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A Facile and Sensitive Near-Infrared Fluorescence Probe for the Detection of Endogenous Alkaline Phosphatase Activity in Vivo Song-Jiao Li, Chunyan Li, Yongfei Li, Junjie FEI, Ping Wu, Bin Yang, Juan Ou-Yang, and Shi-Xin Nie Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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Analytical Chemistry

hydrogen selenide,55 nitroxyl,56 hydrazine,57 hydrogen peroxide,58 hydrogen sulfide,59 β-lactamase60 and 61 nitroreductase. However, the fluorescent probes based on hemicyanine dyes for ALP are still very rare. So, it is interesting and challenging to further broaden the application of hemicyanine-based NIR fluorescent probes in biosensing. Herein, a hemicyanine-based fluorescent probe named CyP is designed and the chemical structure is shown in scheme 1. The probe possesses a simple structure with easy synthetic steps. Probe itself is nonfluorescent because the phosphate group in the probe is a quenching and recognizing moiety. While, in the presence of ALP, the probe show NIR emission with the wavelength at 738 nm and exhibits high sensitivity to ALP with low detection limit. In particular, the NIR fluorescent probe was applied for the detection of endogenous alkaline phosphatase activity in biological samples such as cell, tissue and living animal successfully.

(m, 1H), 6.76 (t, J = 7.6 Hz, 1H), 4.43-4.37 (m, 2H), 2.69-2.67 (m, 4H), 2.09 (s, 6H), 1.79 (m, 2H), 1.47 (t, J = 7.2 Hz, 3H). 13 C NMR (100 MHz, CDCl3, Figure S2): δ 179.0, 158.2, 153.6, 152.0, 141.3, 139.9, 137.8, 133.1, 131.4, 131.0, 130.7, 130.2, 129.0, 128.8, 127.5, 125.9, 124.0, 122.7, 118.5, 114.7, 113.8, 111.1, 103.2, 53.0, 46.4, 29.8, 26.8, 22.8, 21.0, 14.2. 31P NMR (300 MHz, CDCl3, Figure S3) δ -5.27. MS (TOF, Figure S4) m/z calcd. for C31H31NO5P+ [M]+, 528.19; found, 528.19. Elemental analysis (%) calcd. for C31H31INO5P: C, 56.82, H, 4.75, N, 2.15; found: C, 56.81, H, 4.77, N, 2.14. Scheme 1. Synthetic route for CyP O HO P OH O

OH POCl3 O N

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EXPERIMENTAL SECTION CyOH

Reagents and Instruments. 2,3,3Trimethylbenzoindolenine, cyclohexanone, iodoethane, resorcinol, phosphorus oxychloride were purchased from Sigma-Aldrich (St. Louis, USA). ALP (alkaline phosphatase, calf intestine) was purchased from TaKaRa Biotechnology Corporation (Dalian, China). Note, one unit of the enzyme is defined as the amount that produces 1 µmol of 4-nitrophenol from 4-nitrophenylphosphate in a minute at 37 °C. Unless noted, all the chemicals were of analytical reagent grade and used as received without further purification. Nuclear magnetic resonance (NMR) spectra were measured on a Bruker Avance II NMR spectrometer (Germany). 1H NMR, 13C NMR and 31P NMR were conducted at 400, 100 and 300 MHz, respectively. Mass spectra (MS) was recorded on a Bruker Autoflex MALDI-TOF mass spectrometer (Germany). Element analysis was operated on Perkin Elmer 2400 elemental analyzer (USA). HPLC was carried out on a Agilent 1260 LC system with a C18 column (USA). The fluorescence spectra were obtained on a Perkin Elmer LS-55 fluorescence spectrometer (USA). The absorption spectra were collected on a Perkin Elmer Lambda 25 UV/VIS spectrophotometer (USA). Fluorescence imaging of cells and tissues were carried out by an Olympus FV1000 fluorescence microscope (Japan). Fluorescence imaging of mice was performed on an IVIS Lumina XR small animal optical in vivo imaging system (USA). Synthesis. CyOH was prepared as described previously.62,63 CyP was conveniently synthesized following the synthetic route depicted in Scheme 1. Phosphorus oxychloride (61.3 mg, 0.4 mmol) was added dropwise to a solution of CyOH (82.4 mg, 0.2 mmol) in anhydrous pyridine (10 mL). After stirring at room temperature for 4 h, the resulting mixture was poured into ice and stirred overnight. After the removal of solvent under reduced pressure, the resulted residue was suffered from silica gel column chromatography for purification (CH2Cl2:CH3OH = 2:1) to afford a blue solid product. Yield: 70.9 mg (72%). 1H NMR (400 MHz, CDCl3, Figure S1): δ 8.51 (d, J = 8.4 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.91 (t, J = 8.4 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.79 (s, 1H), 7.76 (d, J = 6.8 Hz, 1H), 7.05 (d, J = 11.2 Hz, 1H), 7.03-6.95

