Simultaneous Fluorescence and Chemiluminescence Turned on by

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Simultaneous Fluorescence and Chemiluminescence Turned on by Aggregation-Induced Emission for Real-time Monitoring of Endogenous Superoxide Anion in Live Cells Jinye Niu, Jilin Fan, Xu Wang, Yongsheng Xiao, Xilei Xie, Xiaoyun Jiao, Chuan-Zhi Sun, and Bo Tang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 2, 2017

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Analytical Chemistry

Simultaneous Fluorescence and Chemiluminescence Turned on by Aggregation-Induced Emission for Real-time Monitoring of Endogenous Superoxide Anion in Live Cells Jinye Niu,1,2 Jilin Fan,1 Xu Wang,*1 Yongsheng Xiao,1 Xilei Xie,1 Xiaoyun Jiao,1 Chuanzhi Sun,1 and Bo Tang*1 1

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China 2

School of Chemical Engineering, Shandong University of Technology, Zibo 255049, P. R. China

Fax: +86-531-86180017; E-mail: [email protected]; [email protected] ABSTRACT: Biological sensors with simultaneous turn-on signals of fluorescence (FL) and chemiluminescence (CL) triggered by one single species, are supposed to integrate spatiotemporally-resolved FL imaging with dynamic CL sensing into one luminescent assay. Efficiently increased accuracy can be expected based on complementary information simultaneously obtained from two independent modes, which is crucial in disease detection and diagnosis. However, very few examples can be found to date because of the key challenges in the rational design of sensing structures. Herein, aggregation-induced emission (AIE) was employed to develop a novel organic platform TPE-CLA with simultaneous turn-on FL/CL signals specifically modulated by O2•− in cells, which can be attributed to the activation of AIE resulted from the decreasing solubility after recognition. Using imidazopyrazinone (CLA) as the reactive motif and tetraphenylethene (TPE) as FL/CL enhancing skeleton, TPE-CLA is sensitive enough to image native O2•− in Raw264.7 cells and lipopolysaccharide stimulated O2•− in mice. Endogenous O2•− in HL-7702 cells induced by acetaminophen (APAP) was uninterruptedly monitored for 7200 s with CL and the results were further confirmed by FL imaging. Accordingly, TPE-CLA turns out to be a reliable candidate for real-time and continuous monitoring of endogenous O2•− in live cells. The strategy utilizing AIE to accomplish the FL/CL dual detection is expected to extend the application of AIE as reaction-activated biosensors.

Photoluminescence has been a preferred technique for biological sensing, in view of its superior sensitivity, high selectivity and the simplicity of operation. Moreover, photoluminescence in conjunction with imaging systems, offers direct visualization for on-site and non-invasive assay at the molecular level in real-time, which provides useful insights into complicated biological structures and processes.1-6 Fluorescence (FL) and chemiluminescence (CL) are the two mainly used modes of photoluminescence for biological assays, among which FL features high spatiotemporal resolution in cell imaging 7-9. However, photo-bleaching of the dyes and photo-damage to the living organisms resulted from continuous irradiation badly limit its application in long term and dynamic monitoring.10-16 CL dispensing with external light source is characteristic of fast response, significantly reduced background and high signal-noise ratio.17,18 Nevertheless, there remains substantial stumbling blocks to the routine use of CL, including low emission intensity, short CL time, and short emission wavelength.19 Therefore, it deserves our attention to develop an integrated sensing platform which can be simultaneously turned on in signals of both FL and CL by one single species. High sensitivity, good spatiotemporal resolution and satisfied capability for real-time

monitoring could be obtained in one assay. More importantly, reduced false results and increased accuracy can be expected with the complementary information for one single species simultaneously obtained from two independent modes, which is crucial in disease detection and diagnosis.20-23 Considering the superior cell membrane permeability, it is of great significance to rationally design organic FL/CL platforms and explore their biological applications. However, to the best of our knowledge, very few FL/CL systems have been developed to date as biosensors, except for one semiconducting polymer-based nanosensor applying FL and CL respectively for two different species.24 Turn-on luminescence possesses much lower background, higher resistance to photo-bleaching and superior reliability relative to those conventional turn-off signals.25 The key challenge in the design of an organic FL/CL system for turn-on sensing is to covalently incorporate an activatable moiety that can produce CL into an appropriate fluorophore, which can be transformed from non-emissive structure to a highly fluorescent emitter upon recognition. Aggregation-induced emission (AIE) is characterized by nearly non-luminescent in the isolated state but strongly luminescent in the aggregated state as a

