Synthesis, functionalization and bioimaging applications of highly fluorescent carbon nanoparticles

Abstract

Highly fluorescent crystalline carbon nanoparticles (CNPs) have been synthesized by one step microwave irradiation of sucrose with phosphoric acid at 100 W for 3 min 40 s. This method is very simple, rapid and economical and hence can be used for large scale applications. The average particle sizes are 3 to 10 nm and they emit bright green fluorescence under the irradiation of UV-light. Therefore, the particles can be used as a unique material for bioimaging as well as drug delivery. To further increase the fluorescence property of the synthetic carbon nanoparticles we simply functionalized them by using different organic dyes, such as fluorescein, rhodamine B and α-naphthylamine; the maximum fluorescence intensity was observed for the particles functionalized with fluorescein. It is very interesting to note that all of those compounds show maximum fluorescence intensity at 225 nm excitation wavelength and for any excitation wavelength the peak positions are exactly same the position as that of CNPs itself, which is completely different from the individual precursors (dyes). All of the above compounds, including CNPs, have also been successfully introduced into the erythrocyte enriched fraction of healthy human blood cells with minimum cytotoxicity.

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1. Introduction

Carbon-based nanomaterials, including carbon nanoparticles, nanocrystals, nanotubes, fullerenes, nanofibers, graphene nanosheets and porous carbon materials have promising applications in nanoelectronics,¹ microelectrical devices,² electrochemistry,^3,4 sensors,⁵ catalysis,⁶ ultracapacitors,^7–9 bioimaging,^10,11 and drug delivery.^12,13 Recently, carbon nanoparticles (CNPs) have become greatly important due to their excellent photoluminescence properties.^14–16 Though CNPs induce some blue or green photoluminescence, they are still comparatively very low compared to that of the fluorescent carbon quantum dots.^17,18 However, due to their toxicity, those quantum dots are very limited in biological or cell imaging applications.^19,20 Hence, the synthesis of environmentally benign carbon nanoparticles with high photoluminescence properties is still a great challenge. Carbon nanoparticles has been synthesized by various methods such as the candle soot method,²¹ laser induced pyrolysis of hydrocarbons,²² low temperature solution synthesis,²³ electrochemical oxidation of graphite,²⁴ microwave pyrolysis of sucrose,²⁵ proton-beam irradiation of nanodiamonds,²⁶ thermal decomposition of organic compounds,^27,28 using mesoporous silica nanoparticle as template²⁹ and using polyacrylonitrile (PAN) as a nanoparticle precursor.³⁰ Although those methods are available for the synthesis of CNP, the candle soot and the microwave pyrolysis of sucrose are the useful techniques because they are economically feasible. However, bulk synthesis is still a challenge due to unavailability of any existing method for the large scale preparation of CNPs. Though the microwave pyrolysis of sucrose in polyethylene glycol (PEG) can only be used as the scale up process for the synthesis of carbon nanoparticles, the fluorescence property of its products is too low to apply them in the field of biotechnology and other useful applications. Again, it is very difficult to isolate the synthesized nanoparticles from PEG as its powder form and hence for any application, PEG must be used with CNPs, which is not desirable for all applications. On the other hand, for the candle soot method the quantum efficiency of the resultant nanoparticles is too low due to the production of particles with various diameters. Recently, it was also reported that those particles with various sizes can be separated from each other by ultracentrifugation.³¹ The fluorescence properties of the CNPs increase with increasing rpm of the centrifugation. The highest quantum yield is observed for the CNPs, which is collected by centrifugation at 14 000 rpm. But by this separation procedure, we are able to produce a milligram scale synthesis of highly fluorescent CNPs.³¹ Therefore, economical and facile approaches for the large scale production of highly fluorescent carbon nanoparticles still remain a great challenge.

