A simple one-step hydrothermal route towards water solubilization of carbon quantum dots from soya-nuggets for imaging applications

Abstract

A simple and low-cost pyrolytic carbonization method has been performed for the easy synthesis of carbon quantum dots from soya-nuggets under an insufficient amount of oxygen. Furthermore, hydrothermal functionalization of the carbonized black material after Soxhlet purification with nitric acid leads to the formation of its quantum sized water soluble version. The hydrothermally functionalized, water soluble carbon quantum dots (wsCQDs) are highly fluorescent and self-passivated, having a quantum yield value of ∼3%, with a small range of size distribution. High photostability with high solubility makes these potential candidates for imaging purposes, and we used these for the fluorescent labeling of Escherichia coli cells.

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Introduction

In the past few years, fluorescent nano-carbons (FNCs),^1–9 comprising carbon dots (CDs),^1–4 carbon nano-diamonds (CNDs),⁵ carbon nano-onions (CNOs),^6,7 and graphene quantum dots (GQDs),^8,9 have drawn immense interest, particularly for biological cell imaging^3,4,10–12 with several benefits in comparison with conventional metal-based quantum dots (QDs).^13–15 In terms of bio-compatibility (non-toxic nature),^4,12,16,17 their high values of quantum yield,^4,12,18 combined with their excellent solubility and stability,^1,3,4 make these QDs an effective fluorescent probe for long-term use for biological purposes.^3,4 FNCs are mostly composed of spherical nano-carbon possessing a high surface area to volume ratio, and have further been utilized for surface passivation purposes via simple surface modification (attaching surface functionalities). Surface modification is mainly achieved via the simple organic chemistry of oxidation, followed by the addition of polymeric^2,10 and monomeric amines¹⁹ to achieve high quantum yield values,^4,12,18 comparable with those of conventional metal-based QDs.^13–15 Metal-based QDs show classical quantum confinement effects¹ in their size-dependent multi-colored emission. Theoretically, in comparison with QDs, FNCs do not have any defined band gap for fluorescence emission,¹⁹ and this is an advantageous property for multi-colored emission from the same nano-carbon.^20–22

Among the FNCs, CDs are widely used fluorescent nano-carbons because of their simpler synthetic methodologies with easy reproducibility. It has been widely accepted that the presence of high density surfacial defects (defective centers) on FNCs is responsible for their tunable multi-colored emission profiles.¹² Emission from the defective centers of surfacial defects was first noticed from the surfacial defects of single-walled and multi-walled carbon nanotubes passivated with polymeric amines.^23,24 Since the first report of CDs from laser ablation of a graphitic target,¹ many more top-down^{10–12,25–32} and bottom-up^33–40 synthetic techniques have been explored for their easy production. Along with exciting sophisticated techniques, such as arc-discharge,⁴¹ abundant progress has been reported in the past few years for the cost-effective green synthesis of photoluminescent CDs by using bio-mass/waste bio-mass as a low-cost, compatible and easily available carbon precursor material, including ascorbic acid,⁴² banana juice,⁴³ candle soot,³⁵ chicken eggs,⁴⁴ citrate,⁴⁵ chitosan,⁴⁶ diesel soot,⁴⁷ gelatin,⁴⁸ glucosamine,⁴⁹ saccharides,⁵⁰ juices (orange, sugar cane, and strawberry),^51–53 pomelo,⁵⁴ and watermelon peel.⁵⁵ Because of their simplicity and straightforward synthesis, most of the synthetic approaches involve either hydrothermal or microwave-assisted green methods. As synthesized, CDs and their functionalized versions have shown immense potential for a wide variety of applications, such as bio-imaging agents,^12,56 biosensors,^47,57 chemical sensors,⁵⁸ drug delivery,⁵⁹ electro-catalysts,⁶⁰ photo-catalysts,⁶¹ and photodynamic therapy.⁶² Low-cost synthetic protocols with easy reproducibility and bio-compatibility make these CDs best suited for imaging purposes.^3,12,62,63

