Photosensitizer-Conjugated Ultrasmall Carbon Nanodots as

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Photosensitizer-Conjugated Ultrasmall Carbon Nanodots as Multifunctional Fluorescent Probes for Bioimaging Alexandre Loukanov, Ryota Sekiya, Midori Yoshikawa, Naritaka Kobayashi, Yuji Moriyasu, and Seiichiro Nakabayashi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b11721 • Publication Date (Web): 01 Apr 2016 Downloaded from http://pubs.acs.org on April 4, 2016

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The Journal of Physical Chemistry

Title:

Photosensitizer-conjugated Ultrasmall Carbon Nanodots as Multifunctional Fluorescent Probes for Bioimaging

Authors: Alexandre Loukanov†,§*, Ryota Sekiya†, Midori Yoshikawa†, Naritaka Kobayashi†, Yuji Moriyasu†, Seiichiro Nakabayashi1†

Affiliations: †

Graduate School of Science and Engineering, Saitama University, Shimo–Ohkubo 255, Sakura – Ku, Saitama 338-8570, Japan §

Laboratory of Engineering NanoBiotechnology, Department of Engineering Geoecology, University of Mining and Geology “St. Ivan Rilski”, Sofia, Bulgaria

Correspondence to: Prof. Alexandre Loukanov (E-mail: [email protected]) Prof. Seiichiro Nakabayashi (E-mail: [email protected]) Graduate School of Science and Engineering, Saitama University, Shimo–Ohkubo 255, Sakura – Ku, Saitama 338-8570, Japan Phone: + 81-48-858-3617

ACS Paragon Plus Environment


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ABSTRACT

Highly luminescent ultrasmall carbon nanodots (CDs) have been prepared by one step microwave-assisted pyrolysis and functionalized with fluorescein photosensitizer by diazo-bond. The absorption edge of such prepared Fluorescein–N=N–CDs was red shifted in comparison with the bare one. Nevertheless, the emission signal induced by the nanoparticle quantum-sized graphite structure was quenched due to photo-isomerization of the diazo group at photoexcited state. In order to restrict the photo–isomerization, i.e. rotation around nitrogen – nitrogen bond, the diazo group was fixed by metal cation to form complex compound or chelate. The obtained metal–complex of Fluorescein–N=N–CDs show absorbance maximum same as bare CDs, but recovered emission signal from nanoparticle moiety, which was bathochromic – shifted. They exhibit lower quantum yield in comparison with the bare CDs but better photostability toward emission quenching in nutrition cell culture. The formed photosensitizer-conjugates nanoprobes were proposed as multifunctional fluorophores for intracellular in vivo imaging due to their attractive photophysical attributes, tunable and excitation-dependent emission. The bioapplication of photosensitizer–conjugated CDs was demonstrated as fluorescent tracers for endocytosis pathways in cultured Tobacco cells. Their successful staining and lower toxicity to the plant cells were compared with conventional quantum dots (CdSe/ZnS core – shell type, which caused acute toxicological in vivo effect).

I. INTRODUCTION

Highly luminescent nanoparticle – based imaging probes have advanced current state-of-the-art labeling technology and are expected to generate new medical diagnostic tools, based on their superior brightness and photostability compared with conventional molecular probes. The current common fluorescence probes (organic dyes) have some drawbacks that limit their applications in living cell studies, especially for long-term in vivo imaging. These limitations are often related to the photodegradation, phototoxicity, nonspecific binding, chemical stability, perturbation of analyzed living processes, chromatic and spherical aberration and etc. 1 For example, all available fluorophores are subject to photodegradation, the more hydrophobic fluorophores are notorious for nonspecifically sticking to lipid membranes and any cellular structures. Dye-sensitized phototoxicity to cells and tissues remains a significant problem in many assays, in which cells are under continuous illumination for more than a few seconds. Production of singlet oxygen and its products is the main cause of photo-oxidation and due to this reason the fluorescent probes must be used always at minimally perturbing concentrations. In our investigation the employed conventional dyes as endocytosis markers FM4-64 and Lucifer Yellow stain all intracellular membranes of Tobacco cells. 2 Due to this reason the endocytic pathways remain unrevealed.


