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A fluorescent assay for γ-glutamyltranspeptidase via aggregation induced emission and its applications in real samples
Biosensors and Bioelectronics 85 (2016) 317–323
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A fluorescent assay for γ-glutamyltranspeptidase via aggregation induced emission and its applications in real samples Xianfeng Hou, Fang Zeng n, Shuizhu Wu n College of Materials Science & Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 March 2016 Received in revised form 6 May 2016 Accepted 10 May 2016 Available online 10 May 2016
γ-Glutamyl transpeptidase (GGT) plays crucial roles in some physiological processes. Herein a turn-on fluorescent probe for γ-glutamyl transpeptidase (GGT) assay based on aggregation-induced-emission (AIE) effect and the enzyme-induced transformation of hydrophilicity to hydrophobicity has been developed by functionalizing tetraphenylethylene (TPE) derivative with two γ-glutamyl amide groups, which simultaneously work as recognition units and hydrophilic groups. When the γ-glutamyl amide groups are cleaved through GGT enzymatic reaction, the hydrophobic reaction product readily aggregate and correspondingly strong blue fluorescence can be observed, as a result of activated AIE process. By virtue of the probe's good solubility in totally aqueous solution, high sensitivity and excellent photostability, the probe can be employed to detect GGT level in human serum samples. Furthermore, the probe can be used for imaging endogenous GGT in living A2780 cells. Hence, the probe holds great promise for acting as a convenient one-step straightforward assay for GGT detection in diagnostic-related applications, and also it could provide a useful approach for conducting pathological analysis for diseases involving GGT. & 2016 Elsevier B.V. All rights reserved.
Keywords: γ-Glutamyl transpeptidase Aggregation-induced-emission Fluorescence Imaging
1. Introduction
γ-Glutamyltranspeptidase (GGT), an endogenous enzyme, plays crucial roles in glutathione metabolism and homeostasis, which is also involved in other important physiological processes such as the detoxification of xenobiotics through cleaving γ-glutamyl linkage of glutathione S-conjugates (Hiratake et al., 2004; Taniguchi and Ikeda, 1998; Whitfield, 2001). On the other hand, elevated GGT levels are closely related to diseases of the liver, biliary system, and pancreas as well (Ostapenko et al., 2011; Vandenhaute et al., 2011); in addition, GGT, which is a cell surface-associated (or bound) enzyme, has been reported to be overexpressed in several human tumors including cervical and ovarian cancers (Grimm et al., 2013; Urano et al., 2011). Hence, GGT has been employed as an important biochemical indicator in clinical diagnosis for evaluating diseases involving liver, gallbladder, and bile ducts; and it can also offer an effective means for conducting pathological study concerning the above-mentioned diseases; therefore, robust and sensitive detection of GGT level is of great importance in terms of both diagnostics-related applications and pathological analysis. A number of methods have been employed for detecting GGT n
Corresponding authors. E-mail addresses: [email protected] (F. Zeng), [email protected] (S. Wu).
http://dx.doi.org/10.1016/j.bios.2016.05.036 0956-5663/& 2016 Elsevier B.V. All rights reserved.
levels. For instance, colorimetric assays with γ-glutamyl-3-carboxy-4-nitroanilide as substrate (Tateishi et al., 1976), high-performance liquid chromatography (HPLC) (Hoskins and Davies, 1986), and fluorescence technique (Hou et al., 2014). Currently, there already are commercial GGT assay kits for clinical diagnosis based on colorimetric enzymatic assay using serum samples [e. g. IBL (Germany)]. Among these methods, in particular fluorescence technique are receiving considerable attention owing to their convenience, noninvasive monitoring capability and usability in live cells or biological samples (Buccella et al., 2011; Burns et al., 2010; Chen et al., 2015; Dickinson et al., 2013; Hu et al., 2016; Kim et al., 2012; Ma et al., 2016; Moragues et al., 2014; Ren et al., 2016; Shen et al., 2011; Tao et al., 2013; Todescato et al., 2014; Tong et al., 2015; Wang and Wolfbeis, 2014; Yang et al., 2013; Zhang et al., 2013; Zhang et al., 2014; Zong et al., 2016). Up to now, a few fluorescent probes for GGT detection or imaging have been reported (Hou et al., 2014; Li et al., 2015; Wang et al., 2015; White et al., 1996; Zhang et al., 2016); all of them adopted conventional fluorophores whose emission will experience aggregation-caused quenching (ACQ); in addition, for these probes, the products of the enzymatic reaction between these probes and GGT are poorly water-soluble, which easily results in aggregation in aqueous media and consequently the ACQ effect will interfere with the enzyme detection; this might turn out to be a limitation for these probes in terms of practical applications.
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In recent years, fluorogens with aggregation-induced emission (AIE) characteristics have attracted great interest (Ding et al., 2013; Yan et al., 2016; Yang et al., 2015). Unlike conventional fluorophores, which experience ACQ effect due to the non-radiative relaxation of the aggregates’ excited states (An et al., 2002), AIE fluorogens like tetraphenylethylene (TPE) derivatives exhibit strong fluorescence enhancement when they are aggregated. That is because, in aggregated state, intramolecular rotations are restricted, which suppresses nonradiative energy dissipation of the aggregates’ excited states, resulting in enhanced radiative transition and high quantum yields (Ding et al., 2013). Based on their unique fluorescence behaviour, a number of AIE-based fluorescent turn-on probes have recently been developed for the detection of various analytes ranging from ions (Gui et al., 2015), DNA (Wang et al., 2008), proteins (Wu et al., 2015), pH (Chen et al., 2013), and cell apoptosis processes (Ding et al., 2013), which indicated that AIE-based sensors possess many advantages, such as low background interference, improved imaging contrast, and photostability (Wang et al., 2014). However, many AIE-based fluorescent probes are not suitable for biological applications due to their much too poor water solubility. It is envisioned that, by coupling hydrophilic γ-glutamyl amide groups onto TPE derivative to make the γ-glutamyl-amide-containing TPE derivative, we could, on one hand, greatly improve the water solubility of the AIE-active fluorophore, making it operable in aqueous biological samples; on the other hand, since γ-glutamyl amide group is the specific substrate for GGT enzymatic reaction, this γ-glutamyl-amide-containing TPE derivative could serve as a fluorescent assay for GGT detection via aggregation induced emission for applications in real biological samples like serum; moreover it could avoid the problems associated with the fluorescent probes for GGT which employed conventional fluorophores (interference resulted from aggregation of enzymatic reaction product due to poor water solubility). To this end, herein, we synthesized the γ-glutamyl-amide-containing TPE derivative and investigated its fluorescent response to GGT. As shown in Scheme 1, the probe molecule can function well in totally aqueous media and biosystems due to its quite good water solubility. When γ-glutamyl amide groups are cleaved by GGT, the resultant product molecules readily aggregate due to poor water solubility, thus strong blue fluorescence can be observed because of AIE effect (Scheme S1). Furthermore, this probe's AIE feature overcomes the limitation of conventional fluorescent probes for GGT, and endows it with quite good resistance to photobleaching. In addition, the specific enzymatic reaction ensures the probe's high selectivity for GGT. Hence, the probe can selectively detect GGT levels in real
biological samples like serum. Because of its low cytotoxicity, the probe can be also used for imaging endogenous GGT in ovarian cancer cells (A2780 cells).
