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A fluorescent probe for alkaline phosphatase via excited state intramolecular proton transfer
Accepted Manuscript Title: A fluorescent probe for alkaline phosphatase via excited state intramolecular proton transfer Author: Qinghua Hu Fang Zeng Changmin Yu Shuizhu Wu PII: DOI: Reference:
S0925-4005(15)00756-X http://dx.doi.org/doi:10.1016/j.snb.2015.05.111 SNB 18558
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
5-3-2015 21-5-2015 27-5-2015
Please cite this article as: Q. Hu, F. Zeng, C. Yu, S. Wu, A fluorescent probe for alkaline phosphatase via excited state intramolecular proton transfer, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.05.111 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
・A new fluorescent probe for ALP via excited-state intramolecular proton transfer. ・It exhibits sensitive and very low detection limit for ALP.
ALP imaging.
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・It is capable of detecting ALP in biological fluid like serum.
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・It is of little cytotoxicity and can be easily internalized into cells for endogenous
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A fluorescent probe for alkaline phosphatase via excited state
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intramolecular proton transfer
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Qinghua Hu, Fang Zeng,* Changmin Yu and Shuizhu Wu*
College of Materials Science and Engineering, State Key Laboratory of Luminescent
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Materials and Devices, South China University of Technology, 510640, China E-mail: [email protected], [email protected]
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Tel: +86 20 22236262; fax: +86 20 22236363.
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Abstract: Alkaline phosphatase (ALP) is an important biomarker for diagnostics, and excessive level of ALP is closely related to a variety of pathological processes; hence the
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development of convenient and sensitive methods for detecting ALP is of great significance for medical sciences and diagnostics. Herein, we report a fluorescent
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probe for detecting ALP via excited state intramolecular proton transfer (ESIPT). The
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probe was synthesized by coupling a phosphate group onto flavone-based fluorophore. For this probe, the phosphate group serves both as the blocking agent for ESIPT and
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the responsive moiety toward ALP. Due to the shielding of the hydroxyl group by the
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phosphate moiety, no ESIPT process occurs, the tautomer emission of the probe is therefore quenched; while in the presence of ALP, ALP cleaves phosphate group and
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generates hydroxyl group, thereby enabling the ESIPT and generating strong
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ESIPT-based emission. This probe exhibits high selectivity for ALP detection with a
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very low detection limit of 0.032 U/L. It is capable of detecting ALP in biological fluid like serum. Furthermore, it is of little cytotoxicity and can be easily internalized into cells for endogenous ALP imaging. Therefore, the probe herein displays strong potential for applications in biological diagnostics and pathological analysis. Keyword: Excited state intramolecular proton transfer; Fluorescence technique;
Imaging; Alkaline phosphatase
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1. Introduction Alkaline phosphatase catalyzes dephosphorylation process of proteins, nucleic acids and small molecules, which can be found in a variety of tissues
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(liver, intestine, kidney, bone, and placenta)[1-3]. As an important diagnostic
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enzyme, ALP has long been identified as an important biomarker for clinical
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diagnostics[4]. Excessive level of ALP is closely related to a variety of pathological processes, such as bone diseases, heart failure, ovarian and breast
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cancers, leukemia and liver diseases (liver cancer, hepatitis, and obstructive jaundice)[5-7].
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A number of methods for detecting the activity/level of ALP have been reported in the past years, such as colorimetric, electrochemical, surface enhanced
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resonance Raman scattering (SERRS) methods and fluorescence technique[8-16]. Among them, fluorescent sensing technique is remarkably important due to its
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rapid, simple and sensitive signal transduction, noninvasive monitoring capability and usability in live organism[17-50]. And this technique has been extensively applied in the research of various enzyme-catalyzed processes[51-55]. Excited-state intramolecular proton transfer (ESIPT) process generally
involves a hydroxyl proton transfer to an acceptor (such as carbonyl) in the excited state, resulting in tautomer emission with a relatively large Stokes shift. ESIPT is a significantly important fluorescent phenomenon. Recently, the fluorescent dyes based
on
ESIPT
process
such
as
2-(2-hydroxyphenyl)benzoxazole,
1-aminoanthraquinone, and flavone, have been used as an attractive fluorescent 4
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signal transducer in sensors[56-67]. In comparison with the other fluorescent processes, such as electron transfer and energy transfer, ESIPT process can occur at a much faster rate[68]. As ESIPT compounds can induce fluorescence change
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through intramolecular hydrogen bonding, fluorophores’ ESIPT characteristics
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offer additional advantages, such as a low background signal in aqueous media.
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Moreover, ESIPT dyes generally have large Stokes’ shift, which can reduce the interference from the excitation light[69]. Although the unique photophysical
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properties of ESIPT have been known for decades, only one ESIPT-based probe has been designed for ALP detection[70], which was operable in solution but
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hasn’t been tested for detecting ALP inside living cells.
Among ESIPT dyes, flavone dyes are a broad class of natural products, and
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have been extensively studied for their antioxidant properties and anticancer activities in the food and health sciences [71]. However, few flavone-based
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biosensors have been studied for bioimaging or biodetection applications (Table S3 in the Supporting Information). To make full use of the unique features of ESIPT and further broaden the applications of ESIPT-based fluoropores in biodetection and bioimaging, we herein present a novel flavone-based fluorescent probe (3-hydroxy-2-(p-tolyl)-4H-chromen-4-one) for detecting ALP (3 in Scheme 1), which functions via ESIPT process. For this probe, due to the shielding of the hydroxyl group by the phosphate moiety, no ESIPT process can occur, the tautomer emission of the probe will be quenched; while in the presence of ALP, ALP cleaves phosphate group and generates hydroxyl group, accordingly ESIPT 5
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occurs, and correspondingly the reaction product gives off strong ESIPT emission; and the schematic illustration for the fluorescent detection of ALP is shown in
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Figure 1. 2. Experimental
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2.1. Reagents and Materials.
