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c Ilse Katz Institute for Nanotechnology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel. ABSTRACT: Carbon dots (C-dots) prepared throug...
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Nitric oxide sensing through azo-dye formation on carbon dots Sagarika Bhattacharya, Rhitajit Sarkar, Biswarup Chakraborty, Angel Porgador, and Raz Jelinek ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00356 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 4, 2017

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ACS Sensors

Nitric itric oxide sensing through azoazo-dye formation on carbon dots Sagarika Bhattacharya,a Rhitajit Sarkar,b,⊥, Biswarup Chakraborty,a,⊥, Angel Porgador,b and Raz Jelinek*a,c a Department

of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel. E-mail: [email protected]; Fax: (+) 972-8-6472943 b The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel c Ilse Katz Institute for Nanotechnology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel

ABSTRACT: Carbon dots (C-dots) prepared through heating of aminoguanidine and citric acid enable bimodal (colorimetric and fluorescence) detection of nitric oxide (NO) in aqueous solutions. The C-dots’ retained the functional units of aminoguanidine, which upon reaction with NO produced surface residues responsible for the color and fluorescence transformations. Notably, the aminoguanidine/citric acid C-dots were non-cytotoxic, making possible real-time and high sensitivity detection of NO in cellular environments. Using multi-prong spectroscopic and chromatography analyses we deciphered the molecular mechanism accounting for the NO-induced structural and photophysical transformations of the C-dots, demonstrating for the first time N2 release and azo 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 many biological systems including neuronal, cardiovascular, and immunological processes associated with macrophage and neutrophil activation.1-2 Irregular NO homeostasis is an indication of inflammatory response3 generally associated with various diseases and disorders like hypertension,4 atherosclerosis,4 diabetes,5 neurodegenerative diseases,6 and tumor growth.7 In particular, activation of macrophages - major inflammatory cells participating in host-defense against pathogen infection – is directly linked to NO generation. Specifically, endotoxins like lipopolysaccharide (LPS) induce signaling 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 sensitive detection platforms, particularly the low concentrations and short half-life of NO in physiological environments (5-60 sec).1, 10 Transition metal complexes have been employed as fluorescent NO sensors utilizing the binding specificity of NO towards metal centers.11-12 NO-selective sensors based on fluorescein derivatives have been reported.13-14 Diaminofluoresceins (DAFs) containing aromatic vicinal amines are also known to detect NO via triazole formation.15 Diamino probes based on Boron dipyrromethene (BODIPY), cyanine, and naphthalene scaffolds have been also used for NO analysis in biological systems.16-19 An organic molecule (LysoNINO) functionalized with the NO-capturing moiety ophenylenediamine together with a lysosome-targeting residue was used for endogenous NO detection in MCFcells18. Although this molecule exhibited rapid NO response of around 15 mins, it was 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 multi-walled carbon nanotubes,20-22 reduced graphene oxide,23 gold nanoparticles,24 and inorganic quantum dots.25 These NO sensing strategies, however, have had limited applicability in biological systems, exhibit long reaction times, low sensitivity, or requiring complex 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 applications as they exhibit broad excitationdependent emission spectra that can be readily modulated upon interaction with molecular targets.30-31 In addition, Cdots are generally non-toxic, and can be easily surfacemodified, thereby displaying varied recognition moieties.32-33 Particularly important, previous studies have demonstrated that C-dots could retain the structural and functional units of the carbonaceous building blocks employed in the synthesis, endowing the dots with biomolecular targeting capabilities simply through selection of the starting reagents.34-36 C-dots synthesized from citric acid and ethylene diamine were used as NO sensors.37 A ratiometric C-dot sensor exhibiting phenylenediamine-containing naphthalimide fuctionalization could detect NO via triazole formation.33 SimÕes and coworkers developed a fluorimetric NO sensor based upon N,S-doped carbon dots.37 The sensor achieved rapid NO detection, however has not been applied in cellular environments. Overall, these C-dot systems exhibit either relatively low NO sensitivity, long response times (up to hours), or involve complex synthetic schemes.

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Furthermore, the mechanisms of NO induced-modulation of C-dots’ fluorescence have not been deciphered. Here we describe fabrication of C-dots through a simple one-step hydrothermal treatment of aminoguanidine hydrochloride and citric acid. Aminoguanidine participates in varied enzymatic processes and has been used as a therapeutic substance for treatment of complications in diabetes.38-39 Due to the presence of the guanidino nitrogen, aminoguanidine has been also investigated as a NO synthase (NOS) enzyme inhibitor.40 We demonstrate that the citric acid/aminoguanidine C-dots undergo bimodal (visible and fluorescence) transformations upon addition of NO, both in water and in cells. Importantly, through application of varied analytical techniques we determine the molecular mechanism responsible for NO sensing. Specifically, the experiments reveal for the first time that reaction of NO with amine moieties upon the Cdots’ surface gave rise to azo dye formation and concomitant release of molecular nitrogen.

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subsequently solubilized in water for further characterization and use. Preparation of nitric oxide solution. Saturated NO solutions were prepared as previously reported.20 Briefly, NO gas was generated by slowly pouring 2M H2SO4 into a glass flask containing saturated aqueous solution of NaNO2 under Ar atmosphere. The NO gas produced by the disproportionation of NaNO2 in acidic medium was passed through 5% (w/v) pyrogallol solution in saturated potassium hydroxide followed by 10% (w/v) potassium hydroxide to remove oxygen, other nitrogen oxides, and acid vapors. Before addition of H2SO4, the apparatus and all the solutions required for NO generation were degassed with argon for 30 minutes to exclude O2 in order to avoid the reaction of NO with O2. The NO-saturated solutions were prepared by purging the NO gas in deoxygenated water and keeping under NO atmosphere until use. Concentration of the saturated NO solution was 4.92 mM at 25°C, determined by the Griess reaction.41 All preparation steps were carried out in a fume hood. NO standard solutions were prepared by making successive dilutions of the saturated NO solution. NO standard solutions were made fresh for experiments and were kept in a glass flask with a rubber and light-free septum with wrapped black foils. Quantum yield measurement. Quantum yield of the amine modified carbon dots was determined by comparing integrated photoluminescence intensity (in the range of 380-650 nm) upon excitation 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:

EXPERIMENTAL SECTION Materials. Aminoguanidine hydrochloride, citric acid, sulfanilamide, N-(1naphthyl)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(2aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)) and lipopolysaccharide from Pseudomonas aeruginosa 10 were purchased from Sigma Aldrich, St. Louis, MO, USA. Potassium hydroxide, sodium hydroxide and pyrogallol were purchased from Alfa aesar and Tzamal D-chem. All chemicals were used without further purification. Methanol and sulfuric acid 98% were purchased from carlo Erba, Italy and Bio-Lab Ltd. (Jerusalem, Israel), respectively. RPMI 1640 medium (RPMI), fetal bovine serum (FBS), and 2-marcaptoethanol were bought from Gibco by Life Technologies, USA. Media supplements, antibiotics, and PBS were acquired from Biological Industries, USA. Cell viability reagent 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from ThermoFisher Scientific, USA. Mili-Q water with resistivity of 18.2 MΩ cm at 25°C were used for all the experiments. Synthesis of C-dots. Aminoguanidine hydrochloride 98% was mixed with citric acid in 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 hours. After the reaction was completed, the resultant mixture was allowed to cool to room temperature yielding a brown precipitate indicating the formation of carbon dots. The precipitate was re-dispersed in 2 mL methanol through sonication for 2 mins and centrifuged at 10,000 rpm for 30 min to remove high-weight precipitate and agglomerated particles. Thereafter, methanol was evaporated under reduced pressure to obtain a brown solid. This was dissolved by sonication in 2 mL distilled water and was dialysed against distilled water several times,

in which = Quantum yield of the sample; = Integrated fluorescence intensity; AS = Absorbance; = refractive index. The index R corresponds to the reference and index S the sample. Fluorescence spectroscopy. Fluorescence emission spectra were recorded on an FL920 spectrofluorimeter. A Varioskan plate reader was 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’s aqueous solution were added, fluorescence measurements following titration with NO were carried out by addition of 10 µL of saturated solution to achieve the final concentration. Quenching efficiency (Q. E.) was calculated by using the Stern-Volmer equation (I0 corresponds to the initial fluorescence while I correspond to the fluorescence recorded after NO addition):

High resolution transmission electron microscopy (HR-TEM). A drop of C-dot solution was placed upon a graphene-coated copper grid and HR-TEM images were observed on a 200 kV JEOL JEM-2100F microscope

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ACS Sensors evolved during the reaction of C-dots with freshly prepared NO saturated aqueous solution, the reaction was performed in argon atmosphere in a septa-sealed gas tight quartz cell equipped with 14 ml overhead space. A complete air-free environment was maintained inside the gas-tight quartz cell using a Schlenk line. Before addition of the NO saturated aqueous solutions (2 ml at 4.92 mM), the C-dots suspension (2 ml of 30 mg/ml) was purged with argon for 30 minutes to avoid reaction of NO with dissolved oxygen. NO generation from a disproportionate reaction of NaNO2 and conc. H2SO4 and followed by purification by Pyrogallol and KOH solution was also performed in Schlenk line under Ar atmosphere, and individual solutions used to prepare and purify NO were purged with minimum 20-30 minutes. Furthermore, prior to saturate with NO, the water used was purged with Ar for at least 30 minutes and kept in Ar atmosphere. With the progress of reaction, the evolved gas from headspace of the quartz cell was injected to the GC using a gas-tight syringe (Hamilton). The evolved N2 gas was detected and identified by 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 amount fresh air as sample in the GC. The volume of N2 injected in GC was calculated assuming air composition N2:O2 to be 78.09:20.86 %. The peak area was plotted with respect to the volume of nitrogen injected. The evolved N2 during the reaction was quantified from the linear fit of the plot. From the calibration curve, the volume of nitrogen (in µmol) in 1 mL was calculated. As the injected volume in GC was 1 mL from the 14 mL overhead space of the gas tight cuvette. The total volume of N2 gas evolved from the 4 mL total reaction mixture of 30 mg/mL C-dots solution should be 14 times the calculated value. Based on 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 30 mg/ml C-dots solution, and produced 5.82 µmol of nitrogen. Cell culture and viability. Mouse (Mus musculus) leukaemic monocyte macrophage cell line RAW 264.7 (ATCC® TIB-71™) was grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), antibiotics and other necessary supplements and maintained at 37°C in a humidified atmosphere containing 5% CO2 in CO2 incubator. For the experiments, cells were seeded in a 96‐well flat bottom culture plate at a density of 5×103 cells/well and allowed to settle for 30 min in incubator. The cells were then treated with increasing concentrations (0, 25, 50, 75, 100, 150 and 200 μg/ml) of freshly prepared C-dots in PBS1X (Stock solution 1 mg/ml) and incubated for 18 h. Post incubation, 10 μl 2 mg/ml MTT solution was added to each well followed by 2 h incubation at 37°C. 50 μl DMSO was added to each well to dissolve the formazan crystals and the absorbance measured at 560 nm using a microplate Multiskan Spectrum (Thermo Electron Corporation, USA). Fresh culture medium was used as the background with n=6 considered for measurement of each sample. Obtained data were represented as percentage of viability and IC50 value was determined from the regression analysis curve.

(Japan). The sample was dried for 12 hours prior to measurements. X-ray photoelectron spectroscopy (XPS). Concentrated aqueous solutions of C-dots were dropcasted on silicon wafers and measurements were performed using an X-ray photoelectron spectrometer ESCALAB 250 ultrahigh vacuum (1 × 10−9 bar) apparatus with an AlKα X-ray source and a monochromator. The beam diameter of was 500 μm and with pass energy (PE) of 150 eV survey spectra were recorded, while for high energy resolution spectra the recorded pass energy (PE) was 20 eV. The AVANTGE program was used to process the XPS results. Nuclear magnetic resonance (NMR). C-13 NMR spectra of C-dots before and after addition of NO were recorded using a Brüker DPX 400 MHz spectrometer at 300 K. C-13 NMR of C-dots was performed in D2O, with compound concentrations 30 mg/mL. After addition of the NO solution to the C-dots, the mixture was stirred for 24 hours under argon atmosphere. Red solid isolated upon removal of the water was re-dissolved in D2O for 13C NMR experiment without further purification. Fourier transform-infrared (FT-IR) analyses. FT-IR measurements were performed on a Thermo Scientific Nicolet 6700 spectrometer. The procedure used for extraction of the product was same used for the NMR experiment. Dynamic light scattering. DLS data were collected at 25°C on an ALV-CGS-8F instrument (ALV-GmbH, Germany) at 90°, and the CONTIN method was used to obtain hydrodynamic radii (Rh). Griess reaction and ultraviole-visible (uv-vis) spectroscopy. The Griess reaction was carried out using uv/vis spectrophotometry analysis. The reaction involves a two-step assay based on the observation that the adduct of nitric oxide and sulfanilic acid (1%) interacts with N-(1naphthyl)ethylenediamine (0.l%), to generate an azo derivative product that is readily monitored by uv-vis spectrophotometry.41 The spectrum of the product shows an absorbance band with a maximum at 540 nm. All absorbance measurements were performed on an Agilent 8453 diode array spectrophotometer with 1 cm cells in water as dispersive medium. Optical spectra and kinetics of Azo dye formation. Kinetic measurements were carried out in water at 25°C under argon atmosphere. The C-dot concentrations were varied from 1-10 mg/ml (volume 2 ml) and 300 µL of 4.92 mM of NO was added. The rate of formation of the azo dye was measured by monitoring the absorbance band at 490 nm. In another experiment, different volumes (100-500 µL) of 4.92 mM NO solution were added to 2 ml C-dots solution (conc. 2.5 mg/ml) and azo dye formation was monitored. On comparing the optical density obtained at 540 nm on addition of Griess reagent to the NO treated Cdot solution, we determined that 99% 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 dedicated thermal-conductivity detector (TCD), and argon was used as the carrier gas. To analyze headspace gas