2. Material and methods 2.1. Chemicals and materials 4-Methylbenzophenone, TiCl4, Zn dust, di-tert-butyl discarbonate ((Boc)2O), 2-(4-aminophenyl) ethanol, N-bromosuccinimide (NBS), carbon tetrachloride (CCl4), sodium hydride (NaH), 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloricde (EDC), 4-dimethyaminopyridine (DMAP), glutamic acid, leucine, cysteine, arginine, phenylalanine and N,N-dimethylformamide (DMF) were purchased from Aladdin Reagents. Boc-L-glutamic acid 1-tert-butyl ester (Boc-Glu-OtBu), N,N-diisopropylethylamine (DIEA), alkaline phosphatase (ALP), carboxylesterase, deacetylase and γ-glutamyltranspeptidase (GGT) were purchased from Sigma Aldrich. γ-Glutamyltranspeptidase (GGT) ELISA Kit was purchased from Shanghai Enzyme-linked Biotechnology Co. Ltd.. Human serum samples were supplied by The Sixth Affiliated Hospital, Sun Yat-sen University; and the serum samples were from both healthy and unhealthy human beings with abnormal GGT levels. Petroleum ether, ethyl alcohol, ethyl acetate, ammonia water, methanol, dichloromethane, trithylamine (TEA), benzoyl peroxide (BPO), concentrated sulfuric acid, anhydrous sodium sulfate, sodium chloride and sodium hydroxide were of analytical grade reagents. The water used in the experiments was triple-distilled, treated by ion exchange columns and then by a Milli-Q water purification system. 2.2. Synthesis of 1,2-bis(4-methylphenyl) 1,2-diphenylethene (Compound 1) The compound was prepared according to previously published experimental procedures (Zhao et al., 2010). The synthesis was briefly described as follows: Under nitrogen atmosphere, into a vacuum-evacuated 100 mL round-bottomed flask was added 1.96 g (10 mmol) of 4-methylbenzophenone and 60 mL of THF. The solution was cooled down to 5 °C, then TiCl4 (5.70 g, 30 mmol) and Zn dust (2.62 g, 40 mmol) were added. After being refluxed overnight, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and dissolved in CH2Cl2, and then washed with H2O for several times, the organic layer was dried over anhydrous Na2SO4. The crude product was further
Scheme 1. Schematic illustration of the probe's fluorescent detection for GGT.
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purified by a silica gel column using petroleum ether as eluent. A white solid was obtained in 82.7% yield (1.62 g). 1H NMR (CDCl3, 400 MHz, ppm):2.24 (d, J ¼ 6.8, 6H), 6.88 6.91 (m, 8H), 6.99 7.10 (m, 10H). MS (ESI): m/z 360.1 [MþH] þ .
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dichloromethane: methanol¼ 100: 100: 4 in v/v) to furnish the desired product as a white solid (0.26 g, 43.3%).1H NMR (CDCl3, 400 MHz, ppm): 1.44(s, 18H), 1.46(s, 18H), 1.91(m, 2H), 2.13(m, 2H), 2.33(m, 4H), 2.82(t, J ¼5.2, 4H), 4.21 (m, 10H), 6.52–6.68 (m, 4H), 6.95–7.17 (m, 22H). MS (ESI): m/z 1202.13 [MþH] þ .
2.3. Synthesis of 2-(4-(N-Boc-amino)phenyl)ethanol (Compound 3) The compound was prepared according to previously published experimental procedures (Choi et al., 2006). The synthesis was briefly described as follows: 2-(4-aminophenyl) ethanol (1.37 g, 10.0 mmol) which was dissolved in TEA/MeOH (10 mL /15 mL) was treated with (Boc)2O (2.4 g, 11.0 mmol) at 40 °C overnight. The reaction mixture was evaporated and CH2Cl2 (50 mL) was added. The organic layer was washed with saturated NaCl (20 mL) and H2O (20 mL). The solvent was dried in vacuo and purified by a column chromatography with dichloromethane: methyl alcohol (15: 1) to obtain 3 (1.91 g, 80.6%) as a colourless oil. 1H NMR (CDCl3, 400 MHz, ppm): 1.51(s, 9H), 2.81(t, J ¼4.0, 2H), 3.82(t, J ¼4.4, 2H), 7.14 (d, J ¼ 6.4, 2H), 7.29 (d, J ¼5.6, 2H). MS (ESI): m/z 259.69 [MþNa] þ . 2.4. Synthesis of 1,2-bis(4-(2-oxo-5-(p-(Boc-amino)phenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 4) Into a round-bottom flask was added 0.36 g (1 mmol) of Compound 1, 0.26 g (1.1 mmol) of freshly recrystallized NBS, and a catalytic amount of BPO in 10 mL of CCl4. The solution was refluxed at 80 °C for 6 h. After being cooled to room temperature, the solution was filtered and the filtrate was concentrated. Then the solvent was removed by vacuum-rotary evaporation and the crude product (Compound 2) was obtained. Afterward, to a solution of 60% NaH in THF at 0 °C under a nitrogen atmosphere, Compound 3 in 5 mL THF was added dropwise in 10 min After 1 h, Compound 2 in 5 mL DMF solution was added dropwise to the mixture in 30 min After being refluxed overnight, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and dissolved in CH2Cl2, and then washed with H2O for several times, the organic layer was dried over anhydrous Na2SO4. The crude product was further purified by a silica gel column (ethyl acetate: dichloromethane ¼ 1: 2 in v/v) to obtain Compound 4(0.58 g, 67.4%) as a white solid. 1H NMR (CDCl3, 400 MHz, ppm): 1.39(s, 18H), 2.83(t, J ¼5.2, 4H), 3.82(t, J ¼ 4.4, 4H), 4.69 (d, J ¼6.4, 4H), 6.94–7.14 (m, 26H). MS (ESI): m/z 853.80 [MþNa] þ . 2.5. Synthesis of 1,2-bis(4-(2-oxo-5-(p-aminophenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 5) Compound 4 (0.41 g, 0.5 mmol) was dissolved under stirring in 15 mL CH2Cl2, then hydrogen chloride generated from the reaction of NaCl and H2SO4 was passed into the solution for 1 h. Afterwards the solvent was evaporated under vacuum to get the product (0.30 g, 95%). 1H NMR (d-DMSO, 400 MHz, ppm): 2.73(t, J ¼4.4, 4H), 3.59(t, J ¼ 4.0, 4H), 4.36 (d, J ¼7.2, 4H), 6.88–6.99 (m, 8H), 7.10–7.36 (m, 18H). MS (ESI): m/z 631.52 [M þH] þ . 2.6. Synthesis of 1,2-bis(4-(2-oxo-5-(p-(Boc-L-Glu(otBu)-amino) phenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 6) Under a nitrogen atmosphere, Boc-Glu-OtBu (0.30 g, 1 mmol), EDC (0.23 g, 1.2 mmol) and DMAP (0.006 g, 0.05 mmol) were dissolved in dichloromethane, then the mixture of Compound 5 (0.41 g, 0.5 mmol) and DIEA (0.15 g, 1.2 mmol) in dichloromethane was added. After being stirred at room temperature for 24 h, the solution was washed with slightly acidic water (pH ¼6.5). Then the organic layer solvent was evaporated and the residue was purified by column chromatography on silica gel (hexane:
2.7. Synthesis of Synthesis of 1,2-bis(4-(2-oxo-5-(p-(L-Glu-amino) phenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 7, the probe) Compound 6 (0.26 g, 0.2 mmol) was dissolved under stirring in 10 mL CH2Cl2, then hydrogen chloride generated from the reaction of NaCl and H2SO4 was passed into the solution for 1 h. Afterwards the solvent was evaporated under vacuum to get the product (0.18 g, 95.6%). 1H NMR (CD3OD, 400 MHz, ppm): 2.12(m, 2H), 2.26 (m, 2H), 2.59(m, 4H), 3.03(m, 4H), 4.06(m, 2H), 4.35(t, J ¼ 6.4, 4H), 4.49 (d, J ¼6.8, 4H), 6.98–7.04 (m, 4H), 7.07–7.18 (m, 10H), 7.19– 7.26 (m, 4H), 7.28–7.35 (m, 4H), 7.40–7.44 (m, 4H). MS (ESI): m/z 888.99 [M þH] þ . 2.8. Measurements 1
H NMR spectra were recorded on a Bruker Avance 400 MHz NMR 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. 2.9. Cell viability assay Cell viability assay was employed to evaluate the cytotoxicity of the probe. In the assay, L929 cells (murine aneuploid fibrosarcoma cells) and HeLa cells (human cervical cancer cell) were incubated in DMEM medium that was supplemented with 10% fetal bovine serum (FBS), and allowed to grow for 24 h with 5% CO2 at 37 °C. Upon removal of the medium, the cells were treated with the probe and then incubated for 24 h. MTT assay was adopted to assess the cytotoxicity of the probe against the cells according to ISO 10993-5. For the assays, as for each independent experiment, the assays were performed in eight replicates. And the statistical mean and standard deviation were used to estimate the cell viability. 2.10. Cell incubation and imaging In the experiment, two cell lines, namely A2780 (human ovarian cancer cell) and L929 (murine aneuploid fibrosarcoma cell), were incubated in DMEM medium which was supplemented with 8% and 10% fetal bovine serum (FBS, hyclone) respectively. One day before imaging, cells were passaged and plated on polylysine-coated cell culture glass slides inside the 30 mm glass culture dishes, which were then allowed to grow to 50–70% confluence. As for the experiments, the cells were washed with DMEM, after that the cells were incubated in DMEM medium containing the probe (20 μM) at 37 °C under 5% CO2 for 1 h. Some A2780 cells were incubated in DMEM medium containing inhibitor (DON, 10 μM) for 30 min, and then treated with the probe (20 μM) for 1 h. The control sample was incubated in DMEM medium only. After that, the culture dishes were washed with PBS, the glass slides were taken out, 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.