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Compared to the traditional organic dyes and semiconductor quantum dots, the photoluminescent carbon nanodots (CDs) are superior in terms of high (aqueous) solubility, robust chemical inertness, facile modification and high resistance to photobleaching. 3 CDs are considered to be next generation green nanomaterials with multipurpose applications 4 due to their advantages in stable photoluminescence (PL), broad excitation and tunable emission spectra, low toxicity and etc. Their potential is relatively unexplored yet compared to that of other carbon – based materials, such as fullerenes, carbon nanotubes and graphene. Although numerous reports indicate promising bio – applications, the CDs are still facing two major challenges. The first challenge is labeling efficiency for multimodal imaging purposes, and the second is the quenching effect of the CDs fluorescence in biological culture medium or environment. In the current report these issued are discussed below and we propose solution by developing photosensitizer – conjugated ultrasmall CDs. The use of nanoparticles with different sizes is problematic for high–resolution multimodal imaging, semi–quantitative measurement of epitope numbers, or when epitope density is high. 5 However, the emission color of bare CDs is closely related to their size, shape and structures, i.e. the optical properties are dictated by the quantum confinement effect. Carbon nanodots are described as paracrystalline carbon, composed of angstrom–sized domains (or core), surrounded by amorphous carbon frames. 6,7 Based on the microstructure of the core, CDs can be divided into two classes: class I with a graphitized (crystalline) carbon core and class II with a disorder (amorphous) core. 8 In both classes there is a correlation between the particle size and the emission color. As the size of the CDs with crystalline core increase, the absorption edge, the excitation and the emission maxima are red shifted. 9 In the case of CDs with amorphous core the effect is opposite. The theoretical calculations 10 showed the dependence of the HOMO – LUMO gap on the size of the graphene fragments. As the size of the fragment increases, the gap decreases gradually, and the gap energy in the visible spectral range was obtained from graphene fragments with diameter of 14 – 22 Å, which agrees well with the visible emission of CDs with diameters of < 3 nm. The maximum size limit that yields a fluorescent carbon particle is typically ~ 10 nm; thus, synthetic methods that produce carbon nanoparticles > 10 nm are either non fluorescent or are weakly fluorescent. The best nanoprobes for multimodal imaging purposes are ultrasmall nanoparticles (diameter of 1 – 3 nm) with tunable emission color, because the small size reduces the steric hindrance between antibodies (for immunolabeling bio-application) and leading to higher labeling efficiency. 11 In our report we propose functionalization strategy of bare CDs, which ensure ultrasmall nanoprobes (diameter of 2 – 3 nm) with tunable emission color. The color is result of bathochromic effect due to electronic transition between the quantum-sized graphite structure of nanoparticle and photosensitizer molecule. The PL behavior of CDs is also greatly affected by their surface chemistry. It has reported that the energy gap of CDs might originate also from their surface states. 12 CDs possess a variety of surface functional groups, defects and sometimes even adsorbates and hence, their surface states can hardly be identified in terms of physical and chemical properties. These “defect sites” located on the surface are considered to be responsible for trapping of the excited– state energy, which should be passivated to achieve a high quantum yield. The presence of other



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elements, such as oxygen, nitrogen and their relative ratios in the carbon matrix dictate the quantum yield too. In addition, there exists a correlation between the existence of defect sites and the emission. However, recently, it was found that the presence of some biological relevant metal ions (Fe3+, Cu2+, Co2+ etc.) and certain anions (Cl–, CH3COO–, PO43– etc.) influence fluorescence intensity of CDs. 13 They often occurred in biological nutrition mediums, cells or tissues and might cause fluorescence quenching of CDs due to non-radiative electron transfer from the excited state of the CDs to the d-orbital of the ion (for example Fe3+). In the current report the photosensitizer – conjugated CDs demonstrated in vivo PL activity unlike the preceding bare CDs. The perspective aim of this report is to design and synthesis biocompatible photosensitizer – conjugated carbon nanodots, which can be used as multifunctional fluorescent nanoprobes for multimodal state-of-the-art techniques (with future potential as agents for singlet oxygen or oxygen anion radical generation). Most of the known C-dots have emission centered in the blue regions, which is not preferred for biological applications because of the harm of their wavelength excitation light to living cells or masked due to their autofluorescent. 13 The advantage of proposed nanoprobes as multifunctional fluorophores are their tunable and excitation-dependent emission color. In our synthetic strategy the microwave assisted pyrolysis of proper carbon source was used as a simple, reproducible and facile technique for preparation of bare CDs. We introduced amine – groups during the microwave pyrolysis process as passivation agents, which enhance the fluorescent performance 14 of the resultant CDs. We used inexpensive citric acid as the necessary source of carbon and 1,2–ethylenediamine as the surface passivation agent. To further increase the fluorescent property of resulting CDs they are functionalized by fluorescein as photosensitizer by conjugation trough isothiorea or diazo bond. The photoisomerization was inhibited by formation of chelate metal complex between the diazo bond and electron donor groups on the nanoparticle surface. This design enables a specific electron transitions between the chromophoric groups, which was the general original idea of our report in order to develop the future applications of the functionalized CDs. Photosensitizer – conjugated CDs were entered successful into living Tobacco plant cells (comparable to HeLa cells in medical research), which indicates that in near future these particles could be efficiency applicable in many fields of nano-biotechnology or nanomedicine.

