Nitric Oxide Sensing through Azo-Dye Formation on Carbon DotsSagarika Bhattacharya,† Rhitajit Sarkar,‡,⊥ Biswarup Chakraborty,†,⊥ Angel Porgador,‡

and Raz Jelinek*,†,§

†Department of Chemistry, ‡The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences,and §Ilse Katz Institute for Nanotechnology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel

*S Supporting Information

ABSTRACT: Carbon dots (C-dots) prepared through heating of aminoguanidineand citric acid enable bimodal (colorimetric and fluorescence) detection of nitricoxide (NO) in aqueous solutions. The C-dots retained the functional units ofaminoguanidine, which upon reaction with NO produced surface residuesresponsible for the color and fluorescence transformations. Notably, theaminoguanidine/citric acid C-dots were noncytotoxic, making possible real-timeand high sensitivity detection of NO in cellular environments. Using multiprongspectroscopic and chromatography analyses we deciphered the molecularmechanism accounting for the NO-induced structural and photophysicaltransformations of the C-dots, demonstrating for the first time N2 release andazo dye formation upon the C-dots’ surface.

KEYWORDS: carbon dots, fluorescence quenching, nitric oxide detection, azo dye formation, polymerization of C-dots

Nitric oxide (NO) plays an important role in manybiological systems including neuronal, cardiovascular, and

immunological processes associated with macrophage andneutrophil activation.1,2 Irregular NO homeostasis is anindication of inflammatory response3 generally associatedwith various diseases and disorders like hypertension,4

atherosclerosis,4 diabetes,5 neurodegenerative diseases,6 andtumor growth.7 In particular, activation of macrophagesmajor inflammatory cells participating in host-defense againstpathogen infectionis directly linked to NO generation.Specifically, endotoxins like lipopolysaccharide (LPS) inducesignaling cascades involving genes such as iNOS and NOS-2,8

generating NO in the process.9

While the biological importance of NO is widely recognized,significant barriers exist for development of rapid and sensitivedetection platforms, particularly the low concentrations andshort half-life of NO in physiological environments (5−60s).1,10 Transition metal complexes have been employed asfluorescent NO sensors utilizing the binding specificity of NOtoward metal centers.11,12 NO-selective sensors based onfluorescein derivatives have been reported.13,14 Diaminofluor-esceins (DAFs) containing aromatic vicinal amines are alsoknown to detect NO via triazole formation.15 Diamino probesbased on boron dipyrromethene (BODIPY), cyanine, andnaphthalene scaffolds have been also used for NO analysis inbiological systems.16−19 An organic molecule (Lyso-NINO)functionalized with the NO-capturing moiety o-phenylenedi-amine together with a lysosome-targeting residue was used forendogenous NO detection in MCF-cells.18 Although thismolecule exhibited rapid NO response of around 15 min, itwas limited to lysosomal detection of NO. In addition,

synthesis of the organic molecule was complex. Nanoparticle-based NO detection schemes have also been reported,employing, for example, single-and multiwalled carbon nano-tubes,20−22 reduced graphene oxide,23 gold nanoparticles,24 andinorganic quantum dots.25 These NO sensing strategies,however, have had limited applicability in biological systems,exhibit long reaction times, low sensitivity, or requiringcomplex synthesis routes.Carbon dots (C-dots) have emerged in recent years as a

versatile sensing vehicle for a wide array of target analytes.26−29

C-dots are particularly suited for biological sensing applicationsas they exhibit broad excitation-dependent emission spectrathat can be readily modulated upon interaction with moleculartargets.30,31 In addition, C-dots are generally nontoxic, and canbe easily surface-modified, thereby displaying varied recognitionmoieties.32,33 Particularly important, previous studies havedemonstrated that C-dots could retain the structural andfunctional units of the carbonaceous building blocks employedin the synthesis, endowing the dots with biomolecular targetingcapabilities simply through selection of the starting re-agents.34−36

C-dots synthesized from citric acid and ethylene diaminewere used as NO sensors.37 A ratiometric C-dot sensorexhibiting phenylenediamine-containing naphthalimide fuction-alization could detect NO via triazole formation.33 Simoes andco-workers developed a fluorimetric NO sensor based uponN,S-doped carbon dots.37 The sensor achieved rapid NO

Received: May 27, 2017Accepted: August 3, 2017Published: August 3, 2017

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detection, but has not been applied in cellular environments.Overall, these C-dot systems exhibit either relatively low NOsensitivity or long response times (up to hours), or involvecomplex synthetic schemes. Furthermore, the mechanisms ofNO induced-modulation of C-dots’ fluorescence have not beendeciphered.Here we describe fabrication of C-dots through a simple one-

step hydrothermal treatment of aminoguanidine hydrochlorideand citric acid. Aminoguanidine participates in varied enzymaticprocesses and has been used as a therapeutic substance fortreatment of complications in diabetes.38,39 Due to the presenceof the guanidino nitrogen, aminoguanidine has been alsoinvestigated as a NO synthase (NOS) enzyme inhibitor.40 Wedemonstrate that the citric acid/aminoguanidine C-dotsundergo bimodal (visible and fluorescence) transformationsupon addition of NO, both in water and in cells. Importantly,through application of varied analytical techniques wedetermine the molecular mechanism responsible for NOsensing. Specifically, the experiments reveal for the first timethat reaction of NO with amine moieties upon the C-dots’surface gave rise to azo dye formation and concomitant releaseof molecular nitrogen.