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P-Methyl benzaldehyde, 2-hydroxyacetophenone, sodium hydride and H2O2 aqueous solution were purchased from Aladdin Reagents. Diethyl chlorophosphate,
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bromotrimethylsilane (TMS-Br) and alkaline phosphatase (ALP) were purchased from Sigma Aldrich. Alkaline phosphatase (ALP) ELISA Kit was purchased from
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Shanghai Enzyme-linked Biotechnology Co. Ltd. Human serum samples were supplied by The Sixth Affiliated Hospital, Sun Yat-sen University; and the serum
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samples were from both healthy males and unhealthy males with abnormal ALP levels. Ethyl alcohol, ethyl acetate, methanol, dichloromethane, anhydrous
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magnesium sulfate, sodium chloride and sodium hydroxide were of analytical grade reagents. The water used throughout the experiments was the triple distilled water which was further treated by ion exchange columns and then by a Milli-Q water purification system.
2.2. Synthesis of compound 1. To a solution of 2-hydroxyacetophenone (2.04 g, 15 mmol) and p-methyl
benzaldehyde (1.80 g, 15 mmol) in 60 mL methanol was added NaOH (2.7 g) and the resulting mixture was heated to reflux for 6 h. After cooling to room temperature, the mixture was further stirred overnight. NaOH (0.5 mol L−1, 60 mL) and H2O2 (30%, 8 6
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mL) were then added, and the mixture was stirred for 5 h at room temperature and afterwards poured into ice-water (200 mL). The precipitate was collected by filtration and washed with cold methanol, and dried to afford compound 1 as a yellow solid
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(yield 61%) . 1H NMR (d6-DMSO, 600 MHz, ppm, Figure S1): 2.40 (s, 3H), 7.39 (d,
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2H), 7.47 (t, J=7.2Hz, 1H), 7.77 (d, 1H), 7.81 (t, J=6.6Hz, 1H), 8.12 (d, 1H), 8.14 (d,
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2H), 9.55 (s, 1H). 2.3. Synthesis of compound 2.
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To a solution of 1 (1 mmol, 252 mg) and NaH (2 mmol, 80 mg) in distilled THF (15 mL), diethyl chlorophosphate (0.3 mL, 2.0 mmol) was added dropwise under
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nitrogen. The reaction mixture was stirred at room temperature for 3 h. The resulting solution was quenched with water (2 mL), and the solvent was evaporated under
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reduced pressure. The crude mixture was extracted with ethyl acetate three times. The combined organic layer was dried over MgSO4 and filtered, and the solvent was
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evaporated under reduced pressure. The crude product was purified by silica gel chromatography with PE/ethyl acetate (1:1) as eluent to afford the desired product (248 mg, 0.64 mmol) as colorless gel-like solid. Yield = 64%.1H NMR (CDCl3, 600 MHz, ppm, Figure S2): 1.26 (t, J=7.2Hz, 6H), 2.44 (s, 3H), 4.15 (m, 4H), 7.34 (d, 2H), 7.43 (t, J=7.8Hz, 1H), 7.51 (d, 1H), 7.69 (t, J=7.2Hz, 1H), 7.89 (d, 2H), 8.28 (d, 1H). MS-ESI (Figure S3): m/z = 410.15 [M+Na]+. 31P NMR (δ ppm, in CDCl3, Figure S4):
-4.43. 2.4. Synthesis of compound 3 (The probe). To a solution of 2 (116 mg, 0.3 mmol) in dry DCM (15 mL) was added 7
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bromotrimethylsilane (0.42 mL, 3.0 mmol) dropwise under nitrogen at 0 °C. The reaction mixture was stirred for 48 h at room temperature. The precipitate was collected by filtration and washed with diethyl ether three times and dried to afford 1
H NMR (d6-DMSO, 600 MHz, ppm,
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compound 3 as a white solid (yield 90%).
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Figure S5): 2.41 (s, 3H), 7.38 (d, 2H), 7.53 (t, J=7.2Hz, 1H), 7.74 (d, 1H), 7.69 (t,
31
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J=6.6Hz, 1H), 8.03 (d, 2H), 8.11 (d, 1H). MS-ESI (Figure S6): m/z = 329.88 [M-H]-. P NMR (δ ppm, in d6-DMSO, Figure S7): -5.39. The synthesis of the probe is
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shown in Scheme 1. 2.5. Alkaline phosphatase (ALP) ELISA Kit assay.
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Detection of ALP using the ELISA Kit assay was conducted according to the instructions of the ELISA Kit, which involves 11 steps.
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2.6. Measurements.
H NMR spectra were recorded on a Bruker Avance 600 MHz NMR
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spectrometer. Mass spectra were obtained through a Bruker Esquire HCT Plus mass spectrometer. UV-vis spectra were recorded on a Hitachi U-3010 UV-vis spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. Fluorescence images were obtained using an Olympus IX 71 with a DP72 color CCD (mirror unit U-MNV2, excitation filter 400-410 nm, emission filter ≥ 455 nm). 2.7. Cell viability assay. To evaluate the toxicity of the probe in living cells, L929 cells (murine aneuploid fibrosarcoma cell) and HeLa cells (human cervical cancer cell) were incubated in 8
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DMEM medium supplemented with 10% fetal bovine serum (FBS) and allowed to grow for 24 h at 37 °C with 5% CO2. After the removal of the medium, cells were treated with the probe and incubated for an additional 24 h. The cytotoxicity of the
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probe against the cells was assessed by MTT assay according to ISO 10993-5. As for
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the assays, for each independent experiment, the assays were performed in eight
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replicates. And the statistical mean and standard deviation were used to estimate the cell viability.
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2.8. Cell incubation and imaging.
Two cell lines, HeLa (human cervical cancer cell) and L929 (murine aneuploid
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fibrosarcoma cell) cells, were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS, HyClone) respectively. One day before imaging, cells were
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passaged and plated on the 30 mm glass culture dishes and allowed to grow to 50-70% confluence. For the experiments, the cells were washed with DMEM,
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incubated in DMEM medium containing the probe (2 × 10
-5
M) at 37 °C under 5%
CO2 for 1.5 hour. Some HeLa cells were incubated in DMEM medium containing the ALP inhibitor (Levamisole hydrochloride, 1 × 10-3 M) for 30 min, and then treated
with the probe (2 × 10
-5
M) for 1.5 h. The control sample was incubated in DMEM
medium only. After that, the culture dishes were washed with PBS, the cells were washed with PBS for three times and then imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD. 3. Results and Discussion 3.1. Synthesis. 9
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The probe was prepared by the reaction of Compound 1 with diethyl chlorophosphate in THF under basic conditions at room temperature for two steps as outlined in Scheme 1. Compound 1 can be also easily prepared through the reaction
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between 2-hydroxyacetophenone and p-methyl benzaldehyde. The structure of the 31
P NMR
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immediate compound and the probe has been characterized by 1H /
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spectrometry and mass spectrometry, as shown in Figures S1-S7. The results confirm the successful synthesis of the probe.