II. EXPERIMENTAL SECTION

2.1. Preparation of photosensitizer–conjugated carbon nanodots. All chemicals were used without further purifications, namely citric acid (Wako), 1,2– ethylenediamine (Wako), 5-amino–fluorescein (Sigma–Aldrich), fluorescein isothiocyanate (Sigma–Aldrich), acetone (Wako) sodium nitrite (Wako), hydrochloric acid (Wako), sodium


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hydroxide (Wako), sodium carbonate (Wako), ethylene glycol (Wako) and ruthenium trichloride (Sigma–Aldrich). A. Synthesis of bare carbon nanodots. In a typical synthesis, citric acid (1 g) was diluted with 10 mL MilliQ water and 1,2–ethylenediamine (0.2 mL, 0.18 g) was injected to the solution under vigorous stirring. Then, the clear transparent solution mixture was subject to microwave irradiation for 3 minutes at 600 W microwave oven, and finally a yellowish brown gum was formed. The carbonization proceeded very fast and no inorganic salt or acid was needed. Thus stock solution of bare CDs was obtained by microwave – assisted pyrolysis, according to the chemical equation on Figure 1. < Fig. 1. >

Figure 1. Preparation of highly luminescent carbon nanodots by microwave – assisted pyrolysis.

Purification protocol for carbon nanodots. When cooled down to room temperature, the formed yellowish brown solid was dissolved in MilliQ water and dialyzed against ultra-pure H2O through dialysis membrane (MWCO of 100 – 500) for 3 days. Thus, 300 mL red–brown aqueous solution containing both reaction precursor and ultrasmall CDs was obtained and the nanoparticles were purified by dissolving (ratio 1 : 5) and centrifugation in acetone. The obtained precipitate pellet was washed with acetone, collected and vacuum–dried at ambient temperature to give 0.98 g product. The yield of the reaction was about 80 % purified nanoparticles. B. Conjugation of bare carbon nanodots with fluorescein isothiocyanate (FITC). Carbon nanodots (1 g) were dissolved in 100 mL of 50 mM carbonate buffer, pH 9.5, containing 150 mM NaCl. At this alkaline pH value the amine groups on nanoparticle surface are mainly unprotonated. Then 40 mg of FITC was added and the reaction mixture was stirred gently for 1 hour at ambient temperature. The modified CDs–FITC were purified from the reaction mixture by centrifugation and washing with acetone (as described above). The reaction yield was over 80 %. C. Conjugation of bare carbon nanodots with fluorescein dye trough diazo–bond. The diazotization reaction was performed in ice bath at temperature between 0 and 5 °C. While 20 mL of concentrated hydrochloric acid was diluted with about 60 g of crushed ice to which 2.5 g sodium nitrite dissolved in 10 mL of water was added. 5 – 7 mL of such prepared nitrous acid (blue color) was added to 20 mL solution of 5-amino–fluorescein with concentration 5 mg/mL (the color of precursor solution was changed to orange – yellow). The prepared diazonium ion was slowly added dropwise to 20 mL solution of bare CDs (with concentration 20 mg/mL) at alkaline pH (pH ~ 9). The color mixture was changing from light yellow to dark red. The obtained Fluorescein–N=N–CDs were purified from the reaction mixture by centrifugation and washed with acetone (as described above).