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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 Griess reaction (Guevara et al., 1998), based on the diazotization of sulphanilic acid with nitrite ion and coupling of this product with diamine which results in a spectrophotometrically measurable pink metabolite. RAW 264.7 cells were seeded in a 24-well culture plate as 1.5×106 cells/well and allowed to settle for 30 min in incubator. Then cells were treated with freshly prepared 150 μg/ml C-dots (determined after titration of concentration range below IC50 value; data not shown) in PBS1X, 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 & 3 h) in a 96-well flat bottom culture plate, 100 μl of supernatant cell culture medium was mixed with 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 min at room temp and the absorbance at 540 nm was measured in a microplate reader Multiskan Spectrum (Thermo Electron Corporation, USA). Two triplet sets were taken for measurement of each sample. Fresh culture medium was used as the background correction in all experiments. The nitric oxide content was calculated from a sodium nitrite standard curve. Cell Incubation with C-dots and Confocal Imaging. In cover glass-bottom Confocal Dish, RAW 264.7 cells were seeded as 1×104 cells/dish and allowed to recuperate for 30 min in 37°C CO2 incubator. As before, cells were then treated with freshly prepared 150 μg/ml C-dots in PBS1X, along with necessary blanks and incubated for 18 h. Post incubation, cells were washed with PBS and confocal microscopy images of C-dot labelled cells were acquired on an UltraVIEW system (PerkinElmer Life Sciences, Waltham, MA) equipped with an Axiovert-200 M microscope (Zeiss, Oberkochen, Germany) and a PlanNeofluar 63×/1.4 oil objective. Excitation wavelengths of 405 nm, 440 nm, and 488 nm were produced by an argon/krypton laser. Then, 10 μg/ml LPS was added to the sample and images of the same location were acquired at different time intervals. A parallel blank was also done using ultrapure water instead of LPS.

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Figure 1B. Specifically, prior to addition of NO the C-dots exhibited visible yellow color, and emitted intense blue fluorescence (exc. 360 nm, Figure 1B left). However, a remarkable and rapid yellow-red transition accompanied by fluorescence quenching occurred when the C-dots were incubated with NO (Figure 1B, right).

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

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 nm and 0.21 nm corresponding to the (020) and (110) planes of graphitic carbons, and confirming the formation of crystalline graphite cores of the C-dots.32, 42 A relatively narrow size distribution of 3.8 ± 0.7 nm was determined from the HR-TEM data (Figure 1A,SI). X-ray diffraction (XRD) analysis further confirms the crystallinity of the C-dots (Figure 1B,SI). The excitation-dependent emission spectra are depicted in Figure 2,SI), consistent with C-dot assembly in solution.43

RESULTS AND DISCUSSION Aminoguanidine/citric acid C-dot synthesis and characterization Figure 1 outlines the synthesis scheme of the C-dots, and demonstrates the bimodal NO detection features. Preparation of the C-dots was carried out through hydrothermal treatment of a mixture of aminoguanidine hydrochloride and citric acid (5:1 weight ratio, Figure 1A). Citric acid has been widely-employed as a carbon source in C-dot synthesis, generally yielding uniform C-dots' graphitic cores.32 The dramatic response of the aminoguanidine/citric acid C-dots to NO is depicted in

Figure 2. Characterization of the aminoguanidine/citric acid C-dots. A. High Resolution Transmission electron microscopy image of C-dots. Lattice spacings within the graphitic cores of the C-dots are indicated. Scale bar corresponds to 2 nm. B. C-13 NMR spectrum of the C-dots. The different func-

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ACS Sensors dot concentrations Figure 3B), also consistent with a firstorder pseudo reaction rate.

tional groups are indicated above the peaks: –CH2NH2 (i), – CH2COOH (ii), –COH (iii), –C=NH (iv), –COOH (v), –CH2COOH (vi). C. X-ray photoelectron spectra (XPS) of C1s, N1s and O1s atoms in the C-dots.

Nuclear magnetic resonance (NMR, Figure 2B) and X-ray photoelectron spectroscopy (XPS, Figure 2C) illuminate the functional units upon the C-dots’ surface. In particular, the 13C-NMR spectrum in Figure 2B indicates that the C-dots retained residues from both the citric acid and aminoguanidine precursors. Specifically, the NMR spectrum shows peaks corresponding to the aliphatic carbons at 36 ppm, 43 ppm and 74 ppm, carboxylic carbons at 171 ppm and 176 ppm, and the –C=N group of aminogunanidine at 158 ppm.44 The XPS analysis in Figure 2C complements the 13C NMR experiment, confirming the presence of different C-, N-, and O- containing units upon the C-dots’ surface. Specifically, the deconvoluted C1s spectrum in Figure 2C reveals peaks at 284.8 eV (corresponding to sp2 carbon atoms, C=C), 286.6 eV (C ̶ OH/C ̶ NH), and 288.6 eV (C=N/C=O units).45 The N1s peaks at 399.5, 400.8 and 401.8 eV are assigned to pyridine N (C=N), pyrrolnic N (C-N/N-N), and N-H groups, respectively,45 whereas the deconvoluted O1s spectrum shows peaks at 531.8 eV for C=O and OH–C=O groups, and 532.7 eV corresponding to C ̶ OH units.42 Overall, the XPS and NMR data indicate that residues upon the C-dots’ surface originated from the two carbonaceous precursors used in the synthesis. The quantum yield of the C-dots was 8% when excited with 360 nm using quinine sulfate as reference.

