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28 Jan 2016 - Min-Woo Choi,. †. Jangwon Seo,. ¶,†. Seunghoon Shin,. †,‡ .... at 0 °C and then stirred 30 min at room temperature. After reac...
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Highly sensitive and selective fluorescent probe for ascorbic acid with a broad detection range through dualquenching and bimodal action of nitronyl-nitroxide Haerim Nam, Ji Eon Kwon, Min-Woo Choi, Jangwon Seo, Seunghoon Shin, Sehoon Kim, and Soo Young Park ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.5b00230 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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Highly sensitive and selective fluorescent probe for ascorbic acid with a broad detection range through dual-quenching and bimodal action of nitronyl-nitroxide Haerim Nam,‡a Ji Eon Kwon,‡a Min-Woo Choi,a Jangwon Seo,†a Seunghoon Shin,a,b Sehoon Kim,b and Soo Young Park*a a

Center for Supramolecular Optoelectronic Materials, Department of Materials Science and Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744 Korea.

b

Center for Theragnosis, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136791 Korea. †

Current address: Division of Advanced Materials, Korea Research Institute of Chemical Technology, 141 GajeongRo, Yuseong-Gu, Daejeon 305-600, Republic of Korea. ‡

These authors contributed equally to this work.

KEYWORDS Nitronyl-Nitroxide, Ascorbic Acid, Fluorescence, Electron Spin Resonance, Sensor ABSTRACT: A novel nitronyl-nitroxide derivative for highly sensitive and selective detection of ascorbic acid (AA) is reported. The probe showed 260-fold fluorescence turn-on and diminished electron spin resonance (ESR) signal upon AA addition. The probe could detect AA over broad concentration range from 1 µM to 2 mM and excellent selectivity over various antioxidants and acids through a combination of fluorescence and ESR responses, which originated from the dualreactiveness of dual-quenching group nitronyl-nitroxide. We successfully demonstrated practical application potential of the probe by preparing nanoparticles and sensor papers and determining AA concentration in real samples including Vitamin drink and orange juices.

Ascorbic acid (AA), also known as Vitamin C, is a naturally occurring antioxidant and plays important roles in many biological processes in human body such as a cofactor in enzymatic reactions, collagen synthesis, scurvy prevention, and immune system enhancement.1-3 It has also been widely used as a food additive to prevent oxidation in juices and soft drinks.4, 5 Moreover, it has been reported that AA has a positive effect on cancer therapy.6 Accordingly, there is ever increasing interest in AA detection where its concentration varies from 10 µM in human blood plasma and serum to 20 mM in food and pharmaceuticals.5-9 A lot of analytical detection methods for AA have been developed including electrophoresis,10 UV-Vis absorption spectroscopy,11, 12 liquid chromatography,4, 13 and electrochemistry,5, 14, 15 so far. In recent years, fluorescence-based method has become a popular approach for AA detection because it offers many advantages over other methods in terms of the sensitivity, convenience, and non-invasiveness.16 A number of fluorescent probes for AA have been reported utilizing redox reactions with KMnO4,17 MnO2 nanosheets,18 chromium(VI) ions,19 Au nanoclusters (AuNCs),20 silver nanoparticles,21 DNA,22 electrochromic dyes,23 cobalt oxyhydroxide,24 and nitroxides.25-41 These studies greatly advanced the research on

the fluorescence detection of AA; however, many of these probes suffer from relatively low sensitivity and narrow detection range due to the inefficiency of the AA-reactive fluorescence quenching group. Therefore, particularly considering a wide variety of AA concentration in different analytic samples, new AA probes possessing broad detection range as well as high sensitivity are highly demanded.

