Preparation of biocompatible and antibacterial carbon quantum dots derived from resorcinol and formaldehyde spheres

Abstract

Green or yellow emitting carbon quantum dots (CQDs) were prepared through the combination of bottom-up and top-down approaches from resorcinol and formaldehyde. The prepared CQDs showed characteristic fluorescence behavior depending on the compositional or structural features. Both CQD-S (green emitting) and CQD-P (yellow-emitting) were physiologically stable and biocompatible as cancer cell bioimaging agents. Especially, silver nanoparticle (Ag NP)-decorated CQDs revealed very good antibacterial activity for Gram-positive S. aureus and Gram-negative E. coli.

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Introduction

Recently, carbon quantum dots (CQDs) have been intensively researched as the next generation carbon nanomaterials in addition to zero-dimensional (0D) fullerene, 1D carbon nanotubes and 2D graphene. CQDs’ promising optoelectrical properties are approaching or equivalent to those of their counterparts, inorganic quantum dots (QDs). CQDs have been widely examined in various applications such as bioimaging,^1–4 sensing,^5,6 photo/electrocatalysis,⁷ light-harvesting,⁸ and drug-delivery systems^9,10 through the exploitation their biocompatibility, low toxicity, excellent photochemical stability and facile surface functionalization.

Up to now, two different categories of approach, “top-down” and “bottom-up”, have typically been adopted for the synthesis of CQDs, depending on the type of source or precursor materials.¹¹ In the “top-down” approach, carbon nanomaterials with well-defined morphology such as carbon nanotubes,¹² nanodiamonds,¹³ graphene oxide,¹⁴ graphite¹⁵ and carbon fibers,¹⁶ together with carbon-rich materials with less well-defined morphology such as carbon soot,^17,18 activated carbon,¹⁹ carbon black²⁰ and paper ash²¹ have been broken down to CQDs with high dispersion stability and tuneable fluorescence emissions through discharge,²² laser ablation²³ and chemical/electrochemical oxidation.¹² In the “bottom-up” approach, various small molecules, polymers and even biocompatible materials have been utilized for the synthesis of CQDs through combustion, thermal treatment, thermal/chemical carbonization, electrochemical and acid/alkali-assisted ultrasonic or oxidation reactions. CQDs prepared by the “bottom-up” approach have been successfully utilized for applications such as sensing and bioimaging with or without surface passivation to enhance the quantum yield and brightness of CQD fluorescence. While the “bottom-up” approach using “green” precursors is much more preferable for CQDs with bioapplications where physiological stability of the material is required, the “top-down” approach using carbon-rich materials is much more better for defining the expected structural/compositional/optoelectrical features of the resulting CQDs. Additionally, the use of carbon-rich materials leads to consistent CQDs structures and compositions regardless of the carbon source, while the use of “green” precursors leads to significant variation in the CQDs depending on the source. Therefore, another synthetic approach combining the advantages of both the “bottom-up” and “top-down” approaches could be versatile for the preparation of CQDs with precisely controlled optoelectrical properties while being reproducible and scalabile.²⁴ In our method, resorcinol and formaldehyde, which are cheap and extensively consumed as the monomers for the production of resorcinol–formaldehyde (RF) resin in industry, were used as the starting materials for the synthesis of RF-based CQDs having two different sizes and fluorescence emission colors (green and yellow) with quantum yields up to 1%. The prepared CQDs were used safely for in vitro imaging of A549 human lung cancer cells due to the non-cytotoxicity of the CQDs. In addition, highly anti-bacterial CQDs, toward both Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative), were simply prepared by electroless silver deposition on CQDs through the exploitation of rich oxygen functionalities of the surface of CQDs.²⁵ Another important feature of the RF-based CQDs is their scalability for mass production, grams of CQDs were reproducibly obtained from each reaction.

