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Fluorescent TPA@GQDs Probe for Sensitive Assay and Quantitative Imaging of Hydroxyl Radical in Living Cells Xin Hai, Zhiyong Guo, Xin Lin, Xuwei Chen, and Jian-Hua Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16094 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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ACS Applied Materials & Interfaces

Fluorescent TPA@GQDs Probe for Sensitive Assay and Quantitative Imaging of Hydroxyl Radical in Living Cells Xin Hai, Zhiyong Guo, Xin Lin, Xuwei Chen*, Jianhua Wang* Research Center for Analytical Sciences, Northeastern University, Box 332, Shenyang 110819, China

KEYWORDS: graphene quantum dots, terephthalic acid, hydroxyl radical, fluorescence enhancement, quantitative imaging, living cell ABSTRACT: A fluorescent probe TPA@GQDs is fabricated by the conjugation of terephthalic acid (TPA) on the surface of graphene quantum dots (GQDs). The TPA@GQDs probe not only owns favorable dispersibility, but also exhibits excellent fluorescence stability over wide pH range and high ionic strength, and favorable anti-photobleaching ability. The great fluorescence enhancement of TPA@GQDs induced by the reaction between TPA and hydroxyl radical makes the TPA@GQDs a powerful probe for the sensitive assay of hydroxyl radical, giving rise to a detection limit low down to 12 nmol L-1. Meanwhile, the obtained fluorescent TPA@GQDs probe shows low cytotoxicity and favorable biocompatibility. Its potential in bioimaging is demonstrated by the quantitative fluorescent imaging of hydroxyl radical in living HeLa cells under different circumstances, which enables the opportunities to study hydroxyl radical dynamics in living cells.

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INTRODUCTION Reactive oxygen species (ROS) are generated from both normal and pathological aerobic metabolism,1 including superoxide radical (O2•-), hydrogen peroxide (H2O2), singlet oxygen (1O2), hypochlorous acid (HOCl) and hydroxyl radical (•OH).2-4 Among these ROS, •OH possess intense electron accepting capacity, thus show strong oxidizability and aggressive reactivity.5 In general, the content of •OH maintains homeostasis in normal bodies, due to the existence of various reducing agents, enzymes, and antioxidants.6 However, this balance will be disturbed by the overproduction of •OH associated with damage, pollution, radiation and cancerization etc. The excess •OH tends to cause oxidative damage to organism even cells apoptosis.7 Hence, the accurate and real-time monitoring of endogenous •OH in living systems and environment is of vital importance, as it can be used as an excellent indicator to the state of living cells. Up to now, various methodologies have been developed for •OH detection, including electron spin resonance (ESR) spectrometry,8 electrochemical method,9 chromatography,10 chemiluminescence,11 and fluorescence spectrometry.12, 13 Among these methodologies, fluorescence spectrometry is extensively used due to its high sensitivity, simplicity, and noninvasiveness.14 In recent years, various fluorescent probes have been proposed for the quantitative assay of •OH, based on the fluorescence enhancement or quenching of fluorescent probes after specific combination with •OH.7, 15, 16 For instance, Ganea et al propose a coumarin-neutral red (CONER) nanoprobe for the detection of •OH based on the ratiometric fluorescence


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signal between 7-hydroxy coumarin 3-carboxylic acid and neutral red dyes. This ratiometric nanoparticle is sensitive and selective for •OH as compared to other reactive oxygen species such as superoxide anion (O2•-), hydrogen peroxide (H2O2) and singlet oxygen (1O2) etc.17 By using 2-[6-(4′-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid as the responsor and recognition, and BSA-AuNCs as the reference, the detection limit for •OH could be lower down to 0.68 µmol L-1.5 However, most of these probes are confined to practical applications due to their sophisticated syntheses and purification, poor photostability and toxicity. Terephthalic acid (TPA) is a structurally symmetrical aromatic compound. The non-fluorescent TPA can react with •OH to generate a highly fluorescent hydroxylated product 2-hydroxyl terephthalic acid (hTPA), which makes TPA a favorable fluorescent radical trapping agent suitable for high performance liquid chromatography separation and detection,18, 19 while restricted condition is usually prerequisite due to the hydrophobicity and instability of TPA and hTPA.20 Graphene quantum dots are nanometer-sized graphene pieces,21 which not only possess the nature of graphene, but also own extraordinary optical properties. The strong quantum confinement and edge effects of GQDs make them favorable candidate for the fabrication of nanoscale optical and electronic devices.22-24 Due to their excellent solubility, low toxicity and favorable biocompatibility, the application of GQDs as biological probes are gaining increasing attention. The large delocalized π-electron system of graphene structure provides GQDs strong affinity to species containing aromatic rings, which not only makes it possible to modulate the electronic