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D. Preparation of ruthenium [Fluorescein–N=N–CDs] complex. 200 mg carbon nanodots modified with diazofluorescein (or Fluorescein–N=N–CDs) were dissolved in 2 mL MilliQ water. The prepared aqueous solution was injected in a triple flask, which contains 1 mmol ruthenium (III) chloride in 50 mL solution of ethylene glycol under Ar atmosphere. The reaction mixture was refluxed for 4 hours at 180 – 190 °C. The color changed from a dark black to a bright yellow in approximately two hours. The resulting solution was then cooled down to room temperature and analyzed. The obtained [Fluorescein–N=N–CDs]Ru2+ nanoparticles were purified from the reaction mixture by centrifugation and washing with acetone.

2.2. Carbon nanodots analysis and characterization. For absorption and fluorescence measurements, we used a Jasco UV – Vis absorption spectrophotometer (model № V – 570) and a Jasco analytical spectrofluorometer (model № FP – 6300), respectively. The quantum yields of the samples were measured by absolute PL quantum yield measurement system (Hamamatsu Photonics, C9920 – 03G). The excitation wavelength was selected from output of a xenon lamp by a monochromator. A home-built atomic force microscope with a commercial SPM controller (ARC2, Asylum Research) and a Si cantilever (AC160, Olympus) with a resonance frequency of 300 kHz and a spring constant of 40 N/m were used. Cantilever deflection is detected with optical beam deflection sensor and tip-sample interaction force is detected by tapping mode. The fluorescent images were taken by Inverted Research Microscope ECLIPSE Ti (Nikon) and Biological microscope BX53 (Olympus).

2.3. Staining and bioimaging of Tobacco BY-2 cells. Tobacco BY-2 cells were cultured for 4 days in Murashige and Skoog (MS) culture medium. Then cell suspension at the logarithmic growth phase, were transferred to fresh MS culture medium containing 20 µM of the corresponding labeling marker (quantum dots, bare CDs, Fluorescein–N=N–CDs, [Fluorescein–N=N–CDs]Ru2+ complex) and incubated at 26 °C for 3 – 4 hours. Cells were washed with MS medium to remove the excess dye at 26 °C.

III. RESULTS AND DISCUSSION

3.1. Optical characteristics of ultrasmall carbon nanodots, prepared by microwave – assisted pyrolysis. Microwave – assisted pyrolysis method is one of the easy and efficient methods for the preparation of fluorescent CDs. In the present study, we used citric acid (CA) which contains carboxyl groups as a carbon source to facilitate the dehydration and carbonization, and 1,2– ethylenediamine (EDA) as the surface passivation agent. The mixing CA with EDA under


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microwave irradiation resulted in a light – brown precipitate. Thus, we produced, by microwave pyrolysis, CDs with the best quantum yield value of 56 %, which is comparable to that of common fluorescent quantum dots. 15–17 The reason for this quantum yield value is formation of surface passivation, which is the main factor contributing to the strong fluorescence of CDs. 18 The nitrogen content in CDs is very important for generating strong PL. The absorption of bare CDs in water shows strong absorption band peaking at ~ 350 nm with a tail extended to 500 – 550 nm. This observed band is attributed to the n – π* transition corresponding to the carbonyl/amine functional groups on the surface. The blue color emission is highly related to the C=O and C=N functional groups on CDs surface, which can generate abundant structural configurations and introduce new energy levels into their electronic structures, and consequently result in more electronic transition possibilities. The PL bandwidth of CDs is much wider. The wide peak may result from the inhomogeneity chemical structure and diverse PL centers. The influence of pH value of the CDs solution on the quantum yield was inspected. The photoluminescence intensity was very stable over the pH range 3 – 11 as it is shown on Fig. 2. < Fig. 2. >

Figure 2. Luminescent properties of bare CDs. (A) Photoluminescence intensity and (B) change of emission maximum at different pH. (C) Photo of aqueous solution with various pH (from 2 to 11) of bare CDs under UV light (356 nm excitation).

That attribute is favorable for CDs bio-application as pH sensor. Excitation-dependent PL behavior was not observed and the intensity decreased slightly when the pH value was 3 and 11, respectively (Fig. 2A). In addition, when pH was changed from acid to base, the emitted photoluminescence was a little shifted to shorter wavelength (Fig. 2B). This pH dependent emission comes from the hydrogen bond of CDs surface. When the pH of environment changed to low the carboxyl groups are protonated and a hydrogen bond is formed between them and electron pairs of the amino groups. Thus the conjugated system has been extended and emission maximum peak is shifted to longer wavelength. The PL stability of the bare CDs under exposure to UV at different ionic strength and pH was additional investigated. There were no changes in photoluminescent intensity or peak characteristics at different ionic strengths. The CDs are almost resistance toward photobleaching in the microscopic experiments. Obvious photobleaching in our experiments was found after 5-6 hours of continuous UV excitation (Fig. S1). Similar results are also reported in the literature. 19-20 A photograph of highly luminescent CDs in tubes with various pH under UV – lamp irradiation is shown on Fig. 2C. A bright blue color PL emission with a peak at around 460 nm was observed upon excitation of the aqueous solution of CDs at 365 nm. The luminescent properties of CDs might originate from the recombination of electron – hole pairs generally from oxygen – containing functional groups.



