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An infinite coordination polymer nanoparticles-based near-infrared fluorescent probe with high photostability for endogenous alkaline phosphatase in vivo
Accepted Manuscript Title: An Infinite Coordination Polymer Nanoparticles-Based Near-Infrared Fluorescent Probe with High Photostability for Endogenous Alkaline Phosphatase in Vivo Authors: Juan Ou-Yang, Chun-Yan Li, Yong-Fei Li, Bin Yang, Song-Jiao Li PII: DOI: Reference:
S0925-4005(17)31828-2 https://doi.org/10.1016/j.snb.2017.09.162 SNB 23253
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
27-7-2017 20-9-2017 23-9-2017
Please cite this article as: Juan Ou-Yang, Chun-Yan Li, Yong-Fei Li, Bin Yang, Song-Jiao Li, An Infinite Coordination Polymer Nanoparticles-Based Near-Infrared Fluorescent Probe with High Photostability for Endogenous Alkaline Phosphatase in Vivo, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.09.162 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.
An Infinite Coordination Polymer Nanoparticles-Based Near-Infrared Fluorescent Probe with High Photostability for Endogenous Alkaline Phosphatase in Vivo Juan Ou-Yang,a Chun-Yan Li,*,a,c Yong-Fei Li,b Bin Yang,a Song-Jiao Lia a
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry
of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, PR China. b
College of Chemical Engineering, Xiangtan University, Xiangtan, 411105, PR China
c State
Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
& Chemical Engineering, Hunan University, Changsha, 410082, PR China *Corresponding author, Tel.: +86 731 58292205; fax: +86 731 58292477. E-mail: [email protected] Highlights ► The fluorescence emission of CyOH@Tb-GMP nanoprobe belongs to NIR region. ► The nanoprobe displays better photostability than NIR dyes such as ICG and CyOH. ► The NIR nanoprobe shows high sensitivity and selectivity for ALP activity assay. ► The nanoprobe is used for monitoring endogenous ALP activity in biological samples.
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Abstract Alkaline phosphatase (ALP) is an essential enzyme in phosphate metabolism and widely distributes in mammalian body fluids and tissues. So far, various fluorescent probes for ALP have been well developed, but some disadvantages such as short emission wavelength and low photostability restrict their biological application. Herein, a near-infrared (NIR) fluorescent nanoprobe based on infinite coordination polymer nanoparticles (CyOH@Tb-GMP) is constructed for endogenous ALP activity assay. The characteristics of the nanoprobe are as following: (1) The fluorescence emission of the nanoprobe is at 718 nm belonging to NIR region, which is suitable for bioimaging. (2) The nanoprobe displays better photostability than common NIR dyes such as ICG as well as CyOH. (3) The nanoprobe exhibits high sensitivity for ALP activity with a 7.5-fold fluorescence enhancement when 2.5 U / mL ALP is added. (4) The NIR nanoprobe is employed for monitoring endogenous ALP activity in various biological samples such as cell, tissue and mice with satisfactory results. Keywords: Alkaline phosphatase; Infinite coordination polymer; Near-infrared; Photostability; Biological samples
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1. Introduction Alkaline phosphatase (ALP) is an essential enzyme in phosphate metabolism. It can catalyze the hydrolysis of phosphoryl esters and widely distributes in mammalian body fluids and tissues [1-3]. Moreover, ALP is an important biomarker for diagnostics, and the deviation of ALP from its normal level is closely related to various diseases including breast and prostatic cancer, bone disease, liver dysfunction, and diabetes [4-6]. In this context, it is very essential to develop a sensitive method for ALP level determination in order to understand the role of ALP in related biological processes and provide the clinical diagnoses of the related diseases. On the basis of the ability of ALP to remove phosphate groups from a wide variety of substrates and obtain the signal readout through various mechanisms, numerous assays for ALP activity have been developed, including electrochemistry [7, 8], colorimetric method [9], chromatographic [10], and surface enhancement Raman scattering assay [11]. Although each of these methods possesses its own features and gives the different insights for ALP sensing, they often require sample processing, complex instrumentation and are not suitable for applying in biological systems. Fluorescence technique is extremely attractive in this regard due to their intrinsic advantages of low background noise and high sensitivity, noninvasive monitoring capability and usability in live organism [12-18]. Up to date, a variety of fluorescent assays for ALP have been reported by using organic dyes [17-22], conjugated 3
polyelectrolytes [23-24], quantum dots [25-28], metal nanoclusters [29, 30] as fluorescent materials. Unfortunately, we note that these approaches usually suffer from some disadvantages. For example, organic dyes have poor water solubility, bad photostability and easy diffusion in vivo. The conjugated polyelectrolytes require a complicated synthesis process and quantum dots are toxic. Noble metal nanoclusters are high cost and poor stability in aqueous system. Therefore, exploring novel fluorescent materials which meet the demand of simplicity, high photostability and low toxicity are still necessary. Infinite coordination polymer (ICP) nanoparticles formed from metal ions and synthetic ligand molecules have been emerging as a new family of functional nanomaterials [31-34]. The unique properties of ICP nanoparticles such as easy synthesis, good water solubility, satisfactory biocompatibility and high photostability make them widely be applied for the detection of metal ions [35], anions[36, 37], and various biological molecules [38-40]. Only a few of the ICP nanoparticles-based fluorescent nanoprobes are applied in the determination of ALP activity [41, 42]. In addition, almost all these nanoprobes have relatively short emission wavelengths (< 650 nm), which greatly limits their practical application in biological sample. As is well known, near-infrared (NIR) emission can minimize photo-damage to living cells, enable deep tissue penetration, and circumvent the spectral overlap with biosubstrate autofluorescence [43-46]. Therefore, it is highly necessary to explore the
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capability of ICP nanomaterials to design NIR fluorescent nanoprobes for ALP detection. To meet the requirements mentioned above, an ICP nanoparticles-based NIR fluorescent nanoprobe (CyOH@Tb-GMP) with high photostability is developed in this paper (Scheme 1). The CyOH@Tb-GMP nanoprobe are constructed with Tb3+ as the metal ion and guanosine-5′-monophosphate (GMP) as the bridging ligand, in which a NIR fluorescence dye (CyOH) is encapsulated into the ICP supramolecular network through an adaptive selfassembly chemistry. In the presence of ALP, the phosphate group in the GMP ligand is cleaved, resulting in the destruction of the Tb-GMP and the release of encapsulated CyOH into solution. This process eventually results in the increase of the fluorescence intensity emitted from CyOH, which provides a basis for NIR fluorescent assay for ALP. Moreover, the NIR fluorescent nanoprobe is applied for the detection of endogenous ALP activity in biological samples such as cell, tissue and mice with satisfactory results. 2. Experimental 2.1 Reagents Guanine monophosphate (GMP), IR-780 iodide and resorcinol were purchased from Sigma-Aldrich (St Louis, USA). Terbium nitrate hexahydrate (Tb (NO3)3·6H2O) and sodium orthovanadate (Na3VO4) were all purchased from Aladdin (Shanghai, China). ALP (alkaline phosphatase, calf intestine) was purchased from TaKaRa
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Biotechnology Corporation (Dalian, China). Note, one unit of the enzyme is defined as the amount that produces 1 μmol of 4-nitrophenol from 4-nitrophenylphosphate in a minute at 37 °C. 2.2 Syntheses Synthesis of CyOH: Resorcinol (0.55 g, 5.0 mmol) and K2CO3 (0.69 g, 5.0 mmol) were placed in a flask containing CH3CN (10 mL), and the mixture was stirred for 10 min at room temperature under nitrogen atmosphere. Then IR-780 iodide (1.53 g, 2.5 mmol) in CH3CN (20 mL) was introduced to the mixture followed by heating at 50 °C for 4 h. After the removal of solvent from the reaction mixture under reduced pressure, the crude product was purified by silica gel column chromatography using CH2Cl2/CH3OH (20:1, v/v) as eluent to afford a blue-green solid product. Yield: 1.54 g (82%). 1H NMR (400 MHz, CDCl3, Fig. S1): δ 8.02 (d, 1H, J = 13.2 Hz), 7.25-7.21 (m, 2H), 7.14 (t, 1H, J = 7.6 Hz), 7.06-7.03 (m, 2H), 6.97 (s, 1H), 6.90 (d, 1H, J = 8.0 Hz), 6.81 (d, 1H, J= 8.0 Hz), 5.56 (d, 1H, J = 13.2 Hz), 3.76 (t, 2H, J = 7.2 Hz), 2.67 (t, 2H, J = 6.0 Hz), 2.59 (t, 2H, J = 6.0 Hz), 1.91-1.82 (m, 4H), 1.67 (s, 6H), 1.04 (t, 3H, J = 7.6 Hz). 13C
NMR (100 MHz, CDCl3, Fig. S2): δ 174.5, 166.9, 163.0, 155.6, 143.2, 142.0, 141.3,
137.8, 132.7, 128.9, 125.8, 123.9, 123.8, 122.6, 118.4, 114.8, 111.0, 103.1, 100.1, 49.8, 46.3, 29.6, 28.5, 24.2, 22.6, 20.8, 11.7. MS (TOF, Fig. S3) m/z 412.4. Synthesis of Tb-GMP nanoparticles: Tb-GMP nanoparticles was synthesized by mixing an aqueous solution of Tb(NO3)3·6H2O (10 mM, 1 mL) with a HEPES buffer (0.1 M, pH 7.4, 1mL) containing GMP (10 mM, 1 mL) at room temperature to form a
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white precipitate. The resulting precipitate was then centrifuged and washed with water several times. Synthesis of CyOH@Tb-GMP nanoparticles: Encapsulation of CyOH into the TbGMP nanoparticles to form CyOH@Tb-GMP was carried out by following procedures. An aqueous solution of Tb(NO3)3·6H2O (10 mM, 1 mL) was added into a HEPES buffer (0.1 M, pH 7.4, 1 mL) containing GMP (10 mM, 1 mL) and CyOH (0.1 mM, 1 mL) to form a precipitate. The resulting precipitate was then centrifuged and washed with water several times until no fluorescence of CyOH was detected in the supernatant of the solution. 2.3 Apparatus All fluorescence measurements were recorded with a Perkin Elmer LS-55 fluorescence spectrometer (USA). UV-vis absorption spectra were collected on a Perkin Elmer Lambda 25 spectrophotometer (USA). 1H NMR and 13C NMR spectra were carried out on a Bruker Avance II 400 spectrometer (Germany) operating at 400 and 100 MHz (TMS as internal standard), respectively. MS spectrometry was performed on a Bruker Autoflex MALDI-TOF MS spectrometer (Germany). Transmission Electron Microscopy (TEM) images were performed by a JEM-2100 transmission electron microscope (Japan). Fourier transform infrared (FI-IR) spectra were recorded with KBr pellets on a Nicolet 6700 FT-IR Spectrometer (USA). Fluorescence imaging of cells and tissues were carried out by an Olympus FV1000