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3.2. Fluorescent response of the probe toward ALP.
The sensing ability of probe for ALP was investigated in Tris-HCl buffered
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water (pH 8.0, 10 mM) with 20% DMSO at 37 °C. Under this condition, the probe (10 μM) shows maximum absorption at 335 nm (see Figure S8), and is weakly
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fluorescent (Φ= 0.0031; see Table S1). Upon addition of ALP, the maximum absorption at 335 nm decreases, in the meantime at 410 nm a new absorption peak
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emerges (see Figure S8), and the probe exhibits a strong fluorescent emission (Φ= 0.085; see Table S1) at 484 nm with the excitation wavelength of 405 nm. In order to choose the appropriate excitation wavelength, we investigated four excitation wavelengths at 345 nm, 375 nm, 390 nm and 405 nm; and we found that, the probe incubated with ALP shows two fluorescent emissions upon excited at 345 nm or 375 nm (see the Figure S9 (A, B)), while the probe incubated with ALP displays only one fluorescent emission upon excited at 390 nm or 405 nm (see the Figure S9 (C, D)). Since visible is more beneficial for biological applications, we choose 405 nm as the excitation wavelength in this study. 10
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In the probe herein, the phosphate group serves both as the blocking agent for ESIPT and the responsive moiety toward ALP. We expect that, the protection of the hydroxyl group in compound 3 (the probe) by the phosphate group will block the
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ESIPT process and result in the quenching of the tautomer emission; the addition of
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ALP cleaves phosphate group, and transforms the probe (compound 3) back into
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compound 1, which correspondingly restores the ESIPT process and recovers the strong Tautomer emission (Figure 1). And the probe remains stable in the wide pH
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range of 4.5 to 9.2 (Figure S10), which guarantees its utilization in biological samples.
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We investigated the fluorescent spectra of the probe solution in the absence and presence of ALP, as shown in Figure 2. The fluorescence spectra of the probe were
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periodically recorded during incubation of the probe with ALP (100 U/L) at 37 °C in Tris-HCl buffered water (pH 8.0, 10 mM) with 20% DMSO. In the absence of ALP,
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the probe solution displays almost no emission. Upon addition of ALP, the probe shows rapid response, the fluorescence intensity increases gradually with the prolonged incubation time. For instance, the fluorescence intensity at 484 nm of the ensemble was enhanced by 22 times after ALP (100 U/L) was introduced and the solution was incubated for 70 min. In addition, HPLC was employed to confirm the enzymatic reaction between the probe and ALP. As shown in Figure. S11, the results indicate that, the fluorescence enhancement during enzymatic reaction is the result of the formation of the expected product (Compound 1). Next, we measured the fluorescence spectra of the probe toward ALP at varied 11
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concentrations (reaction at 37 °C for 60 min in Tris-HCl buffer (pH 8.0) with 20% DMSO), as shown in Figure 3(A). It can be seen that the fluorescence intensity increases steadily with the increasing ALP level. The fluorescence intensity ratio of -5
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the probe solution (1 × 10
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Figure 4 displays the variation of the fluorescence intensity at 484 nm of the probe
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after incubation with different amounts of ALP (0.1 - 100 U/L) for different time periods. Obviously, the fluorescence intensity of the probe increases after incubation
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with ALP; moreover, the fluorescence intensity is enhanced more rapidly when the concentration of ALP is higher. This is understandable by considering the fact that the
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hydrolysis of the probe would be facilitated in the presence of a high concentration of ALP.
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The difference in fluorescence intensity for the probe solution before and after incubation with ALP can be easily distinguished by the hand-held UV lamp (365 nm)
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illumination (see insets of Figure 1). Figure S12 shows the plot of 1/V vs 1/[S], and the
corresponding
Lineweaver-Burk
plot.
By
fitting
the
plot
with
the
Michaelis-Menten equation, the kinetic parameters for ALP (Km and Vmax) were
estimated to be 1.919 µM and 1.21 ×10-3 µmol/min respectively. The affinity constant between the probe with ALP was derived by Scatchard plot (Figure S13), the affinity constant Ka were estimated to be 1.84×107 L·mol-1. We also established a working curve by plotting the emission intensity ratio of 484 nm versus ALP levels, and the detection limit has been determined as 0.032 U/L (Figure S14), which is sensitive enough for determining ALP levels in biological assays, since for healthy adults the 12
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ALP level in blood is in the range of 46 ~ 190 U/L [72]. Further, the selectivity of this probe for ALP was also tested. The fluorescence signal change of the probe solution containing ALP was compared with that of the solutions containing other
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proteins/enzymes under the same conditions (Figure S15), including alanine
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aminotransferase (ALT), acetyl cholinesterase (AchE), Bovine serum albumin (BSA),
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β-galactosidase (β-GAL), glutamyl transpeptidase (GGT), hyaluronidase (HAase), nitroreductase and esterase. As shown in Fig. S14, in contrast to the significant
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increase of fluorescence intensity induced by ALP, the addition of other proteins/enzymes trigger negligible fluorescence changes for the probe solution.
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These results indicate that the probe is highly selective for ALP assay. 3.3. ALP detection in serum samples.
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To extend the application of this probe, we applied the probe to detecting the endogenous ALP levels in human serum samples, the serum samples were diluted
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50-fold for the measurement. First, we investigated the probe’s fluorescence response in diluted serum sample in the presence of or absence of ALP inhibitor, and the results are shown in Figure S16. It is clear that the addition of ALP inhibitor levamisole [73] results in obvious decrease of the fluorescence intensity, confirming the probe’s specific interaction with ALP in diluted serum sample. The serum samples detection results were compared with those by using the commonly-used commercial assay Elisa kit, and the results are presented in Table S2. The endogenous ALP levels in the serum samples from both positive and negative clinical samples measured by the probe are close to those measured by using Elisa kit. However, as for the ELISA kit, 13
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the detection processes are quite complex which requires multiple procedures, while for this probe, the detection process is very convenient and could serve as a one-step
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straightforward fluorescence assay for ALP detection in diagnostics. 3.4. Imaging of endogenous ALP in live cells.