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result of restriction of intramolecular motions (RIM).25 The low background endows AIE-based systems with great potential to realize turn-on luminescent assay through triggering strategies such as cross-linking, solubility change, coordination and so on.26 Tetraphenylethene (TPE), as the prototypical AIE motif typical of simple preparation and easy functionalization, has been under extensive investigations for AIE platform construction and AIE effect activation.25 Superoxide anion (O2•−), as a precursor of other reactive oxygen species (ROS) in cells, is either protective or deleterious in a clinical context 27, which means that it is of great importance to monitor its fluctuations continuously as accurately as possible. In this view, O2•− is selected as an example to explore the potential of FL/CL dual sensing platform in real-time monitoring. Cypridina luciferin analogs with imidazopyrazinone moiety (CLA for short) have been proved to be good candidates for CL detection of O2•−, and successfully applied in analyzing and imaging O2•− in vitro and in vivo.19, 28-30 Herein, inspired by the superior turn-on properties of TPE and well-established response of CLA to O2•−, we covalently linked TPE and CLA to develop a conjugated TPE-CLA probe as an example of FL/CL dual sensing platform, expecting simultaneous turn-on FL/CL signals specifically modulated by O2•−. TPE-CLA turned out to be highly sensitive to O2•− with detection limit (LOD) of 0.21 nM for FL and 0.38 nM for CL respectively, which was successfully applied to image native O2•− in Raw264.7 cells and stimulated O2•− in inflamed mice. Endogenous O2•− induced by acetaminophen (APAP) in HL-7702 cells was uninterruptedly monitored with CL sensing and the results were further confirmed by FL imaging. Accordingly, TPE-CLA turns out to be a reliable candidate for real-time and continuous monitoring of endogenous O2•− in live cells. Therefore, by utilizing the unique luminescent properties of AIE, TPE-CLA shows a novel example of dual FL/CL detection of one single species in biological contexts.

EXPERIMENTAL SECTION Materials. All reagents were purchased commercially and used without further purification. (4-Bromophenyl) (phenyl) methanone, n-BuLi, trimethylborate and pinacol were purchased from Taitan Chemical Reagent Company (Shanghai, China). Titanium tetrachloride, zinc powder, potassium carbonate, sodium carbonate (Na2CO3), 2amino-5-bromopy-razine, [1,1'bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl2), acetyl chloride, 1,4bis(diphenylphosphino)butane-palladium(II) chloride and methylglyoxal were purchased from Aladdin Industrial (Shanghai, China). Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), superoxide dismutase (SOD) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron) was purchased from Shanghai Reagent Co. Ltd. (Shanghai, China). All other chemicals

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and solvents used were of analytical grades. Ultrapure water (18.2 MΩ cm-1) was used throughout the analytical experiments. Raw264.7 and HL-7702 cells were purchased from the Committee on Type Culture Collection of the Chinese Academy of Sciences. BALB/C-nude mice were purchased from Changzhou Cavens laboratory animal Co., Ltd. (Jiangsu, China). Reactive Species. Superoxide anion (O2•−) was delivered from potassium hyperoxide (KO2) in DMSO solution, and absorption spectrum was measured to quantify the concentration of O2•− by a TU-1900 UV-vis spectrophotometer. Hydroxyl radical (•OH) was generated from Fenton reaction (Fe2++H2O2 →Fe3++•OH+OH-). H2O2, tertbutyl hydroperoxide (TBHP), and hypochlorite (ClO-) were obtained from directly diluting commercially available 30%, 70%, and 10% aqueous solutions, respectively. Ascorbic acid (Vc), glutathione (GSH), cysteine (Cys), homocysteine (HCy) and nitrite (NO2-) stock solutions were prepared from commercially respective solids. Hydrogen sulfide (H2S) was produced from stock solution of commercially available NaHS. Nitric oxide (NO) was used from stock solution prepared by sodium nitroprusside. Singlet oxygen (1O2) was produced from the ClO-/H2O2 system. Peroxynitrite (ONOO-) was prepared from stock solution, and absorbance was measured at 302 nm to quantify the concentration of ONOO-. Instruments. 1HNMR and 13CNMR spectra were taken on a 400 MHz spectrometer (Bruker Co., Ltd., Germany); δ values are in ppm relative to tetramethylsilane (TMS). HRMS spectra were obtained on a maxis ultra-high resolution-TOF MS system (Bruker Co., Ltd., Germany). Transmission electron microscopy (TEM) photos were taken with a JEM-2100 electron microscope. Absorption spectra were measured on a TU-1900 UV-vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.). FL measurement was carried out with a FLS-980 fluorescence spectrometer (Edinburgh Instruments Ltd., England) and CL spectra were collected using the same instrument with its light off. CL measurement was implemented with a MPI-B multi-parameter chemiluminescence analyzer (Xi'an Remex Analytic Instrument Co., Ltd, China). FL images were gained on a LSM 880 NLO Confocal Laser Scanning Microscope (Zeiss, Germany). CL imaging was performed by an IVIS Lumina II in vivo imaging system. In MTT assay, absorbance was measured using a microplate reader (RT 6000, Rayto, USA). Synthesis of TPE-CLA and TPE-PZA. The specific synthetic route of TPE-CLA and TPE-PZA is shown in Scheme S1. Compound 1 was synthesized according to the previous literature. 31 Synthesis of Compound 2. Under an argon atmosphere, Compound 1 (0.585 g, 1 mmol), 2-amino-5bromopy-razine (0.696 g, 4 mmol), 1 mL 2M aqueous K2CO3, 2 mL ethanol, and 2 mL toluene were added into a 25 mL double-necked round-bottomed flask and stirred for 10 min. After degassed in vacuum, 0.181 g 1,4bis(diphenylphosphino)-butane-palladium(II) chloride was added. The solution was stirred and heated at 85°C