In this paper, we demonstrated a simple one step microwave synthesis of carbon nanoparticles and functionalized CNPs with fluorescence properties much better than that of the CNPs produced by the previously reported methods. This method is cheap, facile and applicable for large scale synthesis. Here, sucrose is used as the carbon precursor and phosphoric acid as the oxidizing agent. To further increase the fluorescence property of the resulting carbon materials, they are functionalized by fluorescein, rhodamine B and α-naphthylamine. However, our approach is not only able to tag the organic dye molecules to the CNPs, but decreases the cytotoxicity of the synthetic CNPs. These CNPs and functionalized CNPs are easily entered into living cells (human red blood corpuscle (RBC)), which indicates that in the near future, these particles will be efficiently applicable in the field of bio-sensing and drug delivery.

2. Experimental procedures

2.1. Synthesis of carbon nanoparticles (CNPs)

1 g sucrose was placed in a 250 mL beaker and dissolved in the minimum volume (4 mL) of water. 20 mL of ortho-phosphoric acid (88%) was then mixed with the above clear solution followed by heating in a microwave oven at 100 W for 3 min 40 s. The colour of the solution gradually changed from colorless to brownish-black through yellow. The brownish-black solution was allowed to stand at room temperature to cool the solution. After cooling, 50 mL distilled water was added and again allowed to stand for few minutes. Then, a brownish-black precipitate (CNPs) was obtained, which was collected by centrifugation at 4000 rpm for 10 min. The CNPs was purified by washing several times with distilled water followed by the centrifugation. The sample was finally dried at 40 °C in a vacuum oven. The purified CNPs was easily dispersed in water at pH ≥ 7 and also in alcohol, acetone, DMSO and DMF.

2.2. Synthesis of α-naphthylamine tagged CNPs (CNP-Naph)

25 mL of a 1 mg mL⁻¹ aqueous dispersion of CNPs was prepared by 30 min sonication of the nanoparticles in water at pH 8. The pH was adjusted by the addition of dilute ammonium hydroxide. Then, 30 mg EDC was added to disperse the CNPs. The pH of the solution was maintained at 6 by adding 0.1 N hydrochloric acid and the whole solution was vigorously stirred for four hours. In another container 10 mg α-naphthylamine was dissolved in a minimum amount of ethanol, and 5 mL distilled water was added. After that, it was gradually mixed with the activated CNP solution with constant stirring. Stirring was again continued for an additional four hours. Finally, a brownish-black product was isolated through the precipitation of the above dispersion by the addition of excess ethanol. The product was separated by centrifugation, washed several times with ethanol and finally dried at 40 °C in vacuum.

2.3. Synthesis of fluorescein tagged CNPs (FCNP)

25 mL of a 1 mg mL⁻¹ aqueous dispersion of CNPs was activated by EDC using the same procedure as above. Then 100 μL ethylenediamine was added and the solution was stirred for 4 h. After that, an excess amount of ethanol was added and the solution allowed to stand for a few hours. A brownish-black precipitate (CNP-en) was obtained, which was collected by centrifugation at 4000 rpm. The precipitate was washed with water and ethanol to remove the impurities. The product (CNPs-en) was dispersed in 20 mL distilled water at pH 6 by sonication. Simultaneously in another container, 10 mL of a 10⁻⁴ M aqueous solution of fluorescein was activated by 20 mg EDC at pH 6, and was added dropwise to the above dispersion of CNPs-en with constant stirring. After continuous stirring for 4 h, it was precipitated by the addition of excess ethanol. Finally, the product (FCNP) was collected by centrifugation followed by washing with water and ethanol and was dried in vacuum at 40 °C.

2.4. Synthesis of rhodamine B tagged CNPs (CNP-Rh)

The procedure was the same as that of the synthesis of FCNP, only instead of fluorescein, we used 10⁻⁴ M rhodamine B for this purpose.

2.5. Cell labeling

A erythrocyte enriched fraction was centrifuged twice (3000 rpm, 15 min) at 4 °C to removed the residual plasma and buffy coat. RBCs were washed 3 times with sodium phosphate buffer (pH 7.4) and resuspended in the same buffer to make a packed cell volume of ∼10% (w/v) as stock. Then, cell labeling was carried out by mixing 690 μL of the above RBC stock suspension with different volumes (100–500 μL) of CNP, CNP-Naph, FCNP and CNP-Rh separately from their stock solution (0.1 mg mL⁻¹) and making up a total volume of 1.5 mL by addition of phosphate buffer saline to maintain a 5.5% cell volume for each and every set of experiment. Afterwards, the erythrocyte cells were concomitantly centrifuged (3000 rpm, 15 min) at 4 °C and washed thrice with sodium phosphate buffer. Finally, 20μL of the cell suspension were taken and used to prepare a smear on glass slides.