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We first tested in vitro cytotoxicity of the probe. The cytotoxicity was evaluated
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using L929 and HeLa cell lines by MTT assay in compliance with ISO 10993-5, and the results are shown in Figure S17. No significant reduction in cell viability can be
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observed for cells treated with the probe even at high concentrations (up to 50 µM), proving that the probe shows little toxicity in vitro and is suitable for fluorescence
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imaging of ALP in live cells.
The probe’s cell imaging capability was evaluated using HeLa cells, in which
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ALP is overexpressed. The imaging for endogenous ALP in the cells is shown in Figure 5. HeLa cells loaded with the probe show a strong blue-green fluorescence
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(Figure 5E), which is caused by the existence of endogenous ALP in HeLa cells. For comparison, we also applied the probe to cellular imaging in L929 cells that do not have overexpression of ALP [74, 75], as shown in Figure S18. It is clear that, as L929 cells were loaded with the probe, no fluorescence can be observed. We suppose that, the probes internalized by L929 cells won’t undergo dephosphorylation due to the lack of ALP in the cells and accordingly no emission can be observed. To further prove that the blue-green fluorescence observed in HeLa cells were triggered by ALP, the cells were pretreated with ALP inhibitor levamisole hydrochloride for 30 minutes, and then incubated with the probe for 1.5 h. As shown 14
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in Figure 5H, the blue-green fluorescence is almost indiscernible. The results indicate that the intracellular fluorescence changes in HeLa cells are indeed triggered by ALP. The above results demonstrate the strong potential of the probe to detect endogenous
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ALP inside cells as well as aid in pathological analysis.
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ALP in live cells, which is of great importance to clarify the physiological roles of
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4. Conclusions
In summary, we have successfully developed a new fluorescent probe for
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detecting ALP, which functions through visible-light excitable excited-state intramolecular proton transfer (ESIPT). The prove features rapid detection process,
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low detection limit and high selectivity. In comparison with other probes for ALP (as shown in Table S4), this probe has several advantages, first this new probe can be
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easily prepared and works effectively under physiological pH and in such biological sample as human serum. Furthermore, this probe is of little cytotoxicity and can be
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easily loaded into cells for endogenous ALP imaging. Therefore, the strategy herein could offer alternative approaches for fabricating practical fluorescent assays or probes for other types of biomolecules.
Acknowledgements
We gratefully acknowledge the financial support by NSFC (21474031, 21174040, 21025415 and 51403065), and the National Key Basic Research Program of China (Project No. 2013CB834702).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://
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Biographies Qinghua Hu received his Bachelor of Engineering degree from Jishou University, Jishou, China, in 2009. He is currently pursuing the PhD degree in materials science
ip t
at South China University of Technology, Guangzhou, China under the supervision of
us
fluorescence spectroscopy for their biological applications.
cr
Prof. Shuizhu Wu. His research interests include materials synthesis, sensors and
Fang Zeng has been a professor at the College of Materials Science and Engineering,
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South China University of Technology, Guangzhou, China since 2004. His primary research interests include fluorescent materials, nanoparticles, energy transfer,
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chemosensors and biosensors, and drug and gene delivery systems. Changmin Yu obtained his PhD degree at the College of Materials Science and
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Engineering, South China University of Technology, Guangzhou, China in 2013. He is now working in South China University of Technology as a lecturer. His current
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research interests include fluorescent materials, energy transfer in materials, chemosensors and biosensors.
Shuizhu Wu has been a professor at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China since 2005. Her primary research interests include fluorescent materials, photophysics, chemosensors and biosensors, nanoparticles, and biomaterials.
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Figure captions: Fig. 1. Schematic illustration of the probe’s structure and fluorescent response toward ALP.
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Fig. 2. Time course of fluorescence spectra of the probe (1×10-5 M) in the presence of
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ALP (100 U/L) in Tris-HCl buffered water (pH 8.0, 10 mM) with 20% DMSO at 37 °C with the excitation wavelength of 405 nm.
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Fig. 3. (A) Fluorescence spectra of the probe (1 × 10 -5 M) in the presence of different
an
amounts of ALP in Tris-HCl buffer (pH 8.0, 10 mM) with 20% DMSO at 37 °C for 60 min. (B) Fluorescence intensity ratio (F/F0) as a function of ALP level.
independent experiments.
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Excitation wavelength: 405 nm. Data represent mean ±SD from three
d
Fig. 4. Fluorescence intensity at 484 nm of the probe (1 × 10
-5
M) in Tris-HCl
te
buffered (pH 8.0, 10 mM) with 20% DMSO at 37 C versus reaction time in the
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presence of different amounts of ALP.
Fig. 5. Fluorescence microscopy images for HeLa cells in DMEM medium with 1% DMSO. (A-C): The control; (D-F): Cells treated with the probe (2 × 10 -5 M) for
1.5 h; (G-I): Cells treated with inhibitor levamisole hydrochloride (1 × 10 for 30 min and then the probe (2 × 10
-5
-3
M)
M) for 1.5 h. (Excitation filter 400-410
nm, emission filter ≥ 455 nm).
Scheme captions: Scheme 1. Synthetic route of the probe. 29
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Biographies Qinghua Hu received his Bachelor of Engineering degree from Jishou University,
ip t
Jishou, China, in 2009. He is currently pursuing the PhD degree in materials science
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at South China University of Technology, Guangzhou, China under the supervision of
us
Prof. Shuizhu Wu. His research interests include synthesis of fluorescent compounds, sensors and their biological applications.
an
Fang Zeng has been a professor at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China since 2004. His primary
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research interests include fluorescent materials, nanoparticles, energy transfer, chemosensors and biosensors, and drug and gene delivery systems.
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Changmin Yu obtained his PhD degree at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China in 2013. He
Ac ce p
is now working in South China University of Technology as a lecturer. His current research interests include fluorescent materials, energy transfer in materials, chemosensors and biosensors.
Shuizhu Wu has been a professor at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China since 2005. Her primary research interests include fluorescent materials, photophysics, chemosensors and biosensors, nanoparticles, and biomaterials.
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S0925-4005(15)00756-X http://dx.doi.org/doi:10.1016/j.snb.2015.05.111 SNB 18558
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
5-3-2015 21-5-2015 27-5-2015
Please cite this article as: Q. Hu, F. Zeng, C. Yu, S. Wu, A fluorescent probe for alkaline phosphatase via excited state intramolecular proton transfer, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.05.111 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
・A new fluorescent probe for ALP via excited-state intramolecular proton transfer. ・It exhibits sensitive and very low detection limit for ALP.
