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A Reaction-based OFF-ON Near-infrared Fluorescent Probe for Imaging Alkaline Phosphatase Activity in Living Cells and Mice Yi Tan, Ling Zhang, Ka Ho Man, Raoul Peltier, Ganchao Chen, Huatang Zhang, Liyi Zhou, Feng Wang, Derek Ho, Shao Q. Yao, Yi Hu, and Hongyan Sun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14176 • Publication Date (Web): 31 Jan 2017 Downloaded from http://pubs.acs.org on January 31, 2017

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ACS Applied Materials & Interfaces

A Reaction-based OFF-ON Near-infrared Fluorescent Probe for Imaging Alkaline Phosphatase Activity in Living Cells and Mice Yi Tan,†,‡,§ Ling Zhang,†,⊥ Ka Ho Man,‡ Raoul Peltier,‡,§ Ganchao Chen,‡,§ Huatang Zhang,‡,§ Liyi Zhou,‡,§ Feng Wang,ǁ Derek Ho,ǁ Shao Q. Yao,# Yi Hu,*,£ and Hongyan Sun*,‡,§ ‡

Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue,

Kowloon, Hong Kong, P. R. China §

Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research

Institute of City University of Hong Kong, Shenzhen 518057, P. R. China ⊥

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy,

Xuzhou Medical University, Xuzhou 221002, P. R. China ǁ

Department of Physics and Materials Science, City University of Hong Kong, 83 Tat Chee

Avenue, Kowloon, Hong Kong, P. R. China #

Department of Chemistry, National University of Singapore, Singapore

£

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Multi-

disciplinary Research Division, Institute of High Energy Physics, Chinese Academy of Sciences (CAS), Beijing 100049, P. R. China

ACS Paragon Plus Environment

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ABSTRACT Alkaline phosphatases are a group of enzymes which play important roles in regulating diverse cellular functions and disease pathogenesis. Hence developing fluorescent probes for in vivo detection of alkaline phosphatase activity is highly desirable for studying the dynamic phosphorylation in living organisms. Here, we developed the very first reaction-based nearinfrared (NIR) probe (DHXP) for sensitive detection of alkaline phosphatase activity both in vitro and in vivo. Our studies demonstrated that the probe displayed up to 66-fold fluorescence increment upon incubation with alkaline phosphatases, and the detection limit of our probe was determined to be 0.07 U/L, which is lower than that of most of alkaline phosphatase probes reported in literature. Furthermore, we demonstrated that the probe can be applied to detecting alkaline phosphatase activity in cells and mice. In addition, our probe possesses excellent biocompatibility and rapid cell internalization ability. In light of these prominent properties, we envision that DHXP will add useful tools for investigating alkaline phosphatase activity in biomedical research.

KEYWORDS: Alkaline phosphatase, near-infrared, fluorescent probe, bioimaging, mice


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INTRODUCTION Alkaline phosphatases (ALP) belong to a subfamily of phosphatases and they are present in many mammalian tissues. ALP is capable of catalyzing the hydrolysis of phosphate ester group in a variety of proteins, nucleic acids and other small molecules.1 Despite extensive research of ALP, the complicated physiological and pathological functions of ALP have not been fully understood. In-depth investigation of ALP activity in vivo is still challenging, partly due to the absence of tools to monitor ALP activity in living organisms in real time. Moreover, accumulating evidence suggests that abnormally elevated ALP levels in blood are linked to various diseases such as cancer, heart disease, bone disorders and hepatic diseases.2-4 Blood test of ALP activity is of great significance for clinical diagnosis of human diseases. In addition, tracking of ALP activity provides information on the cell differentiation and viability, thereby facilitating the identification of abnormality in cell behaviors.5-6 Therefore, it is crucial to develop specific and sensitive probes for ALP activity assay to study the dynamic phosphatase activity in live cells and animals, and these probes may find important application in clinical diagnosis. Several methods have been utilized to detect ALP activity in the last few decades.7-15 Among them, the fluorescent methods are preferred due to their noninvasiveness, high sensitivity and superb spatiotemporal resolution in visualizing bioreactive species in the biological systems.16-19 Until now, a few fluorescent probes have been reported for monitoring ALP activity through various mechanisms, such as assembly of nanoparticles, host-guest interaction and change of solubility.20-24 However, the application of these probes is hampered by their intrinsic drawbacks, such as low sensitivity, laborious synthetic procedures, complicated sensing mechanism as well as short emission and excitation wavelengths. Recently, fluorescent probes based on


