Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe

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Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe Haydar Ali, Susanta Kumar Bhunia, Chumki Dalal, and Nikhil R. Jana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11318 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 28, 2016

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ACS Applied Materials & Interfaces

Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe Haydar Ali, Susanta Kumar Bhunia,# Chumki Dalal and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata700032, India *Corresponding author. E-mail: [email protected] Telephone: +91-33-24734971. Fax: +91-33-24732805 #

Present Address- Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel

ACS Paragon Plus Environment

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ABSTRACT: Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of < 25 nm hydrodynamic size and their application as fluorescent cell labels. Hydrophobic carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e. 400-550 nm) to capture their emission spanning from 550 nm to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labelled cells. Presented method can be extended for the development of carbon nanoparticle based other bioimaging probes.

Keywords:

nanoparticle,

red

fluorescence,

bioconjugation,

coating,

imaging

probe,

nanobioconjugate


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INTRODUCTION Fluorescent carbon nanoparticle (FCN) with tunable emission is considered as promising alternative of cadmium based fluorescent semiconductor nanocrystals.1-6 FCN has been synthesized with tunable visible emission with high photostability and used as a potential bioimaging probe. Common synthetic approaches include laser ablation of graphite,7 oxidation of candle soot,8,9 carbonization of molecule/polymer10-14 and microwave-based pyrolysis of molecule/polymer.15,16 Although these approaches produce FCN with tunable emission, most of the method provide strong blue/green emission with higher fluorescence quantum yield. In contrast red emitting FCN is produced with < 1% fluorescence quantum yield or mixed with blue/green emitting FCN which is very difficult to isolate.1-6 Thus attempts have been made to prepare fluorescent FCN via synthetic modification,17 surface modifications18 or utilize them via hybrid assembly.19 Water-soluble functional nanomaterials are necessary for all biomedical applications.20 This is because nanoparticle can interacts with bioenvironment in their dispersed form via chemical or biological functional groups present on their surface. Thus coating chemistries (or surface chemistry) have been developed for conversion of high quality hydrophobic nanoparticles into water soluble nanoparticle and conjugation chemistries have been implemented for their transformation into functional nanoparticle.20-24 Using these coating and conjugation chemistries variety of functional nanoparticles have been derived from semiconductor nanoparticle,21 metal nanoparticle,22 metal oxide nanoparticle23 and other nanoparticles.24 Attempts have been made to implement similar coating and conjugation approaches for FCN in deriving functional FCN.1-6,25-28 However, they are mostly concentrated to blue or green emitting FCN and functionalization is limited to few selected molecules such as


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amine,25 polyethylene glycol (PEG),26,27 polyethyleneimine,28 peptide,13 folate13 and amino acid.29 Herein we report red fluorescent functional FCN of < 25 nm hydrodynamic size that can be used as fluorescent cell imaging probe and for other labelling applications. Hydrophobic FCN of 3-8 nm size with yellow-red fluorescence and 14 % fluorescence quantum yield has been synthesized first and then transformed into water soluble functional FCN using amphiphillic polymer-based coating and functionalization.30-33 Following this approach we have synthesized red fluorescent FCN functionalized with PEG, primary amine, glucose, arginine, histidine, biotin and folic acid. Resultant functional nanoparticles have high colloidal stability, 3 % fluorescence quantum yield and stable fluorescence under continuous light exposure. Arginine and folate functionaized FCN have been demonstrated as fluorescent cell label and primary amine terminated FCN can be functionalized with desired biomolecule of interest. Advantage of presented functional FCNs is that they have broad emission in the 550 nm to 750 nm ranges with the excitation between 400-550 nm. This property of nanoprobe can be exploited by using green excitation available in conventional fluorescence microscope to minimize the background fluorescence of cell/tissue environments. In addition one pot coating and functionalization has been developed using conventional amphiphilic polymers that made the bioconjugation simple. In contrast common approaches employ coating of nanoparticle followed by conjugation chemistry-based functionalization of coated nanoparticle in two separate steps. Presented FCN synthesis and functionalization approach can produce milligram to gram scale of nanoprobe and can be extended for functionalization of other biomolecules of interest.