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fluorescence microscope (Japan). Fluorescence imaging of mice was performed on an IVIS Lumina XR small animal optical in vivo imaging system (USA). 2.4 General procedure for ALP activity assay A fluorescent ALP activity assay was performed according to the following procedures. ALP with different activities ranging from 0.01 to 2.5 U was added into the mixture (1 mL, pH 8.0) containing 50 mM Tris-HCl, 1 mM MgCl2, and 1.0 mg CyOH@Tb-GMP nanoprobe. After the addition of ALP, the fluorescent spectrum of each of the mixtures was recorded at 37 °C. The fluorescence emission spectra were recorded at excitation wavelength of 685.0 nm with emission wavelength range from 705.0 to 780.0 nm with excitation slit set at 10.0 nm and emission slit set at 10.0 nm. 2.5 Cell incubation and fluorescence imaging The living HeLa cells were provided by XiangYa Central Experiment Laboratory of Central South University (China). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) at 37 °C under an atmosphere of 5 % CO2. The cells were plated on 35 mm culture dish and allowed to adhere for 24 h. Then three confocal dishes were treated differently and imaged. In the first dish, HeLa cells were used as control incubated in DMEM medium only. In the second dish, HeLa cells were incubated with the CyOH@Tb-GMP nanoprobe (1 mg / mL) for 30 min, followed by washing three times with PBS buffer solution before imaging. In the third dish, HeLa cells were treated with ALP inhibitor sodium orthovanadate (Na3VO4, 100 μM) at 37 °C for 30 min, then with CyOH@Tb-
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GMP nanoprobe (1 mg / mL) for another 30 min, followed by washing three times with PBS buffer solution before imaging. Fluorescence imaging was performed by confocal fluorescence microscope with excitation wavelength at 635 nm and emission wavelength at 680-780 nm. 2.6 Preparation and staining of rat liver tissue slices Tissue slices were prepared from rat liver. A side of the tissue was cut flat using a vibrating-blade microtome. Then three tissue slices were treated differently and imaged. The first tissue slice was used as control. The second tissue slice was incubated with CyOH@Tb-GMP nanoprobe (1 mg / mL) at 37 °C for 30 min, followed by washing three times with PBS buffer solution before imaging. The third group of tissue slice was treated with Na3VO4 (100 μM) at 37 °C for 30 min, then with CyOH@Tb-GMP nanoprobe (1 mg / mL) for another 30 min, followed by washing three times with PBS buffer solution before imaging. Fluorescence imaging was performed by confocal fluorescence microscope with excitation wavelength at 635 nm and emission wavelength at 680-780 nm. 2.7 Fluorescent imaging in Vivo 20-25 g Kunming (KM) mice were used and were kindly kept in all the experimental process. All animal experiments were performed in accordance with the guidelines issued by The Ethical Committee of Xiangtan University. The abdominal fur of the mice was removed by an electric shaver. Then, the mice were divided into three groups. The first group was untreated as a control group. The
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second group was given an intraperitoneal injection of CyOH@Tb-GMP nanoprobe (1 mg / mL). The third group was given an intraperitoneal injection of Na3VO4 (100 μM) and then injection of CyOH@Tb-GMP nanoprobe (1 mg / mL). Before imaging, the mice were anesthetized with chloral hydrate (10% in saline) and remained anesthetized throughout the image period. The mice were placed into the imaging chamber and imaged with 660 nm excitation and 740 nm emission channel. 3. Result and discussion 3.1. Synthesis and characterization of CyOH@Tb-GMP nanoparticles A rapid and facile route for preparing CyOH@Tb-GMP nanoparticles was showed as Scheme 1. Initially, a hemicyanine dye (CyOH) with NIR fluorescence emission was selected as the guest molecule and synthesized starting from the reaction between IR-780 and resorcinol. Then, Tb-GMP nanoparticles were chosen as the host and constructed by self-assembly of GMP and Tb3+ in aqueous solution. Subsequently, CyOH was employed to be encapsulated into the Tb-GMP nanoparticles to form the CyOH@Tb-GMP nanoparticles. The morphology of Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles was analyzed by transmission electron microscopy (TEM) and the results were shown in Fig. 1. The Tb-GMP nanoparticles are colloidal spheres with diameters ranging from 30 to 40 nm (Fig. 1a), which is almost consistent with the reported literature value [31, 41]. The CyOH@Tb-GMP nanoparticles have almost the same shape and size (diameters of 30-40 nm) as the Tb-GMP nanoparticles (Fig. 1b), suggesting that
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the encapsulation of CyOH does not change the morphology of the Tb-GMP nanoparticles. Moreover, the UV-visible spectra of CyOH, Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles were measured and descripted in Fig. 1c. CyOH exhibits a strong absorption peak at 695 nm, while Tb-GMP nanoparticles show no absorption. And there is almost no absorption for CyOH@Tb-GMP nanoparticles, reflecting that CyOH is encapsulated into Tb-GMP nanoparticles to form CyOH@TbGMP nanoparticles. The fluorescence spectra of CyOH, Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles were also studied (Fig. 1d). Similarly, CyOH shows a strong NIR fluorescence at 718 nm, whereas Tb-GMP nanoparticles display no emission. Particularly, the CyOH@Tb-GMP nanoparticles exhibit almost no fluorescence and no obvious change is observed in at least 8 h (Fig. S4), which demonstrates the successful encapsulation of CyOH into Tb-GMP nanoparticles to form CyOH@Tb-GMP nanoparticles and the CyOH@Tb-GMP nanoparticles own excellent chemical stability. Furthermore, Fourier transfer infrared (FT-IR) spectra of GMP, Tb-GMP nanoparticles, CyOH@Tb-GMP nanoparticles and CyOH were displayed in Fig. 2. For GMP, peaks at 1632, 1359, 1064 and 982 cm-1 were assigned to the C=O (ν C=O) and N-C (ν N-C) stretching vibration band in the guanosine moiety of GMP, the antisymmetric (νasPO3) and symmetric (νsPO3) vibration bands in the phosphate moiety of GMP, respectively. After the interaction with Tb3+ to form Tb-GMP nanoparticles, the wavenumber of GMP was shifted to 1634, 1358, 1063 and 970 cm-1, respectively. This suggests that both guanosine and phosphate moieties of GMP are coordinated 11
to Tb3+. As for CyOH@Tb-GMP nanoparticles, the wavenumber of the corresponding peaks (1633, 1360, 1065 and 983 cm-1) shifted slightly, as compared with those of Tb-GMP nanoparticles. In addition, the characteristic peaks of CyOH did not be found after its encapsulation into Tb-GMP nanoparticles. These demonstrate that CyOH is localized inside Tb-GMP nanoparticles and the encapsulation of CyOH has almost no effect on the coordination mode between GMP and Tb 3+. 3.2. Spectral response of CyOH@Tb-GMP nanoprobe for ALP The absorption and fluorescence spectra of CyOH@Tb-GMP nanoprobe before and after reacting with ALP were measured in Tris-HCl buffer solution (Fig. 3). With the addition of ALP into Tris-HCl buffer solution containing CyOH@Tb-GMP nanoprobe, the absorption band centered at 695 nm shows an obvious enhancement (Fig. 3a). Meanwhile, the fluorescence signal of CyOH@Tb-GMP nanoprobe displays noticeable changes (Fig. 3b). The emission intensity at 718 nm, which is ascribed to the emission of CyOH, increases greatly upon adding ALP (Fig. 3b). These spectral changes could be due to ALP-catalyzed hydrolysis of the phosphate ester group to convert GMP into a guanosine base. With the cleavage of phosphate group, the structure of Tb-GMP nanoprobe was destructed and the guest molecule (CyOH) was thus released into the solution (Scheme 1). The fluorescence intensity is closely associated with the degree of the destruction of the CyOH@TbGMP nanoprobe caused by ALP. As a consequence, the CyOH@Tb-GMP nanoprobe can be used for the fluorescence assay of ALP activity.
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3.3 Condition optimization In order to obtain excellent ALP sensing, the sensing conditions need to be optimized. First of all, the effect of reaction temperature on fluorescence intensity was examined (Fig. S5). The experimental results show that the change of fluorescence intensity reaches maximum at 37 °C. Therefore, 37 °C is selected in this study, which is consistent with the circumstance in human body. Then, the effect of pH on the fluorescence of the probe was investigated (Fig. S6) and the results show that the optimum pH for ALP is 8.0. Furthermore, the effect of incubation time on the fluorescence of the CyOH@Tb-GMP nanoprobe was studied since time is another important factor for the enzyme reaction. As could be seen from Fig. 4, the fluorescence of the nanoprobe undergoes a gradual increase and reaches the maximum when the probe is incubated in the presence of ALP for 20 min. So 20 min is used for ALP analysis in the subsequent experiments. In addition, the kinetic parameters for the hydrolysis of CyOH@Tb-GMP nanoprobe catalyzed by ALP under different initial concentrations of the nanoprobe were determined. By Lineweaver– Burk analysis, the catalytic constant (kcat) is calculated to be 0.03 s-1, which is close to the data reported in the literature (Fig. S7). [18-20] 3.4 Sensitivity To estimate the sensitivity of the assay, different activities of ALP were added into the Tris-HCl buffer solution containing CyOH@Tb-GMP nanoprobe (Fig. 5). As displayed in Fig. 5, the fluorescence intensity of the nanoprobe gradually enhances
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with increasing activity of ALP. When 2.5 U/mL ALP is added, the fluorescence intensity increases around 7.5-fold. Moreover, the insert of Fig. 5 illustrates that the probe exhibits a good linear relationship with ALP ranging from 0.01 to 2.5 U / mL. The detection limit is 0.0033 U / mL (3σ/slope), which is low enough for ALP detection in biological samples. 3.5 Selectivity To investigate the selectivity of the CyOH@Tb-GMP nanoprobe, a series of enzymes in serum such as glucose dehydrogenase (GDH), galactosidase (GAL), glucose oxidase (GOX), exonuclease (EXO), acetylcholinesterase (AChE), trypsin and thrombin were evaluated. As shown in Fig. 6, none of the enzymes has the ability to recover the fluorescence except ALP. Meanwhile, the selectivity was also investigated by examining various potential interfering substances such as biothiols (Cys, Hcy, GSH), amino acids (Ala, Val, Leu, Ileu, Pro, Phe, Trp, Met, Gly, Tyr, Ser, Thr, Asn, Gln, Lys, Arg, His, Asp, Glu) and metal ions (K+, Na+, Ca2+, Mg2+, and Zn2+) (Fig. S8). The results indicate that no significant fluorescence change of the nanoprobe is observed, demonstrating its high selectivity to ALP. 3.6 ALP inhibitor efficiency evaluation Having demonstrated the validity of the fluorescent nanoprobe based on CyOH@Tb-GMP nanoprobe, the possibility of applying the nanoprobe for the screening of potential ALP inhibitors was also investigated. Sodium orthovanadate (Na3VO4), a well-known inhibitor for ALP [4-6], was used in the experiments. As
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displayed in Fig. 7, the fluorescence intensity decreases with the addition of increasing concentrations of the inhibitor. The insert of Fig. 7 shows that the corresponding IC50 value (the concentration of an inhibitor required to 50% of inhibition efficiency) of Na3VO4 toward ALP is calculated to be 148.6 μM, which is consistent with the value reported previously [41, 47, 48]. The results essentially demonstrate that the nanoprobe can be used not only for the monitoring of ALP but also for the screening of potential ALP inhibitors. 3.7 Photostability Considering the fluorescent nanoprobes for bioimaging in vivo, it is necessary to assess the photostability of the probe. Thus, the photostability of ALP-treated indocyanine green (ICG, the FDA-approved NIR contrast agent), CyOH, Cy 5.5 and CyOH@Tb-GMP nanoprobe were studied by the time-dependent fluorescence measurements upon continuous illumination (Fig. 8). After exposure for about 100 s, the fluorescence of ICG decreased sharply to approximate 5% of the initial value, showing the almost complete decomposition of the scaffold of ICG. Moreover, after irradiation for 600 s, the fluorescence of CyOH and Cy5.5 underwent a decrease to about 49 % and 37 % of the initial fluorescence intensity, respectively. In contrast, the fluorescence of CyOH@Tb-GMP nanoprobe only fell down to 79 % of the initial value. All these data suggest that the CyOH@Tb-GMP nanoprobe on treatment with ALP owns more excellent property in photostability than ICG, CyOH and Cy5.5, which mainly due to the fact that the dense coordination network of Tb-GMP nanoprobe provides an effective barrier against molecular oxygen. The high photostability of 15