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3. Results and discussion 3.1. Probe synthesis and fluorescent response toward GGT With AIE characteristic, TPE derivatives can be used for fabricating “turn-on” fluorescent probes. The chemical structure of the probe and its synthetic process are depicted in Scheme S2. To synthesize the probe, hydrosoluble glutamic acid groups were coupled onto TPE derivative, so as to make the γ-glutamyl-amidecontaining TPE derivative (the probe, Compound 7) and ensure the probe molecules are well soluble in aqueous media (without aggregation). The intermediate products and the probe were characterized by proton nuclear magnetic resonance (1H NMR) and mass spectrometry (MS) (Figs. S1–S12). Fig. S13 shows the excitation and emission spectra of this probe in PBS-buffered water. In the absence of GGT, the probe solution exhibits almost no emission. Upon reaction with GGT for 45 min, the probe exhibits an emission band at about 472 nm when excited at 360 nm. Fluorescent spectra were measured periodically during the incubation of the probe (20 μM) with GGT (60 U/L) at 37 °C in PBSbuffered water (pH 7.4, 10 mM), so as to investigate the probe's fluorescent response toward GGT, and the results are shown in Fig. 1A. In the absence of GGT, the probe solution displays almost no emission. Upon addition of GGT, an emission at about 472 nm emerges, corresponding to the emission of the enzymatic reaction product which is the same as Compound 5 (Fig. S15). MS analysis also suggests that the product of the enzymatic reaction has the same molecular structure as Compound 5 (Fig. S16). As the incubating (reaction) time is lengthened, the emission at around 472 nm increases. This is because, GGT cleaves the γ-glutamyl amide moieties from the probe molecule, this results in the aggregation of the hydrophobic reaction product (1,2-bis (4-(2-oxo5-((p-aminophenyl))pentyl)phenyl) 1,2-disphenylethene) (Compound 5). DLS analysis indicates that large aggregates form with an average diameter of 116 nm, after the probe being incubated with GGT at 37 °C for 45 min (Fig. S17). In addition, the resistance to photobleaching for the aggregates of the enzymatic reaction product molecules was also investigated. As shown in Fig. 1(B), upon continuous illumination at 365 nm (15 W handheld UV lamp) for 3 h, fluorescence intensity of the reaction product aggregates only reduce by 6.4%, while the other two conventional fluorophores (fluorescein and coumarin) show obvious photobleaching phenomenon. This result suggests that the reaction product aggregates have good photostability. The data for enzyme kinetics follow the Michaelis-Menten equation, and the kinetic parameters have been determined as Vmax ¼1.10 μM/min and Km ¼ 15.17 mM (Fig. S18). The Km value is small, indicating the strong affinity between GGT and the probe molecule.
With the addition of a small amount of CTAB (20 μM) into the probe solution, and in the presence of GGT, the system's fluorescence intensity increases significantly; at 60 U/L GGT, the fluorescence intensity increases up to 12-fold (Fig. 2A). To illustrate the role of CTAB for such obviously enhanced fluorescence intensity, fluorescence spectra and DLS measurements were carried out in the absence or presence of CTAB. Fluorescent spectra show that, in the absence of GGT, even with the addition of CTAB, the probe solution does not display obvious fluorescence (Fig. S19A). DLS analyses also confirm that, in the absence of GGT, the addition of CTAB has no obvious impact on the size of the aggregates in the solution, as shown Fig. S19C. However, in the presence of GGT, after the probe solutions being incubated for 45 min at 37 °C, the fluorescence intensity of the solution with CTAB increases much more drastically, compared to the system without CTAB (Fig. S19B); in the meantime, the size of the aggregates in the solution with CTAB is much larger than the system without CTAB (Fig. S19D). These results suggest that CTAB can promote the formation of aggregates in the system and result in the significant enhancement of fluorescence intensity. In addition, the fluorescence intensity of the probe solutions (with or without CTAB) in the presence of GGT was measured as a function of time (Fig. S20). It is clear that, for both the systems (with or without CTAB), the reaction between GGT and the probe is completed at about 45 min; this indicates that CTAB have no influence on the GGT enzymatic reaction; thus it is highly possible CTAB simply promote the aggregation of the product from the reaction between GGT and the probe. Furthermore, the time-course experiments of the probe at different amounts of GGT were recorded, as shown in Fig. 2B. It also shows that the enzymatic reaction is almost completed in 45 min. The fluorescence spectra of the system in the presence of different amounts of GGT are shown in Fig. 2C and the corresponding working curve (the emission intensity at 472 nm versus GGT levels) is shown in Fig. 2D. And the detection limit has been determined to be 0.59 U/L (Fig. S21), which is sensitive enough for determining GGT levels in biological assays, given that for normal adult females, the normal of GGT level in blood is 5–55 U/L, and that for adult males is 15–85 U/L (Berk and Korenblat, 2011). 3.2. Selectivity of the probe toward GGT In order to evaluate the selectivity of the probe, the fluorescence response of probe solution in the presence of different biological substances was measured, and the results are shown in Fig. 3A. It can be seen that, only GGT induces a prominent intensity enhancement, other species (biologically relevant) do not generate a rise in the fluorescence intensity, which include ions (such as
Fig. 1. (A) Time course of fluorescence spectra for the probe (20 μM) in PBS-buffered water (pH 7.4, 10 mM) at 37 °C upon addition of 60 U/L GGT. (B) Photostability of the reaction product aggregates (black), 20 μM fluorescein (red) and 20 μM 7-diethylamino-4-methylcoumarin (green) in PBS (pH ¼ 7.4, 10 mM). IT/I0 is the fluorescence intensity ratio at 472 nm, 520 nm and 460 nm respectively excited at 360 nm after T-minute light irradiation and before light irradiation.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. (A) Time course of fluorescence spectra for the probe (20 μM) with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C upon addition of 60 U/L GGT. (B) Plot of the fluorescence intensity of the probe (20 μM) with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C versus reaction time in the presence of different amounts of GGT. (C) Fluorescence response of the probe (20 μM) with 20 μM CTAB in the presence of different GGT levels in PBS-buffered water (pH 7.4, 10 mM) at 37 °C for 45 min (D) Fluorescence intensity at 472 nm as a function of GGT level. Excitation wavelength: 360 nm.
