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Ratiometric Fluorescent Strategy for Localizing Alkaline Phosphatase Activity in Mitochondria Based on ESIPT Process Peng Zhang, Caixia Fu, Qian Zhang, Shasha Li, and Caifeng Ding Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02917 • Publication Date (Web): 05 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019
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Analytical Chemistry
Ratiometric Fluorescent Strategy for Localizing Alkaline Phosphatase Activity in Mitochondria Based on ESIPT Process Peng Zhang, Caixia Fu, Qian Zhang, Shasha Li and Caifeng Ding* Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China. * E-mail:
[email protected], Fax: +86 53284022681 ABSTRACT: Fluorescent probes are powerful tools for detecting and mapping the species of interest in vitro and vivo. Although the probes always show high selectivity and/or sensitivity, they are usually affected by some factors, such as detecting conditions and the probe concentrations. Ratiometric fluorescent strategies, possessing advantage of low background noise, would solve the problem effectively and lead to a higher sensing performance. Thus, an ESIPT based ratiometric probe (HBTP-mito) was developed on the basis of a phosphorylated 2-(2′-hydroxyphenyl)-benzothiazole derivative for the determination of ALP activity. HBTP-mito is water-solubility and emits green fluorescence in TBS buffer due to the blockage of ESIPT. Upon the introduction of ALP, phosphate ester of HBTP-mito was hydrolyzed and the ESIPT process was restored. Accordingly, the fluorescence at 514 nm decreases, while emission at 650 nm shows a “turn-on” response. The ratio of intensity (I 514 nm/I 650 nm) decreases linearly with ALP activity increasing from 0 to 60 mU/mL, obtained an LOD of 0.072 mU/mL. The favorable performance of the probe enables its application not only in detection of ALP activity in biological samples but also in the localization of the ALP levels in living cells and in vivo.
Molecular fluorescent probes are well-suited tools to visualize the spatial and temporal distribution of species of interest within living cells. 1−3 As specific enzymes usually plays an important role in the biological processes, molecular fluorescent imaging of target enzyme in cells and in vivo is important for basic research and applied medicine. 4,5 ALP is a kind of hydrolase which catalyzes the dephosphorylation process of phosphate substrates, such as carbohydrates, proteins and nucleic acids. 6,7 The activity of ALP with abnormal levels in serum are related to a variety of diseases, such as diabetes, bone disease, liver dysfunction, and prostate cancer. 8−10 Thus, fluorescent sensing assays for ALP with highly selective and sensitive are of great significance for the diagnosis of ALP related diseases. 11
However, only several ratiometric probes for the detection of ALP have been reported. 13, 27, 28 Otherwise, they often encountered disadvantages in complicated process of probe synthesis and/or emitted in ultraviolet region, which limited their practical application and biological imaging. ESIPT based fluorophores having been used to develop ratiometric probes. Upon the photo-excitation, the change of elecron density is trigged, with proton migration between the nearby donor and acceptor atoms (e.g. O vs. N), which makes a typical ESIPT procedure. When an O atom is employed as the donor, the molecule of enol form ascts as the ground state with covalent binding between O and H atom, which is broken at the excited state as an emissive ketone form. Contributed by the different energy levels between the original absorption of enol form and the product emission of ketone tautomer, a considerable Stokes shift could be hence achieved in fluorescence spectra. 29, 30 A typical ESIPT group, 2-(2′Hydroxyphenyl)-benzothiazoles (HBT) was often used to design fluorescence probes to detect many substances in ratiometric manners. 31-33 In particular, extension of HBT πconjugation at ortho or para positions of phenol will provide sensing assays with red or NIR emission. 34-36
In passed years, some fluorescent substrates for ALP activity have been developed, 12–16 for example βnaphtholphosphate, 17 2-carboxy-1-naphthyl phosphate, 18 trifluoromethyl-β-umbelliferone phosphate, 19 8-quinolyl phosphate 20 and cyanine phosphate. 21 Although these fluorescence probes showed highly selectivity or sensitive to ALP, they may be affected by factors such as environmental conditions (eg temperature, pH, solvent polarity, etc.) and the concentration of the probe. 22 Ratiometric probe allows measurement of emission at two wavelengths with inherent advantages of high sensitivity and low background, which make them excellent candidates not only in vitro bioassays, but also in the detection of target analytes in vivo. 23−26
Mitochondria, as the most important organelle for energy metabolism, are semi-autonomous organelles containing DNA and RNA, in which DNA and RNA can be transcribed and replicated. Therefore, mitochondria contain the enzymes needed to catalyze these physiological processes, as well as enzymes that catalyze protein synthesis. It is reported that the activity of ALP in mitochondria is relatively high. 37 ALP can 1
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catalyze the removal of 5'-phosphate groups from DNA, RNA, ribonucleoside triphosphate and deoxyribonucleoside triphosphate, and plays important roles in the synthesis of DNA and RNA in mitochondria. 38 Therefore, it is necessary to detect ALP activity in mitochondria.