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Fluorescent Detection for ALP Activity. The fluorescent detection for ALP activity was carried out according to the following procedures. ALP with different activities from 0.01 to 2.0 U was added into the solution (1 mL, pH 8.0) containing 0.53 mg CyP, 1 mM MgCl2 and 50 mM Tris-HCl. After the addition of ALP, the fluorescent spectrum of each of the solution was measured at 37 °C. The fluorescence emission spectra were measured with the excitation at 690 nm and the emission at 720-850 nm. The excitation slit and emission slit were both set at 10.0 nm. Cell Incubation and Fluorescence Imaging. The living HeLa cells were obtained from the Biomedical Engineering Center of Hunan University (Changsha, China) and cultured in DMEM (Dulbecco’s modiﬁed Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) at 37 ºC in a 5% CO2 atmosphere. Then three group of cells were treated differently and imaged. The first group of cells were used as control incubated in DMEM medium only. The second group of cells were incubated with the CyP (10 µM) at 37 °C for 30 min, followed by washing three times with PBS buffer solution before imaging. The third group of cells were treated with ALP inhibitor sodium orthovanadate (Na3VO4, 100 µM) at 37 °C for 30 min, then with CyP (10 µM) for another 30 min, followed by washing three times with PBS buffer solution before imaging. Fluorescence imaging were performed by confocal fluorescence microscope with excitation wavelength at 635 nm and emission wavelength at 680-780 nm. Preparation and Staining of Rat Liver Tissue Slices. Tissue slices were prepared from rat liver and a side of the tissue was cut flat using a vibrating-blade microtome. Then three tissue slice were treated differently and imaged. The first tissue slice was used as control. The second tissue slice was incubated with the CyP (10 µM) at 37 °C for 30 min, followed by washing three times with PBS buffer solution before imaging. The third group of cells were treated with Na3VO4 (100 µM) at 37 °C for 30 min, then with CyP (10 µM) for another 30 min, followed by washing three times with PBS buffer solution before imaging. Fluorescence imaging were performed by confocal fluorescence microscope with 2