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 the spectra in panel A). The red curve represents the calculated fit corresponding to a pseudo-first order reaction (R2= 0.995). B. Initial rate of product formation vs. C-dots the symbols and the red line represent the observed and the simulated profile with fitting parameter R2= 0.978. C. Fluorescence emission spectra (excitation at 360 nm) of the C-dot solution recorded upon increasing NO concentrations. D. Stern-Volmer graph depicting the fluorescence quenching efficiency vs. NO concentration. The inset shows a linear relationship in low NO concentrations, R2= 0.95.

Nitric oxide sensing with the aminoguanidine/citric acid C-dots Figures 3 and 4 illustrate the optical and fluorescence transformations of the aminoguanidine/citric acid C-dots induced by NO in an aqueous solution and in cell environments. Figure 3 depicts the dramatic color and fluorescence transitions recorded in the C-dots solution upon addition of NO. Figure 3A presents the ultravioletvisible (uv-vis) absorbance spectra of the C-dots, acquired at different times after addition of NO. Specifically, Figure 3A shows that the intensity of the absorbance peak at 295 nm, corresponding to the π–π* transition of aromatic sp2 carbons, gradually increased, while a shoulder at 345 nm assigned to the transition of n–π* transition of the C=O and C=N bonds does not appear to change with time.46 Figure 3A also demonstrates significant increase of visible absorbance at 490 nm upon incubation times of the C-dots and NO. This spectral change reflects the NOinduced yellow-red colorimetric transformation of the Cdots (i.e. Figure 1B). A kinetic analysis depicting the peak intensity at 490 nm vs reaction time displays an excellent agreement with a pseudo first-order reaction (inset in Figure 3A; note that the concentration of NO is in high excess over the C-dot concentration).47 Linearity of the 490 nm peak intensity was also observed upon examining the reaction of NO reaction with solutions having different C-

In parallel with the color transitions of the C-dots (Figures 3A-B), NO gave rise to quenching of the C-dots’ fluorescence (Figures 3C-D). Figure 3C presents the fluorescence emission spectra (excitation 360 nm) recorded following addition of different NO concentrations to the aminoguanidine/citric acid C-dot solution. Figure 3C clearly shows that increasing NO concentrations induced more quenching of the C-dots fluorescence. The quantitative relationship between C-dots’ fluorescence quenching (exc. 360 nm, em. 430 nm) and NO concentration (determined using the Griess method, Figure 3,SI) determined through the Stern-Volmer equation is illustrated in Figure 3D. Notably, a linear correlation is apparent in the physiologically-important sub-micromolar NO concentrations range, with an excellent detection limit of around 80 nM (inset in Figure 3D). The C-dot concentration threshold for NO detection via fluorescence quenching reveals a very low effective Cdot concentration of ~10 µg/mL (Figure 4, SI). The pHdependence of NO detection by the C-dots was also evaluated, indicating optimal sensitivity in lower pH values (Figure 5, SI). Notably, the intrinsic fluorescence of the C-

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dots was pH-dependent as well (Figure 5A, SI). The selectivity of the C-dots for NO detection in comparison with varied analytes, particularly reactive oxygen species (ROS) was excellent (Figure 6, SI). While Figure 3C-D demonstrate the excellent bimodal NO sensing features of the aminogunidine/citric acid Cdots in water, we further investigated the use of the C-dots for cellular NO detection (Figure 4). Figure 4A presents confocal fluorescence microscopy images of macrophage RAW 264.7 cells that were incubated for 18 hours with the C-dots at a concentration of 150 µg/ml. Figure 4A shows effective labeling of the cells by the fluorescent C-dots; the distinct emission colors in Figure 4A correspond to the excitation-dependent emission 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 cell viability (~70% cell viability was determined after 18h incubation with the C-dots, Figure 7,SI). Figure 4B demonstrates NO-induced fluorescence quenching in the C-dot-labeled macrophage cells. Intracellular NO was generated through addition of lipopolysaccharide (LPS, 10 µg/mL) extracted from Pseudomonas aeruginosa 10 to the cell medium.48 The confocal fluorescence images in Figure 4B (excitation 405 nm) demonstrate that the C-dots’ fluorescence gradually diminished after addition of LPS, and was largely quenched after 20 minutes. Quantitative analysis of the cells' fluorescence intensity using image analysis software confirmed that the NO-induced quenching was statistically significant (Figure 8,SI). As control, C-dot-labeled cells to which LPS was not added did not undergo fluorescence quenching (Figure 9,SI). Notably, the C-dot-labeled cells displayed similar viability after LPS addition, compared to non-Cdot-labeled cells (Figure 10,SI), indicating that NOinduced transformation of the cell-internalized C-dots did not interfere with cell processes. It should be cautioned that fluorescence turn-off sensors might provide false positive signals due to interference from the cell environment or fluorescence quenchers present in the cell medium. Moreover, interpretation of data might be problematic as fluorescence from a sample not containing a fluorescent probe occasionally produces the same (low) signal as samples containing the probe + analyte.49-51

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Figure 4. Fluorescence staining and NO sensing in macrophage cells using aminoguanidine/citric acid Cdots. A. Confocal fluorescence images of macrophage cells labeled with C-dots. Bright field image (i), and confocal fluorescence microscopy images recorded upon excitation at 405 nm, emission filter EM 445/60 (ii), excitation at 440 nm, emission filter EM 477/45 (iii); excitation at 488 nm with emission filter EM 525/50 (iv). B. Real time monitoring of LPS-induced NO generation in C-dot-labeled macrophages. Cdot fluorescence images (excitation 405 nm, emission filter EM 445/60) were recorded at the indicated times after LPS addition. C. Imaging of LPS-induced NO generation in C-dotlabeled macrophages pre-loaded with L-NAME, a NOS inhibitor. C-dot fluorescence images (excitation 405 nm, emission filter EM 445/60) were recorded at the indicated times after LPS addition. Scale bars correspond to 10 μm.