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On Fig. 3 is shown an AFM image of the ultrasmall bare CDs prepared by microwaveassisted pyrolysis. In this experiment, the sample was prepared by dropping a diluted CDs solution onto cleaved highly oriented pyrolytic graphite (HOPG) and dried the droplet by N2 gas after the sample was left for 5 min. The height profile in Fig. 3(b) showed that vertical and lateral sizes of the particles in Fig. 3(a) were 1.5-2.0 nm and 50-60 nm, respectively. The nanoscale height of the CDs might be ascribed to the smaller molecular weight of the starting precursors. The lateral size of the particles measured by an AFM tip with a finite curvature radius © apparently becomes larger than the actual particle size by a geometrical reason [Fig. 3(c)]. Assuming that a spherical particle with a radius of r is measured by the tip, the apparent lateral size (D) is simply expressed as D = 4(rR)1/2. In this experiment, R = 20 nm (slightly larger than the nominal value due to tip abrasion) and r = 1.7 nm (measured from the height profile). Thus, D = 23.3 nm. Even after taking the tip effect on apparent particle size into account, the measured particle size is larger than the estimated value. This might be explained by the lateral aggregation of the CDs in the drying process of the CDs droplet. Therefore, CDs are imaged as pancake-like shape.

< Fig. 3. >

Figure 3. (a) AFM topography image of bare carbon nanodots on a HOPG substrate. (b) Height profile measured along line A-B in (b). (c) Schematic model of the tip effect on apparent lateral size of spherical particle in AFM imaging.

3.2. Photosensitizer – conjugated CDs as multifunctional fluorescent nanoprobes. The UV-Vis spectrum of the prepared bare CDs given on Fig. S2 and Fig. S3 shows a peak at around 360 nm. It was determined that surface modifications of CDs were successfully carried out by the conjugation with fluorescein through the formation of (i) isothiourea linkage or (ii) diazo bond directly to the aromatic structure of bare CDs. Azo compounds efficiently undergo photoisomerization in the photoexcited state, and this process is surely the reason for non – fluorescence properties of the quantum-sized graphite CDs-moieties in Fluorescein–N=N–CDs. Photoisomerization accompanies the change in the direction of the lone pair of one nitrogen atom. Restraining the lone pair from changing direction should prevent the photoisomerization. Therefore, suppression of the photoisomerization movement in the Fluorescein–N=N–CDs was considered a key to provide them with a fluorescent nature. The azo compound possess suitable bonding characteristics due to presence of –N=N– group and can form verities of metal complexes with transition metal ion with general structure which is shown on Fig. 4.

< Fig. 4. >


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Figure 4. Concept of molecular design to make Fluorescein–N=N–CDs fluorescent upon photoirradiation by formation of metal complex with ligands (L) to restrict both rotation and photoisomerization. In our experiment Ru2+ potentially participate in chelate complex between diazo, carbonyl and hydroxyl groups.

Comparison of the properties of the bare and functionalized carbon nanodots. The origins of PL in CDs are not completely clear so far. We reasoned that this wavelength – depended phenomena (or multicolor PL) might come from the different distribution of emissive traps on the surface of CDs, as suggested in the literature. 21 Though bare CDs showed the highest fluorescence and extremely broad peak at 450 nm (if 365 nm excitation wavelength is applied), the peak of the functionalized one is lower of intensity. We tried to correlate the origin of the tunable emission with the fluorescence quantum yield values of the nanoparticles. In all experiments the photosensitizer – conjugated CDs possess lower quantum yields in comparison with the initial bare CDs (see Table 1).

< Table 1. >

Table 1. Comparison of the quantum yield (QY) of the bare and photosensitizer – conjugated carbon nanodots.