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EXPERIMENTAL SECTION Materials. L-Ascorbic acid, oleylamine, poly(maleic anhydride alt-1-octadecene), L-arginine, Lhistidine monohydrochloride, D(+) glucosamine hydrochloride, O,O′-bis(2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol with molecular weight 600 (PEG-diamine), folic acid and biotin N-hydroxysuccinimide ester (biotin-NHS) were purchased from Sigma Aldrich. Instrumentation. UV-visible absorption spectra were measured using Shimadzu UV-2550 spectrophotometer in a one cm quartz cell. Fluorescence spectra were recorded on Perkin Elmer fluorescence spectrometer model LS-45. FEI Tecnai G2 transmission electron microscope was used for transmission electron microscopic (TEM) image of sample. X-ray photoelectron spectroscopy (XPS) was performed using an Omicron (series 0571) X-ray photoelectron spectrometer with a drop-casting of nanoparticle solution. Time correlated single photon counting (TCSPC) was carried out through exciting the sample with the picoseconds diode laser (IBH Nanoled) using a Horiba Jobin Yvon IBH Fluorocube apparatus. Raman spectra with a 785 nm excitation laser were collected using an Agiltron R3000 Raman spectrometer. XRD measurements of the samples were performed on a Bruker D8 Advance powder diffractometer, by using Cu Kα (λ=1.54 Ǻ) as the incident radiation. Fourier transform infrared (FTIR) spectroscopy was carried out on Perkin Elmer Spectrum 100 FTIR spectrometer with solid KBr pellets. Dynamic light scattering (DLS) and zeta-potential of the sample were measured by using NanoZS90 (Malvern) instrument with 90° optics and appropriate refractive index for respective solvents (water as solvent for all the functional nanoparticles and chloroform as solvent for hydrophobic FCN). Fluorescence images of cells were captured by using Olympus IX81 microscope with image-pro plus version 7.0 software.


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Synthesis of yellow-red fluorescent hydrophobic FCN. Hydrophobic FCN was prepared using high temperature degradation method as reported earlier with some minor modifications.13 Typically, one gram ascorbic acid was dissolved in 12 mL oleylamine, taken in a three necked round bottom flask. The solution was heated to 280 °C under continuous air flow through a syringe for 4 hrs. Next, reaction was stopped and temperature was lowered to room temperature. Hydrophobic FCN was then purified using acetone based precipitation and chloroform based redispersion. Typically, 700 mg of hydrophobic FCN was synthesized from one gram ascorbic acid. Finally, FCN solution was prepared in chloroform with the concentration of 10-20 mg/mL. Poly(maleic anhydride alt-1-octadecene)-based one pot coating and functionalization of hydrophobic FCN. At first 20 mg poly(maleic anhydride alt-1-octadecene) was dissolved in one mL chloroform through sonication. After that one mL of purified hydrophobic FCN solution was added and sonicated until clear solution appeared. Next, chloroform was evaporated and resultant solid was mixed with 2 mL basic aqueous solution of functional molecules. The solution of functional molecule was prepared by dissolving them in carbonate buffer solution of pH 9.5 with typical concentration of 5-10 mg/mL. Next, the mixture was stirred for 4-5 hrs and centrifuged at 15,000 rpm for 20 mins to separate larger particles or unreacted polymer. Finally, solution was dialyzed using dialysis tube (cut off~ 12000 Da) to separate free functional molecules and reagents. Resultant PEG, histidine, glucosamine, arginine functionalized FCNs were named as FCN-PEG-NH2, FCN-histidine, FCN-glucose, FCN-arginine, respectively. Functionalization of FCN with biotin and folic acid. In separate vials dimethylformamide (DMF) solutions of biotin-NHS and folate N-hydroxysuccinimide (folate-NHS) were prepared with 10 mM concentration. Folate-NHS was prepared using DCC based coupling chemistry, as reported elsewhere.34 Next, two different sets of one mL FCN-PEG-NH2 solution were prepared