ALP imaging.
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・It is capable of detecting ALP in biological fluid like serum.
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・It is of little cytotoxicity and can be easily internalized into cells for endogenous
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Page 1 of 37
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A fluorescent probe for alkaline phosphatase via excited state
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intramolecular proton transfer
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Qinghua Hu, Fang Zeng,* Changmin Yu and Shuizhu Wu*
College of Materials Science and Engineering, State Key Laboratory of Luminescent
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Materials and Devices, South China University of Technology, 510640, China E-mail: [email protected], [email protected]
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Tel: +86 20 22236262; fax: +86 20 22236363.
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Abstract: Alkaline phosphatase (ALP) is an important biomarker for diagnostics, and excessive level of ALP is closely related to a variety of pathological processes; hence the
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development of convenient and sensitive methods for detecting ALP is of great significance for medical sciences and diagnostics. Herein, we report a fluorescent
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probe for detecting ALP via excited state intramolecular proton transfer (ESIPT). The
us
probe was synthesized by coupling a phosphate group onto flavone-based fluorophore. For this probe, the phosphate group serves both as the blocking agent for ESIPT and
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the responsive moiety toward ALP. Due to the shielding of the hydroxyl group by the
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phosphate moiety, no ESIPT process occurs, the tautomer emission of the probe is therefore quenched; while in the presence of ALP, ALP cleaves phosphate group and
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generates hydroxyl group, thereby enabling the ESIPT and generating strong
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ESIPT-based emission. This probe exhibits high selectivity for ALP detection with a
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very low detection limit of 0.032 U/L. It is capable of detecting ALP in biological fluid like serum. Furthermore, it is of little cytotoxicity and can be easily internalized into cells for endogenous ALP imaging. Therefore, the probe herein displays strong potential for applications in biological diagnostics and pathological analysis. Keyword: Excited state intramolecular proton transfer; Fluorescence technique;
Imaging; Alkaline phosphatase
3
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1. Introduction Alkaline phosphatase catalyzes dephosphorylation process of proteins, nucleic acids and small molecules, which can be found in a variety of tissues
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(liver, intestine, kidney, bone, and placenta)[1-3]. As an important diagnostic
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enzyme, ALP has long been identified as an important biomarker for clinical
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diagnostics[4]. Excessive level of ALP is closely related to a variety of pathological processes, such as bone diseases, heart failure, ovarian and breast
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cancers, leukemia and liver diseases (liver cancer, hepatitis, and obstructive jaundice)[5-7].
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A number of methods for detecting the activity/level of ALP have been reported in the past years, such as colorimetric, electrochemical, surface enhanced
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resonance Raman scattering (SERRS) methods and fluorescence technique[8-16]. Among them, fluorescent sensing technique is remarkably important due to its
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rapid, simple and sensitive signal transduction, noninvasive monitoring capability and usability in live organism[17-50]. And this technique has been extensively applied in the research of various enzyme-catalyzed processes[51-55]. Excited-state intramolecular proton transfer (ESIPT) process generally
involves a hydroxyl proton transfer to an acceptor (such as carbonyl) in the excited state, resulting in tautomer emission with a relatively large Stokes shift. ESIPT is a significantly important fluorescent phenomenon. Recently, the fluorescent dyes based
on
ESIPT
process
such
as
2-(2-hydroxyphenyl)benzoxazole,
1-aminoanthraquinone, and flavone, have been used as an attractive fluorescent 4
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signal transducer in sensors[56-67]. In comparison with the other fluorescent processes, such as electron transfer and energy transfer, ESIPT process can occur at a much faster rate[68]. As ESIPT compounds can induce fluorescence change
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through intramolecular hydrogen bonding, fluorophores’ ESIPT characteristics
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offer additional advantages, such as a low background signal in aqueous media.
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Moreover, ESIPT dyes generally have large Stokes’ shift, which can reduce the interference from the excitation light[69]. Although the unique photophysical
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properties of ESIPT have been known for decades, only one ESIPT-based probe has been designed for ALP detection[70], which was operable in solution but
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hasn’t been tested for detecting ALP inside living cells.
Among ESIPT dyes, flavone dyes are a broad class of natural products, and
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have been extensively studied for their antioxidant properties and anticancer activities in the food and health sciences [71]. However, few flavone-based
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biosensors have been studied for bioimaging or biodetection applications (Table S3 in the Supporting Information). To make full use of the unique features of ESIPT and further broaden the applications of ESIPT-based fluoropores in biodetection and bioimaging, we herein present a novel flavone-based fluorescent probe (3-hydroxy-2-(p-tolyl)-4H-chromen-4-one) for detecting ALP (3 in Scheme 1), which functions via ESIPT process. For this probe, due to the shielding of the hydroxyl group by the phosphate moiety, no ESIPT process can occur, the tautomer emission of the probe will be quenched; while in the presence of ALP, ALP cleaves phosphate group and generates hydroxyl group, accordingly ESIPT 5
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occurs, and correspondingly the reaction product gives off strong ESIPT emission; and the schematic illustration for the fluorescent detection of ALP is shown in
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Figure 1. 2. Experimental
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2.1. Reagents and Materials.
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P-Methyl benzaldehyde, 2-hydroxyacetophenone, sodium hydride and H2O2 aqueous solution were purchased from Aladdin Reagents. Diethyl chlorophosphate,
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bromotrimethylsilane (TMS-Br) and alkaline phosphatase (ALP) were purchased from Sigma Aldrich. Alkaline phosphatase (ALP) ELISA Kit was purchased from
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Shanghai Enzyme-linked Biotechnology Co. Ltd. Human serum samples were supplied by The Sixth Affiliated Hospital, Sun Yat-sen University; and the serum
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samples were from both healthy males and unhealthy males with abnormal ALP levels. Ethyl alcohol, ethyl acetate, methanol, dichloromethane, anhydrous
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magnesium sulfate, sodium chloride and sodium hydroxide were of analytical grade reagents. The water used throughout the experiments was the triple distilled water which was further treated by ion exchange columns and then by a Milli-Q water purification system.