2.6. Instrumentation

Fourier transform infrared spectroscopy (FT-IR) was conducted using a Perkin-Elmer spectrum RX-1 IR spectrophotometer. For absorption and fluorescence measurements, we used a Shimadzu absorption spectrophotometer (model no: UV-1700) and a Spex-fluorolog-3 spectrofluorimeter (model no: FL3-11), respectively. ³¹P NMR spectra were obtained at 400 MHz by using a Bruker Avance-II 400 MHz instrument, where DMSO-d6 was used as the solvent. The phase characterization was carried out by X-ray diffractometer (XRD; model no. PW1710). The sample for XRD was prepared by the deposition of well dispersed carbon nanoparticles on a glass slide and, after drying, the analysis was performed by using cobalt as the target material. High-resolution transmission electron microscopy (HRTEM) was carried out by using a Phillips CM 200, operating at an acceleration voltage of 200 kV. The field emission scanning electron microscopy (FE-SEM) was also performed by Jeol Model JSM-6340F. For FE-SEM analysis, samples were prepared by deposition followed by spin coating of the aqueous dispersion on a glass slide. Again for HR-TEM analysis, a very dilute aqueous suspension was prepared, which was then deposited on a copper grid and finally dried in air. Fluorescent microscopic analyses along with their corresponding optical images were carried out by using a Carl Zeiss-Axiolab fluorescent microscope.

3. Results and discussions

It was determined that surface modifications of any carboxylic acid containing nanoparticles were successfully carried out by the conjugation with aminated base through the formation of amide linkage. In this work, carbon nanoparticles were synthesized from sucrose and H₃PO₄. The particles were functionalized by different organic dyes to increase the fluorescence as well as decrease the cytotoxicity of those synthetic nanoparticles. The whole reactions including the synthesis of CNPs and its various functionalizations are summarized in Scheme 1.

Scheme 1 Schematic representation elaborating the synthesis and functionalization of carbon nanoparticles.

Fig. 1 shows the FTIR spectra of CNP, CNP-en, CNP-Naph, FCNP and CNP-Rh powders, in which the peaks at 3427, 2933, 1702, 1656, 1207 and 1018 cm⁻¹ for CNP clearly demonstrate the presence of –OH, C–H, –COOH, C=O, C–O–C and C–O, respectively. Again for CNP-en, CNP-Naph, FCNP and CNP-Rh, we also observed two additional peaks at 1637 and 1558 cm⁻¹ along with the peak at 1656 cm⁻¹ indicating the presence of an amide bond. Since no certain peak at around 1700 cm⁻¹ was observed for all these compounds, it was easily concluded that the formation of such amide bonds were due to the bonding between the –COOH groups of CNP or the dyes (fluorescein and rhodamine B) with the –NH₂ group of ethylenediamine or α-naphthylamine. A band at 1454 cm⁻¹ indicating the amine group was also observed for CNP-en, which was completely absent in the FTIR spectra of both FCNP and CNP-Rh. Furthermore, a common peak at 3434 cm⁻¹ illustrates the presence of the –OH group for all of the above compounds. Therefore, from Fig. 1 it was suggested that the formation of CNP-Naph and CNP-en had been preceded by the bonding between the –COOH groups of CNP with the –NH₂ group of α-naphthylamine and ethylenediamine, respectively. Since ethylenediamine contains two –NH₂ group, one amine group was still present in CNP-en, which was utilized in the formation of amide bonds with the –COOH group of fluorescein and rhodamine B during the synthesis of FCNP and CNP-Rh, respectively.

Fig. 1 FTIR spectra of (a) CNP, (b) CNP-Rh, (c) CNP-Naph (d) CNP-en and (e) FCNP.