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in borate buffer solution of pH 9. In one vial, 30 µL biotin-NHS solution was mixed, in other vial 30 µL folate-NHS solution was mixed. These two solutions were stirred overnight to achieve complete reaction. After that, the solutions were separately dialyzed against basic water followed by normal water to remove excess reagents. Finally, the solutions were preserved at 4 °C. Characterization of functionalized FCN. Confirmation of functionalization and estimation of functional groups were performed using established methods.35-37 For the determination of primary amine, we have used fluorescamine as a probe and free glucosamine as a reference.35 At first acetone solution of fluorescamine, aqueous solution of free glucosamine of different concentration and aqueous solution of FCN-PEG-NH2 in borate buffer of pH 9.5 were prepared separately. Next, equal volumes of fluorescamine solution were mixed with glucosamine solution or nanoparticle solution. Next, fluorescence was measured at an excitation wavelength of 400 nm. Quantification of arginine is performed using 9,10-phenanthrenequinone-based fluorescence product formation.36 At first, free arginine of different concentration was reacted with 9,10-phenanthrenequinone and fluorescence product was used for the preparation of calibration graph. Next, same reaction was preformed between FCN-arginine and 9,10phenanthrenequinone and fluorescence product was used for estimation of FCN bound arginine. In brief, ethanol solution of 9,10-phenanthrenequinone (150 µM) and aqueous solution of arginine of different concentration were prepared. Next, 50 µL of free arginine or FCN-arginine was taken in vial and mixed with 150 µL of 9,10-phenanthrenequinone solution followed by addition of 25 µL NaOH solution (2.0 M). Next, mixture was incubated at 60 °C for 3 hrs and 100 µL solution of that mixture was added to 100 µL 1.2 (N) HCl and allowed to stand for one


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hour at room temperature under dark condition. Next, fluorescence was measured at an excitation wavelength of 312 nm. For the determination of glucosamine, we have used anthrone-based cororimetric method and free glucosamine was used as a reference.37 At first anthrone solution was prepared in 80 % H2SO4 and aqueous solution of glucosamine was prepared with different concentrations. Next, 2 mL anthrone solution was mixed with 200 µL solution of glucosamine or FCN-glucose and heated in water bath for 15 min. Next, temperature of the mixture was lowered in ice cooled water and absorbance spectra were measured. Cellular labeling application of functionalized FCN. Chinese hamster ovary (CHO) cells were cultured in DMEM media supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin streptomycin at 37 °C and 5 % CO2. KB cells were cultured in folate free RPMI-1640 (Invitrogen) media supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin streptomycin at 37 °C and 5 % CO2. For cellular uptake studies, cells were seeded into 24 well plates with 500 µL media. After overnight growth, 50-200 µL of sample solutions were mixed and incubated for one hour. Next, cells were washed with PBS buﬀer solution to remove unbound particles and used for microscopic imaging studies. MTT assay. For cell viability study, Chinese hamster ovary (CHO) cells were cultured in a 24 well plate in DMEM media. After that, cells were treated with different doses of samples for 24 hrs, washed thoroughly with PBS buffer and fresh DMEM media was added. Next, each well plate with attached cells were treated with 50 µL of freshly prepared methylthiazolyldiphenyltetrazolium bromide (MTT) solution (5 mg/mL) and incubated for 4 hrs. Then the supernatant was removed carefully leaving the formazan in the plate. This formazan was dissolved in SDS solution (8 g of SDS dissolved in 40 mL of DMF-H2O mixture), and absorbance was measured


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at 570 nm. Cell viability was estimated assuming 100 % viability for control sample without any nanoparticle. RESULTS AND DISCUSSION Synthesis and functionalization approach of FCN. Synthesis and functionalization approach of yellow-red fluorescent FCN is shown in Scheme 1 and 2. In the first step hydrophobic FCN is synthesized via carbonization of ascorbic acid at 280 °C using oleylamine as reaction medium and capping agent.13 As the pyrolysis proceeds at higher temperatures, the ascorbic acid is consumed for the build-up of the carbogenic core corresponding to photoluminescence component and gradually increases in size at the expenses of ascorbic acid.12,13 At the same time, other elements, such as oxygen and nitrogen (coming from oleylamine) are also incorporated into the carbon matrix to introduce defect sites.13 The fluorescence property of FCN essentially depends on the extent of defect cites and the ratio of sp3 to sp2 carbons.1,3 In particular the increase in sp2 domain increases the conjugated double bonds and the role of doped oxygen and nitrogen is to control this sp2 domain.13 The synthesis is followed from our earlier protocol with significant modification to achieve better red emission. Resultant FCN is hydrophobic in nature due to capping with oleylamine. These nanoparticles are purified from excess reagents by the usual acetone/ethanol-based precipitation and chloroform-based redispersion and finally dissolved in chloroform. As synthesized hydrophobic FCN is characterized by elemental analysis, DLS, XPS, TCSPC, Raman and XRD study. (Supporting Information, Figure S1-S5) Elemental analysis shows 80 wt % carbon, 10 wt % hydrogen, 3.5 wt % nitrogen and rest is mainly oxygen. Considering high carbon %, graphitic nature of carbon (from Raman spectra with D and G bands, Supporting Information, Figure S4), TEM data of particle image (Figure 1) and mass spectroscopic/HPLC data13 on absence of lower molecular weight compounds it can be