band structure and regulate the optical properties of GQDs via non-covalent modification,25 but also make GQDs excellent substrate for on-demand functionalization/decoration.26, 27 In this work, a novel fluorescent probe TPA@GQDs is fabricated via a facile one-pot hydrothermal method. The alkali-cutting of GO with the assistance of H2O2 bring the obtained GQDs many defects in its structure, and TPA is thus facilely conjugated onto the surface of GQDs via π-π interaction. The conjugation of GQDs with TPA offers the final TPA@GQDs probe excellent dispersibility and anti-photobleaching ability, making the as-prepared TPA@GQDs probe potential for •OH detection with improved sensitivity and satisfactory selectivity. Meanwhile, the obtained fluorescent TPA@GQDs probe owns favorable biocompatibility and exhibits great potential in bioimaging as demonstrated by the quantitative fluorescent imaging of endogenous •OH in living HeLa cells under different circumstances.

EXPERIMENTAL SECTION Materials. Graphite powder, terephthalic acid (TPA), potassium hydroxide (KOH), potassium persulfate (K2S2O8), phosphorus pentoxide (P2O5) and sodium nitrite (NaNO2) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Hydrogen peroxide (H2O2), iron chloride tetrahydrate (FeCl2•4H2O) and iron chloride hexahydrate (FeCl3•6H2O) were purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). All the chemical reagents were of analytical grade and used as received. Deionized water of 18 MΩ cm-1 was used throughout.


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Preparation of TPA@GQDs. GO was prepared according to modified Hummers and Offeman’s method. Briefly, 0.1 g GO and 0.56 g KOH were mixed with 10 mL water by ultrasonic dispersion. Then 0.015 g TPA was added into 2 mL GO dispersion. Subsequently, 0.5 mL 30% H2O2 and 2.5 mL water were added into the reaction system. Thereafter, the resultant solution was transferred into a 25-mL Teflon lined stainless autoclave and heated at 200 oC for 5 h. After cooling to room temperature, the products were filtered through a 0.1 µm microporous membrane to remove the precipitate, and the unreacted H2O2 was removed via rotary evaporation. The obtained solution was adjusted to pH 7 and dialyzed over deionized water in a dialysis bag (MWCO membranes 100~500 Da) for 3 days to remove the residual reactants. Finally, the powder of TPA@GQDs was quantified by lyophilization and re-dispersed in deionized water for further use. The optical properties and morphology of the obtained TPA@GQDs were characterized as described in previous work.28 Fluorescent detection of •OH with TPA@GQDs. The fluorescent assay of •OH was achieved as following. Firstly, 100 µL 1.0 mmol L-1 H2O2 solution was added into TPA@GQDs (Abs310nm=0.4), followed by the addition of 100 µL of Fe2+ (final concentration: 0-6 µmol L-1) and 20 µL 0.04 mol L-1 BR buffer (pH 5.0). Thereafter, the mixed solution was allowed to react at 40 oC for 10 min under slight shaking. Finally, the fluorescence spectra of the resultant solution were recorded with a 1.0 cm optical path under an excitation of 310 nm, and the variation of fluorescence emission at 430 nm were adopted for the quantitative assay of •OH. The selectivity of this system was investigated by adding other reactive oxygen species, metal ions, anions