Experimental

Materials and characterization

Resorcinol (C₆H₄-1,3-(OH)₂, ≥ 99.0%), formaldehyde (HCHO, 37%) solution, ammonium hydroxide (NH₄OH, 28–30%), ethanol (C₂H₅OH, ≥ 99.5%), silver nitrate (AgNO₃, ≥ 99.0%), Pluronic F127 (MW: 12 600 Da), and sodium carbonate (Na₂CO₃) were purchased from Sigma-Aldrich and sulfuric acid (H₂SO₄, 95%), and nitric acid (HNO₃, 60%) were purchased from Samchun Chemicals, South Korea. All chemicals were used without further a purification step.

Fourier transform infra-red (FT-IR) spectra were acquired on a Nicolet iS10 FT-IR spectrometer (Thermo Scientific). Atomic force microscopy (AFM) images were acquired using a Multimode-N3-AM nanoscope 3D scanning probe microscopy (SPM) system (Bruker). Field emission scanning electron microscopy (FE-SEM) images were obtained with an JSM-6700F FE-SEM (JEOL). Ultraviolet-visible (UV-Vis) spectra were measured on an Optizen α UV-Vis spectrometer (Mecasys, South Korea). The luminescence properties were examined using a fluorescence spectrometer (SCINCO, FS-2, South Korea) with a xenon lamp excitation source (150 W). The X-ray diffraction (XRD) measurements of the powder samples were recorded on a Bruker D8-Advance X-ray powder diffractometer using Cu Kα radiation (λ = 1.5406 Å). X-ray photoelectron spectroscopy (XPS) spectra were recorded with a Thermo VG Scientific Sigma Probe spectrometer. Transmission electron microscopy (TEM) images were taken on a TECNAI F20 (Philips) at 200 kV. Raman spectroscopy measurements were performed on an ARAMIS Raman spectrometer (Horiba Jobin Yvon, France) by using 514.5 nm laser radiation. A Helios 06 (15 W) double side vacuum exposure unit (South Korea) was used for UV-Vis irradiation.

Preparation of carbon spheres (CS) from resorcinol and formaldehyde (RF)

At first, spherical RF nanoparticles (RF NPs) with diameter of about 100 nm were prepared by the condensation reaction of resorcinol and formaldehyde monomers through a modified Stöber method using Pluronic F127 as the stabilizer in alcohol/water medium. In brief, at first, 0.4 mL of aqueous NH₄OH solution was mixed with 32 mL of absolute ethanol and 80 mL of deionized water (H₂O) under vigorous stirring for 1 h. After that, resorcinol (0.8 g) and Pluronic F127 (1.69 g) was added into the solution with continuous stirring for 30 min. Then, formaldehyde solution (1.12 mL) was added to the reaction mixture and stirred for 24 h at 30 °C and subsequently heated for 24 h at 100 °C under a static condition in a Teflon-lined autoclave. The solid product was recovered by centrifugation and freeze-dried for 48 h. Secondly, thermal treatment of RF NPs between 350 °C for 2 h and 600 °C for 4 h (1° min⁻¹) under nitrogen atmosphere provided black powders of carbon sphere (CS) NPs showing a 67.1% decrease of weight due to the dehydration and carbonization during the thermal treatment process.²⁶

Synthesis of CQD-S and CQD-P

50 mg of the CS NP powder was treated with a mixture of sulfuric acid and nitric acid (volume ratio of 1 : 2) and the reaction mixture was heated at 100 °C for 12 h in a Teflon-lined autoclave after brief sonication in a bath sonicator for 3 h at room temperature. After acid treatment, most of the CS powder was dissolved into the acid medium and a highly transparent red-colored CQD solution was obtained after dilution with water, although there were little noticeable floating particles. The selective isolation of the RF-based CQDs with different fluorescence colors was attempted by controlled ultracentrifugation of the crude CQD solution. After ultracentrifugation for 20 min at 4000 rpm, a red-colored supernatant solution (CQD-S) and a dark brown precipitate (CQD-P) were separately divided and stored. To remove acid residue from the RF-based CQDs, dialysis of CQD solutions was attempted by using molecular weight cut-off membrane (MWCO 1000 Da) for 72 h. Finally, 3 and 6 mg of CQD-S and CQD-P were obtained after freeze-drying of each of their respective dialyzed CQD solutions.