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considered as carbon nanoparticle. The time-resolved fluorescence decay of hydrophobic FCN and FCN-PEG-NH2 indicates similar decay profiles (Supporting Information, Figure S3). The fluorescence decay of hydrophobic FCN and FCN-PEG-NH2 are fitted with three lifetime values with average lifetime 1.81 and 1.97 ns, respectively, which corresponds the presence of multiple radiative species.13 After synthesis, chloroform solution of FCN is mixed with chloroform solution of amphiphilic polymer (poly(maleic anhydride alt-1-octadecene)), sonicated for 5 min and then chloroform is evaporated to dryness either at room temperature or via mild heating. At this stage hydrophobic octadecyl groups of polymer intercalate with hydrophobic oleyl groups around nanoparticle with the resultant formation of polymer coated FCN. In the next step 1-2 mL of basic solution of PEG-diamine/arginine/histidine/glucosamine is mixed with polymer coated solid FCN produced after chloroform evaporation. At this stage anhydride groups of polymer react with primary amine groups of PEG-diamine/arginine/histidine/glucosamine in forming functional FCN and rest of the anhydride groups hydrolyse. This polymer is widely used for coating of hydrophobic nanoparticle, including hydrophobic carbon nanoparticle.13,20 We have selected PEG-diamine conjugation as it is known to decrease the non-specific binding of nanoparticle with bioenvironment38 and one of the unreacted primary amine would offer further functionalization option. Arginine and histidine functionalization are selected as they are known to increase the cell uptake of nanoparticle and induce endosomal escape property to nanoparticle, respectively.39,40 Glucosamine functionalization is attempted as it can provide glucose terminated surface with PEG like property41 and can be used for interaction with glycoprotein.42 PEG diamine, histidine, glucosamine and arginine functionalized FCNs were named as FCN-PEGNH2, FCN-histidine, FCN-glucose, FCN-arginine, respectively.


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Property of functional FCN. UV-visible absorption and fluorescence spectra of as synthesized hydrophobic FCN is shown in Figure 1. FCN solution appears dark brown in colour but absorption spectrum does not show any characteristic absorption band. It exhibits broad emission in the range of 400 nm to 700 nm and emission intensity and emission maxima vary depending on the excitation wavelength. The highest emission is observed under the 450 nm excitation with emission maximum at 550 nm. As the excitation wavelength decreases from 450 nm to 300 nm, emission intensity decreases without changing emission maxima. As the excitation wavelength increases from 450 nm to 550 nm, emission intensity decreases along with the red shifting of emission maximum from 550 nm to 625 nm. Such a broad emission and excitation-dependentemission phenomenon are very common for fluorescent carbon nanoparticles and occurs possibly due to various energy levels associated with different ‘surface states’ formed by various self-passivated functional groups.1 The fluorescence quantum yield (QY) for this sample is measured using fluorescein disodium salt as reference and the obtained value is ~ 14 %. The particle size of the hydrophobic FCN has been determined from TEM study which shows the sizes between 3-8 nm (Figure 1). Hydrophobic nature of this FCN can be interpreted from high solubility in organic solvents such as chloroform, cyclohexane and toluene but complete insolubility in water. Presence of long chain fatty amine in the purified FCN has been confirmed from the FTIR study which suggests that oleyl groups are capped around FCN under the synthetic condition. (Supporting Information, Figure S6) Optical property, hydrodynamic size, surface charge and application potential of functional FCNs are summarized in Table 1. UV-visible absorption and fluorescence spectra of various functional FCN are shown in Figure 1. It is observed that emission spectra red shift after functionalization and each of the functional FCN exhibits deep red emission. In particular all the