Towards cost-effective synthesis of carbon quantum dots (CQDs), we have used the natural, low-cost carbon source of soya-nuggets for the production of carbonized black material via pyrolysis under an insufficient amount of oxygen. To achieve the water-soluble version of CQDs, for the first time we performed the hydrothermal functionalization of CQDs in the presence of 50% nitric acid solution to isolate quantum-sized water soluble CQDs (wsCQDs) in almost quantitative yield (∼85%). The as-synthesized wsCQDs are self-passivated with multi-colored emissive photoluminescence properties and excellent photostability. These hydrothermally functionalized wsCQDs were applied here as a fluorescent marker for multi-colored imaging of E. coli cells.

Results and discussion

FTIR, Raman and TGA

A schematic representation of the simple synthetic approach for wsCQDs is shown in Scheme 1. Controlled pyrolysis of soya-nuggets in an oxygen deficient environment was performed at 550 °C over two hours in a horizontal CVD furnace, resulting in the formation of black hydrophobic materials composed of CQDs. These as-produced CQDs were purified via the Soxhlet extraction technique to remove un-burnt hydrocarbons. For functionalization purposes, we performed for the first time the hydrothermal functionalization of CQDs in 50% nitric acid solution to give quantum-sized wsCQDs.

Scheme 1 Schematic illustration showing the synthesis and water solubilization of CQDs.

As expected, oxidative treatment^{47,7b,64–67} by hydrothermal functionalization leads to the formation of highly self-passivated wsCQDs due to the incorporation of high-density electrophilic groups (hydroxyl and carboxyl type) over the surface of the CQDs as “surface defects”.^47,7b,64,68 The CQDs combine high solubility in aqueous media^7b,64–70 with tunable photoluminescence properties, without using any external surface passivating agents such as polymeric^2,10 and monomeric amines.¹⁹

FTIR and Raman spectroscopic measurements were performed to confirm the presence of negative surface functionalities and high-density surface defects (Raman D bands) over the surface of the wsCQDs.^7b,47 A representative FTIR spectrum of the wsCQDs is shown in Fig. 1(a). The intense peak at ∼1720 cm⁻¹ is attributed to C=O stretching and the peak at around ∼1215 cm⁻¹ is due to C–O stretching of –COOH groups. The broad peak at around 3425 cm⁻¹ is due to the O–H stretching vibrations of the C–OH group, along with residual water.

Fig. 1 (a) FTIR spectrum of wsCQDs; (b and c) comparative Raman and TGA plots, respectively, of Soxhlet-purified soot (black line) and wsCQDs (red line).

Fig. 1(b) compares the Raman spectra of Soxhlet-purified soot (black line) and hydrothermally functionalized (red line) wsCQDs. Two main characteristic peaks of graphitic carbon are observed in both spectra. The D band, a disorder induced peak, is attributed to surfacial defects and dangling bonds of graphitic hexagonal structures. On the other hand, the G band is responsible for in-plane stretching mode vibrations of symmetric sp² hybridized carbon bonds.^7b,47,64,70 Fig. 1(b) shows the disorder peak (D-band) centered at ∼1359 cm⁻¹ and the graphitic peak (G-band) centered at ∼1581 cm⁻¹ for Soxhlet-purified soot. After oxidative hydrothermal treatment, the same peaks appeared at ∼1348 cm⁻¹ (for the D-band) and ∼1573 cm⁻¹ (for the G-band). These shifts of both the D band and G band vibration frequencies towards lower wavenumbers after functionalization further confirm the weakening of the graphitic network of the carbon structure during the hydrothermal functionalization process. In addition, after functionalization, a broadening of the G band was observed, along with a significant increase of the D band intensity that may be caused by the introduction of high-density defective sites in the form of oxygen containing functional groups.^7b,47,64 The high density of surficial negative oxygenous functional groups in the wsCQDs is confirmed by zeta potential measurements (showing a highly negative value of −29.8 mV), which are in agreement with our FTIR and Raman results.