■ EXPERIMENTAL SECTIONMaterials. Aminoguanidine hydrochloride, citric acid, sulfanila-

mide, N-(1-naphthyl)ethylenediaminedihydrchloride, sodium nitrite,phosphoric acid, sodium nitrate, hydrogen peroxide (30% (w/v)),potassium superoxide, L-NAME ≥ 98% (TLC), powder (Nω-nitro-L-argininemethylesterhydrochloride), BAPTA-AM, ≥ 95% (HPLC)(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis-(acetoxymethyl ester)) and lipopolysaccharide from Pseudomonasaeruginosa 10 were purchased from Sigma-Aldrich, St. Louis, MO,USA. Potassium hydroxide, sodium hydroxide and pyrogallol werepurchased from Alfa Aesar and Tzamal D-chem. All chemicals wereused without further purification. Methanol and sulfuric acid 98% werepurchased from Carlo Erba, Italy and Bio-Lab Ltd. (Jerusalem, Israel),respectively. RPMI 1640 medium (RPMI), fetal bovine serum (FBS),and 2-mercaptoethanol were bought from Gibco by Life Technologies,USA. Media supplements, antibiotics, and PBS were acquired fromBiological Industries, USA. Cell viability reagent 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchasedfrom ThermoFisher Scientific, USA. Milli-Q water with resistivity of18.2 MΩ cm at 25 °C were used for all the experiments.Synthesis of C-Dots. Aminoguanidine hydrochloride 98% was

mixed with citric acid in a 4:1 ratio in 200 μL water in a Teflon film-tightened, septum-capped test tube and then heated in an oven to 150°C for 2 h. After the reaction was completed, the resultant mixture wasallowed to cool to room temperature yielding a brown precipitateindicating the formation of carbon dots. The precipitate wasredispersed in 2 mL methanol through sonication for 2 min andcentrifuged at 10 000 rpm for 30 min to remove high-weightprecipitate and agglomerated particles. Thereafter, methanol wasevaporated under reduced pressure to obtain a brown solid. This wasdissolved by sonication in 2 mL distilled water and was dialyzedagainst distilled water several times, subsequently solubilized in waterfor further characterization and use.Preparation of Nitric Oxide Solution. Saturated NO solutions

were prepared as previously reported.20 Briefly, NO gas was generatedby slowly pouring 2 M H2SO4 into a glass flask containing saturatedaqueous solution of NaNO2 under Ar atmosphere. The NO gasproduced by the disproportionation of NaNO2 in acidic medium waspassed through 5% (w/v) pyrogallol solution in saturated potassiumhydroxide followed by 10% (w/v) potassium hydroxide to removeoxygen, other nitrogen oxides, and acid vapors. Before addition ofH2SO4, the apparatus and all the solutions required for NO generationwere degassed with argon for 30 min to exclude O2 in order to avoidthe reaction of NO with O2. The NO-saturated solutions were

prepared by purging the NO gas in deoxygenated water and keepingunder NO atmosphere until use. Concentration of the saturated NOsolution was 4.92 mM at 25 °C, determined by the Griess reaction.41

All preparation steps were carried out in a fume hood. NO standardsolutions were prepared by making successive dilutions of thesaturated NO solution. NO standard solutions were made fresh forexperiments and were kept in a glass flask with a rubber and light-freeseptum with wrapped black foils.

Quantum Yield Measurement. Quantum yield of the aminemodified carbon dots was determined by comparing integratedphotoluminescence intensity (in the range of 380−650 nm) uponexcitation at 360 nm and absorbance value of carbon dots at 360 nm,with the respective values recorded for quinine sulfate in 1N H2SO4(refractive index (η) of 1.33) according to the following equation:

φ φηη

= × × ×II

AAS R

S

R

R

S

S

R

2

2

in which φs = Quantum yield of the sample; Is = Integratedfluorescence intensity; As = Absorbance; ηs = refractive index. Theindex R corresponds to the reference and index S the sample.

Fluorescence Spectroscopy. Fluorescence emission spectra wererecorded on an FL920 spectrofluorimeter. A Varioskan plate readerwas used for the detection of NO through fluorescence quenching.The 96 well flat-bottom microtiter plates were used for the titration.To each well 100 μL of C-dot aqueous solution was added;fluorescence measurements following titration with NO were carriedout by addition of 10 μL of saturated solution to achieve the finalconcentration. Quenching efficiency (QE) was calculated by using theStern−Volmer equation (I0 corresponds to the initial fluorescencewhile I corresponds to the fluorescence recorded after NO addition):

=−I II

QE 0

High Resolution Transmission Electron Microscopy (HR-TEM). A drop of C-dot solution was placed upon a graphene-coatedcopper grid and HR-TEM images were observed on a 200 kV JEOLJEM-2100F microscope (Japan). The sample was dried for 12 h priorto measurements.