Fig. 2a shows the UV-visible spectra of CNP, CNP-Naph, α-naphthylamine and the mixture of CNP and α-naphthylamine in aqueous medium. CNP gave a certain absorption peak at 273.61 nm, whereas two intense peaks at 211.86 and 304.13 nm appeared for α-naphthylamine. Though both CNP and α-naphthylamine gave intense peaks, a very weak absorption peak at 211.86 nm was observed for CNP-Naph. Again, when we simply mixed CNP with α-naphthylamine, the mixture retains the absorption peaks of α-naphthylamine itself. Similarly, Fig. 2b and 2c represented the UV-visible spectra of FCNP and CNP-Rh, respectively, with their precursors. Though fluorescein and rhodamine B showed several intense peaks at 483.47, 316.8, 274.36, 232.7 nm and 554.2, 353.26, 259.48 nm, respectively, no characteristic peak of those precursors were observed for CNP-en, FCNP and CNP-Rh. For both FCNP and CNP-Rh, a single peak was detected at 272.1 and 283.3 nm, respectively. Again, in case of CNP-en, no characteristic peak was obtained.

Fig. 2 Comparison of the UV-visible spectra of (a) CNP, CNP-Naph, α-naphthylamine and the mixture of CNP and α-naphthylamine; (b) CNP, CNPen, FCNP, fluorescein and the mixture of CNP and fluorescein; (c) CNP, CNPen, CNP-Rh and rhodamine B.

CNP, CNPen, CNP-Naph, FCNP and CNP-Rh all exhibited green fluorescence under the irradiation of UV light. It was very interesting to note that all of the above compounds achieved maximum fluorescence intensity when those were excited at 225 nm and the peak position was exactly the same as that of the position of CNP itself, which was completely different from the peak positions as well as the excitation maxima of the individual precursor dyes. Again, this excitation wavelength (225 nm) was completely different from the usual excitation wavelength (340 nm) of the reported carbon nanoparticles, which were amorphous in nature.^21,25,31 The fluorescence property of the CNPs was also dependent upon the irradiation time. Fig. 3a depicts the fluorescence spectra of CNPs, produced by different irradiation times, which revealed that the fluorescence properties of these synthesized nanoparticles increases with the irradiation times, reaching a maximum at 3 min 40 s and then decreasing. From Fig. 3b, it was also observed that when all the compounds (CNP, CNPen, CNP-Naph, FCNP and CNP-Rh) were excited at 225 nm, the fluorescence spectra showed the same peak position at 453 nm for each and every compound and the peak intensity gradually increased from CNP to CNP-Naph and reached a maximum for FCNP. But for CNP-Rh, the peak intensity was about the same as the intensity of CNP. Though the fluorescence intensities for CNP and CNPen were the same at 225 nm excitation, the intensity for CNPen was comparatively much lower than that of the CNP at any other excitation wavelength (Fig. 3c). Fig. 3d and 3e illustrates that with increasing excitation wavelength, the peaks for both CNP and CNPen gradually shifted from lower to higher wavelengths with the peak intensity decreasing after reaching a maximum at 225 nm excitation wavelength. Finally, from Fig. 3f, 3g and 3h we were able to compare the nature and the fluorescence intensity of the functionalized CNPs with their corresponding dye precursors. Fluorescein gave a characteristic broad peak at 515.7 nm and CNP displayed a sharp peak at 454.5 nm when they were excited at 225 nm. But at the same excitation, FCNP retained the peak position of CNP, showing a peak centered at 454.5 nm with increased peak intensity. On the other hand, in case of α-naphthylamine, CNP and CNP-Naph, the peak position for the dye was very close to the position of CNP and CNP-Naph (around 451 nm), though the intensity of the former was comparatively very high and the peak was broad in nature. Again, in the case of rhodamine B, at 225 nm excitation wavelength, the intensity of the corresponding emission peak was comparatively less than that of the CNP. It was also very interesting to note that all of the above dyes gave very intense broad peaks when they were excited at 340 nm. But at this excitation wavelength no characteristic peak was observed for FCNP, CNP-Naph and CNP-Rh, indicating that they were completely different from their corresponding dyes and also they were completely pure, i.e. not a mixture of CNP and dyes. The photograph of CNP and all of its functionalized compounds are shown in Fig. 4. From Fig. 4, it was clearly observed that the aqueous dispersion of all of the compounds including CNP emitted bright green fluorescence light, when they were irradiated under a UV lamp.