and biomolecules into the reaction system under same conditions. Quantitative imaging of •OH in HeLa cells. Standard MTT assay is adopted to evaluate the in vitro cytotoxicity of TPA@GQDs. HeLa cells were incubated with a series concentration of TPA@GQDs (0, 0.05, 0.10, 0.20, 0.40, 1.00, 2.00, 4.00 mg mL-1) for 20 h and 20 µL MTT solution was then added into the system to incubate for another 4 h. Afterwards the supernate was discarded and the sediment was dissolved in 150 µL DMSO. The cell viability was determined by a microplate reader (BioTek, USA). The quantitative imaging of •OH was constructed as following. First, HeLa cells were incubated with TPA@GQDs (1 mg mL-1) for 12 h and washed twice with phosphate buffered saline (PBS, 10 mmol L-1, pH 7.4) to remove residual TPA@GQDs. Then 1 mmol L-1 H2O2 and a series concentration of Fe2+ (0, 1, 5, 10 µmol L-1) were introduced into the cells and incubated for another 30 min. Finally, the cells were washed twice with PBS (10 mmol L-1, pH 7.4) to remove redundant •OH and recorded on a confocal laser scanning microscope (CLSM, Olympus, Japan). The incubated cells were firstly excited with laser of 405 nm and emission ranged from 425 nm to 490 nm was recorded as the blue-channel imaging result. Thereafter, the fluorescence emission from 500 nm to 570 nm excited by a 488 nm laser was recorded as the green-channel results. The parameters of confocal fluorescence imaging were kept consistent in each measurement during the imaging assay. The fluorescence intensity was conducted using Image J software (National Institutes of Health). A linear calibration curve was developed between •OH concentration and the


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optical density of images. The imaging of •OH in normal, external stimulated (treated with 50 µmol L-1 of dexamethasone and 10 µg mL-1 of lipopolysaccharide) were performed under the same conditions. Then the content of •OH in these cells were calculated according to the calibration curve.

RESULTS AND DISCUSSION Preparation and characteristics of TPA@GQDs probe. In this work, TPA@GQDs is prepared through a one-pot hydrothermal method with GO as source material, as illustrated in Scheme 1. During the fabrication of GQDs from GO, KOH is employed as alkali-cutting agent and H2O2 serves as the assistant reagent. This alkali-cutting procedure not only offers a high productive yield of GQDs, but also leads to the production of defects in the structure of obtained GQDs.29 Generally, aromatic molecules can be conjugated onto π-rich rGO plate due to the π-π interactions,30 and this conjugation usually occur on the defect sites of graphene structure for the significant delocalization of π electrons.31 Moreover, the expansion of π-conjugated framework and electron-withdrawing substituents will contribute to π-π interactions.31, 32

As a typical benzene structure with two electron-withdrawing groups, TPA is prone

to immobilize on the defect sites of GQDs via π-π conjugation interactions, and repairs some of the defect of GQDs caused by alkali-cutting.33 Therefore, the final obtained TPA@GQDs probe not only maintains the intrinsic nature of GQDs, but also owns the characteristic properties of TPA.



KOH

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TPA

H2O2

GO

GQDs

TPA@GQDs

Scheme 1. The schematic diagram for the preparation of TPA@GQDs.

TEM images indicate that GQDs are monodisperse with an average diameter of 2.1 nm in lateral size (Figure 1A). HRTEM images show apparent lattice fringe of 0.230 nm, corresponding to (1120) lattice fringes of graphene.34, 35 The average size of TPA@GQDs is 6.8 nm (Figure 1B), which is larger than that of GQDs owning to the conjugation of TPA. The insert of Figure 1B demonstrates a higher crystallinity than that of GQDs, with a same interplanar spacing of 0.230 nm. The result suggests that the conjugation of TPA and GQDs repairs some defects in GQDs, and improve the crystallinity of final product. AFM analyses of GQDs illustrate an average topographic height of 0.820 nm, suggesting that most of GQDs consist of 1-2 graphene layers (Figure 1C).36, 37 After the conjugation with TPA, the average height increases to 1.15 nm (Figure 1D).