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Scheme 1. Proposed sensing mechanism of mitochondriatargeting probe HBTP-mito for ALP, and the enol or keto structure of enzymatic product HBT-mito in the ESIPT process.
Kinetic of probe HBTP-mito. To acquire the kinetic constants, HBTP-mito of varying concentrations (from 1 to 10 μM) was hydrolyzed by ALP respectively. The hydrolysis reaction was monitored by testing I 514 nm/I 650 nm after the incubation of HBTP-mito and ALP in TBS buffer solution (pH = 8.0) for 40 min at 37 °C.The initial reaction velocity was obtained from slopes of process curves, and parameters including Km (1.476 μM) and Vmax (0.625 μM·min-1) for HBTP-mito were provided by Line weaver-Burk plot as shown in Figure S14. Cell cytotoxicity and fluorescent imaging in living cells. The cells were seeded in Petri dishes with high-glucose DMEM, 10 % fetal bovine serum, 1 % penicillin and 1 % streptomycin for 24 h before use. Cell cytotoxicity was studied by CCK8 assay. HeLa cells were seeded in 96-well plates for 1 day before treated with HBTP-mito or HBT-mito of different concentrations (2-50 μM). After being washed with DPBS, the cytotoxic effect was examined by CCK8 assay. For intracellular sensing assay, cells were treated with probe HBTP-mito (10 μM) for 30 min. As for control experiments, cultures were pretreated with inhibitors Na3VO4 (100 μM or 200 μM) and NaH2PO4 (1 mM or 3 mM) for 30 min respectively, and then probe HBTP-mito (10 μM) for another 30 min. Fluorescence imaging was captured on a Leica SP5 laser scanning confocal microscopy with a excitation wavelength set at 405 nm and fluorescence wavelengths at 480 - 540 nm for green channel and 650 - 750 nm for red channel. Fluorescent imaging in vivo. The animal studies were approved by the Medical Ethics Committee of Nanjing University. The animal procedures were carried out in accordance with the guidelines for the care and use of Laboratory Animals of Nanjing University. Female BABLc nude mice were obtained from Shanghai Slac Laboratory Animal Co., Ltd at 5-6 weeks of age. The in vivo imaging experiments were carried out at Jiangsu Keygen Biotech Co., Ltd. using IVIS® Lumina LT Series III PerkinElmer. For in vivo imaging, the probe HBTP-mito (30 μL, 50 μM in water) was injected into the tumored mouse. As a control experiment, another mouse was pretreated by the inhibitor, Na3VO4 (2 μL, 100 mM in water) for 30 minutes, and then the same dose of HBTP-mito was injected. The changes of the fluorescence intensity of the tumored mice were recorded with time. The emission filters were set as 520 nm for green channel and 650 nm for red channel respectively under the excitation of 405 nm.
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RESULTS AND DISCUSSION
Synthesis of HBT-mito and HBTP-mito. Details of the synthesis and characterization of HBT-mito and HBTP-mito are described in the supporting information (SI). General procedures for spectra measurements. The stock solutions of HBT-mito and HBTP-mito (5.0 mM, respectively) were prepared in analytical DMSO and stored in refrigerator at 4 °C. The probe HBTP-mito was diluted to 10 μM with TBS buffer (10 mM) of pH at 8.0 using the stock solution for absorption and fluorescence spectral exploration. Detection limit. The limit of detection (LOD) for ALP was determined by 3σ/k based on the linearity between A 358 nm/A 496 nm or I 514 nm/I 650 nm and ALP concentration, σ represents the standard deviation of the blank samples (n=20) and k stands for the slope of linear regression equation of A 358 nm/A 496 nm or I 514 nm/I 650 nm vs the ALP concentration.