CyOH@Tb-GMP nanoprobe makes it be used as a promising nanomaterial for bioimaging in vivo. 3.8 Fluorescence Imaging in Living Cells Given that the high photostability and NIR fluorescence, the practical utility of CyOH@Tb-GMP nanoprobe for fluorescent imaging of ALP in living cells are investigated. Initially, the cytotoxicity tests of HeLa cells were studied by an MTT assay with the concentrations of nanoprobe from 0 to 2.5 mg / mL (Fig. S9). The results show more than 98% cells are viable, which indicates that the noncytotoxicity of the nanoprobe to cells at our experimental conditions. With the low cytotoxicity, the nanoprobe based on CyOH@Tb-GMP was applied for detecting endogenous ALP activity in living cells. HeLa cells (ALP-positive) and HUVEC cells (ALP-negative) were used for evaluating the nanoprobe’s cell imaging capability, in which they have been reported to express ALP at different levels [18, 49]. The imaging for ALP in the HeLa cells and HUVEC cells was shown in Fig. 9 and Fig. S10, respectively. Both HeLa cells and HUVEC cells in the absence of CyOH@Tb-GMP nanoprobe show no fluorescence. After the incubation with CyOH@Tb-GMP nanoprobe, HeLa cells show strong red fluorescence, indicating a high expression level of ALP inside HeLa cells. In contrast, HUVEC cells display relatively weak red fluorescence after incubation with CyOH@Tb-GMP nanoprobe, suggesting that ALP is expressed at low levels in HUVEC cells. In addition, inhibition experiments show that no noticeable fluorescence signal is observed in both cells after the incubation of inhibitor Na3VO4. This indicates that ALP activity can be efficiently inhibited by treatment of Na3VO4. Taken together, 16
these results indicate that this nanoprobe can function well to monitor endogenous ALP activity in living cells. 3.9 Fluorescence Imaging in Tissues To further indicate the superiority of the CyOH@Tb-GMP nanoprobe, the imaging of rat liver tissue was carried out (Fig. 10). The tissue slices not incubated with CyOH@Tb-GMP nanoprobe exhibit no fluorescence (Fig. 10a), while the tissue slices incubated with CyOH@Tb-GMP nanoprobe show significant fluorescence (Fig. 10b). Meanwhile, only negligible fluorescence is observed from the tissues pretreated with Na3VO4, and then treated with CyOH@Tb-GMP nanoprobe (Fig. 10c). The fluorescence signal of tissue slice with CyOH@Tb-GMP nanoprobe at different tissue depths was collected in the Z-scan mode (Fig. 10d). The CyOH@Tb-GMP nanoprobe possesses satisfactory tissue imaging capability at the depths of 40-120 μm. All these results illuminate that CyOH@Tb-GMP nanoprobe has excellent tissue penetrating and staining ability. 3.10 Visualizing ALP in vivo The absorption and emission of the nanoprobe are in the NIR region, which renders it highly favorable for fluorescence imaging of endogenous ALP in vivo. Thus, the imaging application of CyOH@Tb-GMP nanoprobe in mice was explored. Kunming (KM) mice were selected as our model and divided into three groups. The first group of mice was untreated as a control group, no fluorescence is observed (Fig. 11a, quantitation in Fig. 11d). The second group of mice was given an
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intraperitoneal injection of CyOH@Tb-GMP nanoprobe, strong fluorescence signal is noticed (Fig. 11b, quantitation in Fig. 11d), indicating a high expression level of ALP. The third group of mice was injected with Na3VO4 and then with CyOH@Tb-GMP nanoprobe, only negligible fluorescence is observed (Fig. 11c, quantitation in Fig. 11d). With the above results taken together, it is revealed that CyOH@Tb-GMP nanoprobe is suitable for imaging endogenous ALP in vivo. 4. Conclusion In summary, a kind of near-infrared (NIR) fluorescence nanoprobe based on CyOH@Tb-GMP nanoprobe has been reported for the detection of endogenous alkaline phosphatase (ALP) activity. The nanoprobe exhibits high sensitivity for ALP activity with a 7.5-fold fluorescence enhancement and better photostability than ICG as well as CyOH. Most importantly, the NIR nanoprobe is employed for monitoring endogenous ALP activity in various biological samples such as cell, tissue and mice successfully. On the basis of these results, it is greatly anticipated that our strategy will be a powerful tool for designing a wide range of NIR fluorescence nanoprobe. Acknowledgments This work was supported by the National Natural Science Foundation of China (21775133, 21505113), Hunan Provincial Natural Science Foundation (2015JJ6104), China Postdoctoral Science Foundation funded project (2014M552140, 2015T80876), State Key Laboratory of Chemo/Biosensing and Chemometrics Foundation (2015007), Scientific Research Fund of Hunan Provincial Education Department (15K125, 16B252), Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization, Hunan Provincial Innovation Foundation For Postgraduate (CX2017B333).
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References [1] L. Bjelakovic, G. Kocic, T. Cvetkovic, D. Stojanovic, S. Najman, Z. Pop-Trajkovic, B. Bjelakovic, Alkaline phosphatase activity in human milk during the first month of lactation, Acta Fac. Med. Naiss. 26 (2009) 43-47. [2] J. E. Coleman, Structure and mechanism of alkaline phosphatase, Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 441-483. [3] H. N. Fernley, 18 Mammalian alkaline phosphatases, Enzyme 4 (1971) 417-447. [4] C. M. Li, S. J. Zhen, J. Wang, Y. F. Li, C. Z. Huang, A gold nanoparticles-based colorimetric assay for alkaline phosphatase detection with tunable dynamic range, Biosens. Bioelectron. 43 (2013) 366-371. [5] K. Ooi, K. Shiraki, Y. Morishita, T. Nobori, High‐molecular intestinal alkaline phosphatase in chronic liver diseases, J. Clin. Lab. Anal. 21 (2007) 133-139. [6] U. Sharma, D. Pal, R. Prasad, Alkaline phosphatase: an overview, Indian J. Clin. Biochem. 29 (2014) 269-278. [7] K. Ino, Y. Kanno, T. Arai, K. Y. Inoue, Y. Takahashi, H. Shiku, Novel electrochemical methodology for activity estimation of alkaline phosphatase based on solubility difference, Matsue T. Anal. Chem. 84 (2012) 7593-7598. [8] S. Goggins, C. Naz, B. J. Marsh, C. G. Frost, Ratiometric electrochemical detection of alkaline phosphatase, Chem. Commun. 51 (2015) 561-564.
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[9] H. Jiao, J. Chen, W. Li, F. Wang, H. Zhou, Y. Li, C. Yu, Nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation: a new strategy for colorimetric sensing of alkaline phosphatase activity and inhibitor screening, ACS. Appl. Mater. Interfaces 6 (2014) 1979-1985. [10] T. Hasegawa, M. Sugita, K. Takatani, H. Matsuura, T. Umemura, H. Haraguchi, Assay of alkaline phosphatase in salmon egg cell cytoplasm with fluorescence detection of enzymatic activity and zinc detection by ICP-MS in relation to metallomics research, Bull. Chem. Soc. Jpn. 79 (2006) 1211-1214. [11] A. Ingram, B. D. Moore, D. Graham, Simultaneous detection of alkaline phosphatase and β-galactosidase activity using SERRS. Bioorg, Bioorg. Med. Chem. Lett. 19 (2009) 1569-1571. [12] Z. G. Yang, J. F. Cao, Y. X. He, J. H. Yang, T. Kim, X. J. Peng, J. S. Kim, Macro/micro-environment-sensitive chemosensing and biological imaging, Chem. Soc. Rev. 43 (2014) 4563-4601. [13] X. Sun, Q. Xu, G. Kim, S. E. Flower, J. P. Lowe, J. Yoon, T. D. James, A watersoluble boronate-based fluorescent probe for the selective detection of peroxynitrite and imaging in living cells, Chem. Sci. 5 (2014) 3368-3373. [14] J. Xu, J. Pan, Y. Zhang, J. Liu, L. Zeng, X. Liu, Ultrasensitive near-infrared fluorescence-enhanced probe for discriminative detection of GSH and Cys from different emission channels, Sens. Actuators B 238 (2017) 58-65.
20
[15] D. Lu, L. Zhou, R. Wang, X. B. Zhang, L. He, J. Zhang, W. Tan, A two-photon fluorescent probe for endogenous superoxide anion radical detection and imaging in living cells and tissues, Sens. Actuators B 250 (2017) 259-266. [16] S. Li, M. Dong, R. Li, L. Zhang, Y. Qiao, Y. Jiang, H. Wang, A fluorometric microarray with ZnO substrate-enhanced fluorescence and suppressed “coffee-ring” effects for fluorescence immunoassays, Nanoscale 7 (2015) 18453-18458. [17] F. Y. Cao, Y. Long, S. B. Wang, B. Li, J. X. Fan, X. Zeng, X. Z. Zhang, Fluorescence light-up AIE probe for monitoring cellular alkaline phosphatase activity and detecting osteogenic differentiation, J. Mater. Chem. B 4 (2016) 4534-4541. [18] Y. Tan, L. Zhang, K. H. Man, R. Peltier, G. Chen, H. Zhang, Y. Hu, Reaction-based off-on near-infrared fluorescent probe for imaging alkaline phosphatase activity in living cells and mice, ACS Appl. Mater. Interfaces 9 (2017) 6796-6803. [19] H. Zhang, P. Xiao, Y. T. Wong, W. Shen, M. Chhabra, R. Peltier, Y. Xie, Construction of an alkaline phosphatase-specific two-photon probe and its imaging application in living cells and tissues, Biomaterials 140 (2017) 220-229. [20] J. Park, A. Helal, H. S. Kim, Y. Kim, Fluorogenic assay of alkaline phosphatase activity based on the modulation of excited-state intramolecular proton transfer, Bioorg. Med. Chem. Lett. 22 (2012) 5541–5544. [21] Z. G. Song, R. T. K. Kwok, E. G. Zhao, Z. K. He, Y. N. Hong, J. W. Y. Lam, B. Liu, B. Z. Tang, A ratiometric fluorescent probe based on ESIPT and AIE processes for alkaline
21
phosphatase activity assay and visualization in living cells, ACS Appl. Mater. Interfaces 6 (2014) 17245-17254. [22] S. J. Li, C. Y. Li, Y. F. Li, J. J. Fei, P. Wu, B. Yang, J. Ou-Yang, S. X. Nie, A Facile and Sensitive Near-Infrared Fluorescence Probe for the Detection of Endogenous Alkaline Phosphatase Activity in Vivo, Anal. Chem. 89 (2017) 6854-6860. [23] Y. Liu, K. S. Schanze, Conjugated polyelectrolyte-based real-time fluorescence assay for alkaline phosphatase with pyrophosphate as substrate, Anal. Chem. 80 (2008) 8605-8612. [24] Y. Li, Y. Li, Z. Liu, X. Su, Sensitive fluorometric detection of alkaline phosphatase using a water-soluble conjugated polymer, RSC Advances 4 (2014) 42825-42830. [25] G. Li, H. Fu, X. Chen, P. Gong, G. Chen, L. Xia, Y. Wu, Facile and sensitive fluorescence sensing of alkaline phosphatase activity with photoluminescent carbon dots based on inner filter effect, Anal. Chem. 88 (2016) 2720-2726. [26] L. Jia, J. P. Xu, D. Li, S. P. Pang, Y. Fang, Z. G. Song, J. Ji, Fluorescence detection of alkaline phosphatase activity with β-cyclodextrin-modified quantum dots, Chem. Commun. 46 (2010) 7166-7168. [27] S. Y. Liu, S. Pang, W. D. Na, X. G. Su, Near-infrared fluorescence probe for the determination of alkaline phosphatase, Biosens. Bioelectron. 55 (2014) 249-254. [28] R. Freeman, T. Finder, R. Gill, I. Willner, Probing protein kinase (CK2) and alkaline phosphatase with CdSe/ZnS quantum dots, Nano Lett. 10 (2010) 2192-2196.