A

Fraction (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80

30

B

60

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20

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20 0

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50 nm

nm 2

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µM

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b 500 nm

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6

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500 nm

Figure 1. TEM images of (A) GQDs; (B) TPA@GQDs; Insert: size distributions and HRTEM images of GQDs (A) and TPA@GQDs (B); (C) AFM images of GQDs; (D) AFM image of TPA@GQDs; insert: height profile along the line from a to b. Figure 2 illustrates the X-ray photoelectron spectroscopy (XPS) patterns of GQDs and TPA@GQDs. In GQDs, the deconvoluted peak of 284.5 eV is associated with the sp2 hybridized graphitic carbon (sp2 C=C), while the peak located at 285.1 eV derives from sp2 hybridized C=C with defects. The proportion of sp2 C=C and sp2 hybridized C=C with defects is 0.38 and 0.28, respectively (Figure 2A, Table S1).29, 38 Compared to GQDs, the content of sp2 C=C (284.5 eV) of TPA@GQDs increases to 0.57, whereas the fraction of C=C with defects (285.1 eV) decreases to 0.15, indicating the defects in GQDs is restored after the π-π conjugation of TPA (Figure 2B). Moreover, the peaks of C1s corresponding to C-OH, C=O and COOH are all observed in the XPS patterns of GQDs and TPA@GQDs with the location at 286.1 eV, 287.9 eV and 289.3 eV, respectively, and the proportion of each deconvoluted peak is also well consistent with that of O1s XPS spectra (Figure 2C, D and Table S2). The structure and component of GQDs and TPA@GQDs are further explored by FT-IR



spectra (Figure 2E). The broad absorption band of GQDs at 3422 cm-1 is ascribed to the stretching vibration of O-H,39 which also appears in TPA@GQDs. In GQDs, adsorption band at 1593 cm-1 is assigned to the skeletal vibration of C=C in aromatic ring, while it shifts to lower frequency (1578 cm-1) in TPA@GQDs due to the π-π conjugation.40 In contrast to GQDs (C=O, 1682 cm-1), the intensity of C=O (1630 cm-1) in TPA@GQDs increases and the position shifts to lower frequency, corresponding to the conjugation between C=O bond and aromatic rings.41 Moreover, bands at 1329 cm-1 and 1427 cm-1 are attributed to the symmetrical and antisymmetrical stretching vibrations of COO-. After the π-π conjugation, intense symmetrical stretching vibrations appear at 1391 cm-1 in the spectra of TPA@GQDs. These results clearly reveal that TPA has been conjugated onto GQDs. Raman spectra of GQDs (Figure 2F) exhibits a typical D band (1380 cm-1) corresponding to disordered sp3 defects and a G band (1540 cm-1) relating to ordered sp2 C-C bond.42 Both of the D and G bands are found to be broadened compared with that of graphene (D band: 1350 cm-1; G band: 1580 cm-1),43 and the position of D band shifts to lower wave-number, which is ascribed to the oxidization and hydrothermal cutting processes.44 The marked red-shift of G band in 1540 cm-1 might be relevant to the increased number of graphene layers.45 Generally, the ratio of ID/IG indicates the disorder degree of GQDs. The ID/IG ratio of TPA@GQDs is determined to be 1.08, which is smaller than that of GQDs (1.20), suggesting that TPA@GQDs exhibit a lower disorder degree compared to GQDs due to the conjugated structures.46 Furthermore, a little red-shift of G band in TPA@GQDs (1535 cm-1) is observed


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compared to that of GQDs, indicating a thicker height of TPA@GQDs, which further demonstrates the conjugation of TPA on GQDs.

C=O (287.9 eV) COOH (289.3 eV)

288

sp C=C (284.5eV)

2

sp C=C (284.5 eV)

C-O-C/C-OH (286.1 eV)

290

2

B

defect C=C (285.1 eV)

286

Intensity (a.u.)

Intensity (a.u.)

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defect C=C (285.1 eV) C=O (287.9 eV) C-O-C/C-OH (286.1 eV) COOH (289.3 eV)

290

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Binding Energy (eV)

D C-O-C/C-OH (532.6 eV) C=O (531eV)

O-C=O (533.4 eV)

536

532

Intensity (a.u.)