To design a probe with ratiometric manners for monitoring ALP in mitochondria, the traditional ESIPT group HBT was employed as fluorophore and the pyridinium salt was introduced as a mitochondria-target. Compound HBT-mito and its phosphorylated product HBTP-mito were synthesized as descripted in Schem S1. The synthetic products were characterized by 1H, 13C, 31P NMR and HR-MS (Figures S1S7). The optical properties of HBT-mito and HBTP-mito were first investigated. As shown in Figure S8, just like other ESIPT compounds, the pink solution of HBT-mito (10 μM) in 10 mM TBS with a pH value of 8.0 exhibited two obvious absorption bands at 310 and 496 nm, and two corresponding emission bands at 476 and 650 nm, which were originated from its enol form and keto form, respectively. 40 The ratio of two peaks was determined by content of two structures that
In this work, an HBT based ratiometric fluorescence probe (HBTP-mito) with ESIPT attributed to ALP activity in vitro and in vivo was reported, in which pyridinium salt was designed as the mitochondria-target 39 (because they possess superior membrane permeability and could enter the mitochondria in cells within a time) and HBT served as ESIPT executive group. Upon phosphorylation, HBTP-mito shows a fluorescence at about 514 nm (enol form) because of the blockage of the ESIPT process. ALP can catalyze the hydrolysis of various phosphate esters rapidly with excellent substrate specificity. With the introduction of ALP, HBTPmito was hydrolyzed and P–O bond in HBTP-mito was cleaved. Therefore, HBT was released and the ESIPT process was activated (Scheme 1). The emission of the probe changed from green to red with a rather large Stocks shift about 260 nm. Accordingly, the fluorescence at 514 nm (enol form) decreased, while 650 nm (keto form) showed a “turn-on” response. The ratio of I 514 nm/I 650 nm decreased linearly upon the ALP activity increase to 60 mU/mL. This favorable performance of the probe made us explore that it not only in detection ALP activity in biological samples but also in the localization of ALP levels in live cells (HeLa, A549 and HUVEC) and vivo.
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yellow to red, allowing a “nake-eye” determination for ALP (Figure 1a inset). The corresponding fluorescence at 514 nm underwent agradual decrease alone with an increase at 650 nm incubated from 0 to 120 min (Figure 1c). The ratiometric of absobance (A 496 nm/A 358 nm) and intensity (I 514 nm/I 650 nm) achieved the maximum when the probe was incubated with ALP for about 60 min at 37 oC. This indicated that the enzymatic reaction has reached saturation state. As we all know, enzymatic reaction is always temperature dependent. In order to further understand the temperature effect on ALP activity, the ALP-catalyzed hydrolysis reaction under different temperatures from 0 to 45 °C (0 °C, 10 °C, 20 °C, 37 °C and 45 °C were selected) was studied. As shown in Figure 1b and 1d, the catalytic activity of the enzyme was very weak at a low temperature, such as 0 oC, because the A 496 nm/A 358 nm and I 514 nm/I 650 nm changed slowly. While the temperature was above 37 °C, 45 oC for example, the A 496 nm/A 358 nm or I 514 nm/I 650 nm changes were significantly slower than that of 37 °C, because ALP lost activity gradually at such a high temperature. Therefore, we chose the optimum reaction temperature, 37 °C, as a reaction temperature for subsequent studies. (a)
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undergo ESIPT process. 41,42 Thus, we hypothesized that if the -OH group in HBT-mito was replaced, ESIPT of HBT-mito was blocked and the spectral characteristics should change. Excitingly, the result went as we expected, for compound HBTP-mito, when the hydroxyl radical of HBT-mito was phosphorylated, only a broad absorption band located at 358 nm was observed from its yellow solution, and it emitted a single fluorescent band at 514 nm with a quantum yield of 0.21 in the same conditions with HBT-mito. Therefore, characteristic adjustment of hydroxyl group in HBT-mito can regulate ESIPT to provide huge wavelength shifts in the probe spectra without and with analyte. On the contrary, ALP can hydrolyze phosphate ester and effectively release hydroxyl group. Based on this, the probe HBTP-mito was designed for detecting ALP as a ratiometric fluorescent probe through adjustment of ESIPT process. In order to set up a fluorescent probe with good sensing performance, it is necessary to optimize the sensing conditions. The optical concentration dependent property of the probe HBTP-mito in TBS was first investigated to optimize the concentration of HBTP-mito. As shown in Figure S9, phosphorylated HBTP-mito, in which the ESIPT process was inhibited, exhibits a maximum absorbance at 358 nm (ε = 33420 