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aggregation-induced emission (AIE) mechanism have been reported for detecting ALP in serum and living cells.25-27 Although these probes showed elevated sensitivity and selectivity, the requirement for high probe concentration (50 µM) and short wavelength emission can limit their application in biological systems. Meanwhile, the cell-permeability of these ALP probes might not be good due to the negative charge from the phosphate groups in the probes.28 Near-infrared (NIR) fluorescence probe is widely accepted as an ideal candidate for cell imaging due to its advantage of longer wavelength excitation and emission, lower energy, deeper tissue penetration as well as reduced background interference from auto-fluorescence.29-37 To our best knowledge, no reaction-based NIR probe for imaging ALP in animals has been reported. Herein, we reported a NIR probe (DHXP) based on a derivative of 3-dihydro-1H-xanthene-6-ol (DHX), which displayed high sensitivity, low cytotoxicity and high selectivity. We further demonstrated the application of DHXP to investigate endogenous ALP activity in cells and mice.

RESULTS AND DISCUSSION Probe Design and Synthesis. DHXP consists of two parts: an NIR fluorophore DHX and a phosphatase reactive site (Scheme 1). In our design, DHX is selected as the fluorescence reporter because of its high quantum yield and good cell permeability.38 DHXP itself is non-fluorescent as the hydroxyl group of DHX fluorophore is protected with a phosphate moiety, which diminishes the electron donating ability of OH group and suppresses the intramolecular charge transfer (ICT) process. After enzymatic dephosphorylation occurs, phosphatases remove the phosphate moiety on DHXP, resulting in the recovery of ICT effect and “turn-on” of fluorescence. Although several fluorogenic substrates have been reported to monitor ALP activity in living cells, the cell permeability of these probes might be poor owing to the presence


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of negatively charged phosphate ion. In our design, a positively charged fluorophore DHX is introduced into the probe DHXP. Such positively charged fluorophore will neutralize the negatively charged phosphate ion and therefore improve cell permeability of the probe. The synthesis of DHXP is shown in Scheme 2. Briefly, compound 4 was synthesized via retro-Knoevenagel reaction by reacting compound 3 with resorcinol in the presence of potassium carbonate (K2CO3) at 50 °C for 2 h. Compound 5 was obtained via phosphorylation of compound 4 with diethyl chlorophosphate and the presence of a base. First attempt to use inorganic base K2CO3 only gave unexpectedly low yield (about 15%). We then replaced K2CO3 with an organic base N, N-diisopropylethylamine (DIEA) and the yield was significantly increased to 43%. We speculated that using the organic base might lead to more complete deprotonation of phenolic hydrogen, hence facilitating the nucleophilic reaction. The probe, DHXP, was then obtained by hydrolysis of compound 5 using bromotrimethylsilane and purified by preparative-HPLC. The compounds were characterized with ESI-MS, 1H NMR,

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C NMR

and 31P NMR. More details of the synthetic process can be found in the experimental section.


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Scheme 1. Illustration of the fluorometric assay for ALP activity with DHXP.

Scheme 2. Synthetic scheme of the probe DHXP, according to the literature39-40 with modifications.

Figure 1. (A) UV absorption spectra; (B) fluorescence emission spectra (λex = 680 nm) of DHXP (5 µM) in Tris-HCl buffer (10 mM, pH = 7.4) without (black line) or with (red line) 0.1 U/mL ALP.