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286

C=O (531eV) C-O-C/C-OH (532.6eV)

O-C=O (533.4eV)

528

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532

E

F 1120 1630

1578

1391

1108(C-O) 3422(-OH)

3000

1682 1329 1593 (C=C)1427 (COO )

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-1

Wavenumber (cm )

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Intensity (a.u.)

TPA@GQDs

GQDs

528

Binding Energy (eV)

Binding Energy (eV)

2964

284

Binding Energy (eV)

C

Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1380

1535

TPA@GQDs

1380

1540

GQDs

1200

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-1

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Raman shift (cm )

Figure 2. High-resolution C1s XPS spectra of (A) GQDs; (B) TPA@GQDs; O1s XPS spectra of (C) GQDs; (D) TPA@GQDs; FT-IR spectra (E) and Raman spectra (F) of GQDs and TPA@GQDs. Optical properties of TPA@GQDs. Typically, GQDs possess striking behavior of photology due to quantum confinement effect and edge effect.47 The UV-vis absorbance of GQDs shows an obvious absorption at 229 nm assigned to the π-π* transition of aromatic sp2 domains,21 and a weak shoulder at 260 nm ascribed to the n-π* transition of C=O (Figure 3A, B).48 Compared to GQDs, the adsorption bands of TPA@GQDs corresponding to the π-π* transition of C=C and n-π* transition of C=O shift to 235 nm and 280 nm, respectively. This red shift is attributed to the formation



of conjugation system.49, 50 Similar to GQDs (Figure S1), the TPA@GQDs probe exhibits an excitation-dependent PL behavior (Figure 3C), along with an excitation/emission maximum of 310 nm/430 nm. Due to the molecular aggregation of TPA@GQDs by the ‘‘π-π stacking’’ interaction,51 a shoulder peak around 540 nm is also observed in the fluorescence emission of TPA@GQDs probe. Figure S2 illustrates the fluorescence behaviors of TPA@GQDs under different conditions. No obvious variations on TPA@GQDs fluorescence is observed when pH value changes from 5.0 to 10.0 (Figure S2A). At the same time, TPA@GQDs exhibit excellent tolerance to ionic strength. The fluorescence of TPA@GQDs keep relative stable even KCl concentration is high up to 2.0 mol L-1 (Figure S2B). Moreover, the irradiation of 2 h under xenon lamp causes no change on the fluorescence intensity of TPA@GQDs, while a 25% decrease on fluorescence intensity is observed for fluorescein under the same irradiation conditions (Figure S2C). These results well suggest that the obtained TPA@GQDs is merited with favorable photo-stability, and it might be used as powerful fluorescent probe to handle with real samples. 1.5

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Figure 3. (A, B) UV-visible spectra of GQDs and TPA@GQDs; (C) Fluorescence spectra of TPA@GQDs. Inset: Photographs of TPA@GQDs under 365 nm excitation.


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Fluorescent detection of •OH. Typically, TPA is a non-fluorescent compound, which can react with •OH to produce a fluorescent aromatic hydroxylated product, 2-hydroxyterephthalic acid (hTPA). However, TPA is not soluble in water, which makes the practical application of TPA in •OH detection quite limited. In addition, strict detective condition is usually needed due to the photoinstability of hTPA.20 In present study, TPA is conjugated on the surface of GQDs via π-π conjugation to give a novel TPA@GQDs probe. By merit of the excellent solubility of GQDs, the obtained TPA@GQDs probe can be well dispersed in aqueous phase. At the same time, the non-covalent conjugation strategy provides the final TPA@GQDs probe with the characteristic properties of TPA, and the fluorescence of TPA@GQDs is enhanced greatly in the presence of •OH (Figure 4A), making TPA@GQDs a potential probe for the sensitive detection of •OH. Figure S3 illustrates the fluorescence response of TPA@GQDs towards Fe2+ (0.3 µmol L-1) and H2O2 (1 mmol L-1). The results indicated that the presence of only Fe2+ or H2O2 in the system nearly cause no effect on the fluorescence intensity of TPA@GQDs, while the coexistence of Fe2+ and H2O2 lead to an obvious fluorescence improvement of TPA@GQDs, due to the fact that the producing of •OH via Fenton reaction. Moreover, the TPA@GQDs probe possesses favorable photostability both in the absence and in the presence of •OH (Figure 4B), which enable the practical measurement for quantitative assay.