Fig. 3. (A) Fluorescence response of 20 μM probe with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C for 45 min in the presence of different biologicallyrelevant substances. (B) Fluorescence response of 20 μM probe with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C for 45 min in the presence of 60 U/L GGT and with the addition of different biological substances respectively. The concentration of the ions and amino acids are 100 μM, while the concentration of protein enzymes are 100 U/L.
Na þ , K þ , Mg2 þ , Ca2 þ , Al3 þ , NO3-, CO32 and PO43 ), amino acids (such as glutamic acid, leucine, cysteine, arginine and phenylalanine) and protein enzymes (such as alkaline phosphatase (ALP), carboxylesterase and deacetylase). The interference of these biologically-relevant substances was also investigated. Fig. 3B shows the fluorescence response of the probe towards GGT in the presence of these substances. It is clear that, these species do not interfere with fluorescence response of the probe, indicating these substances have negligible interfering effect on the GGT enzymatic reaction with the probe molecule. The probe herein can
specifically respond towards GGT, owing to the fact that GGT can specifically catalyze the cleavage of γ-glutamyl group. 3.3. Detection of GGT in real biological samples like serum Next, in order to examine the operability of the probe system in real samples, we applied the probe system to detecting the GGT levels in human serum samples. Firstly, we compared the probe's detection for GGT with the Elisa Kit (the commonly-used commercial assay kit for GGT) in PBS-buffered water. As Table S1
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Table 1 Endogenous GGT detection in human serum samples by using Elisa kit assay and this probe. Sample no. GGT amount (U/L) by ELISA Kit assay 1 2 3 4 5 6
53.26 7 4.37 62.337 3.89 42.217 5.34 238.8 7 11.82 205.7 7 12.10 186.17 10.87
GGT amount (U/L) by this probe 55.76 72.78 63.05 73.63 40.02 73.85 250.2 76.76 200.7 76.33 179.9 77.02
Note: The serum samples were 5-fold diluted for measurement; and the values (mean 7 SD) herein represent GGT levels in the undiluted serum samples, which are calculated based on the measurements for the diluted samples. Samples 1 and 2 were obtained from healthy males; Samples 3 was obtained from healthy female, Samples 4 and 5 were obtained from males with hepatic diseases, Samples 6 was obtained from female with hepatic diseases. All the serum samples were provided by The Sixth Affiliated Hospital, Sun Yat-sen University.
shows, the recovery and SD value from these two methods are similar; this suggests the accuracy and reliability of the probe system herein. Then we attempted to detect the endogenous GGT levels in human serum samples (Table 1, by using the calibration curve (Fig. 2D) as standard for this probe). The endogenous GGT levels in the serum samples from both healthy and unhealthy human beings measured by this probe are close to those measured by Elisa Kit. The results indicate that, the probe holds great
potential for serving as an effective detection method for GGT level in diagnostics-related applications. Furthermore, compared to Elisa Kit (the antibody-based colorimetric assay) involving complex procedures in the detection process, the probe system herein is quite convenient and could act as a one-step straightforward fluorescent assay for GGT. 3.4. Imaging of GGT in living cells A2780 cell lines (one of ovarian cancer cells), which is overexpressed with GGT (Grimm et al., 2013; Hanigan, 1995; Urano et al., 2011), were employed in the experiment, so as to further evaluate the applicability of the probe for live-cell imaging. First the cytotoxicity of the probe was measured by MTT assay with L929 and Hela cell lines in compliance with ISO 10993-5. And the results are shown in Figure S22. With these two cell lines, the probe show high cell viability of over 90% even at the high concentration of 80 μM; the results indicate that, the probe has very low cytotoxicity. Hence the probe is suitable for fluorescence imaging of GGT in live cells. And then we applied the probe in GGT imaging in living cells. As shown in Fig. 4B and Fig. S23B, no background fluorescence can be observed in A2780 and L929 cells in the absence of the probe, suggesting that autofluorescence from the cells is insignificant under this condition. When A2780 cells were treated with the probe for 1 h, the cells show a strong blue fluorescence (Fig. 4E);
Fig. 4. Fluorescence microscopy images. A2780 cells incubated in the absence of the probe (A, B and C), in the presence of the probe (D, E and F; the probe concentration: 20 μM), or in the presence of the inhibitor DON and the probe (G, H and I; DON concentration: 10 μM, the probe concentration: 20 μM). D, E and F: the cells were incubated with the probe for 1 h; G, H and I: the cells were first incubated with the inhibitor DON for 30 min, and then incubated with the probe for 1 h.(For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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this suggests that, the product molecules from the reaction between GGT and the probe aggregate inside A2780 cells. For comparison, we also applied the probe to cellular imaging in L929 cells, which do not have overexpression of GGT (Fig. S23). It can be seen that, when L929 cells were treated with the probe, no fluorescence can be observed; this is because, the probe molecules uptake by L929 cells do not aggregate, since no reaction will occur due to the lack of GGT in these cells. In addition, to further prove that the blue fluorescence observed in A2780 cells were indeed triggered by GGT, A2780 cells were first pretreated with GGT inhibitor DON for 30 min, and then treated with the probe for 1 h. As shown in Fig. 4H, no fluorescence can be observed. This indicates that the blue fluorescence in A2780 cells is triggered by GGT. These results demonstrate that the probe is promising for detecting endogenous GGT in live cells, which is useful for pathological analysis for the disease involving GGT. Compared to other fluorescent probes for GGT (as shown in Table S2), this probe has several advantages: first, owing to its good water solubility and biocompatibility, the probe can detect GGT in human serum samples, and imaging endogenous GGT in living cells; second, AIE effect endows this probe excellent photostability and high imaging contrast; third, this probe demonstrates strong potential for serving as a convenient one-step fluorescent assay for ALP.
4. Conclusion In summary, we have developed a new fluorescence turn-on probe for GGT based on the AIE effect, which functions through GGT-induced transformation of hydrophilic-to-hydrophobic property and the corresponding aggregation induced emission. CTAB can enhance fluorescence enhancement of the probe system through promoting the aggregation of the product from the reaction between GGT and the probe. In addition, the probe exhibits good water solubility, excellent photostability and high selectivity for GGT. The sensitivity and reliability ensure this probe can serve as a convenient one-step straightforward assay for GGT detection in serum samples, which holds promise for diagnostic-related applications. Furthermore the probe can be used for imaging endogenous GGT in living A2780 cells. Therefore, the probe would provide a useful approach for further elucidating the physiological roles of GGT as well as for conducting pathological analysis for diseases involving GGT.
Acknowledgment We gratefully acknowledge the financial support by the National Key Basic Research Program of China (Project No. 2013CB834702), NSFC (Grant nos. 21574044, 21474031 and 21174040), the Science and Technology Planning Project of Guangdong Province (Project no. 2014A010105009) and the Fundamental Research Funds for the Central Universities (2015ZY013).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.05.036.