L/mol·cm). Also, it showed increasing linearly with concentration ranging from 1 to 30 μM, from which the good water solubility was proved. The corresponding fluorescence of the enol form of the probe at 514 nm increased linearly when its concentration was below 10 μM (Figure S10). However, when the concentration of HBTP-mito was further raised up to 30 μM, the intensity increased deviated from linear relationship, which may be due to the self-quenching effect of fluorescent dyes. Therefore, we chose 10 μM HBTPmito in the following sensing experiments. The pH effect of fluorescence intensity of HBTP-mito and the mixture of HBTP-mito/ALP was also evaluated. The TBS buffer with pH value from 5.0 to 9.5 were investigated. From the results showed in Figure S11, for HBTP-mito only, the value of I 514 nm/I 650 nm grew very slowly as the solution turned more alkali, indicating that HBTP-mito was relative stability at this condition. The ratiometric fluorescent intensity (I 514 nm/I 650 nm) of the solution changed relatively large when the pH was in the range 5.0–8.0 upon treated with ALP. While the pH value of the reaction solution was above 8.5, the changes of I 514 nm/I 650 nm became very small due to the inhabitation of ESIPT process of HBT-mito in alkaline conditions. These results indicated that HBTP-mito could respond to ALP well under physiological pH. Considering that ALP showed maximum activity at pH range of 8.0–10.5, 29 we chose condition in TBS buffer with pH value of 8.0 for the subsequent experiments. The incubation time of the enzymatic hydrolysis from HBTP-mito to HBT-mito was also investigated. Both of the absorption and fluorescence spectra of HBTP-mito were measured every two min in the presence of ALP (100 mU/mL) at 37 oC. As shown in Figure 1, 10 μM HBTP-mito in TBS buffer (pH 8.0) was yellow and emited green fluorescence before ALP was added. The absorption at 358 nm decreased accompanied by agradual increasing at 496 nm, and an obviously isosbestic point appeared at 390 nm during the incubation. In addition, such a large change of absorption behaviour triggered the color of the mixture turned from
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Figure 1. Time dependence of absorbance (a) and fluorescence (c) of HBTP-mito from 0 to 120 min in TBS at 37 °C with a pH value of 8.0 upon addition of ALP. Plots of A 496 nm/A 358 nm (b) and I 514 nm/I 650 nm (d) of HBTP-mito treated with ALP from 0 to 120 min at different temperatures (0 °C: black line; 10 °C: red line; 20 °C: blue line; 37 °C: green line; 45 °C: pink line). [HBTP-mito] = 10 μM, [ALP] = 100 mU/mL, λex = 390 nm. In order to estimate the capability of this probe for ALP quantitative analysis, a series of concentration-dependent experiments were therefore put forward. Referring to Figure 2a, this ALP-catalysis reaction was first traced by the responsive absorption upon increasing ALP concentrations of 0-200 mU/mL, and the original absorbance located at 358 nm showed a gradual diminution accompanied by an evident increase at 496 nm. Accordingly, ESIPT process of the dephosphorylation product should be responsible for this significant bath ochromic shift of the maximum absorption band. Notably, the absorption response of HBTP-mito to ALP within the concentration range of 0-70 mU/mL showed an excellent linearity, producing an LOD of 0.095 mU/mL as determined by 3σ/k (Figure S12). 3
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Analytical Chemistry In the corresponding fluorescence titration, the emission response of HBTP-mito toward ALP was examined. The addition of ALP to the TBS buffer (10 mM, pH=8.0) of HBTP-mito caused a decrease of the emission maximum at 514 nm (Figure 2c) and an increase at 650 nm upon ALP concentrition increasing. The quantum yield of the HBTPmito/ALP mixture was determined as 0.16. The intensities of both peaks at I 514 nm and I 650 nm were plotted, as shown in Figure 2d, and they were exponential or logarithmic with ALP activity. At the same time, HBTP-mito showed well linearity in wide range from 0 to 60 mU / mL), and an LOD was calculated to be 0.072 mU / mL. (Figure S13), which was much lower than the reported probes (Table S1). The excellent sensitivity should be attributed to the ratiometric mode, which used two fluorescence signal channels to read simultaneously, thus minimizing some interference factors and generating an amplified signal output. 43,44 The fluorescent changes of the detection process could also be excited by near-infrared light (780 nm) due to the two-photon properties of the fluorophores. Moreover, kinetic parameters for enzymatic reaction were determined by using the Lineweaver-Burk plot of 1/V versus the reciprocal of HBTP-mito concentrations, giving rise to a Km (Michaelis constant) of 1.476 μM and Vmax (maximum initial reaction rate) of 0.625 μM·min-1 (Figure S14). (b)