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for 24h. After cooling to room temperature, 30 mL water and 40 mL ethyl acetate were added successively. Then the aqueous phase was separated and extracted twice with ethyl acetate (15 mL). The combined organic phase was washed twice with brine (10 mL), dried over anhydrous MgSO4. After filtered and concentrated in vacuum, the resulting residue was purified by silica gel column chromatography, using petroleum and ethyl acetate (v/v = 1:3) as eluent to obtain a faint yellow solid (80% yield). 1 H NMR (400 MHz, DMSO-d6, δ): 8.44 (s, 2H), 7.90 (s, 2H), 7.74-7.70 (m, 4H), 7.17-7.10 (m, 6H), 7.06- 6.98 (m, 8H), 6.54 (s, 4H). 13C NMR (100 MHz, DMSO-d6, δ): 155.30, 155.25, 143.71, 142.78, 140.77, 139.27, 138.98, 135.68, 131.88, 131.85, 131.60, 131.25, 128.29, 127.03, 124.52. HRMS (m/z): [M+H]+ calcd, 519.2292; found, 519.2220. Synthesis of TPE-CLA. Under an argon atmosphere, compound 2 (0.156 g, 0.3 mmol) was dissolved in 3 mL ethanol, then methylglyoxal (0.072 g, 1 mmol) and 100 μL 37% HCl was added and the solution was stirred and heated at 80°C for 12 h. After cooling to room temperature, the solution was concentrated in vacuum. The resulting residue was purified by silica gel column chromatography, using DCM and methanol (v/v = 1:6) as eluent to obtain TPE-CLA as a yellow solid (40% yield). 1H NMR (400 MHz, DMSO-d6, δ): 8.20-7.72 (m, 4H), 7.66 (d, J = 8.1 Hz, 4H), 7.18-7.12 (m, 6H), 7.12-6.94 (m, 10H), 2.27 (s, 6H). 13C NMR (100 MHz, DMSO-d6, δ): 174.74, 143.68, 143.43, 140.84, 131.65, 131.21, 130.09, 129.66, 128.37, 127.20, 125.42, 63.46, 63.42. HRMS (m/z): [M+H]+ calcd, 627.2503; found, 627.2508. Synthesis of TPE-PZA. Acetyl chloride (0.5 mmol) was added dropwise to a mixed solution of compound 1 and triethylamine (0.5 mmol) that dissolved in DCM (50 mL) at 0°C. Then the solution was stirred at room temperature under argon atmosphere for 12 h. After evaporation of the solvent, the resulting residue was purified by silica gel column chromatography, using DCM as eluent to obtain TPE-PZA as a light yellow solid (60% yield). 1H NMR (400 MHz, DMSO-d6, δ): 8.69 (s, 2H), 8.16 (s, 2H), 7.99-7.95 (m, 4H), 7.39-7.31 (m, 6H), 7.29-7.23 (m, 8H), 6.76 (s, 2H), 2.74 (s, 6H). 13C NMR (100 MHz, DMSO-d6, δ): 167.07, 157.11, 157.36, 142.88, 141.38, 141.09, 137.79, 133.99, 133.96, 133.71, 133.36, 130.40, 129.14, 126.63, 29.78. HRMS (m/z): [M+H]+ calcd, 603.2503; found, 623.2535.