Fig. 3 Comparison among the photoluminescence spectra of (a) carbon nanoparticles prepared using different irradiation (microwave) times; (b) CNP, CNPen, CNP-Rh, CNP-Naph and FCNP at 225 nm excitation wavelength; (c) CNP and CNPen at 220, 225 and 230 nm excitation wavelengths; (d) CNP with progressively longer wavelengths from 200 nm to 250 nm; (e) CNPen at different excitation wavelengths, ranging from 200 nm to 250 nm; (f) CNP-Naph, CNP and α-naphthylamine; (g) FCNP, CNP and fluorescein; (h) CNP-Rh, CNP and rhodamine B.

Fig. 4 Consecutive bright field and UV irradiation images of CNP (A, B); CNP-Naph (C, D); FCNP (E, F) and CNP-Rh (G, H).

The zeta potential of CNP and all of its functionalized compounds were given in Fig. 5. Due to the presence of a –NH₂ group, the zeta potential of CNPen was positive from pH 1 to pH 6 and therefore it was easily dispersed in acidic medium, but not in water at pH 7 or greater than 7. Except CNPen, all other compounds were easily dispersible in basic medium, but quite unstable in acidic medium. At pH 10 to 14, the highest zeta potential was observed for FCNP and lowest for CNP-Naph. Due to the formation of amide bond between CNP and α-naphthylamine, the free –COO⁻ was replaced by amide linkage and hence the zeta potential for CNP-Naph was comparatively less than the potential value of CNP at any pH ranging from 8 to 14. But from pH 8 to 7, the potential of CNP abruptly dropped from −23.8 to −9.2 mV, which was again the lowest value among all of its functionalized compounds, even the potential of CNPen. Therefore at pH 7 it was readily agglomerated, as compared to FCNP and CNP-Rh. For those compounds, they were quite stable at pH 7 and the agglomeration was found with adjusting the pH to 5 or less than 5. Since fluorescein contains –OH group and rhodamine B contains –N(CH₂–CH₃)₂, the higher potential value of FCNP than CNP-Rh was absolutely reasonable. But the higher potential value for both FCNP and CNP-Rh than that of CNP at any pH was also clearly understandable. If just there was only the replacement of –COOH group with an amide bond, without any structural change of the corresponding dyes, the zeta potential must be higher than its functionalized products. Therefore from Fig. 5 it was also concluded that the dyes must be hydrolyzed during the reaction with CNP and therefore the zeta potential values of FCNP and CNP-Rh were greater than that of CNP itself. The UV-visible spectra at different pH for all of the compounds (Fig. 6a and 6b) again supported the above facts. From Fig. 6a and 6b, it was revealed that any organic dyes, such as fluorescein and rhodamine B, showed at least two different characteristic peaks in UV-visible spectra when varying the pH of their aqueous solutions—one was in acid medium and another one was in basic medium. Generally for most of those organic dyes, an acid–base equilibria could exist depending upon the pH of the solution. Therefore the UV-visible adsorption spectrum would be the function of pH for such dye molecules in aqueous solution, owing to their existence as a cationic, neutral, anionic or dianionic species.^32,33 For fluorescein, this is due to the conversion of –OH to O⁻, which decreases the transition energy and hence increases the wavelength in the UV-visible spectrum. Again for rhodamine B, the difference between the peaks obtained from basic and acidic media was comparatively less (552.32 and 556.75 nm) than that of the fluorescein, because there was no hydroxy group present on it, but only the conversion of a single –COOH to –COO⁻ with formation of free –NR₃ from –NHR₃⁺. But for both FCNP and CNP-Rh, there were no effect on their UV-visible peaks with changing the pH of their aqueous dispersion. Again, no characteristic intense peak was observed for the above compounds at any acidic or basic pH, which was very unusual for any fluorescent compound. Therefore some structural change or hydrolysis of those dyes must have originated during the formation of amide bonds with CNP. The stabilities of all these compounds are shown in Fig. 7, which show photographs of CNP, FCNP, CNP-Rh and CNP-Naph dispersing in water at pH 8 and the precipitation, appearing at pH 3.