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In order to confirm that the red fluorescence originates from the catalytic of ALP instead of other effects, we designed the ALP activity inhibition experiments. Na3VO4, a commonly ALP activity inhibitor, was used to coincubate for 0.5 h, before HBTP-mito was added. According to Figure S18a, with addition of increasing concentration of Na3VO4, the ratio of fluorescence at 514 and 650 nm (I 514 nm/I 650 nm) increased gradually and the noticeable inhibitory effect was observed. In addition, as shown in Figure S18b, the inhibition efficiency tended to increase slowly with the increase of inhibitor concentration. Based on the plots between Na3VO4 concentrations and the inhibiting efficiency, the IC50 corresponding to 100 mU/mL ALP was found to be 10 μM. 45 Additional, a competitive inhibitor, phosphate 46 was also served to gradually reduce the response to ALP: an inverse relationship between NaH2PO4 with concentrations from 0 to 1 mM and the value of I 514 nm/I 650 nm was observed (Figure S19). On the other hand, the inhibitors, Na3VO4 and NaH2PO4, exerted little influence on the absorbance or fluorescence spectra of either compound HBT-mito or HBTP-mito in the absence of ALP (Figure S20). The high selectivity of HBTP-mito toward ALP was also shown in extended assays upon addition of various potential interfering species that may coexist, such as anions (F-, Cl-, Br-, I-, CO32-, HCO3-, NO3-, SO42- and AcO-), ions (K+, Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Al3+ and Fe3+) or some biological molecules (inorganic pyrophosphatase, telomerase, lysozyme, thrombin, BSA, acetylcholinesterase, butyrylcholinesterase, protein kinase A, tyrosinase and acid phosphatase). It was found that only ALP influenced the emission and absorption of HBTPmito as expected (Figure S21 and S22), whereas other molecules did not induce any visible spectral change to HBTP-mito. Excess amount (100 μM) of these species could not change the absorbance and fluorescence spectra of probe, claiming a good selectivity of HBTP-mito for ALP in vitro. Competitive tests showed that the coexistence of these species did not change the fluorescence and absorbance of HBTPmito/ALP system either (Figure S23). As expected, these data demonstrated that HBTP-mito was suitable for detecting ALP in physiological environment dur to the high selectivity. Since ALP is one of the most used detection enzymes in actual disease test, to explore the feasibility of detecting alkaline phosphatase in serum by using HBTP-mito, the experiments were conducted in TBS containing human serum of required concentration obtained from a local hospital (Qingdao Central Hospital, Qingdao, China). The red emission of HBTP-mito could be clearly observed, and the relative regularity decrease of I 514 nm/I 650 nm still allowed it to quantify ALP activity, as shown in Figure S25. The ALP activity of human serum sample was determined as 61.4 ± 3.56 mU/mL by using the method described above, which was consisted with the result of standard medical method, the colorimetric method using p-nitrophenol (64.2 mU/mL, Figure S24). 47 Thus, the proposed probe is believed to be promising for ALP activity assessment in serum samples. For intracellular fluorescent imaging agents, cytotoxicity is a critical consideration. Then, the cytotoxicity of HBTP-mito and HBT-mito were evaluated by CCK-8 afterward. Figures S26 and S27 showed that the cell survival rate remained above 80%, after the incubation with 2-50 μM of HBTP-mito or HBT-mito for 5 h. The cells still showed good viability even
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Figure 2. Absorbance (a) and fluorescence (c) spectra of HBTP-mito in TBS (10 mM, pH 8.0) with increasing concentration of ALP from 0 to 200 mU/mL. Plots of (b) A 496 nm/A 358 nm and (d) intensity at 514 nm (black square) and 650 nm (red dot) versus ALP concentration. [HBTP-mito] = 10 μM, λex = 390 nm, incubation time: 60 min. The spectral changes of HBTP-mito induced by ALP were mainly due to the catalytic dephosphorylation process, which was characterized using NMR spectra. Figure S15 shows the 1H NMR that the proton signals of the mixture of HBTPmito/ALP moved upfield compared with HBTP-mito, and the peak positions are similar to that of HBT-mito. 31P NMR spectrum of HBTP-mito shows a single peak at -5.01 ppm with H2PO4- at -0.10 ppm. When the solution of HBTP-mito was treated with ALP for 4 h, a signal at -0.10 ppm appeared while that at -5.01 ppm disappeared (Figure S16), indicating that phosphate ester of HBTP-mito was hydrolyzed and phosphate was generated. The enzymatic dephosphorylation was also confirmed by HR-MS data: a signal at 373.1436 m/z (calcd. 373.1369) for HBT-mito ([C23H22N2OS]+) in the mixed solution of HBTP-mito/ALP in TBS was found (Figure S17). 4