The absorption edge of prepared Fluorescein–N=N–CDs was red shifted in comparison with the bare one. The obtained metal – complex modified Fluorescein–N=N–CDs show absorbance maximum same as bare CDs, but bathochromic fluorescence with yellow color and recovered nanoparticle emission signal (as shown on Fig. 5).

< Fig. 5. >

Figure 5. Comparison of the optical properties of bare carbon nanodots and photosensitizer – conjugated carbon nanodots.

The bare CDs in our experiment possess not excitation-depended emission. Their strong emission comes from the quantum-sized graphite structure instead of the carbon-oxygen surface and the quantum yield is controlled by the surface chemistry. 3 The bare CDs and [Fluorescein– N=N–CDs]Ru2+ show same characteristic absorption at 345 – 350 nm, which can be ascribed to n → p* transitions (of C=O, C=N and –N=N–) with a tail into visible range. The shoulder peak



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of fluorescein moiety disappears in the case of metal complex modified CDs, which can be explained with the photoisomerizaton restriction and the occurred electronic transition between the quantum-sized graphite structure and fluorescein molecule. This effect cause also a higher degree of surface diazotization and the result is red-shifted and excitation-depended emission as shown on Fig. 6.

< Fig. 6. >

Figure 6. Photoluminescence emission spectra of [Fluorescein–N=N–CDs]Ru2+ nanoparticles: (A) with progressively longer excitation wavelength from 340 nm to 480 nm in 20 nm increments and (B) normalized intensities of the emission spectra.

To further explore the optical properties of the as-prepared [Fluorescein–N=N–CDs]Ru2+, we carried out a detailed PL study with different excitation wavelengths ranging from 320 nm to 460 nm (Fig. 6A). Unlike most other luminescent CDs, the as-prepared exhibit an excitation – dependent PL behavior only when the excitation is larger than 380 nm. More specifically, the PL peak shifted from 460 nm to 530 nm by changing the excitation wavelength from 400 to 460 nm. In contrast, the PL peak remained almost unchanged (at 460 nm) for the excitation wavelength over 320 – 400 nm. [Fluorescein–N=N–CDs]Ru2+ complex had preeminent multicolor fluorescent emission depending on different excitations, as shown on Fig. 6B. When the excitation wavelength changed from 340 nm to 480 nm, the maximum emission peak position shifted to longer wavelength, from 450 nm to 550 nm, so called red – shift about 100 nm. The PL intensity decreased remarkably, showing clear excitation wavelength dependence. It should be noted that as the excitation wavelength red – shifted, the emission wavelength could reach red light and can be used in cell imaging, trough relatively weak. In addition, the fluorescence maximum and intensity are depended on the excitation wavelength. The higher intensity was observed at 360 nm excitation and the fluorescence spectra feature small tails extending into the long wavelength region.

3.3. Carbon nanodots vs semiconductor quantum dots for bioimaging. Cell imaging technique is valuable in defining cell behaviors that are necessary for invasion, intravasation, and extravasation. To prove that photosensitizer-conjugated CDs are useful as imaging nanoprobes we investigated the endocytosis pathway in tobacco cells. We chose tobacco BY-2 cells, because they are well studied, nongreen plants, fast growing and comparable to HeLa cells for human research. In an exploratory experiment to assess the potential application of CDs as bioimaging nanoprobe tobacco cells were cultured in the medium containing 100 µg staining reagent for 4 hours. The washed cells were imaged under a fluorescence microscope.


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A control experiment for staining of BY-2 cells with commercial quantum dots was done. It was motivated because the quantum dots have superior luminescence performance, which is attractive for in vitro and in vivo optical imaging in biological applications. 1 In our experiment, however, the quantum dots seemed harmful for BY-2 cells. The endocytosis processes were blocked at the beginning state as in Fig. 7. It is probably because the commercial quantum dots contain cadmium (or other heavy metals). 22 < Fig. 7. >

Figure 7. Labeling of BY – 2 Tobacco cells with commercial quantum dots (Invitrogen) and inhibition of the endocytosis at the beginning state. (A) Fluorescent and (B) light micrographs of a same object. (C) and (D) confocal images of different objects. With arrows are pointed the accumulated autolysosomes in the cultured cells. Scale bar represents 20 µm.

