Efficient Singlet Oxygen Monitoring in Aqueous Media Comprising a Polymer-embedded Eu3+-Complex

Abstract

Singlet dioxygen (¹O₂) plays a pivotal role as the active agent in photodynamic therapy (PDT) for cancer treatment, as well as in the photo-inactivation of antibiotic-resistant microbes (antimicrobial photodynamic therapy, aPDT). The ability to sensitively monitor the production and behavior of ¹O₂ following its photo-catalytic generation is crucial for developing effective therapeutic strategies. Optical sensor molecules that respond to ¹O₂ through changes in absorption or, more sensitively, fluorescence, are suitable choices. While most monitors report ¹O₂ via altered absorption spectra, only few compounds respond by the onset of fluorescence, even fewer based on lanthanide luminescence. By embedding a novel lanthanide complex (Eu³⁺) into polystyrene nanoparticles (beads), we achieved close to a 500-fold emission intensity boost in the presence of ¹O₂, very long decay times of up to 879 µs and unprecedented stability in acidic and basic media. Furthermore, the beads present a high-surface charge (>+30 mV), yielding stable aqueous dispersions, which we exploited in a preliminary "proof of principle" staining experiment of (negatively charged) bacterial surfaces. The straightforward synthesis circumvents intricate preparative steps and restrictive costs. The decay characteristics furthermore pave the road to time-gated measurements, that is, to the suppression of interfering autofluorescence from biological samples.

Keywords: bacteria; lanthanides; polymers; sensors; singlet oxygen detection.

Scheme 1

Synthesis pathway toward polymeric nanoparticles (NPs) loaded with an Eu(III) complex as ¹O₂ sensor, starting from the binary complex to illustrate its application in aqueous dispersions and on bacteria.

Figure 1

Bright Field STEM of PS_Eu (left) and SEM of PMMA_Eu NPs (right). Colloidal dispersions of 0.1 wt% at pH 7.5 were used.

Figure 2

Zeta potentials of the PS_Eu and PMMA_Eu NPs at various pH levels. Colloidal dispersions of 0.01 wt% were used.

Figure 3

Excitation (left) and emission (right) spectra of PS_Eu and PMMA_Eu NPs before and after endoperoxidation. Colloidal dispersions of 0.01 wt% were used at pH 7.5. Endoperoxidation was attained by probe self‐sensitization, through exposure to a 3.5 W, 430 nm LED for 30 minutes. The inset provides a magnified view, showing the spectra of the probes before endoperoxidation at an appropriate scale.

Figure 4

Luminescence rise of PS_Eu (left) and PMMA_Eu (right) upon continuous exposure to ¹O₂ generated by RB in aqueous dispersion. Colloidal dispersions of 0.01 wt% were used at pH 7.5 at an RB concentration of 5 µM. Irradiation proceeded in a photoreactor with 1.0 W 520 nm LED.

Figure 5

(Left) Plot of the luminescence intensity (I) of both PS/PMMA_Eu probes at 614 nm with increasing irradiation time and photosensitized exposure to ¹O₂. The inserted figure shows a plot of ln(1/I)‐ln(1/I₀) versus irradiation time at pH 7.5 RB concentration of 5 µM. Irradiation was conducted in a photoreactor with a 1.5 W / 520 nm LED. (Right) Stability test of the endoperoxidized PS and PMMA_Eu probes at various pH levels. Dispersions of 0.01 wt% were used.

Figure 6

Bright field and fluorescence microscope images of the E. coli and B. megaterium bacteria containing PS‐Eu (top rows) and PMMA_Eu (bottom rows) beads attached to their surface. The fluorescence images show the gradual increase in red emission from the labelled bacteria cells on prolonged exposure to ¹O₂. The test dispersions contained about 10⁷ bacteria cells/mL with roughly 10⁴ beads per bacteria cell (see Supporting Information for calculation) and 5 µM RB, irradiated for up to 30 minutes using a 1.0 W LED in the photoreactor.