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incubated with 10 μM of HBTP-mito or HBT-mito lasting for even 24 h (Figure S28), which clearly suggested that the proposed probe and dephosphorylated product exhibited limited cytotoxicity to cultured cells. Contributed by the introduction of pyridinium salt, this elaborate probe HBTP-mito possesses superior mitochondriatageting capability, as depicted in Figure 3. After co-staining with HBTP-mito and the commercially purchased mitochondrial tracker (Mito Tracker Green), HeLa cells imaging was captured in red and green emissive windows respectively. Impressively, the HBTP-mito staining regions overlapped well with the fluorescent domain of Mito Tracker Green (Figure 3a, b), which could be visualized by the coloroverlay of yellow in Figure 3c. Fluorescence distribution correlation between HBTP-mito and the tracker along the linear region of interest (ROIs, Figure 3a-c) offered a more evident profile for the co-locolization estimation, with a synchronous changes in intensity (Figure 3e, f). Moreover, an overlap coefficient (0.96) and a high Pearson’s coefficient (0.96) were also obtained, suggesting the well-behaved colocolization effect between Mito Tracker Green and HBTPmito, which affirmed the capability of HBTP-mito for staining endogenic ALP activity in mitochondria specifically. (a)
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Figure 4. (a) Fluorescent images of different cell lines. The cell lines were incubated with HBTP-mito (10 μM) for 1 h. 480−540 nm for green channel; 650−750 nm for red channel, λex = 405 nm, scale bar = 40 μm. (b) Fluorescence intensity ratios from (a). To further investigate the fluorescence changes in tumor cells caused by the enzymatic dephosphorylation process, the confocal laser scanning microscopy was used. If the red emission arises from the hydrolysate of HBTP-mito, fluorescence emission of ALP inhibited cells should give a brighter image in green channel than that without treated with inhibitor. As it can be seen from Figure 5, HeLa cells emitted strong emission in red channel after incubation with HBTPmito, because of the over expression of ALP. The relatively weak emission obzerved from green channel would be attributed to the short wavelength portion from HBT-mito. However, HeLa cells pretreated by different concentrations of ALP inhibitors, Na3VO4 or NaH2PO4, exhibited increasing fluorescence intensity correspondingly in green channel, while the fluorescence in red channel decreased. These results indicated that the emission changes from tumor cells were caused by endogenous ALP. All of these results demonstrated that HBTP-mito can specifically detect ALP, which is an improtant biomarker of some related diseases.
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Figure 3. Fluorescent localization of mitochondria. HeLa cells were co-incubated with 100 nM Mito Tracker Green and 10 μM HBTP-mito for about 30 min, respectively. (a) Fluorescence emission of green channel for Mito Tracker Green (λem = 500−540 nm, λex = 488 nm); (b) fluorescence emission of red channel for HBTP-mito (λem = 650−750 nm, λex = 405 nm); (c) merged emission from images (a) and (b); (d) the image of brightfield, (e) fluorescence intensity plots of Mito Tracker Green and HBTP-mito; (f) intensity of ROIs across the selected HeLa cell. Scale bar = 20 μm. For intracellular imaging, HeLa and A549 cell lines were used as models, due to the over express of ALP, while HUVEC cell lines were used as control because the relatively low expression of ALP. The tumor cells incubated with 10 μM HBTP-mito for 30 min presented a significant fluorescent change from green to red in the cell cytoplasm, as shown in Figure 4. However, for HUVEC cells, it still emitted strong green fluorescence accompanied by weak signals in red channel. This result suggested that ALP activity in HeLa and A549 cell lines possibly higher by about the same factor. This means that HBTP-mito can localize endogenic ALP activity in situ. 5
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Figure 6. (a) Fluorescent images of tumored nude mice upon treated with an intratumoral injection of HBTP-mito (30 μL, 2.5 mM in DPBS buffer of pH 7.4) and (b) before the injection of HBTP-mito, the tumor was pre-treated with 20 μL Na3VO4 (5 mM in distilled water) for 0.5 h. (c) Fluorescence intensity ratios (I red / I green) from (a) and (b). (d) Fluorescence intensity ratios (Ired / Igreen) of the main internal organs after anatomy (tumor, heart, liver, spleen, lung and kidney). Inset: Fluorescence images (pseudocolor) of the organs of tumored mice pretreated without or with Na3VO4. 480−540 nm for green channel; 650−750 nm for red channel, λex = 405 nm.