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Figure 4. Fluorescence spectra (A) and photostability (B) of TPA@GQDs in the presence of 1 µmol L-1 •OH. The experimental parameters affecting the detection of •OH, e.g., concentration of TPA@GQDs, pH value, incubating temperature and time, were thoroughly investigated as displayed in Figure S4. The fluorescence of TPA@GQDs rapidly increases upon the attaching of •OH under optimal experimental conditions (Figure 5A). Therefore, a fluorescent approach for •OH detection is developed by illuminating the relationship between •OH concentration and the relative fluorescence intensity of TPA@GQDs. As illustrated in Figure 5B, linear calibration ranges are obtained within 0.018-6 µmol L-1 with regression equation of F/F0=2.90 C·OH+1.91 (R2=0.9972). The limit of detection is derived to be 12 nmol L-1 (3σ/s, n=11), which is much lower than that of reported fluorescent methods (Table S3),5, 17, 52-55 suggesting the improved sensitivity of this proposed method. 20

A 10000 6 µmol

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Figure 5. (A) Fluorescence spectra of TPA@GQDs treated with •OH of different concentration (0-6 µmol L-1); (B) Linear calibration graphs between F/F0 and •OH concentration within 0.018-6 µmol L-1.


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In order to evaluate the selectivity of TPA@GQDs probe in •OH detection, this detection system is treated with some common metal ions (Na+, K+, Mg2+, Al3+, Fe3+, Co2+, Ni2+, Cu2+, Mn2+), anions (PO43-, CO32-, NO3-, NO2-, SO42-, Cl-, F-, Br-, I-), biomolecules including ascorbic acid (AA), citric acid (CA), urea (UR) and glucose (Glu) and other typical ROS species such as single oxygen (1O2), hypochlorite (ClO−), peroxynitrite (ONOO−) in the presence of •OH (0.3 µmol L-1), respectively. The relative fluorescence intensity of TPA@GQDs in the presence of •OH reveals undisturbed fluorescence responses (±5%) toward foreign species (Figure 6), indicating the favorable selectivity of TPA@GQDs in •OH assay. 3

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Figure 6. Relative fluorescence intensity (F/F0) of TPA@GQDs treated with 0.3 µmol L-1 •OH in the coexistence with 100 µmol L-1 different foreign species. Determination of •OH contents in real samples. To assess the practicability of the proposed sensing system, •OH contents in lake water samples and HeLa cell dispersions were determined using TPA@GQDs as fluorescent probe. HeLa cells were first digested by trypsin and collected via centrifugation, then washed with PBS for 2 times. The precipitate was dispersed in 1 mL PBS (OD600=0.7). The detection results were summarized in Table 1. It can be seen that similar results are obtained



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among the different batches of samples. Spiking tests of •OH were also performed for these samples and favorable recoveries for the real samples were achieved. Table 1. Assay results of •OH in lake water of NanHu and HeLa cell dispersions (n=3, at 95% confidence level). Detected

Spiked

Found

Recovery

(µmol L-1)

(µmol L-1)

(µmol L-1)

(%)

1

0.128±0.021

0.5

0.603±0.005

95.0±3.6

Lake water 2

0.144±0.005

0.5

0.620±0.002

95.3±6.2

3

0.141±0.009

0.5

0.620±0.003

95.7±2.2

1

0.489±0.069

1.0

1.514±0.112

102.5±6.0

2.5

2.834±0.090

93.8±3.6

1.0

1.568±0.079

106.7±5.3

2.5

2.843±0.074

93.6±3.0

1.0

1.393±0.101

92.1±3.8

2.5

2.800±0.171

93.1±6.8

Sample

HeLa cell

Time

2

0.501±0.078

dispersions 3

0.471±0.044

n=3, means±t s/ n , ƒ=95%, t=4.303. Quantitative imaging of •OH in living cells. In order to evaluate the feasibility of this fluorescent probe in quantitative imaging of •OH in living cells, the cytotoxicity of TPA@GQDs was first achieved via standard MTT assay. The cell viabilities under a series of TPA@GQDs (0-4 mg mL-1) were shown in Figure S5. Nearly no variation of cell viability is observed when the concentration of TPA@GQDs is less than 2.0 mg mL-1, indicating the low cytotoxicity and favorable biocompatibility of