X-ray Photoelectron Spectroscopy (XPS). Concentratedaqueous solutions of C-dots were drop-casted on silicon wafers andmeasurements were performed using an X-ray photoelectronspectrometer ESCALAB 250 ultrahigh vacuum (1 × 10−9 bar)apparatus with an Al Kα X-ray source and a monochromator. Thebeam diameter of was 500 μm and with pass energy (PE) of 150 eVsurvey spectra were recorded, while for high energy resolution spectrathe recorded pass energy (PE) was 20 eV. The AVANTAGE programwas used to process the XPS results.

Nuclear Magnetic Resonance (NMR). 13C NMR spectra of C-dots before and after addition of NO were recorded using a BrukerDPX 400 MHz spectrometer at 300 K. 13C NMR of C-dots wasperformed in D2O, with compound concentrations 30 mg/mL. Afteraddition of the NO solution to the C-dots, the mixture was stirred for24 h under argon atmosphere. Red solid isolated upon removal of thewater was redissolved in D2O for 13C NMR experiment withoutfurther purification.

Fourier Transform-Infrared (FT-IR) Analyses. FT-IR measure-ments were performed on a Thermo Scientific Nicolet 6700spectrometer. The procedure used for extraction of the product wassame used for the NMR experiment.

Dynamic Light Scattering. DLS data were collected at 25 °C onan ALV-CGS-8F instrument (ALV-GmbH, Germany) at 90°, and theCONTIN method was used to obtain hydrodynamic radii (Rh).

Griess Reaction and UV−vis Spectroscopy. The Griessreaction was carried out using UV−vis spectrophotometry analysis.The reaction involves a two-step assay based on the observation thatthe adduct of nitric oxide and sulfanilic acid (1%) interacts with N-(1-naphthyl)ethylenediamine (0.1%), to generate an azo derivativeproduct that is readily monitored by UV−vis spectrophotometry.41

The spectrum of the product shows an absorbance band with a

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maximum at 540 nm. All absorbance measurements were performedon an Agilent 8453 diode array spectrophotometer with 1 cm cells inwater as dispersive medium.Optical Spectra and Kinetics of Azo Dye Formation. Kinetic

measurements were carried out in water at 25 °C under argonatmosphere. The C-dot concentrations were varied from 1 to 10 mg/mL (volume 2 mL) and 300 μL of 4.92 mM of NO was added. Therate of formation of the azo dye was measured by monitoring theabsorbance band at 490 nm. In another experiment, different volumes(100−500 μL) of 4.92 mM NO solution were added to 2 mL C-dotsolution (conc. 2.5 mg/mL) and azo dye formation was monitored.On comparing the optical density obtained at 540 nm on addition ofGriess reagent to the NO treated C-dot solution, we determined that99% of NO added reacted with the aminoguanidine/citric acid C-dots.Gas Chromatography (GC). Evolved gas was detected in a

Thermo Scientific Focus Gas Chromatograph (GC) with a dedicatedthermal-conductivity detector (TCD), and argon was used as thecarrier gas. To analyze headspace gas evolved during the reaction of C-dots with freshly prepared NO saturated aqueous solution, thereaction was performed in argon atmosphere in a septa-sealed gastightquartz cell equipped with 14 mL overhead space. A complete air-freeenvironment was maintained inside the gastight quartz cell using aSchlenk line. Before addition of the NO saturated aqueous solutions (2mL at 4.92 mM), the C-dot suspension (2 mL of 30 mg/mL) waspurged with argon for 30 min to avoid reaction of NO with dissolvedoxygen. NO generation from a disproportionate reaction of NaNO2and conc. H2SO4 and followed by purification by Pyrogallol and KOHsolution was also performed in Schlenk line under Ar atmosphere, andindividual solutions used to prepare and purify NO were purged withminimum 20−30 min. Furthermore, prior to saturation with NO, thewater used was purged with Ar for at least 30 min and kept in Aratmosphere. With the progress of the reaction, the evolved gas fromheadspace of the quartz cell was injected to the GC using a gastightsyringe (Hamilton). The evolved N2 gas was detected and identifiedby comparing its identical retention time with N2 in air.Calculation of N2 Generated by Reaction of NO with the C-