Scheme 1. (a) Chemical structure of NN-CN-TFFP and (b) proposed reaction mechanism of NN-CN-TFFP with ascorbic acid and their bimodal responses. FL indicates the fluorescence signal. Nitroxides have been reported as a promising reactive unit for AA detection in many previous studies because they are able to quench the excited-state of the covalently linked fluorophore due to their paramagnetic doublet spin.37 The chemical reduction of the nitroxides

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by AA would change the paramagnetic spin into a diamagnetic hydroxylamine, and hence turn on the fluorescence simultaneously with diminished electron spin resonance (ESR) signal.26, 30, 32 In contrast to the typical nitroxides (e.g., TEMPO), it has been suggested that nitronylnitroxides can quench the fluorescence even after losing the doublet spin through photoinduced electron transfer (PET) process due to their imidazole-type structure.42, 43 Thus, it is expected that the fluorescence of molecules containing a nitronyl-nitroxide will be effectively quenched by the cooperative effects of the nitroxide radical and PET from the imidazole structure. Since N. Medvedeva et al. first reported a nitronyl-nitroxide type probe for AA using pyrene,36 M. Baumgarten group achieved a remarkably enhanced fluorescence turn-on response (F/F0 = 80) to AA by introducing a pyrazole bridging group.35 However, detailed analyses of the probes on detection range and bimodal responsiveness have not been studied further. In addition, there is still much room for improvement in selectivity over other antioxidants and acids. Herein, we designed a new AA probe NN-CNTFFP bearing a nitronyl-nitroxide moiety as an AAreactive fluorescence quenching group (see Scheme 1a). As a fluorescence signalling unit, cyanostilbene type πconjugated backbone was chosen. We have previously reported that molecules with cyanostilbene type backbone formed very stable nanoparticles by self-assembly in aqueous condition.44, 45 Based on our design strategy, we hypothesize that both the sensitivity and detection range of NN-CN-TFFP for AA will be greatly improved as a result of dual-quenching effect and bimodal spectral responses originating from the dual-reactiveness of the nitronyl-nitroxide. EXPERIMENTAL SECTION Materials. All commercially available reagents were used without further purification unless otherwise stated. All syringes, magnetic stirring bars, glassware, and needles were completely dried in a convection oven. Thin layer chromatography (TLC) with commercial TLC plates (silica gel 60 F254, Merck Co.) was used for monitoring reactions. Silica gel column chromatography was performed using silica gel 60 (particle size 0.063-0.200 mm, Merck Co.). 1H and 13C NMR spectra were measured with a Bruker Avance 300 spectrometer and a Bruker Avance 500 spectrometer, respectively. Mass spectra (MS) were acquired by employing JEOL JMS-700 and elemental analyses were performed on a Vario Micro Cube and a CE Flash1112. Synthesis of 2,3-Dimethyl-2,3bis(hydroxylamino)butane (BHA, 1). Zn dust (10.4 g) and water (20 mL) were added to THF solution (120 mL) containing 2,3-dimethyl-2,3-dinitrobutane (7.04 g, 40.0 mmol) at 0 °C under Ar atmosphere. Subsequently, an aqueous solution (60 mL) of NH4Cl (17.2 g, 0.325 mol) was added dropwise with vigorous stirring over 2.5 h. The re-