Synthesis of Ag NP decorated CQDs

In a typical preparation, 1.5 mL of AgNO₃ solution (1 mM) and 1.5 mL of an aqueous solution of CQDs were mixed with vigorous stirring. Then, the mixed solution was irradiated using a UV lamp for 40–45 min. After the irradiation, the initially pale-brown solution turned into a dark-yellow solution, a result of the UV-assisted reduction of Ag⁺ ions to Ag NPs. Solid Ag NP decorated CQDs were collected by centrifugation (12 000 rpm, 1 h) and subsequent freeze-drying.

Results and discussion

Both “bottom-up” and “top-down” approaches are sequentially attempted to provide RF-based CQDs from RF. The overall synthetic procedure is illustrated in Scheme 1.

Scheme 1 Schematic illustration for the overall synthetic procedure of CQDs and CQDs/Ag NPs.

In Fig. S1,† while the CS powder was only soluble in isopropyl alcohol, ethanol and tetrahydrofuran, both types of CQDs were soluble only in water, probably due to the rich oxygen functionalities on their surface. The formation of CQDs from RF was firstly confirmed with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) analyses. While both the spherical shape and size distribution of the RF NPs were preserved after carbonization to CS NPs, the SEM images of RF NPs and CS NPs showed a sight volume shrinkage after the carbonization process from 100 ± 20 nm to 90 ± 10 nm (Fig. 1a and b). The AFM images of CQD-S and CQD-P showed that RF-based CQDs have carbon dot-like morphologies. While both CQDs showed particle diameters of 40 ± 0.5 and 50 ± 1.0 nm, respectively, the thickness was about 4 nm in both CQDs (Fig. 1c and d). Therefore, it is regarded that RF-based CQDs have anisotropic 2D disc-like morphology. Considering that typical CQDs obtained from other top-down approaches show dot-like morphology with particle sizes less than 10 nm, the disc-like morphology of the RF-based CQDs is very unique. Only a few studies have reported the preparation of CQDs with similar disc-like morphology through a top-down approach from either single-walled carbon nanotubes (SWNTs) or pitch-based carbon fiber (CF), which has a rich sp² carbon content. The TEM images of RF-based CQDs showed that both CQDs have similar lateral dimensions of between 40 and 50 nm, which corresponds to the previous AFM analysis (Fig. 1e and f).

Fig. 1 SEM images of (a) RF NPs (b) CS NPs; AFM and TEM images of CQD-S (c and e) and CQD-P (d and f), respectively.

Further compositional and structural features of RF-based CQDs were examined by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy and X-ray diffraction (XRD) analysis. The FT-IR spectra of both CQDs showed that RF-based CQDs have rich oxygen functionalities such as hydroxy (–OH) and carboxylic acid (COOH) groups together with an aromatic backbone (Fig. 2a). Both CQDs revealed the occurrence of significant carboxylic acid stretching peaks at around 1720 cm⁻¹, which was not observable in CS NPs.

Fig. 2 (a) FT-IR spectra, (b) XPS full survey scans, deconvoluted C1s peaks of (c) CQD-S and (d) CQD-P, (e) Raman spectra (excited with 514.5 nm laser) and (f) XRD profiles of all the samples [Cu Kα radiation (λ = 1.5406 Å)].