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TPA@GQDs. The biocompatibility of this probe is further demonstrated by fluorescence activated cell sorting (FACS) measurements of HeLa cells after treating with TPA@GQDs. HeLa cells were first incubated with a series of concentration of TPA@GQDs for 12 h. Then, the cells were stained by FITC-Annexin V and propidium iodide (PI) to label the apoptosis cells and necrotic cells, respectively. There is no obvious difference between the cells treated with the present probe and control (Figure S6), demonstrating the high biocompatibility of the TPA@GQDs probe. A-1

B-1

C-1

D-1

A-2

B-2

C-2

D-2

A-3

B-3

C-3

D-3

Figure 7. (A-D) The confocal fluorescence images of HeLa cells incubated with 1 mg mL-1 TPA@GQDs for 12 h and then treated with 0, 1, 5, 10 µmol L-1 •OH for 30 min; The fluorescence images of (1), (2) and (3) are collected in blue channel (425-490 nm, λex 405 nm), green channel (500-570 nm, λex 488 nm) and bright field. The scale bar stands for 50 µm. Then, TPA@GQDs are employed as fluorescent probe for real-time monitoring of endogenous •OH in living HeLa cells by confocal fluorescence imaging. The HeLa



cells incubated with TPA@GQDs (1.0 mg mL-1) for 12 h were treated with a series of Fe2+ with 1 mmol L-1 H2O2 (•OH) for 30 min and recorded from two collection channels (λex405 nm/ λem425-490 nm, λex488 nm/ λem500-570 nm). As the concentration of •OH increased, the uptake of •OH in HeLa cells increased along with an enhancement of fluorescence intensity (Figure 7). The fluorescence intensity was conducted using Image J software (National Institutes of Health). Thus, a linear relationship was proposed between the concentration of •OH and the optical density of images (Figure S7), serving as the standard work curve for the quantification of endogenous •OH in HeLa cells. The level of •OH in normal, external stimulated HeLa cells (by the treatment of dexamethasone (50 µmol L-1) and lipopolysaccharide (LPS, 10 µg mL-1)) were monitored under the same conditions (Figure 8), of which image optical density was acquired using the same method. The detailed results were illustrated in Table 2. The content of •OH in normal HeLa cells was derived to be 0.534±0.055 µmol L-1, which was in accordance with that obtained from above regular fluorometry method. Compared to the images of normal cells, bright imaging results were observed for the cells dealt with dexamethasone. This is because that dexamethasone can induce the apoptosis of normal cells, resulting in the increasing content of •OH.56 Moreover, a brighter image with a •OH content of 8.277 µmol L-1 was also exhibited in cells treated with LPS (10 µg mL-1), due to the endogenously generation of •OH stimulated by LPS in HeLa cells.57, 58 In order to confirm this hypothesis, the LPS stimulated cells were further incubated with DMSO (0.5%) for another 15 min, which served as


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scavenger to inhibit the production of endogenous •OH.59 As shown in Figure 8D, a rather weaker image of HeLa cells was observed and the •OH content decreased to 0.679 µmol L-1. These results well indicate that the fluorescence enhancement is indeed triggered by endogenous •OH stimulated by LPS, and TPA@GQDs could be used as potential fluorescence probe for real-time monitoring and quantitation of endogenous hydroxyl radicals in living cells. A-1

B-1

C-1

D-1

A-2

B-2

C-2

D-2

A-3

B-3

C-3

D-3

Figure 8. The confocal fluorescence images of HeLa cells incubated with 1 mg mL-1 TPA@GQDs for 12 h, (A) cells without any treatment; (B) cells in the exposure of 50 µmol L-1 of dexamethasone; (C) cells treated with 10 µg mL-1 of LPS for 30 min; (D) cells treated with 10 µg mL-1 of LPS for 30 min, followed by 0.5% DMSO for 15 min. The fluorescence images of (1), (2) and (3) are collected in blue channel (425-490 nm, λex 405 nm), green channel (500-570 nm, λex 488 nm) and bright field. The scale bar stands for 50 µm.