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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
Figure 5. (a) Fluorescent images of HeLa cells incubated with HBTP-mito (10 μM) for 1 h or the cells were pretreated with Na3VO4 (0.1 mM, 0.2 mM) or NaH2PO4 (1.0 mM, 3.0 mM) for 0.5 h then treated with HBTP-mito (10 μM) for 1 h. 480−540 nm for green channel; 650−750 nm for red channel, λex = 405 nm, scale bar = 20 μm. (b) Fluorescence intensity ratios from (a). Based on the excellent optical property and the above intracellular ALP imaging, HBTP-mito was further utilized to monitor ALP activity in living animals. Tumored BALB/c nude mice were used as model in this experiment. Fluorescence images of nude mice were taken by monitoring the signal changes of two different fluorescence channels. After intratumoral injection of 30 μL of HBTP-mito (2.5 mM in DPBS buffer of pH 7.4) into the living mice, a significant enhancement at red channel was observed with time accompanied by the weakening of the green channel (Figure 6a). As a control experiment, Na3VO4 was utilized to inhibit the ALP activity in tumor. The tumor was pre-treated with 20 μL Na3VO4 (5 mM in distilled water) for 30 min and then the probe was injected. The fluorescence enhancement in red channel was not obvious for the first 0.5 h. It gradually became stronger after 1 h (Figure 6b), but the fluorescent ratio (Ired / Igreen) was much smaller than that had not been pretreated with inhibitor (Figure 6c). Fluorescence intensity ratios of the main internal organs after anatomy (tumor, heart, liver, spleen, lung and kidney) were also measured. As shown in Figure 6d, consistent with the in vivo results, the emission ratio (Ired / Igreen) of the tumor without pre-treated with Na3VO4 is much larger than that of control sample and other internal organs showed almost no fluorescence. The results indicated that HBTP-mito was competented to monitor endogenous ALP activity in living animals.
CONCLUSIONS In summary, a novel mitochondria-targeted ratiometric fluorescent bioprobe HBTP-mito was developed for ALP detection and visualization based on the regulation of ESIPT process through protection and deprotection of a hydroxyl group. This probe showed good water solubility and emitted green fluorescence in aqueous buffer due to the blockage of ESIPT. In the presence of ALP, phosphate ester was hydrolyzed and the ESIPT process restored, thus the fluorescence changed from green to red. The probe could quantitative detect ALP activity between 0−60 mU/mL with an LOD of 0.072 mU/mL. In additional, the I 514 nm/I 650 nm of the enzymatic product prevented the fluorescence interference from the serum. Therefore, it could be used for detecting ALP in serum samples. Due to the low toxicity of the probe, it was ready for detecting ALP activity in vitro and in vivo. The design strategy in this work provided a comprehensive example for constructing ratiometric probes with redder emission and high sensitivity.
ASSOCIATED CONTENT Supporting Information The Supporting Information (SI) is available free of charge on the ACS Publications website at DOI: xxx. Experimental section, 1H, 13C, 31P NMR and MS spectral copies of the synthesized compounds (Figures S1-S7), and fluorescence, absorption and NMR spectral profiles (Figures S86
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S25), and cell cytotoxicity of HBT-mito and HBTP-mito (Figures S26-S28).
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] ACKNOWLEDGMENT We thank the support of NSFC (21904077, 21422504), and NSF of Shandong Province (ZR2019BB055), and Key Laboratory of Spectrochemical Analysis & Instrumentation (Xiamen University), MOE (SCAI1702, SCAI1703). We also thank Professor Yun-Bao Jiang of Xiamen University for support.
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