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action mixture was stirred for 1.5 h at 0 °C and then stirred 30 min at room temperature. After reaction, the mixture was filtered and washed with THF. The filtrate was concentrated under reduced pressure and the residue was kept in the refrigerator at -15 °C for 1 h. The residue was mixed with Na2CO3 (20.0 g, 0.189 mol) and NaCl (12.0 g, 0.205 mol) and then carefully charged into a Soxhlet apparatus under Ar atmosphere. The Soxhlet extraction was performed overnight using dichloromethane under Ar atmosphere. The extracted solution was concentrated under vacuum to afford 1.8 g of white powder (Yield = 30 %). 1H NMR (300MHz, DMSO-d6) δ [ppm]: 6.92 (s, 2H, -OH), 5.38 (s, 2H, -NH) 1.00 (s, 12H, -CH3). MS (FAB+, m/z): [M+H]+ calcd. for C6H17N2O2, 149.12; found, 149.0. Synthesis of 2-(3',5'-bis(trifluoromethyl)-[1,1'biphenyl]-4-yl)acetonitrile (2). 2-(4bromophenyl)acetonitrile (1.0 g, 5.13 mmol) and (3,5bis(trifluoromethyl)-phenyl)boronic acid (1.58 g, 6.12 mmol) was dissolved in 50 mL THF. Subsequently, tetrakis (triphenylphosphine) palladium(0) (0.3 g, 0.260 mmol) and aqueous 2 N K2CO3 solution (25 mL) was added into the solution under dark condition. The reaction mixture was stirred and refluxed overnight at 80 °C. The cooled crude mixture was poured into water and titrated to pH 7 by 1.0 M hydrochloric acid. The organic layer extracted by dichloromethane was evaporated in vacuo. The product was obtained by column chromatography using ethyl acetate and n-hexane as an eluent. After drying, 1.29 g of white powder was obtained (Yield = 64.1 %). 1H NMR (300 MHz, CDCl3) δ [ppm]: 8.00 (s, 2H, Ar-H), 7.88 (s, 1H, Ar-H) 7.64 (d, 2H, Ar-H, J = 8.3 Hz), 7.49 (d, 2H, Ar-H, J = 8.3 Hz), 3.84 (s, 2H, Vinyl-H). 13C NMR (125 MHz, CDCl3) δ [ppm]: 142.3, 138.1, 132.4, 132.2, 130.8, 128.9, 128.0, 127.1, 121.2, 23.3. Synthesis of (Z)-2-(3',5'-bis(trifluoromethyl)-[1,1'biphenyl]-4-yl)-3-(4-formylphenyl)acrylon-itrile (3). tbutyl alcohol (30 mL) solution containing 2 (1.0 g, 3.04 mmol) and terephthalaldehyde (1.22 g, 9.12 mmol) was stirred at 50 °C under dark condition. Tetrabutylammonium hydroxide 0.1 M solution in methanol was added dropwise into the solution. The mixture was vigorously stirred for 0.5 h. After reaction, the mixture was poured into methanol with stirring for 1 h and filtered off. Flash column chromatography (ethyl acetate and n-hexane) was performed for purification to afford 0.98 g of green powder (Yield = 73.8 %). 1H NMR (300 MHz, CDCl3) δ [ppm]: 10.10 (s, 1H, -CHO), 8.10 (d, 2H, Ar-H, J = 8.3 Hz), 8.05 (s, 2H, Ar-H), 8.02 (d, 2H, Ar-H, J = 8.4 Hz), 7.91 (s, 1H, Ar-H), 7.88 (d, 2H, Ar-H, J = 8.6 Hz), 7.75 (d, 2H, Ar-H, J = 8.6 Hz), 7.67 (s, 1H, Vinyl-H). 13C NMR (125 MHz, CDCl3) δ [ppm]: 191.1, 141.9, 140.9, 139.5, 138.9, 137.3, 134.4, 132.5, 132.3, 130.1, 129.8, 128.0, 127.1, 122.2, 121.6, 117.1, 113.9. MS (FAB+, m/z): [M+H]+ calcd. for C24H14F6NO, 446.1; found, 446.0. Synthesis of (Z)-2-(3',5'-bis(trifluoromethyl)-[1,1'biphenyl]-4-yl)-3-(4-(1,3-dihydroxy-4,4,5,5tetramethylimidazolidin-2-yl)phenyl)acrylonitrile (4).

