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Title:
Facile Size-Controlled Synthesis of Fluorescent Carbon Nanoparticles with Size-Independent Optical Properties

Paper sections:
Introduction
<p>The fine tuning of the nanoparticle size for biomedical applications is incredibly important in terms of its clearance, targeting, and degradation [1][2][3][4][5]. Oftentimes, nanoparticle size and shape determine their chemical, optical, and magnetic properties [6][7][8]. The optical properties of plasmonic nanoparticles and up-conversion quantum dots are easily manipulated through size and shape modifications of the nanoparticles [8][9][10][11][12][13][14]. Fluorescent carbon nanoparticles (CNPs) made from the thermal decomposition of sugars have recently gained in popularity for their tunable optical properties and their biocompatible nature [15][16][17][18][19][20][21][22][23]. However, the precise means and mechanisms affording their unique optical properties are not fully understood. Some studies have indicated that the surface chemistry (oxidization, cross-linking, and functionalization) [17][18][19][20], and the internal bonding (graphitic vs diamond, and point defects) [24,25] within the nanoparticle are directly responsible for the resultant fluorescent properties. However, in these studies, researchers were unable to control for size and thus could not rule out the potential that the size of these nanoparticles may be causal of the resultant fluorescent properties. Herein, we compare optical properties of nanoparticles with various sizes prepared in a controlled manner. By making controlled alterations of the carbon source material, concentration of that source material, and incubation time, we were able to generate CNPs of various sizes without changing the resulting absorbance and fluorescence spectral properties. In order to prepare these nanoparticles, we employed a facile hotplate method (Wu et al. 2013), wherein, six batches of nanoparticles were produced simultaneously. We were able to generate nanoparticles between 10 and 100 nm, finding that neither refractive index, absorbency, nor fluorescence property changes between even the smallest and largest carbon particles. From this observation, we concluded that carbon nanoparticle optical properties are independent of nanoparticle size.</p>

Simultaneous multiple CNP synthesis
<p>To six separate 20 ml scintillation vials 1 ml of 0.58 g/mL agave nectar in water was added. To these vials, a square of aluminum foil (approximately 16 cm 2 ) was tightly wrapped over the tops of each scintillation vial and 6 holes, equidistant from each other and the center, were punctured through the taught foil using a 200 µl pipette tip. The tinfoil-covered scintillation vials were arranged according to Fig. 1, similarly to a "revolver," with a dummy scintillation vial in the center containing no agave or tinfoil wrapping. This "revolver" setup was tightly bound with tinfoil to keep the formation of vials continuous throughout the experiment and a rubber band was placed around the entire setup close to the lip of the vials effectively restraining the tinfoil on the vials. This tightly wrapped revolver setup was placed onto the center of a pre-heated hotplate (200C) with tin-foil wrapped over the top of the hotplate to encourage an even heat distribution and to allow for easy cleanup of potential spills. The revolver was rotated continuously throughout a 30-minute period.</p><p>After 30 minutes of heating, the vials were removed from heat and 3 ml of deionized water was added; the solutions were tip sonicated for 2 minutes using 1 amp, 5 s on, 5 s off, and then these solutions were syringe filtered using a 0.220 µm pore filter.</p>

Simultaneous CNP synthesis with variations in incubation time
<p>Six vials containing 1 ml of 0.58 g/ml agave in water were prepared similarly to the previously mentioned "Simultaneous multiple CNP synthesis," and arranged in the "revolver" style. At 5, 10, 15, 30, 45, and 60 minutes vials were removed from the hotplate and replaced with empty 20 ml vials. When these vials were removed from heat, 3 ml of de-ionized water (DI-H2O) was added and the particles were tip sonicated and filtered similarly to the previous methods.</p>

Simultaneous CNP synthesis with variations in concentration of carbon source material
<p>Six vials containing 1 ml of 0.15, 0.29, 0.43, 0.58, 0.78 and 0.88 g/mL agave in water were prepared similarly the previously mentioned "Simultaneous multiple CNP synthesis," and arranged in the "revolver" style. After 30 minutes, the vials were removed from heat and were then tip sonicated and filtered similarly.</p>

