polarity in the cell wall and inner membrane. The CD aggregation was suspected in certain areas based on FA, and the θ provided information on cytoplasmic heterogeneity due to the aggregation and/or interactions with biomolecules. The combined TR-FAIM/FLIM system allowed for simultaneous monitoring of pH and polarity changes through FLIM and viscosity variations through TR-FAIM.

Keywords: E. coli; carbon dots (CDs); fluorescence lifetime imaging microscopy (FLIM); frequency domain (FD); intracellular sensing; pH sensor; polarity sensor; time-resolved fluorescence anisotropy imaging (TR-FAIM).

Figure 1

The structure of E. coli can be schematically depicted as consisting of distinct layers. These layers include the cytoplasm, plasma membrane, cell wall, and outer membrane, each serving specific functions within the bacterium’s cellular structure.

Figure 2

The FD TR-FAIM system is illustrated schematically. FA measurements are implemented by adding entrance polarizers (horizontal and vertical) at the output of the multi-LED source, along with a PBS at the input of the image intensifier in our FD-FLIM system. A pinhole at the PBS input adjusts the fluorescence image width to prevent overlap between the two polarization components. By using a mirror, the two polarized beams extracted by the PBS are directed parallel to the CCD camera, which divides its field of view between the two beams.

Figure 3

TEM image of CDs; (a) E. coli; (b) CDs bound to E. coli; (c) aggregates of CDs inside E. coli (d). The red circles mark CDs and their aggregates.

Figure 4

UV–VIS absorption spectra (a) and emission spectra (b) of the E. coli, CDs, and the CD–E. coli complex. Clearly, the E. coli autofluorescence intensity is about 1–2 orders of magnitude lower than the FI of CDs and the CD–E. coli complex.

Figure 5

The FI (a) and FLT (b) of CDs at various pH values using the FD-FLIM system. At an acidic pH (2–8), the FI increased, while at an alkaline pH (10–14), the FI decreased (red squares). The FLT gradually increased linearly with the increasing pH values, while it showed a potential decrease when the pH exceeded 8.0 (blue triangles). Due to the difficulty in determining the exponential fitting for the FI decay model at pH values above 8, the corresponding data points were omitted from the analysis.

Figure 6

The zeta potential (a), FI measurements (b), and FLT measurements. (c) Measurements of the CDs in water–dioxane mixtures with increasing dioxane concentrations, from 0% to 100%. In a dioxane concentration of up to 50%, both the FI and apparent FLT increased, while beyond 50%, they both decreased.

Figure 7

FA (a) and θ (b) measurements of CDs in water–dioxane mixtures with increasing dioxane concentrations. Surprisingly, the FA (r) and θ values increased as solvent polarity decreased. This observation suggests the involvement of an additional factor contributing to this trend, potentially viscosity.

Figure 8

FI (a), FLT (b), r (c), and θ (d) images of CDs.

Figure 9

FI (a), FLT (b), r (c), and θ (d) images of the CD–E. coli complex.