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Solution (methanol:dichloromethane = 15 mL:10 mL) containing 3 (0.5 g, 1.12 mmol) and 1 (0.5 g, 3.38 mmol) was stirred overnight under Ar atmosphere at 35 °C. The crude mixture was evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate and n-hexane. Additionally, the product was washed with n-hexane. After drying solvent, 0.26 g of yellow powder was obtained (Yield = 40.2 %). 1H NMR (300 MHz, DMSO-d6) δ [ppm]: 8.42 (s, 2H, Ar-H), 8.21 (s, 1H, Ar-H), 8.14 (s, 1H, Vinyl-H), 8.08 (d, 2H, Ar-H, J = 8.5 Hz), 7.96 (d, 2H, Ar-H, J = 5.0 Hz), 7.93(d, 2H, ArH, J = 5.3 Hz), 7.86 (s, 2H, -OH), 7.67 (d, 2H, Ar-H, J = 8.3 Hz), 4.59 (s, 1H, Vinyl-H), 1.10 (d, 12H, Vinyl-H, J = 10 Hz). 13 C NMR (125 MHz, CDCl3) δ [ppm]: 145.1, 143.5, 141.6, 137.3, 134.3, 132.8, 131.2, 130.9, 128.9, 128.7, 128.1, 127.3, 126.4, 117.8, 109.0, 89.8, 66.3, 24.3. MS (FAB+, m/z): [M+H]+ calcd. for C30H28F6N3O2, 576.2; found, 576.0. Synthesis of (Z)-2-(3',5'-bis(trifluoromethyl)-[1,1'biphenyl]-4-yl)-3-(4-(1-Oxyl-3-oxo-4,4,5,5tetramethylimidazolidin-2-yl)phenyl)acrylonitrile (5, NN-CN-TFFP). The solution of 4 (0.1 g, 0.17 mmol) in dichloromethane was vigorously stirred at room temperature. Tetrapropylammonium perruthenate (3.05 mg, 0.0087 mmol) and 4-methylmorpholine N-oxide (20.4 mg, 0.17 mmol) was added to the solution sequentially. After 1.5 h, the complete reaction was confirmed by TLC. The column chromatography using ethyl acetate and n-hexane was performed to afford 0.051 g of green powder (Yield = 50.9 %). MS (FAB+, m/z): [M+H]+ calcd. for C30H25F6N3O2, 573.5; found, 573.2. Anal. calcd. for C30H24F6N3O2, C 62.94, H 4.23, N 7.34; found, C 62.70, H 4.30, N 7.12. Measurements. UV−vis absorption spectra were collected on a Shimadzu UV-1650PC spectrophotometer at room temperature. Fluorescence spectra were obtained by using a QM-40 spectrophotometer (Photon Technology International, Canada). The photoluminescence quantum efficiencies (ΦPL) in solution were obtained using quinine sulfate in 1.0 N sulfuric acid as a reference (ΦPL = 0.546). Electron spin resonance (ESR) spectra were recorded by JES-TE 200 and JES-TE 300 (JEOL) at room temperature. The g-factor corrections were obtained using the Mn (g = 1.981, 2.034) as a standard. Spectrometer parameters were microwave power, 1.0 mW; modulation frequency, 100 kHz. For HPLC analysis, a Thermo dionex ultimate 3000 (Thermo, USA) was used with C-18 column (inno C18 4.6 x 250 mm, 5 µm, innopia, Korea), pumped at a flow rate of 0.8 mL/min For titration and selectivity test, up to 30 μL of analytes standard solutions (200 mM, 2 mM, 200 μM) was added into 3 mL of methanol solution containing 20 μM of the probe. Analytes were dissolved in methanol for AA, butylated hydroxyl toluene, dopamine, fructose, glucose, and citric acid, and in water for hydroxyquinone, Cysteine, Tyrosol, Glutathione, NADH, NAD+, and uric acid. To detect AA in real samples, Vitamin drinks and orange juices were centrifuged and supernatants were filtered through 0.45 µm-pore syringe filter. Then, the filtrates were separated into two portions. To prepare

unspiked and spiked samples, either 100 µL of water or AA standard solution was added into 900 µL of the one separated portion, respectively. For example, to spike 20 µM and 2 µM of AA, 200 mM and 20 mM of AA standard solution were used for fluorescence and ESR analysis, respectively. Subsequently, 3 µL of the unspiked or spiked samples was added into a 3 mL of 20 µM NN-CN-TFFP solution (total water content of the final solution = 0.1 vol%). Then, we monitored fluorescence and ESR spectra changes of the solution. The AA concentration of the original samples were calculated from the determined AA concentration of the diluted final samples by considering all the dilution. Nanoparticles were prepared by simple precipitation method without surfactants. By this method, water was used as a non-solvent for materials in THF solutions. After 70% volume fractions of water addition, materials (20 µM) in mixture solution started to aggregate into nano-size particles. FE-SEM images of nanoparticles were acquired on a JSM-6330F (JEOL). The samples were obtained by dropping the suspension of nanoparticles solution onto the glass substrates and drying it overnight in ambient conditions. To test AA-responsiveness of the nanoparticles at different pH values, nanoparticles were fabricated in THF/water (3:7 v/v) mixture solution containing KCl (100 mM). pH value of the solution was titrated with a standard HCl or KOH solutions. Calculations. All density functional theory (DFT) calculations were carried out using the Gaussian 09 quantum chemical package. The geometry optimizations for the ground state of materials were performed using B3LYP functionals with 6-31G+(d,p) basis set. Vibration frequency calculations at the same level were performed for the obtained structures to confirm the global minimum. The absorption transition energies were calculated using timedependent DFT (TD-DFT) with B3LYP/6-31G+(d,p). Solvent effects were evaluated by using the polarizable continuum model (PCM) with dielectric constant (ε) of 32.613 for methanol. RESULTS AND DISCUSSION NN-CN-TFFP was synthesized through a series of chemical reactions including reduction, Suzuki coupling, Knoevenagel condensation, and oxidation (see Scheme 2). The probe was fully characterized by 1H NMR, 13C NMR, elemental analysis, MS, and electron spin resonance (ESR) spectroscopy (see experimental section for synthetic details). First we examined the absorption and fluorescence spectra of the probe in MeOH solution. As shown in Figure 1a, two characteristic bands were observed in the absorption spectrum: One is π-π* transition band of the aromatic backbone (ε = 35400 M-1 cm-1) at 350 nm and the other is very weak n-π* transition band of the nitronyl-nitroxide moiety (ε = 200 M-1 cm-1) at 600 nm,46 well corresponding with the theoretical results calculated by DFT/TD-DFT method (see Figure S1 and Table S1 in supporting information SI). As expected, fluorescence of