Elemental analysis (CHN) showed the composition of Soxhlet-purified soot C: 59.1 wt%; H: 1.5 wt%; N: 7.9; O (calculated) 31.5 wt% in comparison with the wsCQDs showing the composition C: 31.8 wt%; H: 2.3 wt%; N: 0 and O (calculated) 65.9 wt%. The increase in the content of oxygen by more than two-fold after functionalization shows strong evidence for the formation of highly oxygen-rich species during the hydrothermal functionalization.

To understand the thermal stability and extent of derivatization of the wsCQDs in comparison to Soxhlet-purified CQDs, we performed thermogravimetric analysis (TGA) of both the Soxhlet-purified CQDs and their hydrothermally functionalized soluble version. Fig. 1(c) shows the TGA thermograms of the Soxhlet-purified CQDs and the wsCQDs. Both samples were analyzed for the comparison of thermal de-functionalization up to 800 °C in an inert atmosphere and analysis of the weight loss with temperature. The TGA results show that the temperature required for degradation of the wsCQDs is lower in comparison to the Soxhlet-purified CQDs, which may be because of the easy decomposition of surface functional groups (carboxylic groups).^7b Furthermore, a high degree of weight loss was observed in the case of the wsCQDs (∼69%) in comparison to the Soxhlet-purified CQDs (∼15%) at 800 °C, which further endorses the presence of high-density surface functionalities in the wsCQDs, which are responsible for their lower thermal stability.

Microscopic studies

The surface morphology of the wsCQDs synthesized by the pyrolysis of soya-nuggets has been investigated by field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM), as illustrated in Fig. 2. The FESEM image shown in Fig. 2(a) shows the aggregated nature of the wsCQDs with nearly spherical morphology and homogeneity. TEM and high-resolution TEM (HRTEM) analysis were further carried out to understand the shape and size distribution of the synthesized wsCQDs. Low magnification and high magnification TEM images are shown in Fig. 2(b, c) and (e), respectively. The results clearly show the narrow size distribution of the wsCQDs. Statistical analyses of the size distributions of the wsCQDs were calculated based on several TEM images at various locations. A representative size distribution histogram (Fig. 2(d)) shows that the majority of the wsCQDs lie in the range of ∼5–10 nm. HRTEM images, as shown in Fig. 2(e), reveal the graphitic nature of the wsCQDs, with a lattice spacing of 0.32 nm similar to other forms of graphitic nano-carbon.^{7,21,22,71,72}

Fig. 2 (a) Low magnification SEM image; (b and c) low magnification TEM images; (d) size distribution histogram showing the average size distribution of wsCQDs (in the range 5–10 nm); (e) HRTEM image showing the graphitic lattice fringes of the wsCQDs.

The small size distribution of the wsCQDs after hydrothermal functionalization is confirmed by TEM analysis of the Soxhlet-purified soot and hydrothermally-oxidized soot, as shown in Fig. 3(a) for Soxhlet-purified and Fig. 3(b) for hydrothermally-oxidized soot. The hydrothermal functionalization causes a significant overall reduction in the size of the wsCQDs (∼5–10 nm). Almost uniform size distribution is obtained via the breaking of larger Soxhlet-purified soot particles (50–70 nm) during the oxidative process.

Fig. 3 TEM image of (a) Soxhlet-purified soot and (b) soot after hydrothermal functionalization, along with their size-distributed histograms.

Absorbance/photoluminescence study

The absorption spectrum of the wsCQDs synthesized by pyrolysis of soya nuggets in aqueous solution is shown in Fig. 4(a). A continuous increase in absorption intensity from 800 to 300 nm was observed, due to the π–π* transitions resulting from the overlapping of the delocalized electrons of sp² hybridized aromatic carbons. A distinct strong absorption peak at ∼295 nm was observed due to the n–π* transitions of C=O of high density surfacial-COOH on the wsCQDs.^7b,47,64,68

Fig. 4 (a) Absorption spectrum in water; (b) combined emission spectra at various excitation wavelengths ranging from 400 to 600 nm with a fixed increment of 20 nm of a dilute aqueous solution of wsCQDs; (c) corresponding PLE spectrum obtained at a 520 nm emission wavelength; (d) photo-stability of wsCQDs excited at 480 nm under continuous excitation for 3 h; (e and f) fluorescence micrographs of wsCQDs under (e) 488 nm and (f) 532 nm band pass filters.