Characterization of the Probe. The photophysical properties of the probe DHXP were investigated in Tris-HCl buffer (10 mM, pH = 7.4) with or without 0.1 U/mL ALP. As shown in


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Figure 1A, 5 µM DHXP in Tris-HCl buffer showed absorption maximum at 600 nm and 650 nm respectively. After the addition of 0.1 U/mL ALP, the absorbance band displayed a slight redshift from 650 nm to 680 nm. This was accompanied with a color change from blue to cyan within several minutes, which enables the fast detection of ALP through colorimetric assay without any sophisticated instrument. Fluorescence studies showed that there is negligible fluorescence of DHXP observed at different pH conditions (Figure S1), and DHXP is relatively photostable (Figure S2). These results suggest that DHXP is a stable fluorescent probe. After incubating DHXP with 0.1 U/mL ALP for 30 min, strong fluorescence emission could be observed (Figure 1B). The fluorescence increment was determined to be 66-fold using an emission wavelength of 700 nm. Subsequently, we conducted HPLC analysis to confirm the enzymatic dephosphorylating process. As shown in Figure 2, DHXP itself displayed a peak at 19.3 min. The signal decreased rapidly with addition of ALP. Meanwhile, a new peak at 25.2 min appeared. The peak is attributed to the formation of the enzymatic dephosphorylation product, which showed the same retention time with standard sample of compound 4. These results together proved that probe DHXP can be dephosphorylated efficiently by ALP and the dephosphorylation process can lead to the “turn on” of fluorescence.


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Figure 2. HPLC chromatograms of DHXP (10 µM) incubated with ALP (0.2 U/mL) at different time intervals.

Next we conducted concentration-dependent studies by various concentrations of ALP (0–0.1 U/mL) while keeping the concentration of DHXP constant at 5 µM. The fluorescence intensity was measured every 2 min for 30 min. As observed in Figure 3A, a sharper fluorescence intensity increase was obtained when high concentrations of ALP were used. This implied that the increase of the ALP concentrations resulted in a higher rate of cleavage reaction, leading to expedited hydrolysis process of DHXP. A good linear relationship was obtained when plotting the fluorescence intensity at 700 nm against ALP concentrations from 0 to 0.1 U/mL within the time period of 2 min (Figure 3B, S3). The regression equation was determined as F700nm = 4000000[ALP] + 23164 with a coefficient R2 = 0.9959. Based on 3σ/s method, the limit of detection (LOD) was calculated to be 0.07 U/L, which was much lower than most of the reported fluorescent probes (Table S1), indicating the high sensitivity of DHXP. When ALP was spiked in HEK 293 (an ALP-negative cell line) cell lysates, the LOD was calculated to be 0.063 U/L (Figure S4). To determine the endogenous ALP activity in HeLa or HEK 293 cells, we assayed DHXP or 4-MUP (a commercial ALP probe) in 0.182 mg/mL cell lysates. According to the standard curves of DHXP or 4-MUP (Figure 3B, S5), we found that 0.013 ± 0.00039 U/mL of ALP was detected by DHXP in HeLa cells, which is comparable to 0.014 ± 0.00051 U/mL of ALP detected by 4-MUP. In HEK 293 cells, 0.0021 ± 0.00013 U/mL of ALP detected by DHXP is similar to 0.0025 ± 0.00011 U/mL of ALP detected by 4-MUP. The kinetic parameters, including the Michaelis constant (KM), the catalytic efficiency constant (kcat/KM), and the turnover number (kcat), were then investigated. To do this, we incubated various concentrations of DHXP (0.5–20 µM) with ALP (0.003 U/mL) and then monitored the fluorescence change over 30 min. The reaction rate was found to increase along


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with increasing DHXP concentrations (Figure 3C, S6). Using the Lineweaver–Burk equation 1/V0 = KM/Kcat[E0][S] + 1/kcat[E0], where [E0] is the concentration of ALP, the slope and intercept were obtained (Figure 3D). KM, kcat and kcat/KM were determined to be 21.2 µM, 2.14 S1

and 1.01 105 M-1s-1, respectively. The value of kcat/KM of DHXP was higher than that of two

commercial ALP probes 4-MUP (kcat/KM = 7.7 103 M-1s-1) and ELF-97 (kcat/KM = 3.0 104 M-1s-1), suggesting high catalytic efficiency of ALP towards DHXP.