To determine whether increased NO concentration inside the macrophage cells was indeed the factor responsible for the fluorescence quenching of the C-dots, we independently assessed NO concentration produced within the cells through the Griess method, confirming that LPS-induced generation of NO did occur (Figure 11,SI). Importantly, detection of intracellular NO in real time and within minutes after LPS induction, as demonstrated in Figure 4B for the C-dot-labeled cells, is unprecedented and is significantly faster compared to currently-employed intracellular NO detection schemes.33 Since intracellular NO is generally produced via the activity of NO synthase (NOS),52-53 we further evaluated application of the C-dot-based NO sensor for monitoring the activity of a NOS inhibitor, L-NAME (Figure 4C). Specifically, the confocal fluorescence microscopy images in Figure 4C were recorded following addition of LPS to Cdot-labeled macrophage cells which were also preincubated with L-NAME, a known NOS inhibitor.54 Importantly, no fluorescence quenching was apparent in the fluorescence microscopy images in Figure 4C, presumably due to the NOS inhibition activity of L-NAME. Similar fluorescence quenching inhibitory action was

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ACS Sensors ance of bubbles, corresponding to N2 gas released following the reaction with the C-dots. D. FT-IR spectra of the C-dots before (blue line), 4 hours (green), and 24 hours (red) after addition of NO to the solution. The left arrow corresponds to adsorbed N2, while the right arrow indicates -C-N/-C-O bands.

recorded upon pre-incubating the cells with BAPTA-AM, a Ca2+ chelator known to interfere with NOS enzymatic activity (Figures 12,SI and 13,SI).55 These results are noteworthy, since they indicate that the new C-dot NO sensor could be employed for screening and assessing the activity of NOS inhibitors.

The N1s XPS data in Figure 5B provide additional information on the reaction between NO and the aminoguanidine/citric acid C-dots. Specifically, 4 hours after addition of NO to the C-dots, a pronounced XPS peak exhibiting binding energy of 406.5 eV emerged, ascribed to molecular nitrogen (N2).62 The N2 peak disappeared after 24 hours, likely reflecting release of the adsorbed nitrogen molecules from the C-dots. Further evidence for formation and release of N2 molecules due to the reaction between NO and the C-dots is obtained from the gas chromatography (GC) results in Figure 5C. The GC peak corresponding to a retention time of 2.2 min, ascribed to nitrogen gas (see Experimental section) increased in intensity with NO and C-dots incubation time. The photograph in Figure 5C, inset shows the N2 bubbles in the C-dot solution following reaction with NO and the transformation of color from yellow to red corresponding to formation of the azo-dye.63 Notably, generation of N2 within less than 5 minutes after addition of NO, apparent in the GC data in Figure 5C attests to the rapid reaction with the C-dots. Quantification of the released N2 (through use of a calibration curve, Figure 14, SI) reveals that approximately 60% of the NO was consumed by the N2generating reaction. Consistent with the kinetic analysis in Figure 3A, nitrogen evolution also followed a pseudo first order rate 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 hours after NO addition (Figure 5D, middle spectrum) reveals an emerging peak at 2185 cm-1, ascribed to N2,64-65 which disappeared after 24 hours. The appearance of a transient N2 peak in the FT-IR experiment corroborates the spectroscopic and chromatography analyses in Figure 5(AC). Notably, Figure 5D also shows that FT-IR peaks around 1150 cm-1 corresponding to -C-N (aliphatic amine) and –CO (carboxylic acid and alcohol units) significantly diminished upon reaction of the C-dots with NO (e.g. the green and red spectra in Figure 5D, acquired after 4 hours and 24 hours, respectively), indicating that these residues likely participated in the reaction with NO.

Mechanistic analysis of NO sensing by aminoguanidine/citric acid C-dots To decipher the underlying molecular mechanism responsible for the bimodal NO sensing by the aminoguanidine/citric acid C-dots we applied a multiprong spectroscopic and chromatography analysis (Figure 5). 13C-NMR spectra in Figure 5A indicate that NO reacted with amine groups upon the C-dots’ surface. Specifically, the 13C peak at 36.5 ppm, corresponding to α-carbon linked to the amine residues, exhibited an experimentallysignificant upfield shift to 35.4 ppm (Figure 5A,i), which is consistent with chemical modification of the carbonbonded amine.56-58 The NMR spectrum in Figure 5A,ii further shows that addition of NO to the C-dots gave rise to a new 13C peak at 147.5 ppm, ascribed to carbon atoms on the C-dots’ surface covalently linked to azo residues (e.g. C-N=N-).59 This interpretation is consistent with the NOinduced yellow-red visual transition (Figure 1B) and the uv-vis data presented in Figure 3A demonstrating the emergence of a peak at 490 nm corresponding to an azo moiety,60-61 and the dependence of the peak intensity upon the NO/C-dot reaction time.

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 hours after addition of NO (red). B. X-ray photoelectron spectroscopy (XPS) analysis of N1s before (blue spectrum), 4 hours (green), and 24 hours after addition of NO (red). C. Gas chromatographs recorded at different times after addition of NO-saturated solution to the C-dots (30 mg/ml). Inset: Digital image of the as-prepared aminoguanidine/citric acid C-dots solution under Ar atmosphere in a gas tight cuvette (left), and 5 minutes after addition of 5 mM NO (right). Note the appear-

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depicted in Figure 6B demonstrates a significant increase of hydrodynamic radii from around 10 nm prior to NO addition to 300 nm after reaction with NO. Azo-induced polymerization of the C-dots likely accounts for the attenuation of C-dots’ fluorescence, as aggregation-induced fluorescence quenching is a well-known phenomenon in Cdot systems.27, 66 CONCLUSIONS C-dots exhibiting highly sensitive and rapid colorimetric and fluorescent response to NO were fabricated through a simple one-step hydrothermal synthesis of aminoguanidine hydrochloride and citric acid. The C-dots were not cytotoxic, facilitating real-time NO detection in living cells. Through application of several analytical techniques we deciphered the molecular mechanism responsible for the NO sensing capabilities; specifically, the experiments reveal that reaction of NO with amine moieties upon the C-dots’ surface gave rise to azo dye formation and concomitant release of molecular nitrogen. The reaction scheme indicates that NO-induced formation of the azo units accounts for the visible color transformation of the C-dots’ solution, and azo-promoted polymerization of adjacent C-dots was the likely underlying process leading to fluorescence quenching. The aminoguanidine/citric acid C-dots are easy to prepare and utilize. The dual signal modalities, high-sensitivity, and feasibility of NO sensing in live cell environment make the C-dots a powerful new platform for NO analysis. Moreover, the detailed mechanistic analysis will aid in laying solid foundations for determination of the molecular factors and transformations responsible for the remarkable photophysical properties of C-dots.