Dots. A calibration curve was prepared by injecting a different amountof fresh air as sample in the GC. The volume of N2 injected in GC wascalculated assuming air composition N2:O2 to be 78.09:20.86%. Thepeak area was plotted with respect to the volume of nitrogen injected.The evolved N2 during the reaction was quantified from the linear fitof the plot. From the calibration curve, the volume of nitrogen (inμmol) in 1 mL was calculated. As the injected volume in GC was 1 mLfrom the 14 mL overhead space of the gastight cuvette. The totalvolume of N2 gas evolved from the 4 mL total reaction mixture of 30mg/mL C-dots solution should be 14 times the calculated value. Basedon this analysis, the amount of evolved nitrogen was 97.03 μmol/gr C-dots. The saturated NO solution (9.84 μmol) was added to 2 mL of 30mg/mL C-dots solution, and produced 5.82 μmol of nitrogen.Cell Culture and Viability. Mouse (Mus musculus) leukemic

monocyte macrophage cell line RAW 264.7 (ATCC TIB-71) wasgrown in RPMI 1640 medium supplemented with 10% (v/v) fetalbovine serum (FBS), antibiotics and other necessary supplements andmaintained at 37 °C in a humidified atmosphere containing 5% CO2 inCO2 incubator. For the experiments, cells were seeded in a 96-well flatbottom culture plate at a density of 5 × 103 cells/well and allowed tosettle for 30 min in incubator. The cells were then treated withincreasing concentrations (0, 25, 50, 75, 100, 150, and 200 μg/mL) offreshly prepared C-dots in PBS 1X (stock solution 1 mg/mL) andincubated for 18 h. Post incubation, 10 μL 2 mg/mL MTT solutionwas added to each well followed by 2 h incubation at 37 °C. 50 μLDMSO was added to each well to dissolve the formazan crystals andthe absorbance measured at 560 nm using a microplate MultiskanSpectrum (Thermo Electron Corporation, USA). Fresh culturemedium was used as the background with n = 6 considered formeasurement of each sample. Obtained data were represented as apercentage of viability and IC50 value was determined from theregression analysis curve.Determination of Cellular NO Generation. NO produced in

culture medium of treated and untreated RAW 264.7 cells were

measured as an indicator of NO production using the Griessreaction,42 based on the diazotization of sulfanilic acid with nitriteion and coupling of this product with diamine, which results in aspectrophotometrically measurable pink metabolite. RAW 264.7 cellswere seeded in a 24-well culture plate as 1.5 × 106 cells/well andallowed to settle for 30 min in incubator. Then cells were treated withfreshly prepared 150 μg/mL C-dots (determined after titration ofconcentration range below IC50 value; data not shown) in PBS 1X,along with necessary blanks and incubated for 18 h. Post treatment,medium was changed in all wells, and treated with 1 μg/mL LPS,except the one to be treated as blank, and incubated in CO2 incubator.Then, at three time points (1, 2, and 3 h) in a 96-well flat bottomculture plate, 100 μL of supernatant cell culture medium was mixedwith 100 μL of Griess reagent [equal volumes of 1% (w/v)sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v)naphthylethylenediamine−HCl]. The plate was incubated for 20 minat room temp and the absorbance at 540 nm was measured in amicroplate reader Multiskan Spectrum (Thermo Electron Corpo-ration, USA). Two triplet sets were taken for measurement of eachsample. Fresh culture medium was used as the background correctionin all experiments. The nitric oxide content was calculated from asodium nitrite standard curve.

Cell Incubation with C-Dots and Confocal Imaging. In coverglass-bottom Confocal Dish, RAW 264.7 cells were seeded as 1 × 104

cells/dish and allowed to recuperate for 30 min in 37 °C CO2incubator. As before, cells were then treated with freshly prepared 150μg/mL C-dots in PBS 1X, along with necessary blanks and incubatedfor 18 h. Post incubation, cells were washed with PBS and confocalmicroscopy images of C-dot labeled cells were acquired on anUltraVIEW system (PerkinElmer Life Sciences, Waltham, MA)equipped with an Axiovert-200 M microscope (Zeiss, Oberkochen,Germany) and a Plan-Neofluar 63×/1.4 oil objective. Excitationwavelengths of 405, 440, and 488 nm were produced by an argon/krypton laser. Then, 10 μg/mL LPS was added to the sample andimages of the same location were acquired at different time intervals. Aparallel blank was also done using ultrapure water instead of LPS.

■ RESULTS AND DISCUSSIONAminoguanidine/Citric Acid C-Dot Synthesis and

Characterization. Figure 1 outlines the synthesis scheme of

the C-dots, and demonstrates the bimodal NO detectionfeatures. Preparation of the C-dots was carried out throughhydrothermal treatment of a mixture of aminoguanidinehydrochloride and citric acid (4:1 weight ratio, Figure 1A).Citric acid has been widely employed as a carbon source in C-dot synthesis, generally yielding uniform C-dot graphiticcores.32 The dramatic response of the aminoguanidine/citric

Figure 1. Synthesis of aminoguanidine/citric acid C-dots and bimodalNO sensing. A. Synthesis scheme. B. Digital photographs showing theyellow-red color change and fluorescence quenching (exc. 365 nm)observed in the C-dot solution (5 mg/mL) upon addition of NO (4.9mM).