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NN-CN-TFFP was very hard to detect (ΦPL < 0.0001) attributed to efficient fluorescence quenching of the nitronyl-nitroxide moiety. After addition of AA, a significant fluorescence increase was observed with the maximum emission wavelength at 460 nm (Figure 1c). The increase of fluorescence intensity reached a maximum of 260-fold (ΦPL = 0.014) when 2 mM of AA is added.

Scheme 2. Synthesis route of NN-CN-TFFP. To gain insights into the mechanism of such high fluorescence turn-on, various spectral changes including UV-Vis absorption, fluorescence, and ESR were closely monitored by addition of various concentrations of AA into the probe solution. As shown in Figure 1a, upon increasing AA concentration from 2 µM to 20 µM, the characteristic absorption band of NN-CN-TFFP at 350 nm and 600 nm gradually decreased whilst a new band at around 430 nm increased. However, it was observed that the isosbestic points were not kept constant at 325 nm and 380 nm but began to move when more than 0.5 eq. of AA (10 µM) was added (see Figure 1a and Figure S2 in SI). In addition, further increase of AA concentration generated a new trend of the absorption spectra change. After addition of more than 20 µM of AA, the absorption bands at around 400 nm started to decrease reversely (see Figure 1b), implying that two different reaction occurs between NN-CN-TFFP and AA depending on AA concentration. On the other hand, the fluorescence intensity of the probe gradually increased upon AA addition. It should be noted that the fluorescence intensity barely increased until the AA concentration reached to 10 µM which is 0.5 eq. concentration to that of NN-CN-TFFP (Figure 1c and Figure S2 in SI). But, after exceeding that concentration, the fluorescence intensity started to increase drastically. We also investigated changes in ESR spectrum of NN-CN-TFFP upon AA addition. The nitrogen atom possessing spin I = 1 interacts with an unpaired electron (i.e., radical), resulting in splitting the electron resonance line of a molecule into three equally intense lines.47, 48 Due to the two equivalent nitrogen nuclei of the imidazole moiety of NN-CN-TFFP, five lines (g = 2.0075, |aN| = 0.764 mT) were clearly observed with an intensity ratio of 1:2:3:2:1 as shown in Figure 1d, which is consistent with the typical ESR spectrum of nitronyl-nitroxides.49 As expected, the

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ESR signal gradually diminished upon AA addition and completely disappeared at 12 µM where the AA concentration is just over 0.5 eq. of the probe concentration.

Figure 1. Various spectral responses of 20 µM NN-CNTFFP in MeOH solution to increasing concentrations of AA: Changes in absorption spectra upon addition of (a) 0–20 µM (1 eq.) and (b) 20 µM (1eq.)–2 mM (100 eq.) AA, and in (c) fluorescence and (d) ESR spectra upon addition of 0-2 mM AA. Inset in (a) presents a magnified absorbance graph of NN-CN-TFFP in the absence (○) and presence (●) of 20 µM AA, respectively. The dashed line in (b) indicates absorption spectrum of pristine NN-CNTFFP solution. Inset in (c) plots the relative fluorescence intensity (F/F0) versus the equivalent of added AA. F and F0 are the integrated fluorescence intensities.