Figure 6. Mechanism of the reaction between NO and guanidine/citric acid C-dots. A. Two routes depicting reaction of NO with either aminoguanidine residue or aliphatic amine displayed upon the C-dots’ surface. Both pathways generate molecular nitrogen and a C-dot network conjugated 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.

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 photophysical transformations of the aminoguanidine/citric acid C-dots and accounts for their NO sensing properties. Specifically, Figure 6 highlights the two reaction pathways of NO and the C-dots. One route (Figure 6A, top) involves reaction of NO with the aminoguanidine residue, forming nitroso-amine product which further generates diazonium ion. The diazonium ion, in turn, produces molecular nitrogen, and also undergoes a diazo-coupling reaction to form a polymeric chain of carbon dots conjugated through the diazo (-N=N-) linkage. A second pathway (Figure 6, bottom route) depicts a reaction between NO and the alkyl amine of C-dots producing nitroso-amine intermediate and diazonium ion. Similar to the first reaction pathway, the diazonium ion generates both N2 as well as a diazo-dye-conjugated C-dot network. The reaction mechanisms illustrated in Figure 6 accounts for both the colorimetric and fluorescence response of the C-dots to NO. Specifically, the azo dye formed is responsible for the visible emission at 490 nm, i.e. the yellow-red transformation of the C-dot solution. Importantly, the reaction scheme in Figure 6 shows that polymerization of the C-dots through the azo units constitutes a primary outcome of the reaction between NO and the C-dots. Indeed, dynamic light scattering (DLS) data

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. HR-TEM and XRD of C-dots, Photoluminescence spectra of Cdots, 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. (Figures S1- S9) (PDF)

AUTHOR INFORMATION Corresponding Corresponding Author *E-mail: [email protected]

Author Contributions ⊥B.C

and R.S. contributed equally to this work.

Notes The authors declare no competing financial interest.

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ACS Sensors 19. Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T., Highly Sensitive Fluorescence Probes for Nitric Oxide Based on Boron Dipyrromethene ChromophoreRational Design of Potentially Useful Bioimaging Fluorescence Probe. J. Am. Chem. Soc. 2004, 126 (10), 3357-3367. 20. Li, C. M.; Zang, J.; Zhan, D.; Chen, W.; Sun, C. Q.; Teo, A. L.; Chua, Y. T.; Lee, V. S.; Moochhala, S. M., Electrochemical Detection of Nitric Oxide on a SWCNT/RTIL Composite Gel Microelectrode. Electroanal. 2006, 18 (7), 713-718. 21. Kan, K.; Xia, T.; Yang, Y.; Bi, H.; Fu, H.; Shi, K., Functionalization of multi-walled carbon nanotube for electrocatalytic oxidation of nitric oxide. J. App. Electrochem. 2010, 40 (3), 593-599. 22. Kim, J.-H.; Heller, D. A.; Jin, H.; Barone, P. W.; Song, C.; Zhang, J.; Trudel, L. J.; Wogan, G. N.; Tannenbaum, S. R.; Strano, M. S., The rational design of nitric oxide selectivity in single-walled carbon nanotube near-infrared fluorescence sensors for biological detection. Nat. Chem. 2009, 1 (6), 473-481. 23. Li, W.; Geng, X.; Guo, Y.; Rong, J.; Gong, Y.; Wu, L.; Zhang, X.; Li, P.; Xu, J.; Cheng, G.; Sun, M.; Liu, L., Reduced Graphene Oxide Electrically Contacted Graphene Sensor for Highly Sensitive Nitric Oxide Detection. ACS Nano 2011, 5 (9), 6955-6961. 24. Yu, A.; Liang, Z.; Cho, J.; Caruso, F., Nanostructured Electrochemical Sensor Based on Dense Gold Nanoparticle Films. Nano Lett. 2003, 3 (9), 1203-1207. 25. Sun, J.; Yan, Y.; Sun, M.; Yu, H.; Zhang, K.; Huang, D.; Wang, S., Fluorescence Turn-On Detection of Gaseous Nitric Oxide Using Ferric Dithiocarbamate Complex Functionalized Quantum Dots. Anal. Chem. 2014, 86 (12), 5628-5632. 26. Ding, C.; Zhu, A.; Tian, Y., Functional Surface Engineering of C-Dots for Fluorescent Biosensing and in Vivo Bioimaging. Acc. Chem. Res. 2014, 47 (1), 20-30. 27. Lim, S. Y.; Shen, W.; Gao, Z., Carbon quantum dots and their applications. Chem. Soc. Rev. 2015, 44 (1), 362-381. 28. Hola, K.; Zhang, Y.; Wang, Y.; Giannelis, E. P.; Zboril, R.; Rogach, A. L., Carbon dots—Emerging light emitters for bioimaging, cancer therapy and optoelectronics. Nano Today 2014, 9 (5), 590-603. 29. Song, Y.; Zhu, S.; Yang, B., Bioimaging based on fluorescent carbon dots. RSC Adv. 2014, 4 (52), 27184-27200. 30. Bhattacharya, S.; Sarkar, R.; Nandi, S.; Porgador, A.; Jelinek, R., Detection of Reactive Oxygen Species by a Carbon-Dot–Ascorbic Acid Hydrogel. Anal. Chem. 2017, 89 (1), 830-836. 31. Zhu, A.; Qu, Q.; Shao, X.; Kong, B.; Tian, Y., Carbon-Dot-Based Dual-Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for In Vivo Imaging of Cellular Copper Ions. Angew. Chem. Int. Ed. 2012, 51 (29), 7185-7189. 32. Zheng, M.; Liu, S.; Li, J.; Qu, D.; Zhao, H.; Guan, X.; Hu, X.; Xie, Z.; Jing, X.; Sun, Z., Integrating Oxaliplatin with Highly Luminescent Carbon Dots: An Unprecedented Theranostic Agent for Personalized Medicine. Adv. Mater. 2014, 26 (21), 3554-3560. 33. Yu, C.; Wu, Y.; Zeng, F.; Wu, S., A fluorescent ratiometric nanosensor for detecting NO in aqueous media and imaging exogenous and endogenous NO in live cells. J. Mat. Chem. B 2013, 1 (33), 4152-4159. 34. Bhunia, S. K.; Maity, A. R.; Nandi, S.; Stepensky, D.; Jelinek, R., Imaging Cancer Cells Expressing the Folate Receptor with Carbon Dots Produced from Folic Acid. ChemBioChem 2016, 17 (7), 614-619. 35. Bourlinos, A. B.; Zbořil, R.; Petr, J.; Bakandritsos, A.; Krysmann, M.; Giannelis, E. P., Luminescent Surface Quaternized Carbon Dots. Chem. Mat. 2012, 24 (1), 6-8. 36. Nandi, S.; Malishev, R.; Parambath Kootery, K.; Mirsky, Y.; Kolusheva, S.; Jelinek, R., Membrane analysis with amphiphilic carbon dots. Chem. Comm. 2014, 50 (71), 10299-10302. 37. Simões, E. F. C.; Leitão, J. M. M.; Esteves da Silva, J. C. G., Sulfur and nitrogen co-doped carbon dots sensors for nitric oxide fluorescence quantification. Anal. Chim. Acta 2017, 960, 117-122. 38. Zhang, G. L.; Wang, Y. H.; Teng, H. L.; Lin, Z. B., Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes. World J Gastroentero. 2001, 7 (3), 331-334.