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acid C-dots to NO is depicted in Figure 1B. Specifically, priorto addition of NO the C-dots exhibited visible yellow color, andemitted intense blue fluorescence (exc. 360 nm, Figure 1B left).However, a remarkable and rapid yellow-red transitionaccompanied by fluorescence quenching occurred when theC-dots were incubated with NO (Figure 1B, right).The aminoguanidine/citric acid C-dots were characterized by

several microscopic and spectroscopic techniques (Figure 2).

The high resolution transmission electron microscopy (HR-TEM) image in Figure 2A reveals the lattice planes of the C-dots, exhibiting d spacings of 0.28 and 0.21 nm correspondingto the (020) and (110) planes of graphitic carbons, andconfirming the formation of crystalline graphite cores of the C-dots.32,43 A relatively narrow size distribution of 3.8 ± 0.7 nmwas determined from the HR-TEM data (Figure 1A, SI). X-raydiffraction (XRD) analysis further confirms the crystallinity of

Figure 2. Characterization of the aminoguanidine/citric acid C-dots. A. High resolution transmission electron microscopy image of C-dots. Latticespacings within the graphitic cores of the C-dots are indicated. Scale bar corresponds to 2 nm. B. 13C NMR spectrum of the C-dots. The differentfunctional groups are indicated above the peaks: −CH2NH2 (i), −CH2COOH (ii), −COH (iii), −CNH (iv), −COOH (v), −CH2COOH (vi). C.X-ray photoelectron spectra (XPS) of C 1s, N 1s, and O 1s atoms in the C-dots.

Figure 3. Detection of NO in water by the aminoguanidine/citric acid C-dots. A. UV−vis spectra recorded at different times after addition of the C-dots to a saturated NO solution. Inset: Intensity of the absorbance at 490 nm recorded at different times after addition of NO (values taken from thespectra in panel A). The red curve represents the calculated fit corresponding to a pseudo-first order reaction (R2 = 0.995). B. Initial rate of productformation vs C-dots the symbols and the red line represent the observed and the simulated profile with fitting parameter R2 = 0.978. C. Fluorescenceemission spectra (excitation at 360 nm) of the C-dot solution recorded upon increasing NO concentrations. D. Stern−Volmer graph depicting thefluorescence quenching efficiency vs NO concentration. The inset shows a linear relationship in low NO concentrations, R2 = 0.95.

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the C-dots (Figure 1B, SI). The excitation-dependent emissionspectra are depicted in SI Figure 2, consistent with C-dotassembly in solution.44

Nuclear magnetic resonance (NMR, Figure 2B) and X-rayphotoelectron spectroscopy (XPS, Figure 2C) illuminate thefunctional units upon the C-dots’ surface. In particular, the 13CNMR spectrum in Figure 2B indicates that the C-dots retainedresidues from both the citric acid and aminoguanidineprecursors. Specifically, the NMR spectrum shows peakscorresponding to the aliphatic carbons at 36, 43, and 74ppm, carboxylic carbons at 171 and 176 ppm, and the −CNgroup of aminogunanidine at 158 ppm.45 The XPS analysis inFigure 2C complements the 13C NMR experiment, confirmingthe presence of different C-, N-, and O-containing units uponthe C-dots’ surface. Specifically, the deconvoluted C 1sspectrum in Figure 2C reveals peaks at 284.8 eV (correspond-ing to sp2 carbon atoms, CC), 286.6 eV (C−OH/C−NH),and 288.6 eV (CN/CO units).46 The N 1s peaks at 399.5,400.8, and 401.8 eV are assigned to pyridine N (CN),pyrrolnic N (C−N/N−N), and N−H groups, respectively,46

whereas the deconvoluted O 1s spectrum shows peaks at 531.8eV for CO and OHCO groups, and 532.7 eVcorresponding to C−OH units.43 Overall, the XPS and NMRdata indicate that residues upon the C-dots’ surface originatedfrom the two carbonaceous precursors used in the synthesis.The quantum yield of the C-dots was 8% when excited with360 nm using quinine sulfate as reference.Nitric Oxide Sensing with the Aminoguanidine/Citric

Acid C-dots. Figures 3 and 4 illustrate the optical andfluorescence transformations of the aminoguanidine/citric acidC-dots induced by NO in an aqueous solution and in cellenvironments. Figure 3 depicts the dramatic color andfluorescence transitions recorded in the C-dots solution uponaddition of NO. Figure 3A presents the UV−vis absorbancespectra of the C-dots, acquired at different times after additionof NO. Specifically, Figure 3A shows that the intensity of theabsorbance peak at 295 nm, corresponding to the π−π*transition of aromatic sp2 carbons, gradually increased, while ashoulder at 345 nm assigned to the transition of n−π*transition of the CO and CN bonds does not appear tochange with time.47