2.2. Synthesis of compound 1. To a solution of 2-hydroxyacetophenone (2.04 g, 15 mmol) and p-methyl
benzaldehyde (1.80 g, 15 mmol) in 60 mL methanol was added NaOH (2.7 g) and the resulting mixture was heated to reflux for 6 h. After cooling to room temperature, the mixture was further stirred overnight. NaOH (0.5 mol L−1, 60 mL) and H2O2 (30%, 8 6
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mL) were then added, and the mixture was stirred for 5 h at room temperature and afterwards poured into ice-water (200 mL). The precipitate was collected by filtration and washed with cold methanol, and dried to afford compound 1 as a yellow solid
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(yield 61%) . 1H NMR (d6-DMSO, 600 MHz, ppm, Figure S1): 2.40 (s, 3H), 7.39 (d,
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2H), 7.47 (t, J=7.2Hz, 1H), 7.77 (d, 1H), 7.81 (t, J=6.6Hz, 1H), 8.12 (d, 1H), 8.14 (d,
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2H), 9.55 (s, 1H). 2.3. Synthesis of compound 2.
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To a solution of 1 (1 mmol, 252 mg) and NaH (2 mmol, 80 mg) in distilled THF (15 mL), diethyl chlorophosphate (0.3 mL, 2.0 mmol) was added dropwise under
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nitrogen. The reaction mixture was stirred at room temperature for 3 h. The resulting solution was quenched with water (2 mL), and the solvent was evaporated under
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reduced pressure. The crude mixture was extracted with ethyl acetate three times. The combined organic layer was dried over MgSO4 and filtered, and the solvent was
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evaporated under reduced pressure. The crude product was purified by silica gel chromatography with PE/ethyl acetate (1:1) as eluent to afford the desired product (248 mg, 0.64 mmol) as colorless gel-like solid. Yield = 64%.1H NMR (CDCl3, 600 MHz, ppm, Figure S2): 1.26 (t, J=7.2Hz, 6H), 2.44 (s, 3H), 4.15 (m, 4H), 7.34 (d, 2H), 7.43 (t, J=7.8Hz, 1H), 7.51 (d, 1H), 7.69 (t, J=7.2Hz, 1H), 7.89 (d, 2H), 8.28 (d, 1H). MS-ESI (Figure S3): m/z = 410.15 [M+Na]+. 31P NMR (δ ppm, in CDCl3, Figure S4):
-4.43. 2.4. Synthesis of compound 3 (The probe). To a solution of 2 (116 mg, 0.3 mmol) in dry DCM (15 mL) was added 7
Page 7 of 37
bromotrimethylsilane (0.42 mL, 3.0 mmol) dropwise under nitrogen at 0 °C. The reaction mixture was stirred for 48 h at room temperature. The precipitate was collected by filtration and washed with diethyl ether three times and dried to afford 1
H NMR (d6-DMSO, 600 MHz, ppm,
ip t
compound 3 as a white solid (yield 90%).
cr
Figure S5): 2.41 (s, 3H), 7.38 (d, 2H), 7.53 (t, J=7.2Hz, 1H), 7.74 (d, 1H), 7.69 (t,
31
us
J=6.6Hz, 1H), 8.03 (d, 2H), 8.11 (d, 1H). MS-ESI (Figure S6): m/z = 329.88 [M-H]-. P NMR (δ ppm, in d6-DMSO, Figure S7): -5.39. The synthesis of the probe is
an
shown in Scheme 1. 2.5. Alkaline phosphatase (ALP) ELISA Kit assay.
M
Detection of ALP using the ELISA Kit assay was conducted according to the instructions of the ELISA Kit, which involves 11 steps.
te
1
d
2.6. Measurements.
H NMR spectra were recorded on a Bruker Avance 600 MHz NMR
Ac ce p
spectrometer. Mass spectra were obtained through a Bruker Esquire HCT Plus mass spectrometer. UV-vis spectra were recorded on a Hitachi U-3010 UV-vis spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. Fluorescence images were obtained using an Olympus IX 71 with a DP72 color CCD (mirror unit U-MNV2, excitation filter 400-410 nm, emission filter ≥ 455 nm). 2.7. Cell viability assay. To evaluate the toxicity of the probe in living cells, L929 cells (murine aneuploid fibrosarcoma cell) and HeLa cells (human cervical cancer cell) were incubated in 8
Page 8 of 37
DMEM medium supplemented with 10% fetal bovine serum (FBS) and allowed to grow for 24 h at 37 °C with 5% CO2. After the removal of the medium, cells were treated with the probe and incubated for an additional 24 h. The cytotoxicity of the
ip t
probe against the cells was assessed by MTT assay according to ISO 10993-5. As for
cr
the assays, for each independent experiment, the assays were performed in eight
us
replicates. And the statistical mean and standard deviation were used to estimate the cell viability.
an
2.8. Cell incubation and imaging.
Two cell lines, HeLa (human cervical cancer cell) and L929 (murine aneuploid
M
fibrosarcoma cell) cells, were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS, HyClone) respectively. One day before imaging, cells were
te
d
passaged and plated on the 30 mm glass culture dishes and allowed to grow to 50-70% confluence. For the experiments, the cells were washed with DMEM,
Ac ce p
incubated in DMEM medium containing the probe (2 × 10
-5
M) at 37 °C under 5%
CO2 for 1.5 hour. Some HeLa cells were incubated in DMEM medium containing the ALP inhibitor (Levamisole hydrochloride, 1 × 10-3 M) for 30 min, and then treated
with the probe (2 × 10
-5
M) for 1.5 h. The control sample was incubated in DMEM
medium only. After that, the culture dishes were washed with PBS, the cells were washed with PBS for three times and then imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD. 3. Results and Discussion 3.1. Synthesis. 9
Page 9 of 37
The probe was prepared by the reaction of Compound 1 with diethyl chlorophosphate in THF under basic conditions at room temperature for two steps as outlined in Scheme 1. Compound 1 can be also easily prepared through the reaction
ip t
between 2-hydroxyacetophenone and p-methyl benzaldehyde. The structure of the 31
P NMR
cr
immediate compound and the probe has been characterized by 1H /
us
spectrometry and mass spectrometry, as shown in Figures S1-S7. The results confirm the successful synthesis of the probe.