RESULTS AND DISCUSSION Design and Synthesis of TPE-CLA. TPE-CLA is composed of two CLA units for specific recognition and a TPE skeleton for AIE activation. The chemical structure of TPE-CLA and the proposed FL/CL turn-on mechanism is illustrated in Scheme 1. It is important to note that the non-luminescence of TPE-CLA is the key point in rational design, which can be realized by the reasonable utilization of AIE effect. Endowed with some hydrophilicity by the relatively strong polarity, TPE-CLA is supposed to well disperse in aqueous media, experiencing no RIM and nearly non-luminescent. Upon recognizing O2•−, the CLA unit is firstly oxidized to form a di-oxetanone that decomposes to generate a singlet-excited amide, which then

decays to its ground state (short for PZA) with concomitant CL emission.32 Simultaneously, FL is also triggered with the generation and synchronous aggregation of the hydrophobic TPE-PZA, which effectively turns on AIE as a result of RIM. TPE-CLA was synthesized from TPE with pyrazin-2-amine group and pyruvic aldehyde. Scheme 1. Chemical Structure and Proposed Turn-on Mechanism of TPE-CLA

Verification of the Turn-on Mechanism. To verify our turn-on hypothesis, HRMS spectra of TPE-CLA in the presence of O2•− were firstly acquired (Figure S1) and the peaks at m/z 603.2535 and 625.2355 observed in Figure S1b were in very good agreement with the theoretically predicted values of [M+H]+ and [M+Na]+ peaks for the oxidation product TPE-PZA. Then, the morphology and the emission of TPE-CLA before and after reaction with O2•− were investigated. As can be seen from Figure 1a and Figure 1b, addition of O2•− to TPE-CLA in phosphate buffer solution (PBS) induced the obvious aggregation and strong emission of the samples. Meanwhile, the effect of water fraction on the quantum yield of TPE-CLA and TPE-PZA was investigated, using methanol (CH3OH) and tetrahydrofuran (THF) as good solvents respectively. The results showed that water with volumetric fractions over 90% dramatically enhanced the quantum yield of TPEPZA (Figure 1c), but no increase was found in TPE-CLA (Figure 1d), which indicated the activation of AIE effect upon recognition. These results forcefully support the reaction-activated FL/CL turn-on mechanism mentioned above. Highly sensitive turn-on response of TPE-CLA toward O2•− was confirmed by the 220 fold enhancing in quantum yield from TPE-CLA to TPE-PZA with water fraction of 98%. Spectral Response of TPE-CLA to O2•−. The spectral response of TPE-CLA to O2•− was investigated and the results are shown in Figure 2. The absorption spectra (Figure 2a) give a maximum absorbance of TPE-CLA in the presence of O2•− (short for TPE-CLA-O2•−) at 340 nm, a slight blue-shift of 14 nm relative to TPE-CLA, which is consistent with the shorter conjugation of TEP-PZA than TPE-CLA. More importantly, the reported CL spectrum of CLA is from 300 nm to 500 nm with the maximum of 380 nm,28 which means large overlap between absorption of the TPE-PZA and CL emission of CLA, indicating possible chemiluminescence resonance energy transfer (CRET) from CLA moiety to TPE-PZA for longer emission wavelength. As a verification, intense CL emission centered at 500 nm was observed upon the addition of O2•− (Figure 2b). Meanwhile, strong FL emission appeared in TPECLA-O2•− with the excitation of 350 nm while the signal of

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TPE-CLA is almost indistinguishable (Figure 2c). Notably, high consistence is found in FL/CL spectra of TPE-CLAO2•− and FL spectrum of TPE-PZA (Figure 2d), which confirms that the turn-on of FL and CL is both attributed to the AIE-active TPE-PZA, as illustrated in Scheme 1.