Figure 3A also demonstrates significant increase of visibleabsorbance at 490 nm upon incubation times of the C-dots andNO. This spectral change reflects the NO-induced yellow-redcolorimetric transformation of the C-dots (i.e., Figure 1B). Akinetic analysis depicting the peak intensity at 490 nm vsreaction time displays an excellent agreement with a pseudo-first-order reaction (inset in Figure 3A; note that theconcentration of NO is in high excess over the C-dotconcentration).48 Linearity of the 490 nm peak intensity wasalso observed upon examining the reaction of NO withsolutions having different C-dot concentrations (Figure 3B),also consistent with a pseudo first-order reaction rate.In parallel with the color transitions of the C-dots (Figure

3A,B), NO gave rise to quenching of the C-dots’ fluorescence(Figure 3C,D). Figure 3C presents the fluorescence emissionspectra (excitation 360 nm) recorded following addition ofdifferent NO concentrations to the aminoguanidine/citric acidC-dot solution. Figure 3C clearly shows that increasing NOconcentrations induced more quenching of the C-dots’fluorescence. The quantitative relationship between C-dots’fluorescence quenching (exc. 360 nm, em. 430 nm) and NOconcentration (determined using the Griess method, Figure 3

in SI) determined through the Stern−Volmer equation isillustrated in Figure 3D. Notably, a linear correlation isapparent in the physiologically important submicromolar NOconcentrations range, with an excellent detection limit ofaround 80 nM (inset in Figure 3D). The C-dot concentrationthreshold for NO detection via fluorescence quenching revealsa very low effective C-dot concentration of ∼10 μg/mL (Figure4, SI). The pH-dependence of NO detection by the C-dots wasalso evaluated, indicating optimal sensitivity in lower pH values(Figure 5, SI). Notably, the intrinsic fluorescence of the C-dotswas pH-dependent as well (Figure 5A, SI). The selectivity ofthe C-dots for NO detection in comparison with variedanalytes, particularly reactive oxygen species (ROS) wasexcellent (Figure 6, SI).While Figure 3C,D demonstrates the excellent bimodal NO

sensing features of the aminogunidine/citric acid C-dots inwater, we further investigated the use of the C-dots for cellularNO detection (Figure 4). Figure 4A presents confocalfluorescence microscopy images of macrophage RAW 264.7cells that were incubated for 18 h with the C-dots at aconcentration of 150 μg/mL. Figure 4A shows effective labelingof the cells by the fluorescent C-dots; the distinct emissioncolors in Figure 4A correspond to the excitation-dependentemission wavelengths of C-dots (e.g., Figure 2, SI).Importantly, application of the MTT assay indicated that C-dot labeling of the macrophages did not adversely impact cellviability (∼70% cell viability was determined after 18 hincubation with the C-dots (Figure 7, SI).

Figure 4. Fluorescence staining and NO sensing in macrophage cellsusing aminoguanidine/citric acid C-dots. A. Confocal fluorescenceimages of macrophage cells labeled with C-dots. Bright field image (i),and confocal fluorescence microscopy images recorded uponexcitation at 405 nm, emission filter EM 445/60 (ii), excitation at440 nm, emission filter EM 477/45 (iii); excitation at 488 nm withemission filter EM 525/50 (iv). B. Real time monitoring of LPS-induced NO generation in C-dot-labeled macrophages. C-dotfluorescence images (excitation 405 nm, emission filter EM 445/60)were recorded at the indicated times after LPS addition. C. Imaging ofLPS-induced NO generation in C-dot-labeled macrophages preloadedwith L-NAME, a NOS inhibitor. C-dot fluorescence images (excitation405 nm, emission filter EM 445/60) were recorded at the indicatedtimes after LPS addition. Scale bars correspond to 10 μm.

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Figure 4B demonstrates NO-induced fluorescence quenchingin the C-dot-labeled macrophage cells. Intracellular NO wasgenerated through addition of lipopolysaccharide (LPS, 10 μg/mL) extracted from Pseudomonas aeruginosa to the cellmedium.49 The confocal fluorescence images in Figure 4B(excitation 405 nm) demonstrate that the C-dots’ fluorescencegradually diminished after addition of LPS, and was largelyquenched after 20 min. Quantitative analysis of the cells’fluorescence intensity using image analysis software confirmedthat the NO-induced quenching was statistically significant(Figure 8, SI). As control, C-dot-labeled cells to which LPS wasnot added did not undergo fluorescence quenching (Figure 9,SI). Notably, the C-dot-labeled cells displayed similar viabilityafter LPS addition, compared to non-Cdot-labeled cells (Figure10, SI), indicating that NO-induced transformation of the cell-internalized C-dots did not interfere with cell processes. Itshould be cautioned that fluorescence turn-off sensors mightprovide false positive signals due to interference from the cellenvironment or fluorescence quenchers present in the cellmedium. Moreover, interpretation of data might be problematicas fluorescence from a sample not containing a fluorescentprobe occasionally produces the same (low) signal as samplescontaining the probe + analyte.50−52