Taken all together, it is deduced that two different reactions took place sequentially upon AA addition (see Scheme 1b). First, from 2 µM to 12 µM of AA addition range, it was brought about the reduction of nitronylnitroxide moiety of the probe. AA is a mild reducing agent and typically reacts with a nitroxide to yield a diamagnetic hydroxylamine.50 The linearly decreased ESR signal and the vanished n-π* absorption band at 600 nm provided corroborative evidence for the occurrence of reduction. Because AA is usually oxidized with the loss of two electrons to form dehydroascorbic acid, it is expected that the addition of 0.5 eq. AA to NN-CN-TFFP solution is enough to reduce all of the nitroxide unit into hydroxylamines.51 But, the ESR spectrum with addition of 10 µM AA still exhibited a weak signal and 0.6 eq. of AA is needed to eliminate the signal completely. Considering together with the shift of isosbestic points in the absorption spectra, it is reasonable to infer that another kind of reaction started to occur subsequently. Although all radicals are reduced by AA, it is noteworthy that the fluorescence intensity of probe remains still very low. It indicates that additional fluorescence quenching site still subsists. The subsequent reaction is most likely the protonation of the probe. It was reported that treatment of the nitronyl-nitroxides with acids led to protonation of the NO group.35, 42, 43, 52 AA is also acidic (pKa1 = 4.1 and pKa2 = 11.97) and would protonate the NO group of NN-

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CN-TFFP. With addition of AA from 20 µM to 2 mM, it is observed that the absorbance and the fluorescence of probe gradually changed (see Figure 1b and c). DFT/TDDFT results also revealed that the protonation of NO moiety leads to the decrease of absorption at longer wavelength (see Figure S1 and Table S1 in SI), consistent with the experimental results shown in Figure 1. It should be noted that the fluorescence of the probe was significantly enhanced, indicating that the fluorescence quenching sites are fully arrested by the protonation. The apparent equilibrium constant of the protonation reaction was calculated to be 1.91 × 103 M-1 (see Figure S3 in SI).

Figure 2. (a) Photoluminescence and (b) ESR spectra of NN-CN-TFFP (black lines) with addition of trifluoroacetic acid (TFA, red lines), hydrochloric acid (HCl, blue lines), hydroxyquinone (HQ, green lines), and ascorbic acid (AA, black dashed lines).

To further evidence the protonation effect on the fluorescence turn-on, we added excessive amount of trifluoroacetic acid (TFA) and hydrochloric acid (HCl) to NN-CN-TFFP solution, respectively. It was observed that the addition of the strong acids led to 117-fold and 29-fold increases of the fluorescence intensity for TFA and HCl, respectively, as shown in Figure 2, while no significant change was observed in their ESR spectra. In contrast, addition of hydroxyquinone (HQ), a well-known reducing agent, induced negligible increase of the fluorescence intensity even though the ESR signal decreased by half (see Figure 2). These experiments clearly suggest that NNCN-TFFP has two fluorescence quenching group, NO (i.e., imidazole structure) and NO• (i.e., nitroxide radical), among which the former is more effective. To gain detailed information about the sensitivity of NN-CN-TFFP to AA, we analyzed the fluorescence intensity changes upon AA addition. It is revealed that the relative fluorescence intensity was linearly related to the AA concentration from 10 µM to 0.2 mM (F/F0 = 0.547 [AA]/µM – 4.80 where R2 = 0.982, see Figure 3a and Figure S4a in SI). Specifically the limit of detection (LOD) was found to be as low as 13.6 µM. In addition, NN-CNTFFP exhibited a linear response toward logarithm of AA concentration from 0.1 mM to 2 mM, and the linear equation was F/F0 = 154 log [AA] + 677 with R2 = 0.991 (see Figure 3a and Figure S4b in SI). It must also be noted that the ESR measurements could detect AA from 2 µM to 12 µM where the concentration is below the LOD of fluorescence measurements (see Figure 3a and Figure S5 in SI). The relative ESR intensity showed good linear relationship toward AA concentration (DI/DI0 = –0.0844

[AA]/µM + 0.991 where R2 = 0.989) with a 0.937 µM of LOD.