Figure 1. Verification of the turn-on mechanism. a-b) TEM photos of TPE-CLA (20 μM, 1.0% CH3OH) before (a) and •− after (b) reaction with O2 (50 μM) in PBS; Insert are photos taken under the ultraviolet lamp (365 nm) from the same samples with TEM characterization. c-d) Quantum yield of TPE-PZA (10 μM) in THF/H2O (c, λex = 350 nm) and TPECLA (10 μM) in CH3OH/H2O (d, λex = 354 nm) with different H2O volumetric fraction.

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Figure 2. Spectral Response of TPE-CLA to O2 . a) Absorption spectra of TPE-CLA (20 μM, 1.0% CH3OH) before •− (black) and after (red) reaction with O2 (50 μM). b) CL spectra of TPE-CLA (10 μM, 0.5% CH3OH) before (black) and •− after (red) reaction with O2 (30 μM). c) FL spectra of TPECLA (10 μM, 0.5% CH3OH) before (black for excitation and blue for emission) and after (red for excitation and pink for •− emission) reaction with O2 (30 μM); λex/λem = 350/500 nm. d) Normalized FL spectrum (blue, λex = 350 nm), CL (black) •− spectrum of TPE-CLA (10 μM, 0.5% CH3OH) toward O2 (30 μM) and FL spectrum (red, λex = 350 nm) of TPE-PZA (10 μM, 0.5% THF). All experiments were done in 50 mM PBS (pH = 7.4).

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Detection of O2•− in Solution. The FL/CL response of TPE-CLA to O2•− was tested under different conditions, including pH values ranging from 4.0 to 10.0 (Figure S2) and TPE-CLA concentrations ranging from 1 μM to 50 μM (Figure S3). Both FL/CL signals of TPE-CLA-O2•− was found to be independent of pH in the physiological range, and TPE-CLA in 10 μM and 20 μM was used for further investigation of FL and CL response respectively. Meanwhile, the specificity of TPE-CLA to O2•− was explored with SOD (superoxide dismutase, a scavenger of O2•−) (Figure S4) and other reactive species (Figure S5). The results show that FL/CL signals can be inhibited by SOD and other reactive species shows negligible FL/CL responses. Subsequently, the ratio of luminescence enhancement toward different concentrations of O2•− was examined in PBS (pH = 7.4) and illustrated in Figure S6. The FL and CL enhancement at 510 nm displayed a similar linear relationship with the concentration of O2•− in the range of 0-60 μM and 0-55 μM respectively. The LOD was estimated to be 0.21 nM for FL and 0.38 nM for CL, as calculated by equation LOD = 3S0/K. Therefore, TPE-CLA turns out to be a sensitive and selective FL/CL probe for O2•−, which would be useful for imaging O2•− in vitro and in vivo. Imaging of O2•− in Live Cells and in Vivo. To explore the potential of TPE-CLA to image O2•− in biological systems, we first studied the cytotoxicity of TPE-CLA. The MTT assays with IC50 value of 218.63 μM suggest that TPE-CLA has low toxicity towards living cells (Figure S7). Thanks for the effective two-photon excitation of TPEPZA (with a two-photon cross section of 23.9 GM in in PBS), two-photon imaging was employed to overcome the limited absorption wavelength. Raw264.7 cells were divided into three groups and incubated in Dulbec-coQs modified EagleQs medium (DMEM) containing PBS (Control), Tiron (a scavenger of O2•−) and PMA (phorbol 12-myristate 13-acetate, a stimulator of O2•−) respectively for 30 min before treated with TPE-CLA. After 15 min, FL images were acquired and the corresponding FL intensity was provided (Figure 3). The results showed that obvious FL signal was obtained in the control that could be effectively quenched by Tiron and significantly enhanced by PMA, which demonstrated that TPE-CLA was not only capable of imaging the endogenously stimulated O2•− but also monitoring native O2•− in live cells. Afterwards, the in vivo imaging ability of TPE-CLA was further investigated. Different groups of mice were treated with intraperitoneally (i.p.) administrated saline (I, Control), lipopolysaccharide (LPS) 34 for inducing acute inflammation (II) and LPS + Tiron (III) respectively. After 4h, TPE-CLA was injected and CL images were acquired as soon as possible. The corresponding CL intensity was provided in Figure 4. The results showed that LPSstimulated mice displayed strong CL and the signal was largely inhibited by Tiron, which demonstrated that TPECLA was able to monitor the endogenous O2•− in inflamed mice. From Figure 3 and Figure 4, it is credible to draw the conclusion that TPE-CLA can be successfully applied in imaging as specific O2•− biosensor in living contexts.