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A fluorescent assay for γ-glutamyltranspeptidase via aggregation induced emission and its applications in real samples Xianfeng Hou, Fang Zeng n, Shuizhu Wu n College of Materials Science & Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 March 2016 Received in revised form 6 May 2016 Accepted 10 May 2016 Available online 10 May 2016
γ-Glutamyl transpeptidase (GGT) plays crucial roles in some physiological processes. Herein a turn-on fluorescent probe for γ-glutamyl transpeptidase (GGT) assay based on aggregation-induced-emission (AIE) effect and the enzyme-induced transformation of hydrophilicity to hydrophobicity has been developed by functionalizing tetraphenylethylene (TPE) derivative with two γ-glutamyl amide groups, which simultaneously work as recognition units and hydrophilic groups. When the γ-glutamyl amide groups are cleaved through GGT enzymatic reaction, the hydrophobic reaction product readily aggregate and correspondingly strong blue fluorescence can be observed, as a result of activated AIE process. By virtue of the probe's good solubility in totally aqueous solution, high sensitivity and excellent photostability, the probe can be employed to detect GGT level in human serum samples. Furthermore, the probe can be used for imaging endogenous GGT in living A2780 cells. Hence, the probe holds great promise for acting as a convenient one-step straightforward assay for GGT detection in diagnostic-related applications, and also it could provide a useful approach for conducting pathological analysis for diseases involving GGT. & 2016 Elsevier B.V. All rights reserved.
Keywords: γ-Glutamyl transpeptidase Aggregation-induced-emission Fluorescence Imaging
1. Introduction
γ-Glutamyltranspeptidase (GGT), an endogenous enzyme, plays crucial roles in glutathione metabolism and homeostasis, which is also involved in other important physiological processes such as the detoxification of xenobiotics through cleaving γ-glutamyl linkage of glutathione S-conjugates (Hiratake et al., 2004; Taniguchi and Ikeda, 1998; Whitfield, 2001). On the other hand, elevated GGT levels are closely related to diseases of the liver, biliary system, and pancreas as well (Ostapenko et al., 2011; Vandenhaute et al., 2011); in addition, GGT, which is a cell surface-associated (or bound) enzyme, has been reported to be overexpressed in several human tumors including cervical and ovarian cancers (Grimm et al., 2013; Urano et al., 2011). Hence, GGT has been employed as an important biochemical indicator in clinical diagnosis for evaluating diseases involving liver, gallbladder, and bile ducts; and it can also offer an effective means for conducting pathological study concerning the above-mentioned diseases; therefore, robust and sensitive detection of GGT level is of great importance in terms of both diagnostics-related applications and pathological analysis. A number of methods have been employed for detecting GGT n
Corresponding authors. E-mail addresses: [email protected] (F. Zeng), [email protected] (S. Wu).
http://dx.doi.org/10.1016/j.bios.2016.05.036 0956-5663/& 2016 Elsevier B.V. All rights reserved.
levels. For instance, colorimetric assays with γ-glutamyl-3-carboxy-4-nitroanilide as substrate (Tateishi et al., 1976), high-performance liquid chromatography (HPLC) (Hoskins and Davies, 1986), and fluorescence technique (Hou et al., 2014). Currently, there already are commercial GGT assay kits for clinical diagnosis based on colorimetric enzymatic assay using serum samples [e. g. IBL (Germany)]. Among these methods, in particular fluorescence technique are receiving considerable attention owing to their convenience, noninvasive monitoring capability and usability in live cells or biological samples (Buccella et al., 2011; Burns et al., 2010; Chen et al., 2015; Dickinson et al., 2013; Hu et al., 2016; Kim et al., 2012; Ma et al., 2016; Moragues et al., 2014; Ren et al., 2016; Shen et al., 2011; Tao et al., 2013; Todescato et al., 2014; Tong et al., 2015; Wang and Wolfbeis, 2014; Yang et al., 2013; Zhang et al., 2013; Zhang et al., 2014; Zong et al., 2016). Up to now, a few fluorescent probes for GGT detection or imaging have been reported (Hou et al., 2014; Li et al., 2015; Wang et al., 2015; White et al., 1996; Zhang et al., 2016); all of them adopted conventional fluorophores whose emission will experience aggregation-caused quenching (ACQ); in addition, for these probes, the products of the enzymatic reaction between these probes and GGT are poorly water-soluble, which easily results in aggregation in aqueous media and consequently the ACQ effect will interfere with the enzyme detection; this might turn out to be a limitation for these probes in terms of practical applications.
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In recent years, fluorogens with aggregation-induced emission (AIE) characteristics have attracted great interest (Ding et al., 2013; Yan et al., 2016; Yang et al., 2015). Unlike conventional fluorophores, which experience ACQ effect due to the non-radiative relaxation of the aggregates’ excited states (An et al., 2002), AIE fluorogens like tetraphenylethylene (TPE) derivatives exhibit strong fluorescence enhancement when they are aggregated. That is because, in aggregated state, intramolecular rotations are restricted, which suppresses nonradiative energy dissipation of the aggregates’ excited states, resulting in enhanced radiative transition and high quantum yields (Ding et al., 2013). Based on their unique fluorescence behaviour, a number of AIE-based fluorescent turn-on probes have recently been developed for the detection of various analytes ranging from ions (Gui et al., 2015), DNA (Wang et al., 2008), proteins (Wu et al., 2015), pH (Chen et al., 2013), and cell apoptosis processes (Ding et al., 2013), which indicated that AIE-based sensors possess many advantages, such as low background interference, improved imaging contrast, and photostability (Wang et al., 2014). However, many AIE-based fluorescent probes are not suitable for biological applications due to their much too poor water solubility. It is envisioned that, by coupling hydrophilic γ-glutamyl amide groups onto TPE derivative to make the γ-glutamyl-amide-containing TPE derivative, we could, on one hand, greatly improve the water solubility of the AIE-active fluorophore, making it operable in aqueous biological samples; on the other hand, since γ-glutamyl amide group is the specific substrate for GGT enzymatic reaction, this γ-glutamyl-amide-containing TPE derivative could serve as a fluorescent assay for GGT detection via aggregation induced emission for applications in real biological samples like serum; moreover it could avoid the problems associated with the fluorescent probes for GGT which employed conventional fluorophores (interference resulted from aggregation of enzymatic reaction product due to poor water solubility). To this end, herein, we synthesized the γ-glutamyl-amide-containing TPE derivative and investigated its fluorescent response to GGT. As shown in Scheme 1, the probe molecule can function well in totally aqueous media and biosystems due to its quite good water solubility. When γ-glutamyl amide groups are cleaved by GGT, the resultant product molecules readily aggregate due to poor water solubility, thus strong blue fluorescence can be observed because of AIE effect (Scheme S1). Furthermore, this probe's AIE feature overcomes the limitation of conventional fluorescent probes for GGT, and endows it with quite good resistance to photobleaching. In addition, the specific enzymatic reaction ensures the probe's high selectivity for GGT. Hence, the probe can selectively detect GGT levels in real
biological samples like serum. Because of its low cytotoxicity, the probe can be also used for imaging endogenous GGT in ovarian cancer cells (A2780 cells).