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Upon the addition of ALP to a solution of CyP, the absorption band centered at 604 nm decreased significantly, and a new absorption band with the peak at 703 nm appeared and increased significantly (Figure 1a). The resulting absorption spectrum was similar to that of CyOH. Furthermore, the fluorescence signal of CyP displayed noticeable changes (Figure 1b). After adding ALP, the emission intensity at 738 nm was enhanced greatly within 20 min, and the resulting fluorescence spectrum was the same as that of CyOH exactly in both the emission intensity and the emission band. The reason for the extremely low background signal of CyP is attributed to the strong fluorescence quenching of the phosphate group. In the presence of ALP, the phosphate group could be cleaved and CyOH is thus produced with strong fluorescent signals. Optimization of Fluorescent Sensing of ALP. To construct a sensitive fluorescent probe, the sensing conditions need to be optimized. Initially, the effect of pH on the fluorescence of CyP was examined, since pH was a significant factor in enzyme reaction (Figure S5). The results show that the optimum pH for ALP is 8.0, so we chose Tris-HCl buffer solution with pH 8.0 in further experiments. Next, the effect of reaction temperature on fluorescence intensity was examined (Figure S6). The experimental results show that the change of fluorescence intensity reaches maximum at 37 °C. Therefore, 37 °C is selected in this study, which is consistent with the circumstance in human body. Furthermore, incubation time is another important factor for the enzyme reaction. The amount of CyOH produced by hydrolysis of CyP with ALP is dependent on the incubation time. As shown in Figure S7, the characteristic signal of CyP arises and increases linearly with time and the enzymatic reaction is almost completed in 20 min. So 20 min is used for ALP analysis owing to the fact that the sensitivity is enough for practical application. In addition, to optimize the concentration of CyP used in the assay, CyP with different concentrations was incubated in a constant ALP concentration. The enzyme kinetics was represented by Michaelis-Menten relation and the curve was showed in Figure S8a. To determine the kinetic parameters, the curve is converted to a linear plot using Lineweaver-Burke analysis (Figure S8b). According to the slope and intercept of the fitted line, Vmax and Km are 0.693 µM/min and 9.32 µM, which are close to those reported for ALP determined using other assay methods57. As is well known, the substrate concentration must be higher than its Km value. Thus, 10 µM was selected as the optimum CyP concentration for ALP determination. The detection of ALP activity. In order to examine the possibility of quantitative analysis of ALP activity, the experiments of concentration-dependent monitoring of enzymatic reaction were conducted. Figure 2 illustrates the fluorescence intensity of the probe gradually enhances with increasing activity of ALP. When 2.0 U/mL ALP is added, the fluorescence intensity increases around 10-fold. Moreover, the insert of Figure 2 illustrates that the probe exhibits a good linear relationship with ALP ranging from 0.01 to 2.0 U/mL. The detection limit is 0.003 U/mL (3σ/slope), which is low enough for ALP detection in biological samples.

excitation wavelength at 635 nm and emission wavelength at 680-780 nm. Fluorescent imaging in living Mice. 20-25 g Kunming (KM) mice were used and were kindly kept in all the experimental process. All animal experiments were carried out according to the regulations issued by The Ethical Committee of Xiangtan University. The abdominal fur of the mice was removed by an electric shaver. Then, the mice were divided into three groups and treated differently. The first group of mice was untreated as a control group. The second group of mice was given an intraperitoneal injection of CyP (10 µM). The third group of mice was given an intraperitoneal injection of Na3VO4 (100 µM) and then injection of CyP (10 µM). All the mice were anesthetized with chloral hydrate (10% in saline) before image and remained anesthetized throughout the image period. Finally, the mice were placed into the imaging chamber and imaged with the excitation at 660 nm and the emission at 740 nm.

RESULTS AND DISCUSSION (a) .18 .16

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Figure 1. (a) Absorption and (b) fluorescence spectra of CyOH (10 µM), CyP (10 µM) before and after reacting with ALP (2.0 U/mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex = 690 nm. Spectral Response of Probe CyP to ALP. Absorption and fluorescence spectra of probe CyP in the absence and presence of ALP were measured in Tris-HCl buffer solution (Figure 1). 3



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ALP Inhibitor Investigation. The probe CyP could also be used for the screening of potential ALP inhibitors. To demonstrate this possibility, sodium orthovanadate (Na3VO4), a well-known inhibitor for ALP, was tested. Figure S10 shows that the fluorescence intensity decreases significantly with the addition of increasing concentrations of the inhibitor. As a control, the effect of inhibitor Na3VO4 on the fluorescence of the reaction product (CyOH) was also examined. The result were shown in Figure S11 and no obvious fluorescent change was seen. These spectral data show that Na3VO4 can inhibit ALP activity rather than the fluorescence of CyOH. As shown in the insert of Figure S10, the IC50 value (the concentration of an inhibitor required to achieve 50% of inhibition efficiency) of Na3VO4 was estimated to be 141.9 µM, which are in good agreement with the reported value determined by other methods.64,65 The results clearly demonstrate that our assay could not only be used for the monitoring of ALP activity but also for the screening of potential ALP inhibitors. Reaction Mechanism. On the basis of the evidence from the fluorescence spectra, a possible reaction mechanism of CyP and ALP was proposed in Scheme 2. ALP-catalyzed cleavage of the phosphate group in CyP induced the transformation of CyP into CyOH. CyP itself is nonfluorescent as the hydroxyl group of fluorophore is protected with a phosphate group, which diminishes the electron donating ability of hydroxyl group and hinderes the intramolecular charge transfer (ICT) process. After the addition of ALP, phosphate group is cleaved by ALP, resulting in the recovery of the ICT process and the production of the strong signal. Scheme 2. Proposed reaction mechanism for CyP and ALP