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Figure 3. Imaging O2 in live cells. a-c) FL images of O2 in Raw264.7 cells treated with PBS (Control, a), Tiron (100 μM, b) and PMA (1 μg/mL, c) respectively for 30 min before incubation with TPE-CLA (20 μM ) for 15 min. d) Quantitative of FL intensity in panels a-c. The provided images of cells are representative (n = 7 fields of cells). FL images were acquired with 800 nm excitation and 420-600 nm emission. Data are *** presented as the mean ± SEM; p< 0.001. Results are representative of three independent experiments.

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during this process, as accurately as possible. Inspired by the simultaneously selective turn-on FL/CL of TPE-CLA, HL-7702 cells overdosed with APAP were used as a model to evaluate the capability of TPE-CLA for continuous monitoring of O2•− in real-time. Firstly, kinetic curves of TPE-CLA and CLA triggered by O2•− in live cells were investigated and the results were shown in Figure 5a. Both TPE-CLA and CLA had stable emission in HL-7702 cells, while the former gave much higher CL signals as a response to native O2•− than the latter with the same concentration. The enhancing effect of AIE is consistent with the previous reports studied in organic media (with water volumetric fraction no more than 10%) 35, which means that TPE-CLA is expected to be a candidate superior to CLA for continuous monitoring of native O2•− in cells. Afterwards, two groups of HL-7702 cells were incubated with PBS (Control) and APAP (20 mg/mL) respectively, and the real-time CL signals were uninterruptedly acquired for 7200 s with TPE-CLA injected at 120 s (Figure 5b). As can be seen, obvious CL signals appeared in both control and APAP-treated cells upon the injection of TPECLA, and no distinguishable change in control since then indicated the stability and reliability of TPE-CLA. The CL signals in APAP-treated cells increased gradually and reached its maximum after stimulation for about 100 min and remained almost unchangeable for the last 20 min.

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Figure 4. Imaging O2 in vivo. a) CL image of O2 in LPStreated mice; (I) Saline + TPE-CLA (200 μM, 200 μL), (II) LPS (1 mg/mL, 200 μL) + TPE-CLA (200 μM, 200 μL), (III) LPS (1 mg/mL, 200 μL) + Tiron (20 mΜ, 200 μL) + TPE-CLA (200 μM, 200 μL). b) Quantitative of CL intensity from groups *** (I−III). Data are presented as the mean ± SEM; p< 0.001. Results are representative of three independent experiments.

Real-time Monitoring of O2•− in Live Cells. To evaluate the potential of TPE-CLA for real-time monitoring in live cells, the effect of AIE on CL emission is investigated firstly. Commercial CLA (2-Methyl-6-phenyl-3,7dihydroimidazo[1,2-a]pyrazin-3-one) is used as control to compare the kinetic curves of TPE-CLA and CLA triggered by O2•−. As seen from Figure S8, longer emission time in aqueous media is observed in TPE-CLA than CLA. Then, O2•− in different concentration was alternately injected into TPE-CLA at intervals of 30 s and the CL signals were given in Figure S9, which presented good reversible response of TPE-CLA toward O2•−. The above results indicate that TPE-CLA shows high potential in dynamic monitoring of O2•−. As one of the most widely used analgesics, acetaminophen (APAP) is safe at therapeutic doses, while overdose leads to severe liver injury and even death.30 ROS, generated from mitochondrial respiration impairment by excessive reactive metabolites of overdose APAP, plays a vital role in APAP toxicity.33 In this view, it is of great importance to monitor O2•−, as a precursor of other ROS

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Figure 5. Real-time monitoring of O2 in live cells. a) Kinetic curves of the TPE-CLA (20 μM, black) or CLA (20 μM, red) injected into HL-7702 cells. b) Real-time CL monitoring of •− O2 in HL-7702 cells stimulated by overdosed APAP (20 mg/mL) with TPE-CLA (200 μM). Red line is for control and •− black line is for APAP stimulation. c-g)FL images of O2 in HL-7702 cells stimulated by overdosed APAP (20 mg/mL) for different time; c) 0 min (Control), d) 30 min, e)60 min, f) 90 min and g) 120 min, before incubation with TPE-CLA (20 μM ) for 15 min. h) Quantitative of FL intensity in panels c-g. FL images were acquired with 800 nm excitation and 420-600 nm emission. Data are presented as the mean ± SEM, (n= 7

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fields of cells); p< 0.001. 0.01