Figure 3. Plots of the relative fluorescence intensity (F/F0) and ESR intensity (DI/DI0) of 20 µM NN-CN-TFFP in MeOH solution versus (a) the concentration of AA and (b) various antioxidants and acids: 1, BHT; 2, HQ; 3, Cys; 4, Tyr; 5, GSH; 6, DA; 7, NADH; 8, NAD+; 9, FRU; 10, Glc; 11, TFA; 12, HCl; 13, CA; 14, UA; 15, AA. The concentration of added antioxidants and acids was 2 mM except for UA (20 μM). F and F0 are the integrated fluorescence intensities, and DI and DI0 are the double integrated ESR intensities of NN-CN-TFFP solution in the presence and absence of antioxidants and acids, respectively.

In order to evaluate the selectivity, NNCN-TFFP solution was treated with excess amounts of various antioxidants (see Figure 3b and Figure S6, S7, and S11 in SI). Butylated hydroxyl toluene (BHT), Cysteine (Cys), Tyrosol (Tyr), Glutathione (GSH), and NAD+ exhibited no significant changes in both fluorescence and ESR spectra of the solution. HQ, dopamine (DA), and NADH decreased the ESR intensity but affected no fluorescence intensity. In addition, fructose (FRU), glucose (Glc), and citric acid (CA) which exists largely in many fruits and juices negligibly affected in both fluorescence and ESR spectra of the probe. Uric acid (UA) also negligibly affected both fluorescence and ESR spectra of the probe solution. It is worth noting that the strong acids such as TFA and HCl turned on the fluorescence of NNCN-TFFP solution, however, one can easily distinguish AA from the acids by taking advantage of dual-channel detection through the ESR signal as described above. These results clearly indicate that the dual-reactiveness and dual-quenching of nitronyl-nitroxide group provides not only high sensitivity with a wide detection range but also high selectivity. To evaluate reliability and accuracy of the probe, we measured the concentration of AA in Vitamin drink

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and orange juice. The original samples were diluted to a suitable concentration for assay by the proposed method (see experimental section). The AA concentration of the original Vitamin drink was successfully calculated to be 28.6 ± 0.6 mM with recoveries of 92 ± 3% by the fluorescence analysis method (see Table 1). The vanished ESR spectra of spiked and unspiked Vitamin drink samples also indicated that the fluorescence responses were mainly attributed to AA (see Figure S8a and b in SI). On the other hand, the AA concentration of the orange juice could not be determined because the fluorescence responses of the diluted samples (F/F0 = 2.40, see Figure S8c in SI) were below the detection limit (LOD = 13.6 µM). But, by the ESR analysis method, we could successfully calculate the AA concentration in the original orange juice to be 1.50 ± 0.1 mM with recoveries of 98 ± 11% (see Table 1). The analytical results of the Vitamin drink and orange juice were in good agreement with those by HPLC method (30.8 and 4.08 mM for Vitamin drink and orange juice, respectively; see Figure S9 and S10 in SI), demonstrating the potential applicability of the probe for quantitative AA detection in real samples.

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but distinct intermolecular interactions between nitronylnitroxide radicals in the nanoparticles (see Figure S12a in SI).28,53 On the other hand, obviously very broad ESR spectrum was observed from bigger NN-CN-TFFP nanoparticles when the water content was increased up to 80% (see Figure S12b-d in SI). Similarly to the methanol solution, upon addition of AA, NN-CN-TFFP nanoparticles (water/THF = 7:3 in vol%) showed bimodal responses in fluorescence and ESR spectra. With increasing AA concentration, ESR signal of the nanoparticles gradually decreased (see Figure 4b). But, in sharp contrast to the methanol solution, the nitroxide moiety remained unreacted until the AA concentration reached to 0.4 mM. The fluorescence intensity of nanoparticles also showed significant increases after 0.4 mM of AA was added(see Figure 4c). This is attributable to that NN-CN-TFFP molecules close to the center of nanoparticles have significantly less chance to react with AA before the nitronyl-nitroxide moieties near the nanoparticle surfaces are completely reduced and protonated by AA. Therefore, more AA molecules are needed to diminish the ESR and to recover the fluorescence signal of nanoparticles than those of methanol solution. Most interestingly, the AA sensing ability of NN-CN-TFFP nanoparticles was virtually maintained at pH between 4 and 10 (see Figure 4d and Figure S13 in SI). Furthermore, a more convenient portable paper sensor for AA was achieved by dipping a filter paper in the NN-CN-TFFP solution. As shown in Figure 5, after writing letters with AA solution as ink on the paper sensor, the fluorescence on the trail of handwriting was instantly and dramatically enhanced.