To determine whether increased NO concentration insidethe macrophage cells was indeed the factor responsible for thefluorescence quenching of the C-dots, we independentlyassessed NO concentration produced within the cells throughthe Griess method, confirming that LPS-induced generation ofNO did occur (Figure 11, SI). Importantly, detection ofintracellular NO in real time and within minutes after LPSinduction, as demonstrated in Figure 4B for the C-dot-labeledcells, is unprecedented and is significantly faster compared tocurrently employed intracellular NO detection schemes.33

Since intracellular NO is generally produced via the activityof NO synthase (NOS),53,54 we further evaluated application ofthe C-dot-based NO sensor for monitoring the activity of aNOS inhibitor, L-NAME (Figure 4C). Specifically, the confocalfluorescence microscopy images in Figure 4C were recordedfollowing addition of LPS to C-dot-labeled macrophage cellswhich were also preincubated with L-NAME, a known NOSinhibitor.55 Importantly, no fluorescence quenching wasapparent in the fluorescence microscopy images in Figure 4C,presumably due to the NOS inhibition activity of L-NAME.Similar fluorescence quenching inhibitory action was recordedupon preincubating the cells with BAPTA-AM, a Ca2+ chelatorknown to interfere with NOS enzymatic activity (Figures 12and 13, SI).56 These results are noteworthy, since they indicatethat the new C-dot NO sensor could be employed for screeningand assessing the activity of NOS inhibitors.

Mechanistic Analysis of NO Sensing by Amino-guanidine/Citric Acid C-Dots. To decipher the underlyingmolecular mechanism responsible for the bimodal NO sensingby the aminoguanidine/citric acid C-dots we applied amultiprong spectroscopic and chromatography analysis (Figure5). 13C NMR spectra in Figure 5A indicate that NO reactedwith amine groups upon the C-dots’ surface. Specifically, the13C peak at 36.5 ppm, corresponding to α-carbon linked to theamine residues, exhibited an experimentally significant upfieldshift to 35.4 ppm (Figure 5A,i), which is consistent withchemical modification of the carbon-bonded amine.57−59 TheNMR spectrum in Figure 5A,ii further shows that addition ofNO to the C-dots gave rise to a new 13C peak at 147.5 ppm,ascribed to carbon atoms on the C-dots’ surface covalentlylinked to azo residues (e.g., −CNN−).60 This interpre-tation is consistent with the NO-induced yellow-red visualtransition (Figure 1B) and the UV−vis data presented in Figure

Figure 5. Analysis of NO reaction with the C-dots. A. 13C NMR spectra showing the aliphatic carbon-attached amine groups (i) and alkene region(ii), before (blue spectrum) and 24 h after addition of NO (red). B. X-ray photoelectron spectroscopy (XPS) analysis of N 1s before (bluespectrum), 4 h (green), and 24 h after addition of NO (red). C. Gas chromatographs recorded at different times after addition of NO-saturatedsolution to the C-dots (30 mg/mL). Inset: Digital image of the as-prepared aminoguanidine/citric acid C-dots solution under Ar atmosphere in agastight cuvette (left), and 5 min after addition of 5 mM NO (right). Note the appearance of bubbles, corresponding to N2 gas released following thereaction with the C-dots. D. FT-IR spectra of the C-dots before (blue line), 4 h (green), and 24 h (red) after addition of NO to the solution. The leftarrow corresponds to adsorbed N2, while the right arrow indicates −C−N/−C−O bands.

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3A demonstrating the emergence of a peak at 490 nmcorresponding to an azo moiety,61,62 and the dependence of thepeak intensity upon the NO/C-dot reaction time.The N 1s XPS data in Figure 5B provide additional

information on the reaction between NO and the amino-guanidine/citric acid C-dots. Specifically, 4 h after addition ofNO to the C-dots, a pronounced XPS peak exhibiting bindingenergy of 406.5 eV emerged, ascribed to molecular nitrogen(N2).

63 The N2 peak disappeared after 24 h, likely reflectingrelease of the adsorbed nitrogen molecules from the C-dots.Further evidence for formation and release of N2 molecules dueto the reaction between NO and the C-dots is obtained fromthe gas chromatography (GC) results in Figure 5C. The GCpeak corresponding to a retention time of 2.2 min, ascribed tonitrogen gas (see Experimental section) increased in intensitywith NO and C-dot incubation time. The photograph in Figure5C inset shows the N2 bubbles in the C-dot solution followingreaction with NO and the transformation of color from yellowto red corresponding to formation of the azo-dye.64 Notably,generation of N2 within less than 5 min after addition of NO,apparent in the GC data in Figure 5C, attests to the rapidreaction with the C-dots. Quantification of the released N2

(through use of a calibration curve, Figure 14, SI) reveals thatapproximately 60% of the NO was consumed by the N2-generating reaction. Consistent with the kinetic analysis inFigure 3A, nitrogen evolution also followed a pseudo-first-orderrate dependence (Figure 15, SI).The Fourier transform-infrared (FT-IR) spectra in Figure 5D

further illuminate the reaction pathway between NO and C-

dots. The FT-IR spectrum recorded 4 h after NO addition(Figure 5D, middle spectrum) reveals an emerging peak at2185 cm−1, ascribed to N2,