Figure 3. (A) Time-dependent fluorescence intensity increment at 700 nm using DHXP (5 µM) with different amount of ALP in 30 min. (B) Linear plot of relative fluorescence intensity against various ALP concentrations (0.001-0.1 U/mL) after 2 min of incubation. (C) Time-dependent fluorescence intensity increment at 700 nm using 0.003 U/mL ALP with different amount of DHXP. (D) Lineweaver-Burke plot of the hydrolysis of DHXP by ALP.


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Figure 4. (A) Fluorescence intensity change of DHXP (5 µM) upon incubation with different proteins (0.17

µg/mL of ALP, acid phosphatase (ACP), phosphodiesterase (PDE), and 100 µg/mL of lysozyme, esterase, BSA, avidin, trypsin) in 10 mM Tris-HCl buffer (pH 7.4). (B) Inhibition assay of ALP with sodium orthovanadate.

Selectivity Experiments and ALP Activity Assays. Selectivity is an important criterion to assess the suitability of the probe in biological application. In order to explore the selectivity of our probe, ALP or control proteins such as acid phosphatase (ACP), phosphodiesterase (PDE), trypsin, esterase, BSA, avidin or lysozyme were incubated with DHXP (5 µM) for 10 min, followed by fluorescence measurement. Figure 4A presents the fluorescence intensity of the probe after incubating with 0.17 µg/mL of ALP, ACP, or PDE or 100 µg/mL of other proteins. As expected, the fluorescence intensity resulting from ALP is significantly higher than that from other proteins. This result clearly indicates that the probe is selective for ALP. Fluorescent probe can be used to screen the inhibitors of enzymes. We next carried out inhibition experiments with DHXP. A common inhibitor for phosphatases, sodium orthovanadate, was used for the experiments.41 ALP was first incubated for 30 min with inhibitor of different concentrations (0.01, 0.05, 0.1, 0.5, 1, 3, 5, 10, 30, 50, 100, 500, 1000 and 2000 µM).


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Probe solution (5 µM) in Tris-HCl buffer was then added. After further incubation for 30 min, fluorescence spectra were recorded and the intensity was plotted as a function of natural logarithm of inhibitor concentrations. As revealed in Figure 4B, the fluorescence intensity was substantially reduced when the inhibitor concentrations were increased. The IC50 for sodium orthovanadate was calculated to be 7.51 µM, which is in good agreement with previous reported data. These results unambiguously show that DHXP can be used for both the detection of ALP activity and the screening assay of ALP inhibitors.

Figure 5. Fluorescence images of DHXP (2 µM) in HeLa cells and HEK 293 cells. Scale bar = 25 µm.


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Cell Imaging. Prior to cell imaging experiment, we first performed standard MTT assay to evaluate the cytotoxicity of DHXP. The cell viability remains almost unchanged after incubating with 20 µM DHXP for 24 h (Figure S7), indicating good biocompatibility of DHXP. Encouraged by this result, we further used the probe to detect endogenous ALP activity in complex biological environment. HEK 293 (ALP-negative) and HeLa cells (ALP-positive) were selected as they have been reported to express ALP at different levels.42 Both cells were put under 10-min incubation with 2 µM DHXP and then washed thoroughly to remove excess DHXP before cell imaging. As shown in Figure 5, HeLa cells showed stronger fluorescence, indicating high expression level of ALP inside HeLa cells. In contrast, HEK 293 cells displayed relatively weak red fluorescence, suggesting ALP is expressed at low levels in this cell line. Time course experiments indicated a rapid cell internalization ability of the probe (Figure S8). In addition, inhibition experiments showed that no noticeable fluorescence signal was observed in both cells after pre-incubation of inhibitor Na3VO4. This indicated that ALP activity can be efficiently inhibited by treatment of Na3VO4. Taken together, these results demonstrated that the probe can be employed to measure the enzymatic activity of ALP in cells. In Vivo Imaging in Mice. Our as-prepared DHXP was demonstrated to display an NIR emission property, which is widely accepted as a powerful strategy for in vivo imaging due to its deeper tissue penetration. It was therefore put under further test and used to probe the ALP activity in a mouse model. The Kunming mice were randomly divided into three groups. Group one received i.p. injection of 200 uL of DMSO to serve as a control. Group two received i.p. injection of 200 µL of DHXP (10 mM). Group three received an i.p. injection of 40 µL of sodium orthovanadate (100 mM) and incubated for 30 min, after which an i.p. injection of 200 µL of DHXP (10 mM) was given. After 20 min, the mice were imaged with a Night OWL IILB


