Luminescent Carbon Dots from Wet Olive Pomace: Structural Insights, Photophysical Properties and Cytotoxicity

Abstract

Carbon nanomaterials endowed with significant luminescence have been synthesized for the first time from an abundant, highly localized waste, the wet pomace (WP), a semi-solid by-product of industrial olive oil production. Synthetic efforts were undertaken to outshine the photoluminescence (PL) of carbon nanoparticles through a systematic search of the best reaction conditions to convert the waste biomass, mainly consisting in holocellulose, lignin and proteins, into carbon dots (CDs) by hydrothermal carbonization processes. Blue-emitting CDs with high fluorescence quantum yields were obtained. Using a comprehensive set of spectroscopic tools (FTIR, Raman, XPS, and ¹H/¹³C NMR) in combination with steady-state and time-resolved fluorescence spectroscopy, a rational depiction of WP-CDs structures and their PL properties was reached. WP-CDs show the up-conversion of PL capabilities and negligible cytotoxicity against two mammalian cell lines (L929 and HeLa). Both properties are excellent indicators for their prospective application in biological imaging, biosensing, and dynamic therapies driven by light.

Keywords: antioxidant activity; carbon dots; cytotoxicity; fluorescence; hydrothermal carbonization; olive mill waste; two-photon absorption.

Scheme 1

Fractioning of wet pomace and its main constituents.

Figure 1

(a) FTIR spectrum of WP-CDs-5 (black line) synthesized using a [P-2] = 0.16 g/mL, an EDA/P-2 mass ratio = 0.08, at 250 °C during 72 h, overlayed with that of WP-CDs-5 obtained after acidification (red line; see text). (b) The same spectra amplified in the region between 1800–1300 cm⁻¹.

Figure 2

Decomposition of Raman spectra into five Gaussian components of (a) WP-CDs-1 and (b) WP-CDs-5, and the corresponding labelled bands.

Figure 3

Wide XPS spectra of WP-CDs-3 and WP-CDs-5. Spectra intensities were offset for clarity’s sake.

Figure 4

XPS spectra of WP-CDs-3 (a,c,e); blue lines and WP-CDs-5 (b,d,f); red lines showing C 1s and K 2p (a,b), O 1s (c,d), and N 1s (e,f) regions.

Figure 5

(a) ¹³C NMR and (b) ¹H NMR spectra of WP-CDs-5 in D₂O (32.5 mg/mL). Insets: Expanded regions of interest. Signals from DMF (5 mM), used as internal standard, are indicated by an asterisk (*).

Figure 6

(a) TEM micrograph of WP-CDs-1 and (b) the corresponding histogram with the blue line representing the normal distribution. (c) STEM (HAADF detector) micrograph of WP-CDs-5 and (d) the corresponding histogram with the red line representing the normal distribution.

Figure 7

(a) UV–Vis (black line), excitation (red line; monitored at 426 nm) and emission (blue line, excitation at 340 nm; dot green line, excitation at 230 nm) spectra of an aqueous solution of WP-CDs-5 (0.1 mg/mL); inset: image of WP-CDs-5 under 365 nm illumination. (b) UV-Vis spectra of WP-CDs-1 to 5 (normalized at 278 nm). (c) Emission spectra of WP-CDs-1 excited at 340 and 380 nm and the corresponding excitation spectra monitored at the corresponding emission maxima; for comparison, the emission and excitation spectra of WP-CDs-5 are overlayed. (d) Time-resolved intensity decays (excitation at 340 nm; observation at 430 nm) of WP-CDs-1 and 5 obtained by single-photon timing (SPT) method.

Figure 8

Time-resolved intensity decays of aqueous solutions of WP-CDs-5 under excitations at 340 nm (a) and at 620 nm (b), both emissions being observed at 430 nm; (c) demonstration of the quadratic dependence of emission characteristic of two-photon absorption by plotting the logarithm of fluorescence intensity (F) vs. the logarithm of the average excitation power (P_exc). Excitation at 620 nm, emission at 430 nm.

Figure 9

(a) Steady-state emission spectra and (b) time-resolved intensity decays of aqueous solutions of WP-CDs-5 at various concentrations, excited at 340 nm. Inset in (a): intensity (monitored at emission maximum) vs. concentration of CDs. Exponential decays observed near the emission maximum at each concentration (430, 435, 445, 460, and 500 nm, respectively).

Figure 10

In vitro cytotoxicity results of WP-CDs-5 at various concentrations (0.5–1000 µg/mL) against (a) L929 and (b) HeLa cells after incubation for 24 h, as evaluated by the resazurin fluorescence viability assay.

Figure 11

(a) Absorption spectra of DPPH in ethanol (0.127 mM) on increasing the concentration of WP-CDs-5 in the range 7.4–138 μg/mL and (b) the scavenging activity of DPPH radicals by WP-CDs-5, having AA for comparison (absorbance monitored at 520 nm). Dotted lines drawn as an eye guide.