Table 2. The •OH contents in HeLa cells under various circumstances derived from quantitative imaging. (n=3, at 95% confidence level). Sample (HeLa cells)

Found (µmol L-1)

Normal

0.534±0.055

Treated with dexamethasone

4.174±0.086

Treated with LPS

8.277±0.167

Treated with LPS, DMSO

0.679±0.093

n=3, means±t s/ n , ƒ=95%, t=4.303.

CONCLUSIONS In summary, we have fabricated a novel fluorescent probe TPA@GQDs by π-π conjugation of TPA on the GQDs. The as-prepared TPA@GQDs probe exhibits favorable dispersibility, and long-term stability against pH, ionic strength and illumination. These favorable features offer this obtained fluorescent probe high sensitivity in the assay of hydroxyl radical and superior selectivity over foreign species as well. The contents of hydroxyl radical in environmental and biological samples have been successfully detected based on the fluorescence enhancement of this probe upon the reaction with hydroxyl radical. Moreover, quantitative fluorescence imaging of intracellular •OH under different circumstance have been achieved, suggesting that the TPA@GQDs probe will enable opportunities to study •OH dynamics and expand the understanding of the physiological roles of hydroxyl radical in living cells.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (X.-W. Chen); [email protected] (J.-H. Wang). Tel: +86-24-83688944. Fax: +86-24-83676698. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors appreciate financial support from National Natural Science Foundation of China (21475017, 21275027, 21235001), and Fundamental Research Funds for the Central Universities (N150502001). Supporting Information Available: Fluorescence spectra of GQDs. Inset: Corresponding photographs of GQDs under 365 nm excitation; Fluorescence intensity of TPA@GQDs at different pH (A), ionic strength (B) and irradiation time of xenon lamp (C); Comparison of TPA@GQDs towards Fe2+ (0.3 µmol L-1), H2O2 (1 mmol L-1) and Fe2+/H2O2 (0.3 µmol L-1/1 mmol L-1, •OH: 0.3 µmol L-1); Relative fluorescence intensity (F/F0) in the presence of •OH (6 µmol L-1) at (A) different concentration of TPA@GQDs (A310nm=0.2-0.8); (B) Different time (10-40 min); (C) Different temperature (20-50℃); (D) Different pH (water, 3, 5, 7, 9, 11); Effect of the TPA@GQDs concentration on the viability of HeLa cells after 12 h incubation with DMEM; Apoptosis assay of HeLa cells after TPA@GQDs treatment. HeLa cells were incubated with TPA@GQDs at concentrations of (A) 0, (B) 0.1, (C) 0.2, (D) 0.4 and



(E) 1 mg mL-1 for 12 h. LL, UL, UR, and LR represent the regions of live cells, dead cells, late apoptotic cells, and early apoptotic cells, respectively; Linear calibration graphs between optical density and •OH concentration within 0-10 µmol L-1 in HeLa cells; Changes of binding energy areas and fraction of C1s XPS spectra of GQDs and TPA@GQDs; Changes of binding energy areas and fraction of O1s XPS spectra of GQDs and TPA@GQDs; Comparisons on the performance of fluorescent nanomaterial-based methods for detection of •OH.


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TOC Graphical FL Intensity (a.u.)

4000

•OH

TPA@GQDs + OH

3000 2000

TPA@GQDs 1000 0 300

400

500

Wavelength (nm)

600

4000

•OH Hydroxylation TPA@GQDs

Turn-On

FL Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3000 TPA@GQDs + OH 2000 1000 TPA@GQDs 0 0

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Fluorescent TPA@GQDs Probe for Sensitive Assay ... - ACS Publications

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