an
3.2. Fluorescent response of the probe toward ALP.
The sensing ability of probe for ALP was investigated in Tris-HCl buffered
M
water (pH 8.0, 10 mM) with 20% DMSO at 37 °C. Under this condition, the probe (10 μM) shows maximum absorption at 335 nm (see Figure S8), and is weakly
te
d
fluorescent (Φ= 0.0031; see Table S1). Upon addition of ALP, the maximum absorption at 335 nm decreases, in the meantime at 410 nm a new absorption peak
Ac ce p
emerges (see Figure S8), and the probe exhibits a strong fluorescent emission (Φ= 0.085; see Table S1) at 484 nm with the excitation wavelength of 405 nm. In order to choose the appropriate excitation wavelength, we investigated four excitation wavelengths at 345 nm, 375 nm, 390 nm and 405 nm; and we found that, the probe incubated with ALP shows two fluorescent emissions upon excited at 345 nm or 375 nm (see the Figure S9 (A, B)), while the probe incubated with ALP displays only one fluorescent emission upon excited at 390 nm or 405 nm (see the Figure S9 (C, D)). Since visible is more beneficial for biological applications, we choose 405 nm as the excitation wavelength in this study. 10
Page 10 of 37
In the probe herein, the phosphate group serves both as the blocking agent for ESIPT and the responsive moiety toward ALP. We expect that, the protection of the hydroxyl group in compound 3 (the probe) by the phosphate group will block the
ip t
ESIPT process and result in the quenching of the tautomer emission; the addition of
cr
ALP cleaves phosphate group, and transforms the probe (compound 3) back into
us
compound 1, which correspondingly restores the ESIPT process and recovers the strong Tautomer emission (Figure 1). And the probe remains stable in the wide pH
an
range of 4.5 to 9.2 (Figure S10), which guarantees its utilization in biological samples.
M
We investigated the fluorescent spectra of the probe solution in the absence and presence of ALP, as shown in Figure 2. The fluorescence spectra of the probe were
te
d
periodically recorded during incubation of the probe with ALP (100 U/L) at 37 °C in Tris-HCl buffered water (pH 8.0, 10 mM) with 20% DMSO. In the absence of ALP,
Ac ce p
the probe solution displays almost no emission. Upon addition of ALP, the probe shows rapid response, the fluorescence intensity increases gradually with the prolonged incubation time. For instance, the fluorescence intensity at 484 nm of the ensemble was enhanced by 22 times after ALP (100 U/L) was introduced and the solution was incubated for 70 min. In addition, HPLC was employed to confirm the enzymatic reaction between the probe and ALP. As shown in Figure. S11, the results indicate that, the fluorescence enhancement during enzymatic reaction is the result of the formation of the expected product (Compound 1). Next, we measured the fluorescence spectra of the probe toward ALP at varied 11
Page 11 of 37
concentrations (reaction at 37 °C for 60 min in Tris-HCl buffer (pH 8.0) with 20% DMSO), as shown in Figure 3(A). It can be seen that the fluorescence intensity increases steadily with the increasing ALP level. The fluorescence intensity ratio of -5
M) as a function of ALP level is shown in Figure 3(B).
ip t
the probe solution (1 × 10
cr
Figure 4 displays the variation of the fluorescence intensity at 484 nm of the probe
us
after incubation with different amounts of ALP (0.1 - 100 U/L) for different time periods. Obviously, the fluorescence intensity of the probe increases after incubation
an
with ALP; moreover, the fluorescence intensity is enhanced more rapidly when the concentration of ALP is higher. This is understandable by considering the fact that the
M
hydrolysis of the probe would be facilitated in the presence of a high concentration of ALP.
te
d
The difference in fluorescence intensity for the probe solution before and after incubation with ALP can be easily distinguished by the hand-held UV lamp (365 nm)
Ac ce p
illumination (see insets of Figure 1). Figure S12 shows the plot of 1/V vs 1/[S], and the
corresponding
Lineweaver-Burk
plot.
By
fitting
the
plot
with
the
Michaelis-Menten equation, the kinetic parameters for ALP (Km and Vmax) were
estimated to be 1.919 µM and 1.21 ×10-3 µmol/min respectively. The affinity constant between the probe with ALP was derived by Scatchard plot (Figure S13), the affinity constant Ka were estimated to be 1.84×107 L·mol-1. We also established a working curve by plotting the emission intensity ratio of 484 nm versus ALP levels, and the detection limit has been determined as 0.032 U/L (Figure S14), which is sensitive enough for determining ALP levels in biological assays, since for healthy adults the 12
Page 12 of 37
ALP level in blood is in the range of 46 ~ 190 U/L [72]. Further, the selectivity of this probe for ALP was also tested. The fluorescence signal change of the probe solution containing ALP was compared with that of the solutions containing other
ip t
proteins/enzymes under the same conditions (Figure S15), including alanine
cr
aminotransferase (ALT), acetyl cholinesterase (AchE), Bovine serum albumin (BSA),
us
β-galactosidase (β-GAL), glutamyl transpeptidase (GGT), hyaluronidase (HAase), nitroreductase and esterase. As shown in Fig. S14, in contrast to the significant
an
increase of fluorescence intensity induced by ALP, the addition of other proteins/enzymes trigger negligible fluorescence changes for the probe solution.
M
These results indicate that the probe is highly selective for ALP assay. 3.3. ALP detection in serum samples.
te
d
To extend the application of this probe, we applied the probe to detecting the endogenous ALP levels in human serum samples, the serum samples were diluted
Ac ce p
50-fold for the measurement. First, we investigated the probe’s fluorescence response in diluted serum sample in the presence of or absence of ALP inhibitor, and the results are shown in Figure S16. It is clear that the addition of ALP inhibitor levamisole [73] results in obvious decrease of the fluorescence intensity, confirming the probe’s specific interaction with ALP in diluted serum sample. The serum samples detection results were compared with those by using the commonly-used commercial assay Elisa kit, and the results are presented in Table S2. The endogenous ALP levels in the serum samples from both positive and negative clinical samples measured by the probe are close to those measured by using Elisa kit. However, as for the ELISA kit, 13
Page 13 of 37
the detection processes are quite complex which requires multiple procedures, while for this probe, the detection process is very convenient and could serve as a one-step
ip t
straightforward fluorescence assay for ALP detection in diagnostics. 3.4. Imaging of endogenous ALP in live cells.
cr
We first tested in vitro cytotoxicity of the probe. The cytotoxicity was evaluated
us
using L929 and HeLa cell lines by MTT assay in compliance with ISO 10993-5, and the results are shown in Figure S17. No significant reduction in cell viability can be
an
observed for cells treated with the probe even at high concentrations (up to 50 µM), proving that the probe shows little toxicity in vitro and is suitable for fluorescence
M
imaging of ALP in live cells.