ACKNOWLEDGMENT Financial assistance from the Ministry of Science, grant number 2015-243 is acknowledged. S.B. is grateful to Prof. Ira Weinstock assistance with the GC and UV experiments.

REFERENCES 1. Jiang, S.; Cheng, R.; Wang, X.; Xue, T.; Liu, Y.; Nel, A.; Huang, Y.; Duan, X., Real-time electrical detection of nitric oxide in biological systems with sub-nanomolar sensitivity. Nat. Comm. 2013, 4, 2225. 2. Bartberger, M. D.; Liu, W.; Ford, E.; Miranda, K. M.; Switzer, C.; Fukuto, J. M.; Farmer, P. J.; Wink, D. A.; Houk, K. N., The reduction potential of nitric oxide (NO) and its importance to NO biochemistry. Proc. Nat. Acad. Sci. 2002, 99 (17), 10958-10963. 3. Pacher, P.; Beckman, J. S.; Liaudet, L., Nitric Oxide and Peroxynitrite in Health and Disease. Physiol. Rev. 2007, 87 (1), 315424. 4. Heitmeyer, M. R.; Corbett, J. A., Nitric Oxide Biology and Pathobiology. Academic: San Diego, 2000; pp 785–810. 5. Gonzales-Zulueta, M.; Dawson, V. L.; Dawson, T. M., Nitric Oxide Biology and Pathobiology. Academic: San Diego, 2000; pp 785– 810. 6. Giulivi, C.; Poderoso, J. J.; Boveris, A., Production of Nitric Oxide by Mitochondria. J. Biol. Chem. 1998, 273 (18), 11038-11043. 7. Thejass, P.; Kuttan, G., Allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) inhibit tumour-specific angiogenesis by downregulating nitric oxide (NO) and tumour necrosis factor-α (TNFα) production. Nitric Oxide 2007, 16 (2), 247-257. 8. Boscá, L.; Zeini, M.; Través, P. G.; Hortelano, S., Nitric oxide and cell viability in inflammatory cells: a role for NO in macrophage function and fate. Toxicology 2005, 208 (2), 249-258. 9. Rosenfeld, Y.; Shai, Y., Lipopolysaccharide (Endotoxin)-host defense antibacterial peptides interactions: Role in bacterial resistance and prevention of sepsis. Biochim. Biophys. Acta 2006, 1758 (9), 1513-1522. 10. Gryglewski, R. J.; Palmer, R. M. J.; Moncada, S., Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 1986, 320 (6061), 454-456. 11. Ford, P. C.; Lorkovic, I. M., Mechanistic Aspects of the Reactions of Nitric Oxide with Transition-Metal Complexes. Chem. Rev. 2002, 102 (4), 993-1018. 12. Lim, M. H.; Lippard, S. J., Metal-Based Turn-On Fluorescent Probes for Sensing Nitric Oxide. Acc. Chem. Res. 2007, 40 (1), 41-51. 13. McQuade, L. E.; Lippard, S. J., Fluorescent probes to investigate nitric oxide and other reactive nitrogen species in biology (truncated form: fluorescent probes of reactive nitrogen species). Curr. Opin. Chem. Biol. 2010, 14 (1), 43-49. 14. Sun, C.; Shi, W.; Song, Y.; Chen, W.; Ma, H., An unprecedented strategy for selective and sensitive fluorescence detection of nitric oxide based on its reaction with a selenide. Chem. Comm. 2011, 47 (30), 8638-8640. 15. Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T., Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators:  Diaminofluoresceins. Anal. Chem. 1998, 70 (13), 2446-2453. 16. Chan, J.; Dodani, S. C.; Chang, C. J., Reaction-based smallmolecule fluorescent probes for chemoselective bioimaging. Nat. Chem. 2012, 4 (12), 973-984. 17. Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata, Y.; Nagano, T., Highly Sensitive Near-Infrared Fluorescent Probes for Nitric Oxide and Their Application to Isolated Organs. J. Am. Chem. Soc. 2005, 127 (11), 3684-3685. 18. Yu, H.; Xiao, Y.; Jin, L., A Lysosome-Targetable and TwoPhoton Fluorescent Probe for Monitoring Endogenous and Exogenous Nitric Oxide in Living Cells. J. Am. Chem. Soc. 2012, 134 (42), 1748617489.