The conclusion from this experiment is that the presence of heavy metals such as cadmium and the toxicity issues, associated with heavy metals limited the bio–application potential of well used commercial quantum dots. Nevertheless, tobacco BY-2 cells incubated with CDs seemed healthy (Fig. 8). The microscopic observation revealed that BY-2 cells stained with bare CDs had very low emission signal, which is at the same level as unlabeled cells, and practically were not imaged on the micrograph (Fig. 8 A and B). In the case of both Fluorescein–N=N–CDs and [Fluorescein–N=N–CDs]Ru2+, BY-2 cells were labeled within 2 hours and the stained cells are easily visualized in the conventional fluorescence microscope. Moreover, green and yellow color images could be obtained because of the excitation dependent fluorescence feature of the photosensitizer-conjugated CDs (Fig. 8 C and D). In both cases, cells seemed healthy during the microscopic observation. We found that the fluorescent intensity and photobleaching of Fluorescein–N=N–CDs are better (intensity is higher and photobleaching is slower) than those of FITC–CDs. This is due to the higher number of the surrounded dye molecules, which are linked to the aromatic structure of the nanoparticle trough diazo–bond. FITC molecules are bound to smaller number amino groups on the nanoparticles surface shell whereas Fluorescein–N=N– are bound to large number on the nanoparticle quantum-sized graphite structure.

< Fig. 8. >

Figure 8. Light microscopic observation of tobacco BY-2 cells stained with (A) control experiment with no CDs, (B) bare CDs, (C) Fluorescein–N=N–CDs and (D) [Fluorescein–N=N– CDs]Ru2+. The down fluorescence micrographs were taken with low pass emission filters (LP 510), exposure time 1/6 second in a commercial biological microscope BX53 Olympus). In the case of staining with bare CDs the observation was only cellular autofluorescence or noise. Scale bar represents 20 µm.



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BY-2 cells stained with Fluorescein–N=N–CDs (Fig. 8C) possessed significantly stronger emission signal than those stained with [Fluorescein–N=N–CDs]Ru2+ (Fig. 8D). This effect was explained with the different quantum yield of the nanoprobes (Table 1), which effect their emission signal in the fluorescence microscope. In addition, in the staining with [Fluorescein– N=N–CDs]Ru2+ we observed some labeled details surrounding cell walls, which do not appear in the staining with Fluorescein–N=N–CDs. We also noticed that after longer incubation, a strong fluorescence was observed at the nucleus. In addition, non – homogeneous staining of dividing cells was discovered in the case of staining with Fluorescein–N=N–CDs. The investigation of these phenomena is still in progress All these features demonstrate that the photosensitizerconjugated CDs have good biocompatibility and make them appropriate candidates for cell imaging.

IV. CONCLUSION

Here, we report a fluorescent carbon nanoparticle – based alternative imaging probes that are suitable for biological staining and potential diagnostics. A simple large scale synthesis for the preparation of photosensitizer – conjugated carbon nanodots in chelate complex with metal cation, leading to tunable visible emission color has been developed. The obtained yellow emitting [Fluorescein–N=N–CDs]Ru2+ show absorbance maximum same as naled CDs but do not contain mixture of blue and blue–green emitting nanoparticles. Their average sizes are around 3 nm and therefore easily entered BY-2 cells, giving a bright yellow emission in the fluorescence microscope. The fluorescent micrographs reveals that the dye functionalized CDs are non–toxic for in vivo imaging applications at doses that are required for cell labeling studies. These carbon–based nontoxic fluorescent nanoprobes are powerful alternative to cadmium– based toxic quantum dots. As new fluorescent probes, the advantages of the photosensitizer – conjugated CDs include highly water solubility, tunable emission color, good photostability in physiological pH range and nutrition medium, make them suitable candidates as probes for the applications in bio–imaging field.

Associated content The supporting information is available free of charge on the ACS Publication website at DOI: Stability of CDs emission by ionic strength and continuous UV excitation (Figure S1). Optical properties of FITC–conjugated carbon nanodots (Figure S2). Optical properties of Fluorescein– N=N–CDs (Figure S3).


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microwave, 3 min 600 W

citric acid

ethylene diamine

Figure 1


80 % yield carbon nanodots


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Figure 2


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Figure 3



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C-dot non-fluorescent moiety

C-dot fluorescent moiety

Figure 4


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1

1

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Figure 5



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Figure 6


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Figure 7



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Figure 8


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D

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photoisomerization

restriction of photoisomerization L

C-dot

C-dot

Abstract Picture


L

Photosensitizer-Conjugated Ultrasmall Carbon Nanodots as

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