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functional FCN shows strong emission between 550 nm to 750 nm with emission maxima in the range of 620 nm to 670 nm and they can be excited by light of wavelength between 400 nm to 500 nm. Origin of fluorescence red shifting after functionalization can be due to change of surface capping molecules and change of dispersing medium.43,44 Hydrophobic FCN is capped with hydrophobic ligand and dispersed in organic phase. In contrast functionalized FCNs are capped by polymer with polar functional groups and dispersed in water. So the presence of polar functional groups and change in dispersion medium are possibly contributing to the red shifting of emission. Polymer coating possibly introduces additional surface defect states and polar solvents are known to contribute to red shifting of emission via decreasing energy gap.43,44 The fluorescence quantum yield (QY) for these samples is measured using fluorescein disodium salt as reference and the obtained value is ~ 3 %. This result suggests that surface modification leads to significant decrease of original fluorescence quantum yield of 14 %. The decrease in fluorescence quantum yield is due to the introduction of polar functional groups and polar dispersion medium (water).43,44 However, the remaining emission is strong enough for imaging applications. The ratio of absorbance at 400 nm and 600 nm (E4/E6) (Table 1) has been measured which correlates to aromaticity and degree of condensation.46 Functionalization of FCN has been determined using FTIR study and by selective testing of primary amine, glucose and arginine. (Figure 2 and Supporting Information, Figure S7-S9) Fluorescamine test has been used for qualitative and quantitative estimation of primary amine present in FCN-PEG-NH2. Figure 2a shows strong blue emission after reaction with fluorescamine, confirming the presence of primary amine. Quantitative estimation of primary amine has been performed using the calibration graph prepared using a reference primary amine and it is found that about 25 mg of PEG-diamine is bound per gram of FCN-PEG-NH2.


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(Supporting Information, Figure S7) 9,10-Phenanthrenequinone test has been applied for qualitative and quantitative estimation of arginine in FCN-arginine. Figure 2b shows the appearance of blue fluorescence after reaction with 9,10-phenanthrenequinone, confirming that arginine is successfully attached with the FCN surface. Quantitative estimation of arginine has been performed using the calibration graph prepared from reference arginine and about 33 mg of arginine is present per gram of FCN-arginine. (Supporting Information, Figure S8) Anthrone test has been used for qualitative detection of glucose in FCN-glucose. Figure 2c shows that polymer coated FCN is successfully modified with glucosamine as evidenced by appearance of absorption band at 600 nm after reaction with anthrone. FTIR spectra of FCN before and after various functionalizations also indicate the presence of characteristic vibration band of functional groups. (Supporting Information, Figure S6) Hydrodynamic size of the nanoparticles is measured by dynamic light scattering and the average values ranges between 18 nm to 24 nm. (Figure 3) This size includes 3-8 nm hydrophobic FCN, polymer shell and functional groups. The zeta-potential of functional FCNs has been measured at pH 7.4 and 10.0 and varies between -10 mV to -41 mV depending on solution pH and nature of functionalization. More importantly surface charge is low negative at physiological pH of 7.4 and varies between -10 mV to -22 mV depending on functionalization. Such anionic surface charge arises due to the presence of carboxylate groups that are formed during hydrolysis of anhydride groups present on polymer backbone. Among all the functional FCN, FCN-arginine shows lowest value of -10 mV due to the presence of cationic guanidine group that partially balance the carboxylate anions. As the pH is increased to 10.0, the surface charge of all functional FCN becomes more negative and value becomes -31 mV to -41 mV.