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Figure 2. Fluorescence spectra of CyP (10 µM) upon the addition of ALP (0, 0.01, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 U/mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex = 690 nm. Inset: the plot of fluorescence intensity versus ALP activity. Selectivity of CyP toward ALP. Selectivity is an important factor to evaluate the performance of probe. To verify that this probe is selective to ALP, a number of enzymes in serum such as acid phosphatase (ACP), phosphodiesterase (PDE), glucose oxidase (GOX), glucose dehydrogenase (GDH), galactosidase (GAL), acetylcholinesterase (AChE), trypsin, thrombin were tested. As shown in Figure 3, none of the enzymes had the ability to recover the fluorescence except ALP. Meanwhile, the selectivity was also investigated by examining various potential interfering substances such as biothiols (cysteine, homocysteine, glutathione), amino acids (Pro, Tyr, Asn, Met, Phe, Leu, Asp, Orn, Ala, Trp, His, Ser, Gly, Val, Lys, Glu, Arg, Ileu, Thr) and metal ions (K+, Ca2+, Zn2+, Na+, and Mg2+) (Figure S9). The results indicated that no significant fluorescence changes of the probe were observed, demonstrating its high selectivity to ALP.

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In order to prove the reaction mechanism, HPLC experiments were carried out and the results were shown in Figure S12. In the HPLC chromatogram, CyP itself displayed a signal peak at 3.7 min. Upon adding ALP, a decrease in the CyP signal along with the appearance of a signal at 6.6 min was seen. The peak is attributed to the formation of the enzymatic dephosphorylation product, which shows the same retention time with standard sample of CyOH (6.6 min). These results together prove that probe CyP can be dephosphorylated efficiently by ALP causing the transformation of CyP into CyOH. Meanwhile, 31P NMR experiments were conducted to prove the mechanism. The 31P signals of CyP disappeared, indicating that ALP catalyzes the dephosphorylation process of the probe. Furthermore, the mass spectrometry analysis was also investigated to confirm the proposed mechanism. For CyP, the unique peak was at m/z = 528.19. Upon the addition of ALP, the peak at m/z = 528.19 disappeared and the main peak at m/z = 448.23 corresponding to CyOH occurred, which indicates the ALP-induced transformation of CyP into CyOH (Figure S13).

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Figure 3. Fluorescence response of CyP (10 µM) to ALP (2 U/mL), ACP (2 U/mL), PDE (10 mg/mL), GOX (10 mg/mL), GDH (2 U/mL), GAL (2 U/mL), AChE (2 U/mL), trypsin (10 mg/mL), thrombin (2 U/mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded within 20 min. λex / λem = 690 / 738 nm. 4



observed from the tissues pre-treated with Na3VO4, and then treated with probe CyP (Figure 5c). The fluorescence signal of the tissue slice with probe CyP at different tissue depths was collected in the Z-scan mode (Figure 5d). Probe CyP is successfully applied for imaging in tissue, with imaging depths of 40-120 µm. All these results indicate that probe CyP has outstanding tissue penetrating and staining ability.