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983 small animal in vivo imaging system. As observed in Figure 6, mice treated with DHXP only exhibited notably higher fluorescence (pseudo color) than untreated mice (Figure 6A) or mice pretreated with phosphatase inhibitor (Na3VO4) (Figure 6B). In time-dependent experiments, the mice were given an i.p. injection of 200 µL of DHXP (10 mM). Images were then taken at specific time intervals. As illustrated in Figure 7, fluorescence increased gradually with time, which can be attributed to the interaction of probe DHXP with endogenous ALP. The strong fluorescence signals of the probe unambiguously demonstrated that it could act as a good imaging agent for in vivo ALP assay.

Figure 6. (A) Representative fluorescent images (pseudo color) of the mice: (A) negative control, no Na3VO4 or probe DHXP was injected; (B) Na3VO4 was injected into the intraperitoneal (i.p.) cavity of mice. Probe DHXP was subsequently injected into the i.p. cavity; (C) probe DHXP was injected into the i.p. cavity of the mice; (D) Quantification of the fluorescence intensity of group A- group C.


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Figure 7. Representative fluorescence images (pseudo color) of a Kunming mouse that received an i.p. injection of DHXP. Images were taken after incubation for (A) 0 min; (B) 1 min; (C) 5 min; (D) 20 min; (E) 30 min; and (F) 40 min; respectively. (G) Relative fluorescence intensity of A-F.

CONCLUSION

We have successfully synthesized a reaction-based NIR fluorescent probe for sensitively and selectively detecting ALP activity. The probe is capable of detecting ALP activity in NIR region. The LOD of the probe is as low as 0.07 U/L, which is superior to most of the probes reported previously. The probe possesses unique properties, including good solubility, high reactivity, high cell permeability and remarkable NIR emission. We envision that the probe can be highly applicable for in vivo imaging and ALP assay.


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EXPERIMENTAL SECTION Materials and Chemicals. All chemicals were purchased and used directly without purification, unless otherwise mentioned. The reactions that used air- or moisture sensitive reagents were carried out in dried glassware under N2 atmosphere. The reaction progress was examined by thin-layer chromatography (TLC; Merck 60F-254). Merck silica gel 60 (70−200 mesh) was employed for column chromatography purification. Mass spectra were recorded by PC Sciex API 150EX ESI-MS system. 1H NMR, 13C NMR, or 31P NMR spectra were recorded by a Bruker 300 MHz or 400 MHz NMR spectrometer. pH values were determined with a Five Easy FE20 pH meter. UV absorption spectra and fluorescence spectra of the probe were measured by Shimadzu 1700 UV/vis Spectrometer and Fluoro-Max-4 fluorescence photometer, respectively. Fluorescence images of cells were recorded by a Leica TCS SP5 Confocal Scanning Microscope. Measurement of Absorption and Fluorescence. Suitable amount of DMSO was added to DHXP to prepare stock solution of 10 mM. For the measurement studies, the DHXP stock solution was diluted in 10 mM Tris-HCl buffer at pH 7.4 to obtain a final solution of 5 µM. For absorbance studies, 5 µM DHXP and 0.1 U/mL ALP were mixed together, and the absorbance was measured using Shimadzu 1700 UV/vis Spectrometer. For selectivity experiments, 0.1 U/mL ALP or other proteins (2 U/mL) were mixed with 5 µM DHXP for 30 min in buffer. Fluorescence spectra of the probe were recorded by a FluoroMax-4 fluorescence photometer in a 10-mm quartz cuvette. The excitation and emission wavelength was set at 680 nm and 690−850 nm, respectively. Detection limit. The detection limit studies were carried out following published procedures. Briefly, detection limit was estimated with the formula below:


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LOD =3σ/s, σ=

∑()

whereby x represents the mean of the blank samples; xi represents the values of blank samples ; n is the number of blank measurements and s is the calculated slope of linear regression equation. Fluorescence Microscope Experiments. HEK 293 cells and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). For the microscopy experiment, approximately 1 105 cells were cultured in confocal 20-mm dishes at 37 °C for one day prior to the addition of DHXP. The cells were incubated with DHXP (2 µM) for 10 min at 37 °C, and then the cells were washed twice with PBS in order to remove excess DHXP in the medium. For inhibition experiments, 10 mM Na3VO4 was pre-incubated for 20 min with the cells. DHXP was then added and the mixture was further incubated for 10 min before cell imaging. Fluorescence images were recorded by a Leica TCS SP5 Confocal Scanning Microscope using 633 nm as the excitation wavelength and 700 to 800 nm as the emission wavelength. Cell viability measurement with HeLa cells. HeLa cells were cultured at a density of 1 104 cells/well in a 96-well plate at 37 °C, and exposed to various concentrations of DHXP for 24 h in 200 µL of DMEM medium containing 10 % FBS. After the supernatant in each well was removed and the cells were washed with PBS buffer twice, 200 µL of MTT solution (0.5 mg/mL in medium) was added. The mixture was then incubated for another 4 h. The medium was removed with care. 200 µL of DMSO was subsequently added to dissolve the precipitates. Plate


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reader (Powerwave XS MQX200R) was used to record the absorbance at 570 nm (A570). All experiments were carried out in triplicate. Relative cell viability (%) was determined as a percentage corresponding to the untreated control. Kinetic studies. To determine the kinetic parameters of hydrolysis reaction by ALP, we dissolved DHXP in Tris-HCl buffer at pH 7.4 to prepare DHXP solution in different concentrations (0.5, 1.0, 2.5, 5.0, 10 and 20 µM), followed by the addition of ALP (0.003 U/mL) for dephosphorylation reaction. The enzymatic reaction was monitored through the change of fluorescence at 700 nm at different time intervals. The kinetic parameters (e.g., KM and kcat) of the hydrolysis reaction of DHXP were determined from Lineweaver-Burke plot. Animals. Adult male Kunming mice of 20˗25 g in weights were obtained from the Experimental Animal Centre of Xuzhou Medical University. All the animal experiments were approved by the Institutional Animal Care and Use of Xuzhou Medical College, according to the Chinese legislation on the use and care of laboratory animals. The animals had ad libitum access to standard animal feed and water in a room with temperature (22 ± 2 °C) and humidity (50 ± 10%) on a 12 h light/dark cycle. Before the experiments, the mice used in this study were acclimatized for one week. Fluorescence imaging in mice. The mice were anesthetized by i.p. injection of 10% chloral hydrate (0.04 mL/10 g), and their abdominal fur was removed. The mice were randomly selected and put into several groups. Subsequently, group one received i.p. injection of 200 uL of DMSO as the control group. Group two received i.p. injection of 200 µL of DHXP (10 mM). Group three received i.p. injection of 40 µL of sodium orthovanadate (100 mM) for 30 min and then incubated for 30 min, after which an i.p. injection of 200 µL of DHXP (10 mM) was given. After


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20 min, the mice were imaged with a Night OWL IILB 983 small animal in vivo imaging system. In the time-dependent experiment, an i.p. injection of 200 µL of DHXP (10 mM) was given to the mice. Images were then taken at specific time intervals.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Compound synthesis, photophysical properties of the probe DHXP, fluorescence intensity of DHXP with ALP, standard curve of 4-MUP, Michaelis-Menten plot, selectivity


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experiments, cellular toxicity of DHXP, time course cell imaging, NMR/ESI-MS spectra, examples of fluorescent probes for ALP AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected].

Author Contributions †

Y.T. and L.Zhang contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by the Research Grants Council of Hong Kong (11302415 and 21300714) and the National Natural Science Foundation of China (21202137, 21572190 and 21390411).


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