Dynamic light scattering (DLS)
<p>Hydrodynamic diameters were determined using a Malvern Zetasizer ZS90 particle size analyzer, while the scattered light was collected at a fixed angle of 90°. A photomultiplier aperture of 400 mm was used, with the incident laser power adjusted to obtain a photon counting rate between 200 and 300 kcps. Measurements for which the measured and calculated baselines of the intensity autocorrelation function agreed to within +0.1% were used to calculate hydrodynamic diameter values. Hydrodynamic diameter was analyzed using number distribution. All determinations were made in 3 consecutive measurements with 15 runs each.</p>

UV-Vis absorption studies
<p>For UV-Vis, 1 µL of the CNPs were diluted with 999 µl of carbon-filtered deionized water. For calibration, the blank consisted of carbon-filtered deionized water alone. The absorption spectra was acquired in scanning mode for the range of 200-800 nm using a GENESYS 10S UV-Vis Spectrophotometer (Thermo Fisher Scientific, Rockford, IL). Measurements were taken at every 0.1 nm interval.</p>

Fluorescent studies
<p>For fluorescence measurements, the emission spectra of CNPs were obtained using TECAN Infinite F200 PRO using a Corning® 384 Well Optical Imaging Flat Clear Bottom Black Polystyrene TC-Treated microplates. Measurements were done at different excitation-emission wavelengths, with gain set to 100 for all measurements throughout this work. Furthermore, integration time per each well was set as 20 µs, and the temperature was kept at 25ºC.</p>

Refractive index (RI) measurements
<p>For refractive index measurements, a handheld AR200 refractometer (Reichert technologies) was used. The refractometer was calibrated with deionized water and refractive index of samples were acquired at 1:1000 fold dilution in deionized water.</p>

Transmission electron microscopy (TEM) measurements
<p>For TEM, 10 µL of the CNPs were diluted with 990 µl of carbon-filtered deionized water. With this diluted carbon nanoparticle solution, 2.5 μL were placed on a 300-mesh carbon film supported by a copper grid and allowed to stabilize for 2 min. A filter paper was then used to remove liquid to form thin liquid films, which were consequently allowed to air dry while covered. Images were obtained using a Jeol 2010 cryo-electron microscope operating at 200 kV, and using different degrees of defocus to obtain an adequate bright contrast. Images were recorded on a Gatan UltraScan 2kx2k CCD.</p>

Variations in Carbon Source
<p>In making fluorescent CNPs, a primary component of consideration is the carbon source. While typically sugars are used, it has been established that surfactants containing various functional groups can be added to the synthesis protocol to modify the size and fluorescent properties of the resultant nanoparticles. We aimed to determine how much variation of the physical and optical properties we could induce in our resultant nanoparticle system without the addition of surfactants, maintaining a simplistic singular component system. We compared three commercially available food grade sugar sources (agave, honey, and molasses), with technical grade sucrose. Under identical reaction conditions (0.58 g/ml, 30 min incubation, 200ºC), we observed that nanoparticles produced using technical grade sucrose resulting in significantly smaller nanoparticles (3 ± 2 nm), compared to commercial food-grade sources (Fig. 2a). From these sugar sources, agave nectar resulted in the smallest CNPs at 19 ± 4 nm, with honey slightly larger (41 ± 38 nm), and molasses forming the largest CNPs at 461 ± 79 nm (Fig. 2b). While these CNPs were dramatically different in hydrodynamic diameter, their optical properties were surprisingly similar (Fig. 2b). For all carbon source variants utilized for nanoparticle synthesis, a common absorbance maxima (at 285 nm) was recorded with a fluorescence maxima at or near 510 nm (± < 5 nm). With slight variations in the absorption peak tails (molasses having the broadest tail), there are maximally 5 nm variations between fluorescence peaks. This slight variation in fluorescence cannot be due to nanoparticle size, as there exists a 500 nm variation of nanoparticles size between the largest and smallest nanoparticle formulations. This slight variation in fluorescence could be due to subtle changes in surface oxidation of the nanoparticles, as the thermal decomposition could result in slight oxidation differences between sugars based on oxygen and hydrogen saturation between them.</p>