To further understand the response of CyP to ALP, we performed density functional theory (DFT) calculation with the B3LYP/6-311G method by using the Gaussian 09 package. The optimized structures of CyP and CyOH were presented in Figure S14. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of CyP and CyOH were presented in Figure S15. CyOH is composed of hydroxyl donor (push) linking with hemicyanine acceptor (pull), which makes an ICT from the donor to the acceptor construct a push-pull system resulting in the fluorescence turned on. In contrast, the phosphate moiety (electronwithdrawing group) in CyP hinders the ICT process, which enables the fluorescence turn off. The HOMO-LUMO energy gaps of CyP and CyOH were calculated as 2.37 and 2.37 eV, respectively. The theory calculations are in good agreement with the experimental results, which prove the proposed mechanism. Fluorescence Imaging in Living Cells. The cytotoxicity of CyP was performed by MTT assay in HeLa cells. Figure S16 shows that the cell viability is more than 98% with different concentrations of CyP from 0 to 30.0 µM, suggesting that CyP exhibits almost no cytotoxicity in cells. With the low cytotoxicity, the probe was used for detecting endogenous alkaline phosphatase activity in living cells. The probe’s cell imaging capability was evaluated using HeLa cells, in which ALP is overexpressed.66,67 The imaging for ALP in the cells was shown in Figure 4. HeLa cells in the absence of CyP showed no fluorescence, while significant red fluorescence was observed after incubation with CyP, which was caused by the existence of ALP in HeLa cells. To further prove the fact that the fluorescence was triggered by ALP in HeLa cells, the cells was pretreated with ALP inhibitor (Na3VO4), and then incubated with CyP. As expected, only negligible fluorescence was seen, indicating that this probe can work well to monitor endogenous ALP activity in living cells. Probe

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Figure 5. Fluorescence images of tissues. (a) Control. (b) Tissue slice was incubated only with probe CyP. (c) Tissue slice was pretreated with Na3VO4, and then treated with probe CyP. (d) The confocal z-scan imaging sections at different depths for 0, 20, 40, 60, 80, 100, 120, 140, 160 µm. Visualizing ALP in vivo. Near-infrared light can penetrate tissue deeply, has low background autofluorescence interference and cause little damage to biosample. Thus, the potential application of NIR probe CyP in living animal imaging was explored. Kunming (KM) mice were selected as our model and allocated into three groups. The first group of mice was untreated as a control group, no fluorescence was observed (Figure 6a). The second group of mice was given an intraperitoneal injection of CyP, strong fluorescence signal was noticed (Figure 6b). The third group of mice was injected with Na3VO4 and then CyP, only negligible fluorescence was observed (Figure 6c). With the above results taken together, it can be concluded that CyP could image endogenous ALP in vivo.

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Figure 4. Fluorescence images of HeLa cells. From left to right: unstained control, cells treated with CyP only, cells treated with Na3VO4 and then CyP. Fluorescence Imaging in Tissues. To further show the advantage of NIR fluorescent probe CyP, we then proceeded to investigate the applicability of CyP for the detection of ALP in tissues. As shown in Figure 5a, the tissue slice not incubated with CyP showed no fluorescence, while the tissue slice incubated with CyP, demonstrated strong fluorescence (Figure 5b). Meanwhile, only negligible fluorescence was

Figure 6. Fluorescence images in vivo. (a) Control. (b) a mouse given an injection of CyP. (c) a mouse was given an injection injected with Na3VO4 and then probe CyP.

CONCLUSIONS In summary, we reported a new NIR fluorescent probe based on hemicyanine dye for the detection of endogenous alkaline 5


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phosphatase (ALP) activity. The probe possesses a facile structure, which can be obtained by easy synthetic steps. Moreover, the fluorescence emission of the sensing system is at NIR region that is suitable for potential applications in bioimaging. In addition, the probe exhibits high sensitivity to ALP with 10-fold enhancement when 2.0 U/mL ALP is added. The reaction mechanism is that ALP-catalyzed cleavage of the phosphate group in CyP induced the transformation of CyP into CyOH, and is proved by HPLC, 31P NMR, MS and DFT calculation. Especially, this probe can be used to detect and image endogenous ALP in living cells, tissue and mouse. It is envisioned that the NIR probe will be widely applied in the other animals and even in humans for detection of ALP in the future.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. NMR and MS spectra, supplementary spectral data, DFT calculations, cell cytotoxicity, and comparison table (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +86-731-58292477.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21005068, 21475114, 21505113), Hunan Provincial Natural Science Foundation (2015JJ6104), China Postdoctoral Science Foundation funded project (2014M552140, 2015T80876), State Key Laboratory of Chemo/Biosensing and Chemometrics Foundation (2015007), Scientific Research Fund of Hunan Provincial Education Department (15K125, 16B252), Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization, Hunan Provincial Innovation Foundation For Postgraduate.

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for TOC only

690 nm

690 nm

Fluorescence Off

Fluorescence On

738 nm

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A Facile and Sensitive Near-Infrared Fluorescence ... - ACS Publications

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