22
[29] J. OakaKeem, B. HyunaChung, Smart nanoprobes for the detection of alkaline phosphatase activity during osteoblast differentiation, Chem. Commun. 51 (2015) 3270-3272. [30] X. L. Hu, X. M. Wu, X. Fang, Z. J. Li, G. L. Wang, Switchable fluorescence of gold nanoclusters for probing the activity of alkaline phosphatase and its application in immunoassay, Biosens. Bioelectron. 77 (2016) 666-672. [31] C. Aime, R. Nishiyabu, R. Gondo, N. Kimizuka, Switching on luminescence in nucleotide/lanthanide coordination nanoparticles via synergistic interactions with a cofactor ligand, N. Chem.-Eur. J. 16 (2010) 3604-3607. [32] R. Nishiyabu, N. Hashimoto, T. Cho, K. Watanabe, T. Yasunaga, A. Endo, Y. Katayama, Nanoparticles of adaptive supramolecular networks self-assembled from nucleotides and lanthanide ions, J. Am. Chem. Soc. 131 (2009) 2151-2158. [33] Q. An, J. Liu, M. Yu, J. Wan, D. Li, C. Wang, J. Guo, Multifunctional magnetic Gd3+-based coordination polymer nanoparticles: combination of magnetic resonance and multispectral optoacoustic detections for tumor-targeted imaging in vivo, Small 11 (2015) 5675-5686. [34] H. Tan, B. Liu, Y. Chen, Lanthanide coordination polymer nanoparticles for sensing of mercury (II) by photoinduced electron transfer, ACS nano 6 (2012) 1050510511. [35] H. Tan, Y. Chen, Ag+-enhanced fluorescence of lanthanide/nucleotide coordination polymers and Ag+ sensing, Chem. Commun. 47 (2011) 12373-12375. 23
[36] N. Lin, J. Li, Z. Lu, L. Bian, L. Zheng, Q. Cao, Z. Ding, A turn-on coordination nanoparticle-based fluorescent probe for phosphate in human serum, Nanoscale 7 (2015) 4971-4977. [37] B. Chen, L. Wang, F. Zapata, G. Qian, E. B. Lobkovsky, A luminescent microporous metal−organic framework for the recognition and sensing of anions, J. Am. Chem. Soc. 130 (2008) 6718-6719. [38] Y. Huang, B. Liu, Q. Shen, X. Zhu, Y. Hao, Z. Chang, M. Xu, Lanthanide coordination polymer probe for time-gated luminescence sensing of pH in undiluted human serum, Talanta 164 (2017) 427-431. [39] H. H. Zeng, W. B. Qiu, L. Zhang, R. P. Liang, J. D. Qiu, Lanthanide coordination polymer nanoparticles as an excellent artificial peroxidase for hydrogen peroxide detection, Anal. Chem. 88 (2016) 6342-6348. [40] L. Zhang, J. Zhao, M. Duan, H. Zhang, J. Jiang, R. Yu, Inhibition of dsDNAtemplated copper nanoparticles by pyrophosphate as a label-free fluorescent strategy for alkaline phosphatase assay, Anal. Chem. 85 (2013) 3797-3801. [41] J. Deng, P. Yu, Y. Wang, L. Mao, Real-time ratiometric fluorescent assay for alkaline phosphatase activity with stimulus responsive infinite coordination polymer nanoparticles, Anal. Chem. 87 (2015) 3080-3086. [42] X. Zhang, J. Deng, Y. Xue, G. Shi, T. Zhou, Stimulus response of Au-NPs@GMP-Tb core–shell nanoparticles: toward colorimetric and fluorescent dual-mode sensing of
24
alkaline phosphatase activity in algal blooms of a freshwater lake, Environ. Sci. Technol. 50 (2015) 847-855. [43] F. Yu, M. Gao, M. Li, L. Chen, A dual response near-infrared fluorescent probe for hydrogen polysulfides and superoxide anion detection in cells and in vivo, Biomaterials 63 (2015) 93-101. [44] Y. J. Gong, X. B. Zhang, G. J. Mao, L. Su, H. M. Meng, W. Tan, G. Zhang, A unique approach toward near-infrared fluorescent probes for bioimaging with remarkably enhanced contrast, Chem. Sci. 7 (2016) 2275-2285. [45] H. Zhang, S. Xue, G. Feng, A colorimetric and near-infrared fluorescent turn-on probe for rapid detection of sulfite, Sens. Actuators B 231 (2016) 752-758. [46] Y. Wei, D. Cheng, T. Ren, Y. Li, Z. Zeng, L. Yuan, Design of NIR chromenyliumcyanine fluorophore library for “switch-on” and ratiometric detection of bio-active species in vivo, Anal. Chem. 88 (2016) 1842-1849. [47] I. R. Gibbons, M. P. Cosson, J. A. Evans, B. H. Gibbons, B. Houck, K. H. Martinson, W. J. Tang, Potent inhibition of dynein adenosinetriphosphatase and of the motility of cilia and sperm flagella by vanadate, Proc. Natl. Acad. Sci. 75 (1978) 2220-2224. [48] N. Malashikhina, G. Garai-Ibabe, V. Pavlov, Unconventional application of conventional enzymatic substrate: first fluorogenic immunoassay based on enzymatic formation of quantum dots, Anal. Chem. 85 (2013) 6866-6870.
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[49] C. Fan, S. Luo, H. Qi, A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited-state intramolecular proton transfer, Luminescence 31 (2016) 423-427.
Author Biographies Juan Ou-Yang received her BS degree in 2015 at College of Chemical Engineering, Xiangtan University. Now she is working toward a MS degree in Analytical Chemistry at Xiangtan University. Her research fields focus on the design and synthesis of fluorescent materials and their applications as fluorescent probe. Chun-Yan Li received her PhD degree in 2008 at College of Chemistry & Chemical Engineering, Hunan University. Now she is an associate professor of Xiangtan University. Her research fields focus on the design and synthesis of fluorescent materials and their applications as fluorescent probe. Yong-Fei Li received his PhD degree in 2010 at College of Chemistry & Chemical Engineering, Hunan University. Now he is an instructor of Xiangtan University. His research fields focus on the synthesis of fluorescent materials. Bin Yang received his PhD degree in 2014 at College of Chemistry & Chemical Engineering, Hunan University. Now he is an instructor of Xiangtan University. His research fields focus on the synthesis of fluorescent materials and their applications as fluorescent probe. Song-Jiao Li received her BS degree in 2015 at College of Chemistry, Hengyang Normal University. Now she is working toward a MS degree in Analytical Chemistry at Xiangtan University. Her research fields focus on the design and synthesis of fluorescent materials and their applications as fluorescent probe.
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Figure captions. Fig. 1 TEM image of (a) Tb-GMP nanoparticles and (b) CyOH@Tb-GMP nanoparticles. (c) Absorption and (d) fluorescence spectra of CyOH, Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles in Tris-HCl buffer solution (50 mM, pH 8.0). Fig. 2 FT-IR spectra for GMP, Tb-GMP nanoparticles, CyOH@Tb-GMP nanoparticles and CyOH. Fig. 3 Fluorescence response of CyP (10 µM) to ALP (2 U/mL), GOX (10 mg/mL), GDH (2 U/mL), GAL (2 U/mL), AChE (2 U/mL), trypsin (10 mg/mL), thrombin (2 U/mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded within 20 min. λex / λem = 690 / 738 nm. Fig. 4 Fluorescence spectra of CyOH@Tb-GMP nanoprobe (1 mg / mL) upon the addition of ALP (2.5 U / mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded every 2.5 min. λex = 685 nm. Inset: the plot of fluorescence intensity versus time. Fig. 5 Fluorescence spectra of CyOH@Tb-GMP nanoprobe (1 mg /mL) in the presence of different activities of ALP (0, 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5 U / mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex = 685 nm. Insert: the plot of fluorescence intensity versus ALP activity. Fig. 6 Fluorescence response of CyOH@Tb-GMP nanoprobe (1 mg / mL) to ALP (2.5 U / mL), GDH (10 mg/mL), GAL (2.5 U / mL), GOX (10 mg/mL), EXO (2.5 U / mL), AChE
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(2.5 U / mL), Trypsin (10 mg/mL) or Thrombin (2.5 U / mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex / λem = 685 / 718 nm. Fig. 7 Fluorescence spectra of CyOH@Tb-GMP nanoprobe (1 mg /mL) in the presence of ALP (2.5 U / mL) at different Na3VO4 concentrations (0, 50, 100, 200, 300, 400, 500, 600 μM) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex = 685 nm. Insert: the plot of the inhibition efficiency versus Na3VO4 concentrations. Fig. 8 Time-dependence fluorescence intensity of ICG (treated with 2.5 U / mL ALP for 20 min, monitored at 812 nm), CyOH (treated with 2.5 U / mL ALP for 20 min, monitored at 718 nm), Cy5.5 (treated with 2.5 U / mL ALP for 20 min, monitored at 707 nm) and CyOH@Tb-GMP nanoprobe (treated with 2.5 U / mL ALP for 20 min, monitored at 718 nm) under constant illumination for 0-600 s. Fig. 9 Fluorescence images of HeLa cells. From left to right: control, cells treated with CyOH@Tb-GMP nanoprobe only, cells treated with Na3VO4 and then CyOH@Tb-GMP nanoprobe. Fig. 10 Fluorescence images of tissues. (a) Control. (b) Tissue slice was incubated with CyOH@Tb-GMP nanoprobe. (c) Tissue slice was pre-treated with Na3VO4, and then treated with CyOH@Tb-GMP nanoprobe. (d) The confocal z-scan imaging sections at different depths for 0, 20, 40, 60, 80, 100, 120, 140, 160 μm. Fig. 11 Fluorescence images in vivo. (a) Control. (b) a mouse given an injection of CyOH@Tb-GMP nanoprobe. (c) a mouse injected with Na3VO4 and then with 28
CyOH@Tb-GMP nanoprobe. (d) quantification of fluorescence emission intensity from the selected circle region of (a–c) was averaged and plotted as a ratio to blank.
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(b)
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Scheme Captions. Scheme 1. Schematic illustration of the formation of CyOH@Tb-GMP nanoparticles and the response mechanism of CyOH@Tb-GMP nanoprobe to ALP.
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S0925-4005(17)31828-2 https://doi.org/10.1016/j.snb.2017.09.162 SNB 23253
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
27-7-2017 20-9-2017 23-9-2017
Please cite this article as: Juan Ou-Yang, Chun-Yan Li, Yong-Fei Li, Bin Yang, Song-Jiao Li, An Infinite Coordination Polymer Nanoparticles-Based Near-Infrared Fluorescent Probe with High Photostability for Endogenous Alkaline Phosphatase in Vivo, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.09.162 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.
An Infinite Coordination Polymer Nanoparticles-Based Near-Infrared Fluorescent Probe with High Photostability for Endogenous Alkaline Phosphatase in Vivo Juan Ou-Yang,a Chun-Yan Li,*,a,c Yong-Fei Li,b Bin Yang,a Song-Jiao Lia a
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry
of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, PR China. b
College of Chemical Engineering, Xiangtan University, Xiangtan, 411105, PR China
c State
Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
& Chemical Engineering, Hunan University, Changsha, 410082, PR China *Corresponding author, Tel.: +86 731 58292205; fax: +86 731 58292477. E-mail: [email protected] Highlights ► The fluorescence emission of CyOH@Tb-GMP nanoprobe belongs to NIR region. ► The nanoprobe displays better photostability than NIR dyes such as ICG and CyOH. ► The NIR nanoprobe shows high sensitivity and selectivity for ALP activity assay. ► The nanoprobe is used for monitoring endogenous ALP activity in biological samples.