2. Material and methods 2.1. Chemicals and materials 4-Methylbenzophenone, TiCl4, Zn dust, di-tert-butyl discarbonate ((Boc)2O), 2-(4-aminophenyl) ethanol, N-bromosuccinimide (NBS), carbon tetrachloride (CCl4), sodium hydride (NaH), 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloricde (EDC), 4-dimethyaminopyridine (DMAP), glutamic acid, leucine, cysteine, arginine, phenylalanine and N,N-dimethylformamide (DMF) were purchased from Aladdin Reagents. Boc-L-glutamic acid 1-tert-butyl ester (Boc-Glu-OtBu), N,N-diisopropylethylamine (DIEA), alkaline phosphatase (ALP), carboxylesterase, deacetylase and γ-glutamyltranspeptidase (GGT) were purchased from Sigma Aldrich. γ-Glutamyltranspeptidase (GGT) ELISA Kit was purchased from Shanghai Enzyme-linked Biotechnology Co. Ltd.. Human serum samples were supplied by The Sixth Affiliated Hospital, Sun Yat-sen University; and the serum samples were from both healthy and unhealthy human beings with abnormal GGT levels. Petroleum ether, ethyl alcohol, ethyl acetate, ammonia water, methanol, dichloromethane, trithylamine (TEA), benzoyl peroxide (BPO), concentrated sulfuric acid, anhydrous sodium sulfate, sodium chloride and sodium hydroxide were of analytical grade reagents. The water used in the experiments was triple-distilled, treated by ion exchange columns and then by a Milli-Q water purification system. 2.2. Synthesis of 1,2-bis(4-methylphenyl) 1,2-diphenylethene (Compound 1) The compound was prepared according to previously published experimental procedures (Zhao et al., 2010). The synthesis was briefly described as follows: Under nitrogen atmosphere, into a vacuum-evacuated 100 mL round-bottomed flask was added 1.96 g (10 mmol) of 4-methylbenzophenone and 60 mL of THF. The solution was cooled down to 5 °C, then TiCl4 (5.70 g, 30 mmol) and Zn dust (2.62 g, 40 mmol) were added. After being refluxed overnight, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and dissolved in CH2Cl2, and then washed with H2O for several times, the organic layer was dried over anhydrous Na2SO4. The crude product was further
Scheme 1. Schematic illustration of the probe's fluorescent detection for GGT.
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purified by a silica gel column using petroleum ether as eluent. A white solid was obtained in 82.7% yield (1.62 g). 1H NMR (CDCl3, 400 MHz, ppm):2.24 (d, J ¼ 6.8, 6H), 6.88 6.91 (m, 8H), 6.99 7.10 (m, 10H). MS (ESI): m/z 360.1 [MþH] þ .
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dichloromethane: methanol¼ 100: 100: 4 in v/v) to furnish the desired product as a white solid (0.26 g, 43.3%).1H NMR (CDCl3, 400 MHz, ppm): 1.44(s, 18H), 1.46(s, 18H), 1.91(m, 2H), 2.13(m, 2H), 2.33(m, 4H), 2.82(t, J ¼5.2, 4H), 4.21 (m, 10H), 6.52–6.68 (m, 4H), 6.95–7.17 (m, 22H). MS (ESI): m/z 1202.13 [MþH] þ .
2.3. Synthesis of 2-(4-(N-Boc-amino)phenyl)ethanol (Compound 3) The compound was prepared according to previously published experimental procedures (Choi et al., 2006). The synthesis was briefly described as follows: 2-(4-aminophenyl) ethanol (1.37 g, 10.0 mmol) which was dissolved in TEA/MeOH (10 mL /15 mL) was treated with (Boc)2O (2.4 g, 11.0 mmol) at 40 °C overnight. The reaction mixture was evaporated and CH2Cl2 (50 mL) was added. The organic layer was washed with saturated NaCl (20 mL) and H2O (20 mL). The solvent was dried in vacuo and purified by a column chromatography with dichloromethane: methyl alcohol (15: 1) to obtain 3 (1.91 g, 80.6%) as a colourless oil. 1H NMR (CDCl3, 400 MHz, ppm): 1.51(s, 9H), 2.81(t, J ¼4.0, 2H), 3.82(t, J ¼4.4, 2H), 7.14 (d, J ¼ 6.4, 2H), 7.29 (d, J ¼5.6, 2H). MS (ESI): m/z 259.69 [MþNa] þ . 2.4. Synthesis of 1,2-bis(4-(2-oxo-5-(p-(Boc-amino)phenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 4) Into a round-bottom flask was added 0.36 g (1 mmol) of Compound 1, 0.26 g (1.1 mmol) of freshly recrystallized NBS, and a catalytic amount of BPO in 10 mL of CCl4. The solution was refluxed at 80 °C for 6 h. After being cooled to room temperature, the solution was filtered and the filtrate was concentrated. Then the solvent was removed by vacuum-rotary evaporation and the crude product (Compound 2) was obtained. Afterward, to a solution of 60% NaH in THF at 0 °C under a nitrogen atmosphere, Compound 3 in 5 mL THF was added dropwise in 10 min After 1 h, Compound 2 in 5 mL DMF solution was added dropwise to the mixture in 30 min After being refluxed overnight, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and dissolved in CH2Cl2, and then washed with H2O for several times, the organic layer was dried over anhydrous Na2SO4. The crude product was further purified by a silica gel column (ethyl acetate: dichloromethane ¼ 1: 2 in v/v) to obtain Compound 4(0.58 g, 67.4%) as a white solid. 1H NMR (CDCl3, 400 MHz, ppm): 1.39(s, 18H), 2.83(t, J ¼5.2, 4H), 3.82(t, J ¼ 4.4, 4H), 4.69 (d, J ¼6.4, 4H), 6.94–7.14 (m, 26H). MS (ESI): m/z 853.80 [MþNa] þ . 2.5. Synthesis of 1,2-bis(4-(2-oxo-5-(p-aminophenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 5) Compound 4 (0.41 g, 0.5 mmol) was dissolved under stirring in 15 mL CH2Cl2, then hydrogen chloride generated from the reaction of NaCl and H2SO4 was passed into the solution for 1 h. Afterwards the solvent was evaporated under vacuum to get the product (0.30 g, 95%). 1H NMR (d-DMSO, 400 MHz, ppm): 2.73(t, J ¼4.4, 4H), 3.59(t, J ¼ 4.0, 4H), 4.36 (d, J ¼7.2, 4H), 6.88–6.99 (m, 8H), 7.10–7.36 (m, 18H). MS (ESI): m/z 631.52 [M þH] þ . 2.6. Synthesis of 1,2-bis(4-(2-oxo-5-(p-(Boc-L-Glu(otBu)-amino) phenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 6) Under a nitrogen atmosphere, Boc-Glu-OtBu (0.30 g, 1 mmol), EDC (0.23 g, 1.2 mmol) and DMAP (0.006 g, 0.05 mmol) were dissolved in dichloromethane, then the mixture of Compound 5 (0.41 g, 0.5 mmol) and DIEA (0.15 g, 1.2 mmol) in dichloromethane was added. After being stirred at room temperature for 24 h, the solution was washed with slightly acidic water (pH ¼6.5). Then the organic layer solvent was evaporated and the residue was purified by column chromatography on silica gel (hexane:
2.7. Synthesis of Synthesis of 1,2-bis(4-(2-oxo-5-(p-(L-Glu-amino) phenyl)pentyl) phenyl) 1,2- disphenylethene (Compound 7, the probe) Compound 6 (0.26 g, 0.2 mmol) was dissolved under stirring in 10 mL CH2Cl2, then hydrogen chloride generated from the reaction of NaCl and H2SO4 was passed into the solution for 1 h. Afterwards the solvent was evaporated under vacuum to get the product (0.18 g, 95.6%). 1H NMR (CD3OD, 400 MHz, ppm): 2.12(m, 2H), 2.26 (m, 2H), 2.59(m, 4H), 3.03(m, 4H), 4.06(m, 2H), 4.35(t, J ¼ 6.4, 4H), 4.49 (d, J ¼6.8, 4H), 6.98–7.04 (m, 4H), 7.07–7.18 (m, 10H), 7.19– 7.26 (m, 4H), 7.28–7.35 (m, 4H), 7.40–7.44 (m, 4H). MS (ESI): m/z 888.99 [M þH] þ . 2.8. Measurements 1
H NMR spectra were recorded on a Bruker Avance 400 MHz NMR 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. 2.9. Cell viability assay Cell viability assay was employed to evaluate the cytotoxicity of the probe. In the assay, L929 cells (murine aneuploid fibrosarcoma cells) and HeLa cells (human cervical cancer cell) were incubated in DMEM medium that was supplemented with 10% fetal bovine serum (FBS), and allowed to grow for 24 h with 5% CO2 at 37 °C. Upon removal of the medium, the cells were treated with the probe and then incubated for 24 h. MTT assay was adopted to assess the cytotoxicity of the probe against the cells according to ISO 10993-5. For the assays, as for each independent experiment, the assays were performed in eight replicates. And the statistical mean and standard deviation were used to estimate the cell viability. 2.10. Cell incubation and imaging In the experiment, two cell lines, namely A2780 (human ovarian cancer cell) and L929 (murine aneuploid fibrosarcoma cell), were incubated in DMEM medium which was supplemented with 8% and 10% fetal bovine serum (FBS, hyclone) respectively. One day before imaging, cells were passaged and plated on polylysine-coated cell culture glass slides inside the 30 mm glass culture dishes, which were then allowed to grow to 50–70% confluence. As for the experiments, the cells were washed with DMEM, after that the cells were incubated in DMEM medium containing the probe (20 μM) at 37 °C under 5% CO2 for 1 h. Some A2780 cells were incubated in DMEM medium containing inhibitor (DON, 10 μM) for 30 min, and then treated with the probe (20 μM) for 1 h. The control sample was incubated in DMEM medium only. After that, the culture dishes were washed with PBS, the glass slides were taken out, 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.