Variations in Incubation time
<p>In several nanoparticle synthesis systems, nanoparticle size can be controlled through variations made in the incubation time. Using agave nectar (0.58 g/ml) as our sugar source we prepared nanoparticles with 5, 10, 15, 30, 45, and 60 minutes of thermal decomposition (200 ºC). From UV-Vis analysis, we found identical absorption maxima, with differences in peak tails resultant of formed particle concentration (Fig. 3a). From the fluorescence spectra afforded through 360 nm excitation we found that the fluorescence maxima would shift no more than 5 nm (Fig. 3a). From DLS analysis, we found that the hydrodynamic diameter decreased from 511 ± 222 nm (for 5 min incubation) to 19 ± 4 nm as incubation time approached 30 minutes (Fig. 3b). With continued incubation, both size and variability of the particle size distribution increased (Fig. 3b). While we were able to prepare fluorescent nanoparticles of varied size distributions (20 -500 nm) through variations in incubation time, these variations were not reflected through the resultant optical properties (Fig. 3c and d).</p>

Variations in Concentration
<p>In several nanoparticle synthesis systems, nanoparticle size can be controlled through variations made in the reactant concentration. Using agave nectar as our sugar source at concentrations between 0.15-0.88 g/mL we prepared nanoparticles with 30 minutes of thermal decomposition (200 ºC). All of the nanoparticles formed through variations of agave concentration resulted in identical absorption and fluorescence maxima (Fig. 4a). From size analysis through DLS and TEM, we found a general trend that with higher concentrations of the source material (agave), resultant nanoparticles would be larger (Fig. 4b and c). Again, while we find high variability in nanoparticle size through variation of the source material concentration, these variations are not reflected in the optical properties (Fig. 4d-f).</p>

Conclusion
<p>Within these works, we have demonstrated the capacity for size-controlled synthesis of fluorescent CNPs, through variations in incubation duration, sugar source material, and the concentration of that sugar source. From the resulting optical properties of these nanoparticles, we demonstrated that carbon nanoparticle size is independent of the resulting fluorescent spectra in terms of location and maxima. While we found no change in fluorescent properties in nanoparticles of various sizes, as controlled through sugar source concentration, we did find subtle (< 5 nm) shifts between nanoparticles prepared with varied sugar sources and incubation times. These small shifts in fluorescent spectra could possibly be due to differences in surface oxidation resultant of incubation resultant of differences in oxidation state of the starting materials. The incubation process involving thermal decomposition is directly responsible for the oxidation of the sugar source material and variations of sugar source materials could have different degradation products.</p>
Nanoparticle imaging probes and drug delivery systems have distinct advantages over free or diffusive delivery in that they can concentrate the imaging contrast agent and/or drug, elicit controlled release/degradation, and have a high surface area to volume ratio beneficial for attaching targeting ligands. The size of these nanoparticles affects delivery, retention, degradation rate, and sometimes the radiological properties of the particles. For many optically active nanoparticles (such as gold, silver, and quantum dots), the optical properties are directly dependent on the size and shape of the nanoparticles. While this provides a simplistic outlet for modifying the optical properties of those nanoparticles, it is limiting in that their applications are also dependent on morphology. In these works, we aim to determine if the optical properties of fluorescent carbon nanoparticles are dependent on size through variations in synthetic parameters.Fluorescent carbon nanoparticles with hydrodynamic diameters ranging from 10 -500 nm were prepared through variations of sugar source, concentration of agave (as a sugar source) and incubation time. Through comparisons made between these nanoparticles, we found no change in the local absorbance maxima and refractive index, with < 5 nm shifting in fluorescence maxima location. We have observed that fluorescent carbon nanoparticles can be prepared within a large range of sizes (10 -500 nm) without considerable shift in optical properties. Because of this observation, we can infer that the optical properties of fluorescent carbon nanoparticles are largely size independent.