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Abstract Alkaline phosphatase (ALP) is an essential enzyme in phosphate metabolism and widely distributes in mammalian body fluids and tissues. So far, various fluorescent probes for ALP have been well developed, but some disadvantages such as short emission wavelength and low photostability restrict their biological application. Herein, a near-infrared (NIR) fluorescent nanoprobe based on infinite coordination polymer nanoparticles (CyOH@Tb-GMP) is constructed for endogenous ALP activity assay. The characteristics of the nanoprobe are as following: (1) The fluorescence emission of the nanoprobe is at 718 nm belonging to NIR region, which is suitable for bioimaging. (2) The nanoprobe displays better photostability than common NIR dyes such as ICG as well as CyOH. (3) The nanoprobe exhibits high sensitivity for ALP activity with a 7.5-fold fluorescence enhancement when 2.5 U / mL ALP is added. (4) The NIR nanoprobe is employed for monitoring endogenous ALP activity in various biological samples such as cell, tissue and mice with satisfactory results. Keywords: Alkaline phosphatase; Infinite coordination polymer; Near-infrared; Photostability; Biological samples
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1. Introduction Alkaline phosphatase (ALP) is an essential enzyme in phosphate metabolism. It can catalyze the hydrolysis of phosphoryl esters and widely distributes in mammalian body fluids and tissues [1-3]. Moreover, ALP is an important biomarker for diagnostics, and the deviation of ALP from its normal level is closely related to various diseases including breast and prostatic cancer, bone disease, liver dysfunction, and diabetes [4-6]. In this context, it is very essential to develop a sensitive method for ALP level determination in order to understand the role of ALP in related biological processes and provide the clinical diagnoses of the related diseases. On the basis of the ability of ALP to remove phosphate groups from a wide variety of substrates and obtain the signal readout through various mechanisms, numerous assays for ALP activity have been developed, including electrochemistry [7, 8], colorimetric method [9], chromatographic [10], and surface enhancement Raman scattering assay [11]. Although each of these methods possesses its own features and gives the different insights for ALP sensing, they often require sample processing, complex instrumentation and are not suitable for applying in biological systems. Fluorescence technique is extremely attractive in this regard due to their intrinsic advantages of low background noise and high sensitivity, noninvasive monitoring capability and usability in live organism [12-18]. Up to date, a variety of fluorescent assays for ALP have been reported by using organic dyes [17-22], conjugated 3
polyelectrolytes [23-24], quantum dots [25-28], metal nanoclusters [29, 30] as fluorescent materials. Unfortunately, we note that these approaches usually suffer from some disadvantages. For example, organic dyes have poor water solubility, bad photostability and easy diffusion in vivo. The conjugated polyelectrolytes require a complicated synthesis process and quantum dots are toxic. Noble metal nanoclusters are high cost and poor stability in aqueous system. Therefore, exploring novel fluorescent materials which meet the demand of simplicity, high photostability and low toxicity are still necessary. Infinite coordination polymer (ICP) nanoparticles formed from metal ions and synthetic ligand molecules have been emerging as a new family of functional nanomaterials [31-34]. The unique properties of ICP nanoparticles such as easy synthesis, good water solubility, satisfactory biocompatibility and high photostability make them widely be applied for the detection of metal ions [35], anions[36, 37], and various biological molecules [38-40]. Only a few of the ICP nanoparticles-based fluorescent nanoprobes are applied in the determination of ALP activity [41, 42]. In addition, almost all these nanoprobes have relatively short emission wavelengths (< 650 nm), which greatly limits their practical application in biological sample. As is well known, near-infrared (NIR) emission can minimize photo-damage to living cells, enable deep tissue penetration, and circumvent the spectral overlap with biosubstrate autofluorescence [43-46]. Therefore, it is highly necessary to explore the
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capability of ICP nanomaterials to design NIR fluorescent nanoprobes for ALP detection. To meet the requirements mentioned above, an ICP nanoparticles-based NIR fluorescent nanoprobe (CyOH@Tb-GMP) with high photostability is developed in this paper (Scheme 1). The CyOH@Tb-GMP nanoprobe are constructed with Tb3+ as the metal ion and guanosine-5′-monophosphate (GMP) as the bridging ligand, in which a NIR fluorescence dye (CyOH) is encapsulated into the ICP supramolecular network through an adaptive selfassembly chemistry. In the presence of ALP, the phosphate group in the GMP ligand is cleaved, resulting in the destruction of the Tb-GMP and the release of encapsulated CyOH into solution. This process eventually results in the increase of the fluorescence intensity emitted from CyOH, which provides a basis for NIR fluorescent assay for ALP. Moreover, the NIR fluorescent nanoprobe is applied for the detection of endogenous ALP activity in biological samples such as cell, tissue and mice with satisfactory results. 2. Experimental 2.1 Reagents Guanine monophosphate (GMP), IR-780 iodide and resorcinol were purchased from Sigma-Aldrich (St Louis, USA). Terbium nitrate hexahydrate (Tb (NO3)3·6H2O) and sodium orthovanadate (Na3VO4) were all purchased from Aladdin (Shanghai, China). ALP (alkaline phosphatase, calf intestine) was purchased from TaKaRa
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Biotechnology Corporation (Dalian, China). Note, one unit of the enzyme is defined as the amount that produces 1 μmol of 4-nitrophenol from 4-nitrophenylphosphate in a minute at 37 °C. 2.2 Syntheses Synthesis of CyOH: Resorcinol (0.55 g, 5.0 mmol) and K2CO3 (0.69 g, 5.0 mmol) were placed in a flask containing CH3CN (10 mL), and the mixture was stirred for 10 min at room temperature under nitrogen atmosphere. Then IR-780 iodide (1.53 g, 2.5 mmol) in CH3CN (20 mL) was introduced to the mixture followed by heating at 50 °C for 4 h. After the removal of solvent from the reaction mixture under reduced pressure, the crude product was purified by silica gel column chromatography using CH2Cl2/CH3OH (20:1, v/v) as eluent to afford a blue-green solid product. Yield: 1.54 g (82%). 1H NMR (400 MHz, CDCl3, Fig. S1): δ 8.02 (d, 1H, J = 13.2 Hz), 7.25-7.21 (m, 2H), 7.14 (t, 1H, J = 7.6 Hz), 7.06-7.03 (m, 2H), 6.97 (s, 1H), 6.90 (d, 1H, J = 8.0 Hz), 6.81 (d, 1H, J= 8.0 Hz), 5.56 (d, 1H, J = 13.2 Hz), 3.76 (t, 2H, J = 7.2 Hz), 2.67 (t, 2H, J = 6.0 Hz), 2.59 (t, 2H, J = 6.0 Hz), 1.91-1.82 (m, 4H), 1.67 (s, 6H), 1.04 (t, 3H, J = 7.6 Hz). 13C
NMR (100 MHz, CDCl3, Fig. S2): δ 174.5, 166.9, 163.0, 155.6, 143.2, 142.0, 141.3,
137.8, 132.7, 128.9, 125.8, 123.9, 123.8, 122.6, 118.4, 114.8, 111.0, 103.1, 100.1, 49.8, 46.3, 29.6, 28.5, 24.2, 22.6, 20.8, 11.7. MS (TOF, Fig. S3) m/z 412.4. Synthesis of Tb-GMP nanoparticles: Tb-GMP nanoparticles was synthesized by mixing an aqueous solution of Tb(NO3)3·6H2O (10 mM, 1 mL) with a HEPES buffer (0.1 M, pH 7.4, 1mL) containing GMP (10 mM, 1 mL) at room temperature to form a
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white precipitate. The resulting precipitate was then centrifuged and washed with water several times. Synthesis of CyOH@Tb-GMP nanoparticles: Encapsulation of CyOH into the TbGMP nanoparticles to form CyOH@Tb-GMP was carried out by following procedures. An aqueous solution of Tb(NO3)3·6H2O (10 mM, 1 mL) was added into a HEPES buffer (0.1 M, pH 7.4, 1 mL) containing GMP (10 mM, 1 mL) and CyOH (0.1 mM, 1 mL) to form a precipitate. The resulting precipitate was then centrifuged and washed with water several times until no fluorescence of CyOH was detected in the supernatant of the solution. 2.3 Apparatus All fluorescence measurements were recorded with a Perkin Elmer LS-55 fluorescence spectrometer (USA). UV-vis absorption spectra were collected on a Perkin Elmer Lambda 25 spectrophotometer (USA). 1H NMR and 13C NMR spectra were carried out on a Bruker Avance II 400 spectrometer (Germany) operating at 400 and 100 MHz (TMS as internal standard), respectively. MS spectrometry was performed on a Bruker Autoflex MALDI-TOF MS spectrometer (Germany). Transmission Electron Microscopy (TEM) images were performed by a JEM-2100 transmission electron microscope (Japan). Fourier transform infrared (FI-IR) spectra were recorded with KBr pellets on a Nicolet 6700 FT-IR Spectrometer (USA). Fluorescence imaging of cells and tissues were carried out by an Olympus FV1000
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fluorescence microscope (Japan). Fluorescence imaging of mice was performed on an IVIS Lumina XR small animal optical in vivo imaging system (USA). 2.4 General procedure for ALP activity assay A fluorescent ALP activity assay was performed according to the following procedures. ALP with different activities ranging from 0.01 to 2.5 U was added into the mixture (1 mL, pH 8.0) containing 50 mM Tris-HCl, 1 mM MgCl2, and 1.0 mg CyOH@Tb-GMP nanoprobe. After the addition of ALP, the fluorescent spectrum of each of the mixtures was recorded at 37 °C. The fluorescence emission spectra were recorded at excitation wavelength of 685.0 nm with emission wavelength range from 705.0 to 780.0 nm with excitation slit set at 10.0 nm and emission slit set at 10.0 nm. 2.5 Cell incubation and fluorescence imaging The living HeLa cells were provided by XiangYa Central Experiment Laboratory of Central South University (China). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) at 37 °C under an atmosphere of 5 % CO2. The cells were plated on 35 mm culture dish and allowed to adhere for 24 h. Then three confocal dishes were treated differently and imaged. In the first dish, HeLa cells were used as control incubated in DMEM medium only. In the second dish, HeLa cells were incubated with the CyOH@Tb-GMP nanoprobe (1 mg / mL) for 30 min, followed by washing three times with PBS buffer solution before imaging. In the third dish, HeLa cells were treated with ALP inhibitor sodium orthovanadate (Na3VO4, 100 μM) at 37 °C for 30 min, then with CyOH@Tb-
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GMP nanoprobe (1 mg / mL) for another 30 min, followed by washing three times with PBS buffer solution before imaging. Fluorescence imaging was performed by confocal fluorescence microscope with excitation wavelength at 635 nm and emission wavelength at 680-780 nm. 2.6 Preparation and staining of rat liver tissue slices Tissue slices were prepared from rat liver. A side of the tissue was cut flat using a vibrating-blade microtome. Then three tissue slices were treated differently and imaged. The first tissue slice was used as control. The second tissue slice was incubated with CyOH@Tb-GMP nanoprobe (1 mg / mL) at 37 °C for 30 min, followed by washing three times with PBS buffer solution before imaging. The third group of tissue slice was treated with Na3VO4 (100 μM) at 37 °C for 30 min, then with CyOH@Tb-GMP nanoprobe (1 mg / mL) for another 30 min, followed by washing three times with PBS buffer solution before imaging. Fluorescence imaging was performed by confocal fluorescence microscope with excitation wavelength at 635 nm and emission wavelength at 680-780 nm. 2.7 Fluorescent imaging in Vivo 20-25 g Kunming (KM) mice were used and were kindly kept in all the experimental process. All animal experiments were performed in accordance with the guidelines issued by The Ethical Committee of Xiangtan University. The abdominal fur of the mice was removed by an electric shaver. Then, the mice were divided into three groups. The first group was untreated as a control group. The
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second group was given an intraperitoneal injection of CyOH@Tb-GMP nanoprobe (1 mg / mL). The third group was given an intraperitoneal injection of Na3VO4 (100 μM) and then injection of CyOH@Tb-GMP nanoprobe (1 mg / mL). Before imaging, the mice were anesthetized with chloral hydrate (10% in saline) and remained anesthetized throughout the image period. The mice were placed into the imaging chamber and imaged with 660 nm excitation and 740 nm emission channel. 3. Result and discussion 3.1. Synthesis and characterization of CyOH@Tb-GMP nanoparticles A rapid and facile route for preparing CyOH@Tb-GMP nanoparticles was showed as Scheme 1. Initially, a hemicyanine dye (CyOH) with NIR fluorescence emission was selected as the guest molecule and synthesized starting from the reaction between IR-780 and resorcinol. Then, Tb-GMP nanoparticles were chosen as the host and constructed by self-assembly of GMP and Tb3+ in aqueous solution. Subsequently, CyOH was employed to be encapsulated into the Tb-GMP nanoparticles to form the CyOH@Tb-GMP nanoparticles. The morphology of Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles was analyzed by transmission electron microscopy (TEM) and the results were shown in Fig. 1. The Tb-GMP nanoparticles are colloidal spheres with diameters ranging from 30 to 40 nm (Fig. 1a), which is almost consistent with the reported literature value [31, 41]. The CyOH@Tb-GMP nanoparticles have almost the same shape and size (diameters of 30-40 nm) as the Tb-GMP nanoparticles (Fig. 1b), suggesting that