Fig. 5 Zeta potential of CNP, CNPen, CNP-Rh, CNP-Naph and FCNP as a function of pH in aqueous dispersions at a concentration of ∼0.02 mg mL⁻¹.

Fig. 6 (a) UV-visible absorption spectra for rhodamine B, showing the red shift when changing the solution from basic to acidic. The inset images are the UV-visible absorption spectra for the aqueous dispersion of CNP (left) and CNP-Rh (right) as a function of pH; (b) UV-visible absorption spectra for fluorescein, showing the blue shift when altering the pH from basic to acidic. The inset image is the UV-visible absorption spectra for the aqueous dispersion of FCNP as a function of pH.

Fig. 7 Effect of the addition of acid on the aqueous dispersion of CNP and all of its functionalized compounds. At pH < 5, agglomeration has been found for each and every case. The photograph shows the stable dispersion in base (pH ≈ 9) as well as their agglomeration in acid (pH ≈ 4) for CNP (A,B); CNP-Naph (C,D); FCNP (E,F) and CNP-Rh (G,H).

Now we would like to compare the properties of CNP produced by our present method with the CNP synthesized by the previous microwave irradiation of sucrose in PEG²⁵ (CNP-PEG), which was the main accomplishment of this present research work. Though CNP-PEG showed the highest fluorescence peak at 380 nm excitation wavelength and the peak was extremely broad, the peak intensity as well as the peak area was still exceptionally less than that of the CNP at 225 nm excitation wavelength (Fig. 8). When we excited the CNP-PEG at 225 nm, the peak position was very close (454 nm) to the CNP, suggesting the similar nature of these compounds, but the peak intensity was negligibly small as compared to the fluorescence intensity of the CNP. Again the zeta potential of CNP-PEG was relatively less negative than that of the CNP (Fig. 9). The highest zeta potential for CNP-PEG was −23.9 mV at pH 14 and it was also comparatively very less at any pH ranging from 8 to 14. This was probably due to the presence of a greater amount of hydroxy and carboxylic acid groups in CNP and also the generation of the –C–PO₄³⁻ bond during the microwave irradiation of sucrose in the presence of phosphoric acid. The existence of such –C–PO₄³⁻ bonds in CNP was confirmed from ³¹P NMR spectra of the CNP (Fig. 10) in DMSO-d6 as the solvent. In ³¹P NMR spectra, the sharp peak at 3.372 ppm exhibited the presence of P–O bonds (PO₄³⁻) and the absence of any P–H bond due to non-splitting pattern of the peak. Hence the phosphorous atom of PO₄³⁻ must be bonded with the carbon atom to fulfil the valency of the phosphorous.

Fig. 8 Comparison between the photoluminescence spectra of CNP by using phosphoric acid and the CNP by using PEG (reported) at 225 and 340 nm excitation wavelengths.

Fig. 9 Comparison between the zeta potential for CNP by using phosphoric acid and PEG (reported) as a function of pH.

Fig. 10 ³¹P NMR spectrum of carbon nanoparticles.

The TEM image of the CNP, prepared by 3 min 40 s irradiation, is shown in Fig. 11. The average particle size ranged from 3 to 10 nm. The FE-SEM image of CNP, FCNP, CNP-Rh and CNP-Naph are shown in Fig. 12, which revealed that CNP, CNP-Rh and CNP-Naph are completely spherical in nature. On the other hand the morphology of the FCNP was completely different, containing very small particles with several agglomerations. Again for CNPen, agglomeration was also found due is lower zeta potential value. The X-ray diffraction pattern (Fig. 13) conveyed some sharp peaks for CNP, illustrating the crystalline nature of the sample. For CNP the 2θ value located at 19.3, 27.55, 33.8 and 39.35°, indicating the 100, 110, 111 and 200 planes, respectively, was determined by analytical method.³⁴ This diffraction pattern was completely different from the other reported semi-crystalline or amorphous carbon nanoparticles,^23,25,35 indicating the generation of a new class of carbon nanomaterials. CNP and all of its functionalized compounds were easily dispersed in water at pH 7 and readily enter into the human RBC. After entering into the cells, all those particles preserved their fluorescence properties and hence they turned bright green under UV excitation. But the control RBC, in which no particles were introduced, remained colourless under such UV excitation. Fig. 14a, 14b, 14c, 14d, 14e, 14f, 14g and 14h show the optical microscopy images with corresponding fluorescence microscopy images of control, CNP-Naph, FCNP, and CNP encapsulated RBC, respectively, wherein CNP-Naph displayed the brightest fluorescence among all of them.