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3. Results and discussion 3.1. Probe synthesis and fluorescent response toward GGT With AIE characteristic, TPE derivatives can be used for fabricating “turn-on” fluorescent probes. The chemical structure of the probe and its synthetic process are depicted in Scheme S2. To synthesize the probe, hydrosoluble glutamic acid groups were coupled onto TPE derivative, so as to make the γ-glutamyl-amidecontaining TPE derivative (the probe, Compound 7) and ensure the probe molecules are well soluble in aqueous media (without aggregation). The intermediate products and the probe were characterized by proton nuclear magnetic resonance (1H NMR) and mass spectrometry (MS) (Figs. S1–S12). Fig. S13 shows the excitation and emission spectra of this probe in PBS-buffered water. In the absence of GGT, the probe solution exhibits almost no emission. Upon reaction with GGT for 45 min, the probe exhibits an emission band at about 472 nm when excited at 360 nm. Fluorescent spectra were measured periodically during the incubation of the probe (20 μM) with GGT (60 U/L) at 37 °C in PBSbuffered water (pH 7.4, 10 mM), so as to investigate the probe's fluorescent response toward GGT, and the results are shown in Fig. 1A. In the absence of GGT, the probe solution displays almost no emission. Upon addition of GGT, an emission at about 472 nm emerges, corresponding to the emission of the enzymatic reaction product which is the same as Compound 5 (Fig. S15). MS analysis also suggests that the product of the enzymatic reaction has the same molecular structure as Compound 5 (Fig. S16). As the incubating (reaction) time is lengthened, the emission at around 472 nm increases. This is because, GGT cleaves the γ-glutamyl amide moieties from the probe molecule, this results in the aggregation of the hydrophobic reaction product (1,2-bis (4-(2-oxo5-((p-aminophenyl))pentyl)phenyl) 1,2-disphenylethene) (Compound 5). DLS analysis indicates that large aggregates form with an average diameter of 116 nm, after the probe being incubated with GGT at 37 °C for 45 min (Fig. S17). In addition, the resistance to photobleaching for the aggregates of the enzymatic reaction product molecules was also investigated. As shown in Fig. 1(B), upon continuous illumination at 365 nm (15 W handheld UV lamp) for 3 h, fluorescence intensity of the reaction product aggregates only reduce by 6.4%, while the other two conventional fluorophores (fluorescein and coumarin) show obvious photobleaching phenomenon. This result suggests that the reaction product aggregates have good photostability. The data for enzyme kinetics follow the Michaelis-Menten equation, and the kinetic parameters have been determined as Vmax ¼1.10 μM/min and Km ¼ 15.17 mM (Fig. S18). The Km value is small, indicating the strong affinity between GGT and the probe molecule.
With the addition of a small amount of CTAB (20 μM) into the probe solution, and in the presence of GGT, the system's fluorescence intensity increases significantly; at 60 U/L GGT, the fluorescence intensity increases up to 12-fold (Fig. 2A). To illustrate the role of CTAB for such obviously enhanced fluorescence intensity, fluorescence spectra and DLS measurements were carried out in the absence or presence of CTAB. Fluorescent spectra show that, in the absence of GGT, even with the addition of CTAB, the probe solution does not display obvious fluorescence (Fig. S19A). DLS analyses also confirm that, in the absence of GGT, the addition of CTAB has no obvious impact on the size of the aggregates in the solution, as shown Fig. S19C. However, in the presence of GGT, after the probe solutions being incubated for 45 min at 37 °C, the fluorescence intensity of the solution with CTAB increases much more drastically, compared to the system without CTAB (Fig. S19B); in the meantime, the size of the aggregates in the solution with CTAB is much larger than the system without CTAB (Fig. S19D). These results suggest that CTAB can promote the formation of aggregates in the system and result in the significant enhancement of fluorescence intensity. In addition, the fluorescence intensity of the probe solutions (with or without CTAB) in the presence of GGT was measured as a function of time (Fig. S20). It is clear that, for both the systems (with or without CTAB), the reaction between GGT and the probe is completed at about 45 min; this indicates that CTAB have no influence on the GGT enzymatic reaction; thus it is highly possible CTAB simply promote the aggregation of the product from the reaction between GGT and the probe. Furthermore, the time-course experiments of the probe at different amounts of GGT were recorded, as shown in Fig. 2B. It also shows that the enzymatic reaction is almost completed in 45 min. The fluorescence spectra of the system in the presence of different amounts of GGT are shown in Fig. 2C and the corresponding working curve (the emission intensity at 472 nm versus GGT levels) is shown in Fig. 2D. And the detection limit has been determined to be 0.59 U/L (Fig. S21), which is sensitive enough for determining GGT levels in biological assays, given that for normal adult females, the normal of GGT level in blood is 5–55 U/L, and that for adult males is 15–85 U/L (Berk and Korenblat, 2011). 3.2. Selectivity of the probe toward GGT In order to evaluate the selectivity of the probe, the fluorescence response of probe solution in the presence of different biological substances was measured, and the results are shown in Fig. 3A. It can be seen that, only GGT induces a prominent intensity enhancement, other species (biologically relevant) do not generate a rise in the fluorescence intensity, which include ions (such as
Fig. 1. (A) Time course of fluorescence spectra for the probe (20 μM) in PBS-buffered water (pH 7.4, 10 mM) at 37 °C upon addition of 60 U/L GGT. (B) Photostability of the reaction product aggregates (black), 20 μM fluorescein (red) and 20 μM 7-diethylamino-4-methylcoumarin (green) in PBS (pH ¼ 7.4, 10 mM). IT/I0 is the fluorescence intensity ratio at 472 nm, 520 nm and 460 nm respectively excited at 360 nm after T-minute light irradiation and before light irradiation.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. (A) Time course of fluorescence spectra for the probe (20 μM) with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C upon addition of 60 U/L GGT. (B) Plot of the fluorescence intensity of the probe (20 μM) with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C versus reaction time in the presence of different amounts of GGT. (C) Fluorescence response of the probe (20 μM) with 20 μM CTAB in the presence of different GGT levels in PBS-buffered water (pH 7.4, 10 mM) at 37 °C for 45 min (D) Fluorescence intensity at 472 nm as a function of GGT level. Excitation wavelength: 360 nm.