65,66 which disappeared after 24 h.The appearance of a transient N2 peak in the FT-IR experimentcorroborates the spectroscopic and chromatography analyses inFigure 5A−C. Notably, Figure 5D also shows that FT-IR peaksaround 1150 cm−1 corresponding to −C−N (aliphatic amine)and −C−O (carboxylic acid and alcohol units) significantlydiminished upon reaction of the C-dots with NO (e.g., thegreen and red spectra in Figure 5D, acquired after 4 and 24 h,respectively), indicating that these residues likely participated inthe reaction with NO.The reaction mechanisms outlined in Figure 6, based upon

the spectroscopic and chromatography analyses in Figures 1−5,provide the molecular basis for the structural and photophysicaltransformations of the aminoguanidine/citric acid C-dots andaccount for their NO sensing properties. Specifically, Figure 6highlights the two reaction pathways of NO and the C-dots.One route (Figure 6A, top) involves reaction of NO with theaminoguanidine residue, forming the nitroso-amine productwhich further generates a diazonium ion. The diazonium ion, inturn, produces molecular nitrogen, and also undergoes a diazo-coupling reaction to form a polymeric chain of carbon dotsconjugated through the diazo (−NN−) linkage. A secondpathway (Figure 6, bottom) depicts a reaction between NO andthe alkyl amine of C-dots producing a nitroso-amineintermediate and diazonium ion. Similar to the first reactionpathway, the diazonium ion generates both N2 as well as adiazo-dye-conjugated C-dot network.

Figure 6. Mechanism of the reaction between NO and guanidine/citric acid C-dots. A. Two routes depicting reaction of NO with eitheraminoguanidine residue or aliphatic amine displayed upon the C-dots’ surface. Both pathways generate molecular nitrogen and a C-dot networkconjugated through the diazo dye (polymerized network at right). B. Dynamic light scattering (DLS) profiles of the C-dots before (blue) and after(red) reaction with NO in water.

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The reaction mechanisms illustrated in Figure 6 accounts forboth the colorimetric and fluorescence response of the C-dotsto NO. Specifically, the azo dye formed is responsible for thevisible emission at 490 nm, i.e., the yellow-red transformation ofthe C-dot solution. Importantly, the reaction scheme in Figure6 shows that polymerization of the C-dots through the azounits constitutes a primary outcome of the reaction betweenNO and the C-dots. Indeed, dynamic light scattering (DLS)data depicted in Figure 6B demonstrates a significant increaseof hydrodynamic radii from around 10 nm prior to NOaddition to 300 nm after reaction with NO. Azo-inducedpolymerization of the C-dots likely accounts for the attenuationof C-dots’ fluorescence, as aggregation-induced fluorescencequenching is a well-known phenomenon in C-dot systems.27,67

■ CONCLUSIONS

C-dots exhibiting highly sensitive and rapid colorimetric andfluorescent response to NO were fabricated through a simpleone-step hydrothermal synthesis of aminoguanidine hydro-chloride and citric acid. The C-dots were not cytotoxic,facilitating real-time NO detection in living cells. Throughapplication of several analytical techniques we deciphered themolecular mechanism responsible for the NO sensingcapabilities; specifically, the experiments reveal that reactionof NO with amine moieties upon the C-dots’ surface gave riseto azo dye formation and concomitant release of molecularnitrogen. The reaction scheme indicates that NO-inducedformation of the azo units accounts for the visible colortransformation of the C-dots’ solution, and azo-promotedpolymerization of adjacent C-dots was the likely underlyingprocess leading to fluorescence quenching. The amino-guanidine/citric acid C-dots are easy to prepare and utilize.The dual signal modalities, high sensitivity, and feasibility ofNO sensing in live cell environment make the C-dots apowerful new platform for NO analysis. Moreover, the detailedmechanistic analysis will aid in laying solid foundations fordetermination of the molecular factors and transformationsresponsible for the remarkable photophysical properties of C-dots.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssen-sors.7b00356.

HR-TEM and XRD of C-dots, Photoluminescencespectra of C-dots, Calibration curve for NO, cell viability,real time monitoring of C-dots on addition of water,estimation of NO release from macrophage cell,calibration curve for nitrogen, and time scan of the N2

evolution on addition of 2 mL 4.9 mM NO solution(PDF)

■ AUTHOR INFORMATION

Corresponding Author*E-mail: razj@bgu.ac.il. Fax: (+) 972-8-6472943.

ORCIDRaz Jelinek: 0000-0002-0336-1384Author Contributions⊥B.C. and R.S. contributed equally to this work.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

Financial assistance from the Ministry of Science, grant number2015-243 is acknowledged. S.B. is grateful to Prof. IraWeinstock assistance with the GC and UV experiments.

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