To examine the detailed optical properties of the wsCQDs, their photoluminescence (PL) spectra with varying excitation wavelength from 400 to 600 nm with a 20 nm continuous increase have been analyzed and are presented in Fig. 4(b). The tunable emission of the wsCQDs shows broad and excitation wavelength-dependent PL properties, and the maximum emission intensity was observed at 420 nm excitation wavelength. Consistent with the general observations from various researchers, the wsCQDs showed a progressive red shift of their emission spectra with the increase in the excitation wavelength, a generic feature of photoluminescent carbon nanoparticles.^47,64 The as-prepared wsCQDs sample showed a quantum yield value of ∼3%. This quantum yield can be further increased up to 36% via surface functionalization with organic moieties, such as with PEG-1500.^1,2 However, in the present work we focused on the isolation of self-passivated uniformly distributed wsCQDs without using any external passivation.

Our synthetic strategy does not involve any surface passivating functional group such as amines. Hence, the tunable PL emission properties of the wsCQDs can be attributed to the varied fluorescence characteristics of different sized carbon particles and the various energy levels associated with different emissive sites on the carbon surface.^19–22 The PL excitation (PLE) spectrum corresponding to the strongest luminescence at 520 nm emission wavelengths for the wsCQDs has been recorded and is shown in Fig. 4(c). Three PLE bands at ∼342 nm (3.63 eV), ∼393 nm (3.15 eV) and ∼439 nm (2.82 eV) have been observed. These may be due to the presence of different types of emission sites over the synthesized wsCQDs' surface.^7b,70 The observed energy difference between excitation bands was less than 1.5 eV, which arises due to the presence of carbon radicals on the surface of the wsCQDs in the form of the triplet ground state which are responsible for the PL properties, along with the radiative recombination.^{1–4,7b,47,64} The photo-stability of the synthesized wsCQDs has been tested upon continuous excitation at 480 nm for 3 hours, and the fluorescence remains intact without any photo-bleaching effect, which corroborates the reasonably good photo-stability of the wsCQDs. A fluorescence micrograph of the wsCQDs under 488 and 532 nm band pass filters is shown in Fig. 4(e) and (f), which clearly shows the presence of green and red emitting wsCQDs.

Optical imaging of E. coli

The wsCQDs were used as a fluorescent probe for imaging E. coli cells. Fluorescence imaging has been performed by incubating the wsCQD solution in a growth medium of the DH5α strain of E. coli cells using a previously described method.^5,22,7b The wsCQD labelled E. coli cells show multicolor photoluminescence, as illustrated in Fig. 5(a) and (b), imaged under 488 and 532 nm band pass filters, respectively. The wsCQDs could be successfully used as a fluorescent probe for imaging other cell lines and cellular systems.

Fig. 5 Fluorescence microscope images of E. coli treated with wsCQDs; under (a) 488 and (b) 532 nm band pass filters.

Experimental

Chemicals

All of the chemicals, including toluene, methanol, acetone and nitric acid (HNO₃, 70%), were of analytical grade and used as received. The carbon precursor (soya-nuggets), purchased from a local market, was purified by repetitive washing with deionized (DI) water and finally air dried before use.