Interestingly, the aromatic C=C stretching peak of the CS NPs at 1576 cm⁻¹ was significantly shifted to 1594 and 1600 cm⁻¹, in the cases of CQD-S and CQD-P, respectively. Both CQDs revealed strong –OH stretching peaks at 3410 cm⁻¹ and CS NPs showed only a weak signal in this region. More detailed compositional information of RF-based CQDs was obtained from XPS analysis (Fig. 2b). The three peaks observed at 284.0, 401.0 and 533 eV are considered as the C1s, N1s and O1s binding peak, respectively. While CS NPs had a carbon-dominant composition (C 93.2%, O 5.6%, N 1.2%), the RF-based CQDs had an increased oxygen content of more than 27%. CQD-S showed a slightly higher inclusion of oxygen (C 63.3%, O 35.6%, N 1.0%) compared with CQD-P (C 71.0%, O 27.1%, N 1.8%). This higher oxygen content of the CQD-S compared with CQD-P explains the higher dispersion stability of CQD-S during the centrifugation step because there is little structural inconsistency between them as confirmed from the previous discussions of AFM and TEM analysis. Deconvoluting the C1s peaks of both CQDs showed the presence of significant oxygenated carbons. Four overlapping peaks 281.09, 284.44, 286.11 and 288.25 eV for CQD-S and 282.08, 284.53, 286.58, 288.28 eV for CQD-P correspond to the C1s peaks of C–H, C=C, C=O and COOH groups of CQDs, respectively. It is clear that the peak intensities of the oxygen functionalities (OH, C=O and COOH) are much higher compared with pristine CS NPs (Fig. 2c and d). Raman spectra of CS NPs and CQDs were compared (Fig. 2e). Both showed characteristic D and G band peaks, which support the presence of significant sp² carbons (G band) together with sp³ defects (D band) both in CS NPs and RF-based CQDs. The D band to G band intensity ratio (I_D/I_G) was significantly increased after the formation of CQDs (0.90) from CS NPs (0.74), from which we can assume that the observed structural defects might be introduced into CQDs by various forms of oxygen functionalities including carbonyl, carboxyl, hydroxyl and epoxy groups being incorporated into the sp² carbon skeletal of CS NPs after the acid treatment. Interestingly, D band peaks of CQD-S and CQD-P were significantly shifted to 1381 and 1382 cm⁻¹, respectively, from 1356 cm⁻¹ in the case of CS NPs while G band peaks didn’t show a remarkable shift from 1600 cm⁻¹.²⁷ While the origin of this low-frequency D band peak shift of RF-based CQDs is not clear at this stage, it is believed that the oxygen rich environments near the sp³ defect sites on the CQDs compared with carbon rich environment of the CS NPs might contribute to this shift. Then, crystalline structures of both CS NPs and CQDs were exploited by XRD analysis (Fig. 2f). Pristine CS NPs showed a sharp graphitic diffraction peak (002) at 2θ of 27.7° (d = 3.22 Å), which indicates the preservation of the partial graphitic structure in CS NPs. This graphitic scattering peak of the CS NPs was significantly diminished in CQD-S and preserved in CQD-P, respectively. This different crystallinity of CQD-S compared with CQD-P might come from the much highly oxygenated compositional feature of CQD-S.

The quasi 2D disk-like morphology of RF-based CQDs and their rich oxygen functionalities prompt us to investigate the optical properties of CQDs and their application toward cell imaging based on fluorescence. Ultraviolet-visible (UV-Vis) absorption spectra of CQD-S and CQD-P exhibited two strong absorption peaks at 220 and 229 nm, which are attributed to the π–π* transition peaks of the isolated sp² conjugated domains in CQDs together with the overlapping peaks at 280 nm, which are attributed to n–π* transition peaks of the carbonyl (C=O) groups in CQDs (Fig. 3a).²⁷ With an excitation at 460 nm, CQD-S and CQD-P showed definite green and yellow emissions, respectively. Fluorescence spectra of both CQDs were examined with varying excitation wavelengths from 380 to 500 nm (Fig. 3b and c). The normalized fluorescence spectra of CQD-S definitely showed maximum fluorescence emission between 501 and 555 nm (greenish emission), while CQD-P showed a maximum fluorescence emission between 523 and 581 nm (yellowish emission). Any up conversion fluorescence was not observed in both CQDs. The above multicolour emissions of RF-based CQDs are typical features of CQDs whose fluorescence emission mechanism is based not on the band gap transition of conjugated π domains but on the surface defect-derived origin. The presence of different particle sizes of CQDs and different emissive trap sites in CQDs might contribute to the above tunable fluorescence emissions of RF-based CQDs. RF-based CQDs showed very good biocompatibility as cancer cell imaging agents. After the incubation of A549 lung cancer cells with CQDs for 24 h, most A549 cells were completely labeled with either green-emitting CQD-S or yellow-emitting CQD-P (Fig. 3d and e). A MTT-mediated cell viability assay demonstrated more than 92% in the concentration range of 0–0.1 mg mL⁻¹, revealing the presence of a strong candidate for biological applications as non-toxic materials (Fig. S2†). These results promoted the materials as strong candidates to use in cellular imaging for diagnosis purposes.