Figure 4. (a) FE-SEM images of NN-CN-TFFP nanoparticles. Scale bar in the inset figure indicates 200 nm. (b) ESR and (c) fluorescence responses of NN-CN-TFFP nanoparticles (20 μM in water/THF = 7:3 in vol %) upon addition of AA. (d) Plots of the relative fluorescence intensity (F/F0,pH7) of NN-CN-TFFP nanoparticles at various pH. F0,pH7 is integrated fluorescence intensity in the absence of AA at pH 7. The added AA concentration was 2 mM.

Finally, we examined whether NN-CN-TFFP can detect AA in aqueous condition for more practical uses. As shown in Figure 4a, the probe molecules could form 100 nm-sized nanoparticles in aqueous condition (water/THF = 7:3 in vol%) by self-assembly without any surfactants. ESR spectrum with clear five line pattern indicates that the nitronyl-nitroxide radicals were completely preserved in the nanoparticles; however a broader and distorted spectral shape (∆g = 0.0190) in comparison with that of solution (∆g = 0.0179) can be ascribed to the weak

Figure 5. Fluorescence photoimages of the fluorescent paper sensor for AA under 365 nm handheld UV lamp before (left) and after (right) writing letters with AA solution as ink.

CONCLUSIONS In summary, we could demonstrate a novel fluorescent AA probe bearing nitronyl-nitroxide as an AA-reactive unit. The fluorescence enhancement of probe by AA addition was as high as 260-fold in methanol solution. The probe could detect AA from as low as 0.937 µM up to 2 mM through a combination of fluorescence and ESR responses, which satisfies the needs in various analytic samples, and showed excellent selectivity over various antioxidants and acids. The high selectivity and sensitivity with a broad detection range of the probe originated from the dual-reactiveness of dual-quenching group nitronylnitroxide. Furthermore, we successfully determined AA concentration in Vitamin drink and orange juices by the

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proposed method. We also prepared nanoparticles and a sensor paper using the probe, and examined AA detection capabilities in aqueous condition and solid-state, respectively, for practical applications.

ASSOCIATED CONTENT Supporting Information Calculated absorption energies and spectra by DFT/TD-DFT method. Absorption, photoluminescence, and ESR spectra changes of NN-CN-TFFP with addition of AA, various antioxidants, Vitamin drink, orange juices. The non-linear regression fit for calculating association constants. Linear relationship of the relative fluorescence and double integrated ESR intensity with AA concentration. HPLC analysis results of Vitamin drink and orange juice. This material is available free of charge via the Internet at http://pubs.acs.org.

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

Present Addresses † Current address: Division of Advanced Materials, Korea Research Institute of Chemical Technology, 141 Gajeong-Ro, Yuseong-Gu, Daejeon 305-600, Republic of Korea.

Author Contributions ‡These authors contributed equally.

ACKNOWLEDGMENT This work was supported by the National Research Foundation of Korea (NRF) through a grant funded by the Korean Government (MSIP; no. 2009-0081571) and by the Air Force Office of Scientific Research (AFOSR: Grant No. FA23861214008).

Table 1. Measured results of AA concentration in real samples (mean ± s.d., n = 3) Sample

AA found in diluted samples (µM) unspiked

Vitamin drink

e

orange juice (FL) orange f (ESR)

a

25.7 ± 0.6 e

juice

spiked

Recovery

b

calculated

44.2 ± 0.2

92 ± 3%

n.d.

19.5 ± 0.2

g

1.35 ± 0.09

3.31 ± 0.28

g

AA in original samples (mM) c

found

28.6 ± 0.6

30.8

n.d.

< 13.6

4.08

98 ± 11

1.50 ± 0.10

4.08

d

a 9/10000-fold diluted for spike and recovery test. b 200 and 20 mM of AA standard solution were added to the original samples of Vitamin drink and orange juice to spike 20 µM and 2 µM of AA for fluorescence and ESR analysis, respectively. c Calculated from measured concentration in diluted samples. d Measured by HPLC. e Measured by fluorescence analysis. f Measured by ESR analysis. g Not determined due to detection limit.

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