Fig. 3. (A) Fluorescence response of 20 μM probe with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C for 45 min in the presence of different biologicallyrelevant substances. (B) Fluorescence response of 20 μM probe with 20 μM CTAB in PBS-buffered water (pH 7.4, 10 mM) at 37 °C for 45 min in the presence of 60 U/L GGT and with the addition of different biological substances respectively. The concentration of the ions and amino acids are 100 μM, while the concentration of protein enzymes are 100 U/L.
Na þ , K þ , Mg2 þ , Ca2 þ , Al3 þ , NO3-, CO32 and PO43 ), amino acids (such as glutamic acid, leucine, cysteine, arginine and phenylalanine) and protein enzymes (such as alkaline phosphatase (ALP), carboxylesterase and deacetylase). The interference of these biologically-relevant substances was also investigated. Fig. 3B shows the fluorescence response of the probe towards GGT in the presence of these substances. It is clear that, these species do not interfere with fluorescence response of the probe, indicating these substances have negligible interfering effect on the GGT enzymatic reaction with the probe molecule. The probe herein can
specifically respond towards GGT, owing to the fact that GGT can specifically catalyze the cleavage of γ-glutamyl group. 3.3. Detection of GGT in real biological samples like serum Next, in order to examine the operability of the probe system in real samples, we applied the probe system to detecting the GGT levels in human serum samples. Firstly, we compared the probe's detection for GGT with the Elisa Kit (the commonly-used commercial assay kit for GGT) in PBS-buffered water. As Table S1
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Table 1 Endogenous GGT detection in human serum samples by using Elisa kit assay and this probe. Sample no. GGT amount (U/L) by ELISA Kit assay 1 2 3 4 5 6
53.26 7 4.37 62.337 3.89 42.217 5.34 238.8 7 11.82 205.7 7 12.10 186.17 10.87
GGT amount (U/L) by this probe 55.76 72.78 63.05 73.63 40.02 73.85 250.2 76.76 200.7 76.33 179.9 77.02
Note: The serum samples were 5-fold diluted for measurement; and the values (mean 7 SD) herein represent GGT levels in the undiluted serum samples, which are calculated based on the measurements for the diluted samples. Samples 1 and 2 were obtained from healthy males; Samples 3 was obtained from healthy female, Samples 4 and 5 were obtained from males with hepatic diseases, Samples 6 was obtained from female with hepatic diseases. All the serum samples were provided by The Sixth Affiliated Hospital, Sun Yat-sen University.
shows, the recovery and SD value from these two methods are similar; this suggests the accuracy and reliability of the probe system herein. Then we attempted to detect the endogenous GGT levels in human serum samples (Table 1, by using the calibration curve (Fig. 2D) as standard for this probe). The endogenous GGT levels in the serum samples from both healthy and unhealthy human beings measured by this probe are close to those measured by Elisa Kit. The results indicate that, the probe holds great
potential for serving as an effective detection method for GGT level in diagnostics-related applications. Furthermore, compared to Elisa Kit (the antibody-based colorimetric assay) involving complex procedures in the detection process, the probe system herein is quite convenient and could act as a one-step straightforward fluorescent assay for GGT. 3.4. Imaging of GGT in living cells A2780 cell lines (one of ovarian cancer cells), which is overexpressed with GGT (Grimm et al., 2013; Hanigan, 1995; Urano et al., 2011), were employed in the experiment, so as to further evaluate the applicability of the probe for live-cell imaging. First the cytotoxicity of the probe was measured by MTT assay with L929 and Hela cell lines in compliance with ISO 10993-5. And the results are shown in Figure S22. With these two cell lines, the probe show high cell viability of over 90% even at the high concentration of 80 μM; the results indicate that, the probe has very low cytotoxicity. Hence the probe is suitable for fluorescence imaging of GGT in live cells. And then we applied the probe in GGT imaging in living cells. As shown in Fig. 4B and Fig. S23B, no background fluorescence can be observed in A2780 and L929 cells in the absence of the probe, suggesting that autofluorescence from the cells is insignificant under this condition. When A2780 cells were treated with the probe for 1 h, the cells show a strong blue fluorescence (Fig. 4E);
Fig. 4. Fluorescence microscopy images. A2780 cells incubated in the absence of the probe (A, B and C), in the presence of the probe (D, E and F; the probe concentration: 20 μM), or in the presence of the inhibitor DON and the probe (G, H and I; DON concentration: 10 μM, the probe concentration: 20 μM). D, E and F: the cells were incubated with the probe for 1 h; G, H and I: the cells were first incubated with the inhibitor DON for 30 min, and then incubated with the probe for 1 h.(For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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this suggests that, the product molecules from the reaction between GGT and the probe aggregate inside A2780 cells. For comparison, we also applied the probe to cellular imaging in L929 cells, which do not have overexpression of GGT (Fig. S23). It can be seen that, when L929 cells were treated with the probe, no fluorescence can be observed; this is because, the probe molecules uptake by L929 cells do not aggregate, since no reaction will occur due to the lack of GGT in these cells. In addition, to further prove that the blue fluorescence observed in A2780 cells were indeed triggered by GGT, A2780 cells were first pretreated with GGT inhibitor DON for 30 min, and then treated with the probe for 1 h. As shown in Fig. 4H, no fluorescence can be observed. This indicates that the blue fluorescence in A2780 cells is triggered by GGT. These results demonstrate that the probe is promising for detecting endogenous GGT in live cells, which is useful for pathological analysis for the disease involving GGT. Compared to other fluorescent probes for GGT (as shown in Table S2), this probe has several advantages: first, owing to its good water solubility and biocompatibility, the probe can detect GGT in human serum samples, and imaging endogenous GGT in living cells; second, AIE effect endows this probe excellent photostability and high imaging contrast; third, this probe demonstrates strong potential for serving as a convenient one-step fluorescent assay for ALP.
4. Conclusion In summary, we have developed a new fluorescence turn-on probe for GGT based on the AIE effect, which functions through GGT-induced transformation of hydrophilic-to-hydrophobic property and the corresponding aggregation induced emission. CTAB can enhance fluorescence enhancement of the probe system through promoting the aggregation of the product from the reaction between GGT and the probe. In addition, the probe exhibits good water solubility, excellent photostability and high selectivity for GGT. The sensitivity and reliability ensure this probe can serve as a convenient one-step straightforward assay for GGT detection in serum samples, which holds promise for diagnostic-related applications. Furthermore the probe can be used for imaging endogenous GGT in living A2780 cells. Therefore, the probe would provide a useful approach for further elucidating the physiological roles of GGT as well as for conducting pathological analysis for diseases involving GGT.
Acknowledgment We gratefully acknowledge the financial support by the National Key Basic Research Program of China (Project No. 2013CB834702), NSFC (Grant nos. 21574044, 21474031 and 21174040), the Science and Technology Planning Project of Guangdong Province (Project no. 2014A010105009) and the Fundamental Research Funds for the Central Universities (2015ZY013).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.05.036.
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