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This is due to more ionization of carboxylic acid groups with increasing pH that produces more anionic carboxylate functional groups. Colloidal stability of functional FCNs has been investigated under physiological conditions that include phosphate buffer, cell culture media and other buffer solutions. Figure 4 shows digital image of colloidal solution of functional FCNs under normal and UV light which have been prepared one month before. Results show that functional FCNs do not precipitate and maintain good colloidal stability. It has also been investigated that fluorescence property of functional FCN colloid remain intact after one hour UV light exposure and fluorescence of drop casted film of functional FCN remain intact after 15 mins UV exposure. (Figure 4) Red fluorescent FCN as cell imaging probe. Cell labelling application potential of functionalized FCNs has been extensively studied. In particular red emission of FCN extending from 550 nm to 750 nm, which are excitable by both blue and green light has been exploited for imaging application using the available options in conventional fluorescence microscope. For example, we have used a set up that provide blue excitation in the range between 420 nm to 490 nm and emissions are collected beyond 520 nm. The other set provide green excitation in the range between 480 nm to 550 nm and emission is collected after 590 nm. In addition we have investigated the nature of interaction of various functional FCN with cell and studied the role of functionalization on this interaction. Labelling is performed by mixing the functional FCN solution with cell culture media along with the cells present on the culture plate. Next, cells are washed to remove unbound FCN and then imaged under fluorescence microscope. Results are shown in Figure 5. It is observed that FCN-arginine has stronger interaction with cell as compared to other functional FCN. This result can be explained based on the nature of functionalization and their role to modulate the surface charge of FCN.20 Anionic surface charge


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of functional FCN induces electrostatic repulsion from anionic cell membrane and minimizes their interaction with cell.20 PEG and glucose functionalization are known to decrease the nonspecific binding interaction with cell membrane.38,41 Labelling by FCN-arginine can be explained due to their relatively lower anionic surface charge and specific interaction of guanidium cation with anionic cell membrane.39 Although the overall surface charge of FCN-arginine is negative, arginine can provide localized cationic surface charge via guanidine groups, and offer interaction with cell membrane. Cytotoxicity study shows that all the functional FCN are nontoxic in the studied concentration range. (Supporting Information, Figure S10) In order to extend the application potential, FCN-PEG-NH2 has been transformed into folate functionalized FCN (FCN-folate) and their application as selective fluorescent label has been explored. Folate receptors are over expressed in variety of cancer cells and thus folate functionalized nanoparticles are widely used for selective targeting and imaging of cancer cells.34 Here we have used KB cells having over expressed folate receptors for FCN-folate based labelling and imaging study. Results show that FCN-folate efficiently labels KB but does not label CHO cells having low folate receptor and FCN-PEG-NH2 cannot label any of these cells. (Figure 6 and Supporting Information, Figure S11-S13) Fluoresence image of FCN-folate labelled KB cells was taken at different z planes and result shows that FCN-folate mostly localizes inside the cells. (Supporting Information, Figure S12) This result shows that amine terminated FCN-PEG-NH2 can be used for different functionalization and selective biolabeling application. Fluorescence microscopic image of FCN-folate labelled KB cells has been captured under blue (FITC) and green (Texas red) excitations which show that both conditions generate similar quality images. As our probe can provide similar quality images under blue or green excitation, the green excitation can be used under critical situation (when there is high


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background signal under blue excitation).45 We have also synthesized biotinylated FCN from FCN-PEG-NH2 (FCN-biotin) and found that FCN bound biotin is active in binding with Avidin (Supporting Information, Figure S14). All the results suggest that different functional FCN can be derived from FCN-PEG-NH2 and resultant nanobioconjugate can be used for selective biolabeling applications.

CONCLUSION We report successful synthesis of red fluorescent carbon nanoparticle-based functional nanoparticle and demonstrated their application potential as fluorescent cell label. Reported functional carbon nanoparticle has < 25 nm hydrodynamic size, high colloidal stability under physiological condition and they can be excited with blue and green light to capture their red emission. Polyethylene glycol and primary amine terminated carbon nanoparticle reported here can be transformed into desired nanobioconjugate using the commercially available reagents and protocols. Presented carbon nanoparticle synthesis and functionalization approach can produce milligram to gram scale of nanoprobe and can be extended for functionalization of other biomolecules of interest.

ASSOCIATED CONTENT Supporting Information Image of solid and soluble FCN, characterization and quantification of functionalization, cytotoxicity data and additional fluorescence image of FCN labelled cells. This material is available free of charge via Internet at http://pubs.acs.org AUTHOR INFORMATION


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Corresponding Authors *Email: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We acknowledge the financial support from DST, Government of India. (Grant number SB/S1/IC-13/2013) HA and CD thank CSIR, India, for providing research fellowship.

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