Fig. 11 HR-TEM image of the carbon nanoparticles.

Fig. 12 FE-SEM images of (a) CNP, (b) CNP-Naph, (c) CNPen (d) CNP-Rh and (e) FCNP.

Fig. 13 XRD pattern for the CNPs.

Fig. 14 Optical and their corresponding fluorescence microscopic images of control RBC cells (a, b), and the RBC cells treated with CNP-Naph (c, d), FCNP (e, f) and CNP (g, h).

Although it was also reported that carbon nano materials were biologically toxic in nature,³⁶ from the extent of haemolysis, it was proposed that all of the above particles, including CNP, showed minimal toxicity after entering into the human RBC and the cytotoxicity was least for FCNP among all of them (Fig. 15). To determine the cytotoxicity or the extent of haemolysis, 690 μL of 10% RBC in buffered saline was added to 1 mg mL⁻¹ aqueous dispersions of each and every particle and the reaction mixtures were incubated at 37 °C for 3 h. The extent of haemolysis was determined spectrophotometrically^37,38 by taking the absorbance of the supernatant at 540 nm:

Percentage (%) of haemolysis = (A/B) × 100

where ‘A’ and ‘B’ are the absorbance for the sample (reaction mixture) in PBS and the RBC suspension in 20 mL cold distilled water, respectively, at 540 nm. For normal erythrocyte (control), the percentage of haemolysis was ∼8%, which was very close (∼11%) after introduction of 300 μL (1 mg mL⁻¹) aqueous dispersion FCNP, to that of the 690 μL sample of 10% RBC in buffered saline. When FCNP was replaced by CNP-Naph, the percentage of haemolysis was ∼14.7%, indicating that both FCNP and CNP-Naph were bio-compatible in nature. On the contrary the cytotoxicity was highest for CNP (∼22.96%) among all its functionalized compounds. This was probably due to the presence of carboxylic acid groups in CNPs, which was replaced by an amide bond after functionalization.

Fig. 15 Percentage of cell haemolysis after introduction of CNP and its functionalized nanoparticles as a function of their used volume.

Conclusions

A simple large scale synthesis for the preparation of highly fluorescent carbon nanoparticles has been developed. It was again functionalized by various organic dyes to increase the fluorescence property as well as decrease the cytotoxicity of the synthesized carbon nanoparticles. The average sizes of the CNPs are 3 to 10 nm and therefore easily enter into the erythrocyte enriched fraction of human blood cells, giving a bright green fluorescence under UV excitation. FCNP and CNP-Naph also give the bright green fluorescence under the UV excitation, but their intensities are comparatively much higher than that of the CNP itself. However the increasing intensity is not only due to the individual dyes, because after binding with CNP, they lose their own fluorescence properties and hence they do not give any photoluminescence peak at 340 to 450 nm excitation wavelengths. Since CNP-Naph shows the least cytotoxicity (negligible amount) among all the materials and its preparation procedure is simpler than that of the FCNP and again its green fluorescence after entering living cells is the brightest, in future it can be used as a unique material for bio-imaging and drug delivery.

Acknowledgements

The authors acknowledge Technology, Information, Forecasting and Assessment Council (TIFAC) and Department of Science and Technology, Government of India for funding and Authority of Indian Institute of Technology, Kharagpur for providing working facilities. We also express sincere thanks to Mr. Shouvik Mitra for his assistance (AERU, Biological Sciences Division, ISI-Kolkata) during the optical and fluorescent imaging of human RBC cell.

Notes and references

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