Synthesis of wsCQDs

As produced carbon soot was collected by pyrolyzing soya-nuggets in a horizontal CVD setup under an insufficient amount of oxygen. Under typical experimental conditions, a known amount of purified soya-nuggets (natural carbon-rich precursor) was taken in a rectangular quartz boat and placed in the middle portion of a horizontal CVD furnace. The furnace was initialized to keep warming with a ramp of 5 °C per minute and the temperature was set at 550 °C for 2 hours under the flow of N₂ : O₂ (95 : 5). The pyrolyzed as-synthesized black mass (carbon soot) was taken out of the furnace, and ground using a mortar and pestle. The as-obtained fine powder was subjected to sequential Soxhlet purification with toluene, methanol and acetone to remove smaller un-burnt soluble impurities. Afterward, the Soxhlet-purified carbon soot was finally washed with DI water and vacuum dried for 48 h. About 200 mg of Soxhlet-purified soot was treated with 50% nitric acid (100 mL) under hydrothermal conditions at 120 °C for 4 hours to obtain a brownish black colored solution. The soluble portion of the mixture was further separated by high-speed centrifugation and subjected to evaporation on a water bath to obtain wsCQDs with a yield of ∼85% (with respect to ∼200 mg starting material). Further purification and removal of residual nitrate within the wsCQDs was performed as described previously.^7b,47,64

Straining

The DH5α strain of E. coli cells was cultured on a nutrient plate in Luria–Bertani (LB) medium at 37 °C and further used for imaging purposes. In a laminar hood, the cells were cultured in LB medium and a mixed aqueous solution of 100 mL of wsCQDs (1 mg mL⁻¹ water). Then, a portion of the incubated cells was separated and washed with phosphate-buffered saline (PBS), smeared on glass slides and subsequently imaged.

Measurements

The initial morphological and microstructural characterization of the wsCQDs was performed by field-emission scanning electron microscopy (FESEM) in high-vacuum mode on a SUPRA 40VP FESEM (Carl Zeiss NTS GmbH, Oberkochen, Germany) microscope operated at an accelerating voltage of 10 kV. An ethanolic dispersion of the sample was deposited onto the surface of a brass stub, then evaporated at room temperature. Before imaging, the sample was gold coated. The quality internal characterization and structural parameters of the wsCQDs were analysed using a Tecnai 20 G2 200 kV TEM/HRTEM (transmission electron microscopy/high resolution TEM). The sample was prepared by drying 10 μL of the ethanolic solution of wsCQDs on a 300 mesh carbon coated Cu grid. Fluorescence microscopy images of the wsCQDs and E. coli cells were obtained on a Leica inverted microscope (Leica DC 200, microscopy system ltd, CH-9435, Heerbrugg). UV-Vis spectroscopic measurements were analyzed using a PerkinElmer Lambda 35 UV-Vis spectrophotometer in aqueous solution. Photoluminescence spectral measurements were analyzed on a PerkinElmer LS55 fluorescence spectrophotometer. Zeta potential measurements were carried out on a Beckman Coulter Delsa™ Nano in aqueous medium. Infrared spectra were obtained on a Bruker Fourier transform infrared spectrometer (FTIR, Vector 22 model) using a KBr disc. Raman measurements were performed using a Raman spectrometer (WITEC Model) with an Ar⁺ laser (λ_e: 532 nm). The elemental composition for the detection of the percentage of carbon (C), hydrogen (H), nitrogen (N) and oxygen (O) (calculated) was obtained using a PerkinElmer 2400 micro analyser. Thermogravimetric analyses (TGA) were performed on a Mettler Toledo Star System (heating rate of 10 °C min⁻¹) in a nitrogen atmosphere.

Conclusions

Quantum-sized fluorescent wsCQDs have been synthesized here via the pyrolysis of soya-nuggets under an oxygen-deficient atmosphere. The nano-sized water soluble carbogenic particles derived from hydrothermal treatment in acid solution exhibit self-passivated tunable multi-colored emissive photoluminescent cores with narrow size distribution. The synthesized wsCQDs were successfully applied for imaging E. coli cells. In the future, the wsCQDs can be easily scaled up and will show potential for accomplishing applications in cell labeling and drug delivery.

Acknowledgements

P.D. thanks UGC, New Delhi for major research project [F. no. 39-868/2010 (SR)] and SERB, New Delhi for financial support through the fast track scheme (SB/FT/CS-190/2011). K.M.T. thanks the European Union for a MEET fellowship and UBS for infrastructure. A.B. and A.S. thank MNIT-Jaipur for doctoral fellowships and S.K.S. thanks DST New Delhi for funding [SB/EMEQ-383/2014].

Notes and references

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