Fig. 3 (a) UV-Vis spectra (insets are photographic images of CQD solutions with a concentration of 0.25 mg mL⁻¹) and normalized fluorescence spectra of (b) CQD-S, (c) CQD-P at different excitation wavelengths (insets are photographic images of CQD solutions with an excitation of 365 nm UV), confocal microscopy images (488 nm light excitation) of A549 cells incubated with (d) CQD-S, (e) CQD-P.

Further hybridization of RF-based CQDs with silver nanoparticles (Ag NPs) was attempted by exploiting the rich oxygen functionalities on the surface of CQDs. Addition of silver nitrate (AgNO₃) into the aqueous dispersion of either CQD-S or CQD-P and the subsequent irradiation with UV light (365 nm) induced the growth of Ag NPs on RF-based CQDs within 1 h. UV-Vis spectra of Ag NP-decorated CQD solutions clearly showed the surface plasmon resonance (SPR) peaks of Ag NPs at 460 nm (Fig. 4a and b). TEM and energy dissipate X-ray (EDX) analysis clearly revealed the presence of several Ag NPs on CQDs (Fig. S3†). It is reported that the photo-excited electrons from CQDs enable the reduction of Ag⁺ ions on the surface of CQDs.²⁵ XPS spectrum of Ag NP-decorated CQDs clearly showed the incorporation of significant Ag atoms (Fig. S4 and S5†). The presence of Ag NPs on RF-based CQDs presented very good antimicrobial activities for two model bacteria, Gram-positive S. aureus and Gram-negative E. coli.^28,29 Clear inhibition zones were observed after incubation of the above model bacteria only with Ag NP-decorated CQDs, confirming the prevention both Gram-negative and Gram-positive bacterial growth (Fig. 3c and d).

Fig. 4 UV-Vis spectra and schematic illustration of (a) CQD-S/Ag NP and (b) CQD-P/Ag NP before and after UV-irradiation. Antimicrobial activities of as prepared sample against (c) Gram positive S. aureus and (d) Gram negative E. coli.

Conclusion

In summary, green or yellow emitting CQDs were prepared through the combination of bottom-up and top-down approaches from RF resin. The prepared CQDs showed characteristic fluorescence behavior dependant on the compositional or structural features. Both CQD-S (green emitting) and CQD-P (yellow-emitting) were physiologically stable and biocompatible as cancer cell bioimaging agents. Especially, Ag NP-decorated CQDs revealed very good antibacterial activity for Gram-positive S. aureus and Gram-negative E. coli. Through the combination of both bottom-up and top-down approaches, CQDs with precisely controlled optoelectrical properties, which were applicable as both bioimaging and antibacterial agents, were simply obtained from resorcinol and formaldehyde, cheap commodity chemicals, in a reproducible and scalable manner. Work towards further control of the size distribution of RF-based CQDs, the enhancement of fluorescence intensity through surface passivation and hybridization of RF-CQDs with other nanomaterials to accomplish the preparation of multi-functional CQDs is on-going.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (no. 2014055946), a grant from the Fundamental R&D Program for Core Technology of Materials funded by the Ministry of Knowledge Economy, Republic of Korea (1415120175), and Chungcheong Institute for Regional Program Evaluation (CIRPE) Promotion Project of the MOTIE (Ministry of Trade, Industry and Energy) Republic of Korea (A004600100).

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Footnote

† Electronic supplementary information (ESI) available: Details of the experimental procedure for in vitro and antibacterial activity studies, solubility test of CS, TEM images, EDX, full and deconvoluting C1s, O1s, N1s and Ag3d XPS spectra of CQD-S/Ag NP and CQD-P/Ag NP. See DOI: 10.1039/c5ra01506e

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