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39. Griffiths, M. J. D.; Messent, M.; MacAllister, R. J.; Evans, T. W., Aminoguanidine selectively inhibits inducible nitric oxide synthase. Br. J. Pharmacol. 1993, 110 (3), 963-968. 40. Corbett, J. A.; Tilton, R. G.; Chang, K.; Hasan, K. S.; Ido, Y.; Wang, J. L.; Sweetland, M. A.; Lancaster, J. R.; Williamson, J. R.; McDaniel, M. L., Aminoguanidine, a Novel Inhibitor of Nitric Oxide Formation, Prevents Diabetic Vascular Dysfunction. Diabetes 1992, 41 (4), 552-556. 41. Mesároš, Š.; Grunfeld, S.; Mesárošová, A.; Bustin, D.; Malinski, T., Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrine microelectrode. Anal. Chim. Acta 1997, 339 (3), 265-270. 42. Bhunia, S. K.; Zeiri, L.; Manna, J.; Nandi, S.; Jelinek, R., Carbon-Dot/Silver-Nanoparticle Flexible SERS-Active Films. ACS Appl. Mater. Interfaces 2016, 8 (38), 25637-25643. 43. Khan, S.; Gupta, A.; Verma, N. C.; Nandi, C. K., Time-Resolved Emission Reveals Ensemble of Emissive States as the Origin of Multicolor Fluorescence in Carbon Dots. Nano Lett. 2015, 15 (12), 8300-8305. 44. Zhu, S.; Meng, Q.; Wang, L.; Zhang, J.; Song, Y.; Jin, H.; Zhang, K.; Sun, H.; Wang, H.; Yang, B., Highly Photoluminescent Carbon Dots for Multicolor Patterning, Sensors, and Bioimaging. Angew. Chem. Int. Ed. 2013, 52 (14), 3953-3957. 45. Li, W.; Zhang, Z.; Kong, B.; Feng, S.; Wang, J.; Wang, L.; Yang, J.; Zhang, F.; Wu, P.; Zhao, D., Simple and Green Synthesis of NitrogenDoped Photoluminescent Carbonaceous Nanospheres for Bioimaging. Angew. Chem. Int. Ed. 2013, 52 (31), 8151-8155. 46. Zhu, X.; Zhao, T.; Nie, Z.; Miao, Z.; Liu, Y.; Yao, S., Nitrogendoped carbon nanoparticle modulated turn-on fluorescent probes for histidine detection and its imaging in living cells. Nanoscale 2016, 8 (4), 2205-2211. 47. Rahaman, R.; Chakraborty, B.; Paine, T. K., Mimicking the Aromatic-Ring-Cleavage Activity of Gentisate-1,2-Dioxygenase by a Nonheme Iron Complex. Angew. Chem. Inter. Ed. 2016, 55 (44), 13838-13842. 48. Brunetti, J.; Roscia, G.; Lampronti, I.; Gambari, R.; Quercini, L.; Falciani, C.; Bracci, L.; Pini, A., Immunomodulatory and Antiinflammatory Activity in Vitro and in Vivo of a Novel Antimicrobial Candidate. J. Biol. Chem. 2016, 291 (49), 25742-25748. 49. Kumar, P.; Kaushik, R.; Ghosh, A.; Jose, D. A., Detection of Moisture by Fluorescent OFF-ON Sensor in Organic Solvents and Raw Food Products. Analytical Chemistry 2016, 88 (23), 11314-11318. 50. Liu, H.; Li, M.; Xia, Y.; Ren, X., A Turn-On Fluorescent Sensor for Selective and Sensitive Detection of Alkaline Phosphatase Activity with Gold Nanoclusters Based on Inner Filter Effect. ACS Applied Materials & Interfaces 2017, 9 (1), 120-126. 51. New, E. J., Harnessing the Potential of Small Molecule Intracellular Fluorescent Sensors. ACS Sensors 2016, 1 (4), 328-333. 52. Murad, F., Discovery of Some of the Biological Effects of Nitric Oxide and Its Role in Cell Signaling (Nobel Lecture). Angew. Chem., Int. Ed. 1999, 38 (13-14), 1856-1868. 53. Zhuang, J. C.; Wogan, G. N., Growth and viability of macrophages continuously stimulated to produce nitric oxide. Proc. Nat. Acad. Sci. 1997, 94 (22), 11875-11880. 54. Pfeiffer, S.; Leopold, E.; Schmidt, K.; Brunner, F.; Mayer, B., Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NGnitro-L-arginine. Br. J. Pharmacol. 1996, 118 (6), 1433-1440. 55. Sutton, M. V.; McKinley, M.; Kulasekharan, R.; Popik, V. V., Photo-cleavable analog of BAPTA for the fast and efficient release of Ca2+. Chemical Communications 2017, 53 (41), 5598-5601. 56. Chiang, C.-H.; Liu, N.-I.; Koenig, J. L., Magic-angle crosspolarization carbon 13 NMR study of aminosilane coupling agents on silica surfaces. J. Colloid Interface Sci 1982, 86 (1), 26-34. 57. Tasic, L.; Abraham, R. J.; Rittner, R., Substituent effects on 1H and 13C NMR chemical shifts in α-monosubstituted ethyl acetates: principal component analysis and 1H chemical shift calculations. Magn. Reson. Chem. 2002, 40 (7), 449-454. 58. Liu, P.; Han, J.; Chen, C. P.; Shi, D. Q.; Zhao, Y. S., Palladiumcatalyzed oxygenation of C(sp2)-H and C(sp3)-H bonds under the assistance of oxalyl amide. RSC Adv. 2015, 5 (36), 28430-28434.

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59. Gann, A. W.; Amoroso, J. W.; Einck, V. J.; Rice, W. P.; Chambers, J. J.; Schnarr, N. A., A Photoinduced, Benzyne Click Reaction. Org. Lett. 2014, 16 (7), 2003-2005. 60. Pinheiro, H. M.; Touraud, E.; Thomas, O., Aromatic amines from azo dye reduction: status review with emphasis on direct UV spectrophotometric detection in textile industry wastewaters. Dyes and Pigments 2004, 61 (2), 121-139. 61. Butler, R. N., Diazotization of heterocyclic primary amines. Chem. Rev. 1975, 75 (2), 241-257. 62. Iyer, G. R. S.; Maguire, P. D., Metal free, end-opened, selective nitrogen-doped vertically aligned carbon nanotubes by a single step in situ low energy plasma process. J. Mater. Chem. 2011, 21 (40), 16162-16169. 63. Nagano, T.; Takizawa, H.; Hirobe, M., Reactions of nitric oxide with amines in the presence of dioxygen. Tetrahedron Lett. 1995, 36 (45), 8239-8242. 64. Enemark, J. H.; Davis, B. R.; McGinnety, J. A.; Ibers, J. A., The structure of a molecular nitrogen compound of cobalt and evidence for CoH(N2)(PPh3)3. Chem. Comm. (London) 1968, (2), 96-97. 65. Bertino, M.; Steinhögl, W.; Range, H.; Hofmann, F.; Witte, G.; Hulpke, E.; Wöll, C., The low-energy thermal excitation spectrum of nitrogen molecules adsorbed on Ni(110): Implications for molecular adsorption sites. App. Phys. A 1996, 62 (2), 95-101. 66. Qian, Z. S.; Chai, L. J.; Huang, Y. Y.; Tang, C.; Jia Shen, J.; Chen, J. R.; Feng, H., A real-time fluorescent assay for the detection of alkaline phosphatase activity based on carbon quantum dots. Biosens. Bioelectron. 2015, 68, 675-680.

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C-dots produced from aminoguanidine make possible bimodal NO sensing through reaction with amine residues, generating azo dye and N2. Table of Contents (TOC) graphics

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