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the encapsulation of CyOH does not change the morphology of the Tb-GMP nanoparticles. Moreover, the UV-visible spectra of CyOH, Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles were measured and descripted in Fig. 1c. CyOH exhibits a strong absorption peak at 695 nm, while Tb-GMP nanoparticles show no absorption. And there is almost no absorption for CyOH@Tb-GMP nanoparticles, reflecting that CyOH is encapsulated into Tb-GMP nanoparticles to form CyOH@TbGMP nanoparticles. The fluorescence spectra of CyOH, Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles were also studied (Fig. 1d). Similarly, CyOH shows a strong NIR fluorescence at 718 nm, whereas Tb-GMP nanoparticles display no emission. Particularly, the CyOH@Tb-GMP nanoparticles exhibit almost no fluorescence and no obvious change is observed in at least 8 h (Fig. S4), which demonstrates the successful encapsulation of CyOH into Tb-GMP nanoparticles to form CyOH@Tb-GMP nanoparticles and the CyOH@Tb-GMP nanoparticles own excellent chemical stability. Furthermore, Fourier transfer infrared (FT-IR) spectra of GMP, Tb-GMP nanoparticles, CyOH@Tb-GMP nanoparticles and CyOH were displayed in Fig. 2. For GMP, peaks at 1632, 1359, 1064 and 982 cm-1 were assigned to the C=O (ν C=O) and N-C (ν N-C) stretching vibration band in the guanosine moiety of GMP, the antisymmetric (νasPO3) and symmetric (νsPO3) vibration bands in the phosphate moiety of GMP, respectively. After the interaction with Tb3+ to form Tb-GMP nanoparticles, the wavenumber of GMP was shifted to 1634, 1358, 1063 and 970 cm-1, respectively. This suggests that both guanosine and phosphate moieties of GMP are coordinated 11
to Tb3+. As for CyOH@Tb-GMP nanoparticles, the wavenumber of the corresponding peaks (1633, 1360, 1065 and 983 cm-1) shifted slightly, as compared with those of Tb-GMP nanoparticles. In addition, the characteristic peaks of CyOH did not be found after its encapsulation into Tb-GMP nanoparticles. These demonstrate that CyOH is localized inside Tb-GMP nanoparticles and the encapsulation of CyOH has almost no effect on the coordination mode between GMP and Tb 3+. 3.2. Spectral response of CyOH@Tb-GMP nanoprobe for ALP The absorption and fluorescence spectra of CyOH@Tb-GMP nanoprobe before and after reacting with ALP were measured in Tris-HCl buffer solution (Fig. 3). With the addition of ALP into Tris-HCl buffer solution containing CyOH@Tb-GMP nanoprobe, the absorption band centered at 695 nm shows an obvious enhancement (Fig. 3a). Meanwhile, the fluorescence signal of CyOH@Tb-GMP nanoprobe displays noticeable changes (Fig. 3b). The emission intensity at 718 nm, which is ascribed to the emission of CyOH, increases greatly upon adding ALP (Fig. 3b). These spectral changes could be due to ALP-catalyzed hydrolysis of the phosphate ester group to convert GMP into a guanosine base. With the cleavage of phosphate group, the structure of Tb-GMP nanoprobe was destructed and the guest molecule (CyOH) was thus released into the solution (Scheme 1). The fluorescence intensity is closely associated with the degree of the destruction of the CyOH@TbGMP nanoprobe caused by ALP. As a consequence, the CyOH@Tb-GMP nanoprobe can be used for the fluorescence assay of ALP activity.
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3.3 Condition optimization In order to obtain excellent ALP sensing, the sensing conditions need to be optimized. First of all, the effect of reaction temperature on fluorescence intensity was examined (Fig. S5). The experimental results show that the change of fluorescence intensity reaches maximum at 37 °C. Therefore, 37 °C is selected in this study, which is consistent with the circumstance in human body. Then, the effect of pH on the fluorescence of the probe was investigated (Fig. S6) and the results show that the optimum pH for ALP is 8.0. Furthermore, the effect of incubation time on the fluorescence of the CyOH@Tb-GMP nanoprobe was studied since time is another important factor for the enzyme reaction. As could be seen from Fig. 4, the fluorescence of the nanoprobe undergoes a gradual increase and reaches the maximum when the probe is incubated in the presence of ALP for 20 min. So 20 min is used for ALP analysis in the subsequent experiments. In addition, the kinetic parameters for the hydrolysis of CyOH@Tb-GMP nanoprobe catalyzed by ALP under different initial concentrations of the nanoprobe were determined. By Lineweaver– Burk analysis, the catalytic constant (kcat) is calculated to be 0.03 s-1, which is close to the data reported in the literature (Fig. S7). [18-20] 3.4 Sensitivity To estimate the sensitivity of the assay, different activities of ALP were added into the Tris-HCl buffer solution containing CyOH@Tb-GMP nanoprobe (Fig. 5). As displayed in Fig. 5, the fluorescence intensity of the nanoprobe gradually enhances
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with increasing activity of ALP. When 2.5 U/mL ALP is added, the fluorescence intensity increases around 7.5-fold. Moreover, the insert of Fig. 5 illustrates that the probe exhibits a good linear relationship with ALP ranging from 0.01 to 2.5 U / mL. The detection limit is 0.0033 U / mL (3σ/slope), which is low enough for ALP detection in biological samples. 3.5 Selectivity To investigate the selectivity of the CyOH@Tb-GMP nanoprobe, a series of enzymes in serum such as glucose dehydrogenase (GDH), galactosidase (GAL), glucose oxidase (GOX), exonuclease (EXO), acetylcholinesterase (AChE), trypsin and thrombin were evaluated. As shown in Fig. 6, none of the enzymes has the ability to recover the fluorescence except ALP. Meanwhile, the selectivity was also investigated by examining various potential interfering substances such as biothiols (Cys, Hcy, GSH), amino acids (Ala, Val, Leu, Ileu, Pro, Phe, Trp, Met, Gly, Tyr, Ser, Thr, Asn, Gln, Lys, Arg, His, Asp, Glu) and metal ions (K+, Na+, Ca2+, Mg2+, and Zn2+) (Fig. S8). The results indicate that no significant fluorescence change of the nanoprobe is observed, demonstrating its high selectivity to ALP. 3.6 ALP inhibitor efficiency evaluation Having demonstrated the validity of the fluorescent nanoprobe based on CyOH@Tb-GMP nanoprobe, the possibility of applying the nanoprobe for the screening of potential ALP inhibitors was also investigated. Sodium orthovanadate (Na3VO4), a well-known inhibitor for ALP [4-6], was used in the experiments. As
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displayed in Fig. 7, the fluorescence intensity decreases with the addition of increasing concentrations of the inhibitor. The insert of Fig. 7 shows that the corresponding IC50 value (the concentration of an inhibitor required to 50% of inhibition efficiency) of Na3VO4 toward ALP is calculated to be 148.6 μM, which is consistent with the value reported previously [41, 47, 48]. The results essentially demonstrate that the nanoprobe can be used not only for the monitoring of ALP but also for the screening of potential ALP inhibitors. 3.7 Photostability Considering the fluorescent nanoprobes for bioimaging in vivo, it is necessary to assess the photostability of the probe. Thus, the photostability of ALP-treated indocyanine green (ICG, the FDA-approved NIR contrast agent), CyOH, Cy 5.5 and CyOH@Tb-GMP nanoprobe were studied by the time-dependent fluorescence measurements upon continuous illumination (Fig. 8). After exposure for about 100 s, the fluorescence of ICG decreased sharply to approximate 5% of the initial value, showing the almost complete decomposition of the scaffold of ICG. Moreover, after irradiation for 600 s, the fluorescence of CyOH and Cy5.5 underwent a decrease to about 49 % and 37 % of the initial fluorescence intensity, respectively. In contrast, the fluorescence of CyOH@Tb-GMP nanoprobe only fell down to 79 % of the initial value. All these data suggest that the CyOH@Tb-GMP nanoprobe on treatment with ALP owns more excellent property in photostability than ICG, CyOH and Cy5.5, which mainly due to the fact that the dense coordination network of Tb-GMP nanoprobe provides an effective barrier against molecular oxygen. The high photostability of 15
CyOH@Tb-GMP nanoprobe makes it be used as a promising nanomaterial for bioimaging in vivo. 3.8 Fluorescence Imaging in Living Cells Given that the high photostability and NIR fluorescence, the practical utility of CyOH@Tb-GMP nanoprobe for fluorescent imaging of ALP in living cells are investigated. Initially, the cytotoxicity tests of HeLa cells were studied by an MTT assay with the concentrations of nanoprobe from 0 to 2.5 mg / mL (Fig. S9). The results show more than 98% cells are viable, which indicates that the noncytotoxicity of the nanoprobe to cells at our experimental conditions. With the low cytotoxicity, the nanoprobe based on CyOH@Tb-GMP was applied for detecting endogenous ALP activity in living cells. HeLa cells (ALP-positive) and HUVEC cells (ALP-negative) were used for evaluating the nanoprobe’s cell imaging capability, in which they have been reported to express ALP at different levels [18, 49]. The imaging for ALP in the HeLa cells and HUVEC cells was shown in Fig. 9 and Fig. S10, respectively. Both HeLa cells and HUVEC cells in the absence of CyOH@Tb-GMP nanoprobe show no fluorescence. After the incubation with CyOH@Tb-GMP nanoprobe, HeLa cells show strong red fluorescence, indicating a high expression level of ALP inside HeLa cells. In contrast, HUVEC cells display relatively weak red fluorescence after incubation with CyOH@Tb-GMP nanoprobe, suggesting that ALP is expressed at low levels in HUVEC cells. In addition, inhibition experiments show that no noticeable fluorescence signal is observed in both cells after the incubation of inhibitor Na3VO4. This indicates that ALP activity can be efficiently inhibited by treatment of Na3VO4. Taken together, 16
these results indicate that this nanoprobe can function well to monitor endogenous ALP activity in living cells. 3.9 Fluorescence Imaging in Tissues To further indicate the superiority of the CyOH@Tb-GMP nanoprobe, the imaging of rat liver tissue was carried out (Fig. 10). The tissue slices not incubated with CyOH@Tb-GMP nanoprobe exhibit no fluorescence (Fig. 10a), while the tissue slices incubated with CyOH@Tb-GMP nanoprobe show significant fluorescence (Fig. 10b). Meanwhile, only negligible fluorescence is observed from the tissues pretreated with Na3VO4, and then treated with CyOH@Tb-GMP nanoprobe (Fig. 10c). The fluorescence signal of tissue slice with CyOH@Tb-GMP nanoprobe at different tissue depths was collected in the Z-scan mode (Fig. 10d). The CyOH@Tb-GMP nanoprobe possesses satisfactory tissue imaging capability at the depths of 40-120 μm. All these results illuminate that CyOH@Tb-GMP nanoprobe has excellent tissue penetrating and staining ability. 3.10 Visualizing ALP in vivo The absorption and emission of the nanoprobe are in the NIR region, which renders it highly favorable for fluorescence imaging of endogenous ALP in vivo. Thus, the imaging application of CyOH@Tb-GMP nanoprobe in mice was explored. Kunming (KM) mice were selected as our model and divided into three groups. The first group of mice was untreated as a control group, no fluorescence is observed (Fig. 11a, quantitation in Fig. 11d). The second group of mice was given an
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intraperitoneal injection of CyOH@Tb-GMP nanoprobe, strong fluorescence signal is noticed (Fig. 11b, quantitation in Fig. 11d), indicating a high expression level of ALP. The third group of mice was injected with Na3VO4 and then with CyOH@Tb-GMP nanoprobe, only negligible fluorescence is observed (Fig. 11c, quantitation in Fig. 11d). With the above results taken together, it is revealed that CyOH@Tb-GMP nanoprobe is suitable for imaging endogenous ALP in vivo. 4. Conclusion In summary, a kind of near-infrared (NIR) fluorescence nanoprobe based on CyOH@Tb-GMP nanoprobe has been reported for the detection of endogenous alkaline phosphatase (ALP) activity. The nanoprobe exhibits high sensitivity for ALP activity with a 7.5-fold fluorescence enhancement and better photostability than ICG as well as CyOH. Most importantly, the NIR nanoprobe is employed for monitoring endogenous ALP activity in various biological samples such as cell, tissue and mice successfully. On the basis of these results, it is greatly anticipated that our strategy will be a powerful tool for designing a wide range of NIR fluorescence nanoprobe. Acknowledgments This work was supported by the National Natural Science Foundation of China (21775133, 21505113), Hunan Provincial Natural Science Foundation (2015JJ6104), China Postdoctoral Science Foundation funded project (2014M552140, 2015T80876), State Key Laboratory of Chemo/Biosensing and Chemometrics Foundation (2015007), Scientific Research Fund of Hunan Provincial Education Department (15K125, 16B252), Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization, Hunan Provincial Innovation Foundation For Postgraduate (CX2017B333).