The probe’s cell imaging capability was evaluated using HeLa cells, in which
te
d
ALP is overexpressed. The imaging for endogenous ALP in the cells is shown in Figure 5. HeLa cells loaded with the probe show a strong blue-green fluorescence
Ac ce p
(Figure 5E), which is caused by the existence of endogenous ALP in HeLa cells. For comparison, we also applied the probe to cellular imaging in L929 cells that do not have overexpression of ALP [74, 75], as shown in Figure S18. It is clear that, as L929 cells were loaded with the probe, no fluorescence can be observed. We suppose that, the probes internalized by L929 cells won’t undergo dephosphorylation due to the lack of ALP in the cells and accordingly no emission can be observed. To further prove that the blue-green fluorescence observed in HeLa cells were triggered by ALP, the cells were pretreated with ALP inhibitor levamisole hydrochloride for 30 minutes, and then incubated with the probe for 1.5 h. As shown 14
Page 14 of 37
in Figure 5H, the blue-green fluorescence is almost indiscernible. The results indicate that the intracellular fluorescence changes in HeLa cells are indeed triggered by ALP. The above results demonstrate the strong potential of the probe to detect endogenous
cr
ALP inside cells as well as aid in pathological analysis.
ip t
ALP in live cells, which is of great importance to clarify the physiological roles of
us
4. Conclusions
In summary, we have successfully developed a new fluorescent probe for
an
detecting ALP, which functions through visible-light excitable excited-state intramolecular proton transfer (ESIPT). The prove features rapid detection process,
M
low detection limit and high selectivity. In comparison with other probes for ALP (as shown in Table S4), this probe has several advantages, first this new probe can be
te
d
easily prepared and works effectively under physiological pH and in such biological sample as human serum. Furthermore, this probe is of little cytotoxicity and can be
Ac ce p
easily loaded into cells for endogenous ALP imaging. Therefore, the strategy herein could offer alternative approaches for fabricating practical fluorescent assays or probes for other types of biomolecules.
Acknowledgements
We gratefully acknowledge the financial support by NSFC (21474031, 21174040, 21025415 and 51403065), and the National Key Basic Research Program of China (Project No. 2013CB834702).
15
Page 15 of 37
Appendix A. Supplementary data
Ac ce p
te
d
M
an
us
cr
ip t
Supplementary data associated with this article can be found, in the online version, at http://
16
Page 16 of 37
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Biographies Qinghua Hu received his Bachelor of Engineering degree from Jishou University, Jishou, China, in 2009. He is currently pursuing the PhD degree in materials science
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at South China University of Technology, Guangzhou, China under the supervision of
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fluorescence spectroscopy for their biological applications.
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Prof. Shuizhu Wu. His research interests include materials synthesis, sensors and
Fang Zeng has been a professor at the College of Materials Science and Engineering,
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South China University of Technology, Guangzhou, China since 2004. His primary research interests include fluorescent materials, nanoparticles, energy transfer,
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chemosensors and biosensors, and drug and gene delivery systems. Changmin Yu obtained his PhD degree at the College of Materials Science and
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Engineering, South China University of Technology, Guangzhou, China in 2013. He is now working in South China University of Technology as a lecturer. His current
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research interests include fluorescent materials, energy transfer in materials, chemosensors and biosensors.
Shuizhu Wu has been a professor at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China since 2005. Her primary research interests include fluorescent materials, photophysics, chemosensors and biosensors, nanoparticles, and biomaterials.
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Figure captions: Fig. 1. Schematic illustration of the probe’s structure and fluorescent response toward ALP.
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Fig. 2. Time course of fluorescence spectra of the probe (1×10-5 M) in the presence of
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ALP (100 U/L) in Tris-HCl buffered water (pH 8.0, 10 mM) with 20% DMSO at 37 °C with the excitation wavelength of 405 nm.
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Fig. 3. (A) Fluorescence spectra of the probe (1 × 10 -5 M) in the presence of different
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amounts of ALP in Tris-HCl buffer (pH 8.0, 10 mM) with 20% DMSO at 37 °C for 60 min. (B) Fluorescence intensity ratio (F/F0) as a function of ALP level.
independent experiments.
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Excitation wavelength: 405 nm. Data represent mean ±SD from three
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Fig. 4. Fluorescence intensity at 484 nm of the probe (1 × 10
-5
M) in Tris-HCl
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buffered (pH 8.0, 10 mM) with 20% DMSO at 37 C versus reaction time in the
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presence of different amounts of ALP.
Fig. 5. Fluorescence microscopy images for HeLa cells in DMEM medium with 1% DMSO. (A-C): The control; (D-F): Cells treated with the probe (2 × 10 -5 M) for
1.5 h; (G-I): Cells treated with inhibitor levamisole hydrochloride (1 × 10 for 30 min and then the probe (2 × 10
-5
-3
M)
M) for 1.5 h. (Excitation filter 400-410
nm, emission filter ≥ 455 nm).
Scheme captions: Scheme 1. Synthetic route of the probe. 29
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Biographies Qinghua Hu received his Bachelor of Engineering degree from Jishou University,
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Jishou, China, in 2009. He is currently pursuing the PhD degree in materials science
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at South China University of Technology, Guangzhou, China under the supervision of
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Prof. Shuizhu Wu. His research interests include synthesis of fluorescent compounds, sensors and their biological applications.
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Fang Zeng has been a professor at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China since 2004. His primary
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research interests include fluorescent materials, nanoparticles, energy transfer, chemosensors and biosensors, and drug and gene delivery systems.
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Changmin Yu obtained his PhD degree at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China in 2013. He
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is now working in South China University of Technology as a lecturer. His current research interests include fluorescent materials, energy transfer in materials, chemosensors and biosensors.
Shuizhu Wu has been a professor at the College of Materials Science and Engineering, South China University of Technology, Guangzhou, China since 2005. Her primary research interests include fluorescent materials, photophysics, chemosensors and biosensors, nanoparticles, and biomaterials.
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