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References [1] L. Bjelakovic, G. Kocic, T. Cvetkovic, D. Stojanovic, S. Najman, Z. Pop-Trajkovic, B. Bjelakovic, Alkaline phosphatase activity in human milk during the first month of lactation, Acta Fac. Med. Naiss. 26 (2009) 43-47. [2] J. E. Coleman, Structure and mechanism of alkaline phosphatase, Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 441-483. [3] H. N. Fernley, 18 Mammalian alkaline phosphatases, Enzyme 4 (1971) 417-447. [4] C. M. Li, S. J. Zhen, J. Wang, Y. F. Li, C. Z. Huang, A gold nanoparticles-based colorimetric assay for alkaline phosphatase detection with tunable dynamic range, Biosens. Bioelectron. 43 (2013) 366-371. [5] K. Ooi, K. Shiraki, Y. Morishita, T. Nobori, High‐molecular intestinal alkaline phosphatase in chronic liver diseases, J. Clin. Lab. Anal. 21 (2007) 133-139. [6] U. Sharma, D. Pal, R. Prasad, Alkaline phosphatase: an overview, Indian J. Clin. Biochem. 29 (2014) 269-278. [7] K. Ino, Y. Kanno, T. Arai, K. Y. Inoue, Y. Takahashi, H. Shiku, Novel electrochemical methodology for activity estimation of alkaline phosphatase based on solubility difference, Matsue T. Anal. Chem. 84 (2012) 7593-7598. [8] S. Goggins, C. Naz, B. J. Marsh, C. G. Frost, Ratiometric electrochemical detection of alkaline phosphatase, Chem. Commun. 51 (2015) 561-564.
19
[9] H. Jiao, J. Chen, W. Li, F. Wang, H. Zhou, Y. Li, C. Yu, Nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation: a new strategy for colorimetric sensing of alkaline phosphatase activity and inhibitor screening, ACS. Appl. Mater. Interfaces 6 (2014) 1979-1985. [10] T. Hasegawa, M. Sugita, K. Takatani, H. Matsuura, T. Umemura, H. Haraguchi, Assay of alkaline phosphatase in salmon egg cell cytoplasm with fluorescence detection of enzymatic activity and zinc detection by ICP-MS in relation to metallomics research, Bull. Chem. Soc. Jpn. 79 (2006) 1211-1214. [11] A. Ingram, B. D. Moore, D. Graham, Simultaneous detection of alkaline phosphatase and β-galactosidase activity using SERRS. Bioorg, Bioorg. Med. Chem. Lett. 19 (2009) 1569-1571. [12] Z. G. Yang, J. F. Cao, Y. X. He, J. H. Yang, T. Kim, X. J. Peng, J. S. Kim, Macro/micro-environment-sensitive chemosensing and biological imaging, Chem. Soc. Rev. 43 (2014) 4563-4601. [13] X. Sun, Q. Xu, G. Kim, S. E. Flower, J. P. Lowe, J. Yoon, T. D. James, A watersoluble boronate-based fluorescent probe for the selective detection of peroxynitrite and imaging in living cells, Chem. Sci. 5 (2014) 3368-3373. [14] J. Xu, J. Pan, Y. Zhang, J. Liu, L. Zeng, X. Liu, Ultrasensitive near-infrared fluorescence-enhanced probe for discriminative detection of GSH and Cys from different emission channels, Sens. Actuators B 238 (2017) 58-65.
20
[15] D. Lu, L. Zhou, R. Wang, X. B. Zhang, L. He, J. Zhang, W. Tan, A two-photon fluorescent probe for endogenous superoxide anion radical detection and imaging in living cells and tissues, Sens. Actuators B 250 (2017) 259-266. [16] S. Li, M. Dong, R. Li, L. Zhang, Y. Qiao, Y. Jiang, H. Wang, A fluorometric microarray with ZnO substrate-enhanced fluorescence and suppressed “coffee-ring” effects for fluorescence immunoassays, Nanoscale 7 (2015) 18453-18458. [17] F. Y. Cao, Y. Long, S. B. Wang, B. Li, J. X. Fan, X. Zeng, X. Z. Zhang, Fluorescence light-up AIE probe for monitoring cellular alkaline phosphatase activity and detecting osteogenic differentiation, J. Mater. Chem. B 4 (2016) 4534-4541. [18] Y. Tan, L. Zhang, K. H. Man, R. Peltier, G. Chen, H. Zhang, Y. Hu, Reaction-based off-on near-infrared fluorescent probe for imaging alkaline phosphatase activity in living cells and mice, ACS Appl. Mater. Interfaces 9 (2017) 6796-6803. [19] H. Zhang, P. Xiao, Y. T. Wong, W. Shen, M. Chhabra, R. Peltier, Y. Xie, Construction of an alkaline phosphatase-specific two-photon probe and its imaging application in living cells and tissues, Biomaterials 140 (2017) 220-229. [20] J. Park, A. Helal, H. S. Kim, Y. Kim, Fluorogenic assay of alkaline phosphatase activity based on the modulation of excited-state intramolecular proton transfer, Bioorg. Med. Chem. Lett. 22 (2012) 5541–5544. [21] Z. G. Song, R. T. K. Kwok, E. G. Zhao, Z. K. He, Y. N. Hong, J. W. Y. Lam, B. Liu, B. Z. Tang, A ratiometric fluorescent probe based on ESIPT and AIE processes for alkaline
21
phosphatase activity assay and visualization in living cells, ACS Appl. Mater. Interfaces 6 (2014) 17245-17254. [22] S. J. Li, C. Y. Li, Y. F. Li, J. J. Fei, P. Wu, B. Yang, J. Ou-Yang, S. X. Nie, A Facile and Sensitive Near-Infrared Fluorescence Probe for the Detection of Endogenous Alkaline Phosphatase Activity in Vivo, Anal. Chem. 89 (2017) 6854-6860. [23] Y. Liu, K. S. Schanze, Conjugated polyelectrolyte-based real-time fluorescence assay for alkaline phosphatase with pyrophosphate as substrate, Anal. Chem. 80 (2008) 8605-8612. [24] Y. Li, Y. Li, Z. Liu, X. Su, Sensitive fluorometric detection of alkaline phosphatase using a water-soluble conjugated polymer, RSC Advances 4 (2014) 42825-42830. [25] G. Li, H. Fu, X. Chen, P. Gong, G. Chen, L. Xia, Y. Wu, Facile and sensitive fluorescence sensing of alkaline phosphatase activity with photoluminescent carbon dots based on inner filter effect, Anal. Chem. 88 (2016) 2720-2726. [26] L. Jia, J. P. Xu, D. Li, S. P. Pang, Y. Fang, Z. G. Song, J. Ji, Fluorescence detection of alkaline phosphatase activity with β-cyclodextrin-modified quantum dots, Chem. Commun. 46 (2010) 7166-7168. [27] S. Y. Liu, S. Pang, W. D. Na, X. G. Su, Near-infrared fluorescence probe for the determination of alkaline phosphatase, Biosens. Bioelectron. 55 (2014) 249-254. [28] R. Freeman, T. Finder, R. Gill, I. Willner, Probing protein kinase (CK2) and alkaline phosphatase with CdSe/ZnS quantum dots, Nano Lett. 10 (2010) 2192-2196.
22
[29] J. OakaKeem, B. HyunaChung, Smart nanoprobes for the detection of alkaline phosphatase activity during osteoblast differentiation, Chem. Commun. 51 (2015) 3270-3272. [30] X. L. Hu, X. M. Wu, X. Fang, Z. J. Li, G. L. Wang, Switchable fluorescence of gold nanoclusters for probing the activity of alkaline phosphatase and its application in immunoassay, Biosens. Bioelectron. 77 (2016) 666-672. [31] C. Aime, R. Nishiyabu, R. Gondo, N. Kimizuka, Switching on luminescence in nucleotide/lanthanide coordination nanoparticles via synergistic interactions with a cofactor ligand, N. Chem.-Eur. J. 16 (2010) 3604-3607. [32] R. Nishiyabu, N. Hashimoto, T. Cho, K. Watanabe, T. Yasunaga, A. Endo, Y. Katayama, Nanoparticles of adaptive supramolecular networks self-assembled from nucleotides and lanthanide ions, J. Am. Chem. Soc. 131 (2009) 2151-2158. [33] Q. An, J. Liu, M. Yu, J. Wan, D. Li, C. Wang, J. Guo, Multifunctional magnetic Gd3+-based coordination polymer nanoparticles: combination of magnetic resonance and multispectral optoacoustic detections for tumor-targeted imaging in vivo, Small 11 (2015) 5675-5686. [34] H. Tan, B. Liu, Y. Chen, Lanthanide coordination polymer nanoparticles for sensing of mercury (II) by photoinduced electron transfer, ACS nano 6 (2012) 1050510511. [35] H. Tan, Y. Chen, Ag+-enhanced fluorescence of lanthanide/nucleotide coordination polymers and Ag+ sensing, Chem. Commun. 47 (2011) 12373-12375. 23
[36] N. Lin, J. Li, Z. Lu, L. Bian, L. Zheng, Q. Cao, Z. Ding, A turn-on coordination nanoparticle-based fluorescent probe for phosphate in human serum, Nanoscale 7 (2015) 4971-4977. [37] B. Chen, L. Wang, F. Zapata, G. Qian, E. B. Lobkovsky, A luminescent microporous metal−organic framework for the recognition and sensing of anions, J. Am. Chem. Soc. 130 (2008) 6718-6719. [38] Y. Huang, B. Liu, Q. Shen, X. Zhu, Y. Hao, Z. Chang, M. Xu, Lanthanide coordination polymer probe for time-gated luminescence sensing of pH in undiluted human serum, Talanta 164 (2017) 427-431. [39] H. H. Zeng, W. B. Qiu, L. Zhang, R. P. Liang, J. D. Qiu, Lanthanide coordination polymer nanoparticles as an excellent artificial peroxidase for hydrogen peroxide detection, Anal. Chem. 88 (2016) 6342-6348. [40] L. Zhang, J. Zhao, M. Duan, H. Zhang, J. Jiang, R. Yu, Inhibition of dsDNAtemplated copper nanoparticles by pyrophosphate as a label-free fluorescent strategy for alkaline phosphatase assay, Anal. Chem. 85 (2013) 3797-3801. [41] J. Deng, P. Yu, Y. Wang, L. Mao, Real-time ratiometric fluorescent assay for alkaline phosphatase activity with stimulus responsive infinite coordination polymer nanoparticles, Anal. Chem. 87 (2015) 3080-3086. [42] X. Zhang, J. Deng, Y. Xue, G. Shi, T. Zhou, Stimulus response of Au-NPs@GMP-Tb core–shell nanoparticles: toward colorimetric and fluorescent dual-mode sensing of
24
alkaline phosphatase activity in algal blooms of a freshwater lake, Environ. Sci. Technol. 50 (2015) 847-855. [43] F. Yu, M. Gao, M. Li, L. Chen, A dual response near-infrared fluorescent probe for hydrogen polysulfides and superoxide anion detection in cells and in vivo, Biomaterials 63 (2015) 93-101. [44] Y. J. Gong, X. B. Zhang, G. J. Mao, L. Su, H. M. Meng, W. Tan, G. Zhang, A unique approach toward near-infrared fluorescent probes for bioimaging with remarkably enhanced contrast, Chem. Sci. 7 (2016) 2275-2285. [45] H. Zhang, S. Xue, G. Feng, A colorimetric and near-infrared fluorescent turn-on probe for rapid detection of sulfite, Sens. Actuators B 231 (2016) 752-758. [46] Y. Wei, D. Cheng, T. Ren, Y. Li, Z. Zeng, L. Yuan, Design of NIR chromenyliumcyanine fluorophore library for “switch-on” and ratiometric detection of bio-active species in vivo, Anal. Chem. 88 (2016) 1842-1849. [47] I. R. Gibbons, M. P. Cosson, J. A. Evans, B. H. Gibbons, B. Houck, K. H. Martinson, W. J. Tang, Potent inhibition of dynein adenosinetriphosphatase and of the motility of cilia and sperm flagella by vanadate, Proc. Natl. Acad. Sci. 75 (1978) 2220-2224. [48] N. Malashikhina, G. Garai-Ibabe, V. Pavlov, Unconventional application of conventional enzymatic substrate: first fluorogenic immunoassay based on enzymatic formation of quantum dots, Anal. Chem. 85 (2013) 6866-6870.
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[49] C. Fan, S. Luo, H. Qi, A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited-state intramolecular proton transfer, Luminescence 31 (2016) 423-427.
Author Biographies Juan Ou-Yang received her BS degree in 2015 at College of Chemical Engineering, Xiangtan University. Now she is working toward a MS degree in Analytical Chemistry at Xiangtan University. Her research fields focus on the design and synthesis of fluorescent materials and their applications as fluorescent probe. Chun-Yan Li received her PhD degree in 2008 at College of Chemistry & Chemical Engineering, Hunan University. Now she is an associate professor of Xiangtan University. Her research fields focus on the design and synthesis of fluorescent materials and their applications as fluorescent probe. Yong-Fei Li received his PhD degree in 2010 at College of Chemistry & Chemical Engineering, Hunan University. Now he is an instructor of Xiangtan University. His research fields focus on the synthesis of fluorescent materials. Bin Yang received his PhD degree in 2014 at College of Chemistry & Chemical Engineering, Hunan University. Now he is an instructor of Xiangtan University. His research fields focus on the synthesis of fluorescent materials and their applications as fluorescent probe. Song-Jiao Li received her BS degree in 2015 at College of Chemistry, Hengyang Normal University. Now she is working toward a MS degree in Analytical Chemistry at Xiangtan University. Her research fields focus on the design and synthesis of fluorescent materials and their applications as fluorescent probe.
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Figure captions. Fig. 1 TEM image of (a) Tb-GMP nanoparticles and (b) CyOH@Tb-GMP nanoparticles. (c) Absorption and (d) fluorescence spectra of CyOH, Tb-GMP nanoparticles and CyOH@Tb-GMP nanoparticles in Tris-HCl buffer solution (50 mM, pH 8.0). Fig. 2 FT-IR spectra for GMP, Tb-GMP nanoparticles, CyOH@Tb-GMP nanoparticles and CyOH. Fig. 3 Fluorescence response of CyP (10 µM) to ALP (2 U/mL), GOX (10 mg/mL), GDH (2 U/mL), GAL (2 U/mL), AChE (2 U/mL), trypsin (10 mg/mL), thrombin (2 U/mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded within 20 min. λex / λem = 690 / 738 nm. Fig. 4 Fluorescence spectra of CyOH@Tb-GMP nanoprobe (1 mg / mL) upon the addition of ALP (2.5 U / mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded every 2.5 min. λex = 685 nm. Inset: the plot of fluorescence intensity versus time. Fig. 5 Fluorescence spectra of CyOH@Tb-GMP nanoprobe (1 mg /mL) in the presence of different activities of ALP (0, 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5 U / mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex = 685 nm. Insert: the plot of fluorescence intensity versus ALP activity. Fig. 6 Fluorescence response of CyOH@Tb-GMP nanoprobe (1 mg / mL) to ALP (2.5 U / mL), GDH (10 mg/mL), GAL (2.5 U / mL), GOX (10 mg/mL), EXO (2.5 U / mL), AChE
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(2.5 U / mL), Trypsin (10 mg/mL) or Thrombin (2.5 U / mL) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex / λem = 685 / 718 nm. Fig. 7 Fluorescence spectra of CyOH@Tb-GMP nanoprobe (1 mg /mL) in the presence of ALP (2.5 U / mL) at different Na3VO4 concentrations (0, 50, 100, 200, 300, 400, 500, 600 μM) in Tris-HCl buffer solution (50 mM, pH 8.0) at 37 °C recorded at 20 min. λex = 685 nm. Insert: the plot of the inhibition efficiency versus Na3VO4 concentrations. Fig. 8 Time-dependence fluorescence intensity of ICG (treated with 2.5 U / mL ALP for 20 min, monitored at 812 nm), CyOH (treated with 2.5 U / mL ALP for 20 min, monitored at 718 nm), Cy5.5 (treated with 2.5 U / mL ALP for 20 min, monitored at 707 nm) and CyOH@Tb-GMP nanoprobe (treated with 2.5 U / mL ALP for 20 min, monitored at 718 nm) under constant illumination for 0-600 s. Fig. 9 Fluorescence images of HeLa cells. From left to right: control, cells treated with CyOH@Tb-GMP nanoprobe only, cells treated with Na3VO4 and then CyOH@Tb-GMP nanoprobe. Fig. 10 Fluorescence images of tissues. (a) Control. (b) Tissue slice was incubated with CyOH@Tb-GMP nanoprobe. (c) Tissue slice was pre-treated with Na3VO4, and then treated with CyOH@Tb-GMP nanoprobe. (d) The confocal z-scan imaging sections at different depths for 0, 20, 40, 60, 80, 100, 120, 140, 160 μm. Fig. 11 Fluorescence images in vivo. (a) Control. (b) a mouse given an injection of CyOH@Tb-GMP nanoprobe. (c) a mouse injected with Na3VO4 and then with 28
CyOH@Tb-GMP nanoprobe. (d) quantification of fluorescence emission intensity from the selected circle region of (a–c) was averaged and plotted as a ratio to blank.
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(b)
(a)
100 nm
.6 Tb-GMP CyOH CyOH@GMP
.5
Absorbance
100 nm
(d)
.4
Fluorescence Intensity
(c)
.3 .2 .1 0.0
Tb-GMP CyOH CyOH@Tb-GMP
600
400
200
0 720
560 580 600 620 640 660 680 700 720 740
ν (C=O)
GMP
740
760
780
Wavelength (nm)
Wavelength (nm)
νas (PO3) νs (PO3)
ν (N-C)
Tb-GMP
CyOH@Tb-GMP
CyOH
.4
Absorbance
(b)
.5 CyOH@GMP CyOH@GMP+ALP
Fluorescence Intensity
(a)
.3 .2 .1 0.0 560 580 600 620 640 660 680 700 720 740
Wavelength (nm)
600
CyOH@GMP CyOH@GMP+ALP
500 400 300 200 100 0 700
720
740
760
Wavelength (nm)
30
780
25 min
Fluorescence Intensity
500
400
0
Fluorescence Intensity
600
600
500 400 300 200 100
300
0
5
10
15
20
25
Time (min) 200
100
720
740
760
780
Wavelength (nm)
Fluorescence Intensity
600
600 ALP
Fluorescence Intensity
500
400
500 400 300 200 100 0.0
.5
300
1.0
1.5
2.0
ALP (U / mL)
200
100
720
740
Wavelength (nm)
31
760
780
2.5
3.0
8
F / F0
6
4
Th rom bin
Try ps in
AC
hE
O EX
GO X
GA L
GD H
P AL
Bla nk
2
Fluorescence Intensity
600 Na3VO4
500
400
Inhibition Efficiency (%)
100
300
80 60 40 20 0 0
100
200
300
Na3VO4 (
200
100
720
740
760
Wavelength (nm)
32
780
400
)
500
600
1.2
ICG+ALP CyOH+ALP Cy 5.5+ALP CyOH@Tb-GMP+ALP
1.0
.6
.4
.2
0.0 0
100
200
300
400
500
Time (s)
CyOH@Tb-GMP
Fluorescence
Bright-filed
Control
Overlay
I / I0
.8
33
Inhibitor + CyOH@Tb-GMP
600
(a)
(b)
(c)
60
80 100 120 140 160 μm
(d)
Imaging depth 0
(a)
(b)
20
40
(c)
34
(d)
Scheme Captions. Scheme 1. Schematic illustration of the formation of CyOH@Tb-GMP nanoparticles and the response mechanism of CyOH@Tb-GMP nanoprobe to ALP.
35