Real-time Ratiometric Fluorescent Assay for Alkaline Phosphatase

Jan 29, 2015 - In aqueous media containing no ALP, the coumarin@Tb-GMP nanoparticles are water-stable and emit green light. Upon the presence of ALP, ...
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Real-time Ratiometric Fluorescent Assay for Alkaline Phosphatase Activity with Stimulus Responsive Infinite Coordination Polymer Nanoparticles Jingjing Deng, Ping Yu, Yuexiang Wang, and Lanqun Mao* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences (CAS), Beijing 100190, China S Supporting Information *

ABSTRACT: This study demonstrates a novel ratiometric fluorescent method for real-time alkaline phosphatase (ALP) activity assay with stimulus responsive infinite coordination polymer (ICP) nanoparticles as the probe. The ICP nanoparticles used in this study are composed of two components; one is the supramolecular ICP network formed with guanine monophosphate (GMP) as the ligand and Tb3+ as the central metal ion, and the other is a fluorescent dye, i.e., 7-amino-4-methyl coumarin (coumarin) encapsulated into the ICP network. Upon being excited at 315 nm, the ICP network itself emits green fluorescence at 552 nm. Coumarin dye encapsulated in the ICP network emits weak fluorescence at 450 nm upon excitation at the same wavelength (315 nm), and this fluorescence emission becomes strong when the encapsulated dye is released from the network into the solution phase. Hence, we develop a ratiometric fluorescent assay based on the ALP-induced destruction of the supramolecular ICP network and the release of coumarin. This mechanism can be used for real-time ratiometric fluorescent monitoring of ALP activity by continuously measuring the ratio of fluorescent intensity at the wavelength of 552 nm (F552) to that at 450 nm (F450) (F552/F450) in the time-dependent fluorescent spectra of the coumarin@Tb-GMP suspension containing ALP with different activities. Under the experimental conditions employed here, the F552/F450 value is linear with the ALP activity within a range from 0.025 U/mL to 0.2 U/mL. The detection limit is down to 0.010 U/mL (S/N = 3). Moreover, the assay developed here is employed for ALP inhibitor evaluation. This study offers a simple yet sensitive method for real-time ALP activity assay. single change (increase or decrease) in fluorescence intensity could be easily affected by various factors including fluctuations in light source intensity and/or environmental effects, such as pH, polarity of the media, and photobleaching27−31 and are therefore subjected to some limitations for practical applications.32−36 This study demonstrates a ratiometric fluorescent method for real-time ALP activity assay, which could overcome the problems inherent in the existing methods mentioned above. This is because the ratiometric method uses the ratio of intensity readings at two different wavelengths, and thus the environmental fluctuations could be largely canceled out, which is more suitable for the practical applications. On the other hand, high sensitivity could be achieved by the amplified signal readout through simultaneous readings of two channels.37−39 The mechanism underlying our ratiometric assay is essentially based on the uses of stimulus responsive infinite coordination polymer (ICP) nanoparticles constructed with Tb3+ as the metal ion and GMP as the bridging ligand, in which fluorescent dye (i.e., 7-amino-4-methyl coumarin) is used as a guest

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s an essential enzyme in phosphate metabolism, alkaline phosphatase (ALP, EC 3.1.3.1) nonspecifically catalyzes the hydrolysis of phosphoryl esters in alkaline media and is widely distributed in mammalian body fluids and tissues.1−3 In biological systems, the expression level of ALP plays important roles in various biological processes. For instance, an abnormal level of serum ALP can be used as an important indicator for several diseases, such as bone diseases, liver dysfunction, breast and prostatic cancer, and diabetes.4−7 In this context, developing a method for sensitive ALP assay is very essential to understanding the molecular mechanism of phosphate hydrolysis and thereby provides the clinical diagnoses of the related diseases. So far, several methods have been reported for ALP assay mostly based on the ability of ALP to remove phosphate groups from a wide variety of substrates to obtain the signal readout through various mechanisms.8−15 Among these methods, fluorescent assays have been used widely because of their simplicity, high sensitivity, and real-time detection capability.16−24 Most of these fluorescent assay methods are based on measuring the absolute change (e.g., “turn-on” or “turn-off”) of the fluorescence intensity triggered by ALP-catalyzed detachment of the phosphate group, some of which even achieved a high sensitivity.24−26 However, these methods relying on a © XXXX American Chemical Society

Received: December 22, 2014 Accepted: January 29, 2015

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DOI: 10.1021/ac504773n Anal. Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Illustration of Ratiometric ALP Activity Assay with Stimulus Responsive Coumarin@Tb-GMP as Fluorescent Probe

6H2O (10 mM, 1 mL) with a HEPES buffer (0.1 M, pH 7.4, 1 mL) containing GMP (10 mM) at room temperature to form a white precipitate. The resulting precipitate was then centrifuged and washed with water several times. Encapsulation of 7-amino4-methyl coumarin (coumarin) into the Tb-GMP network to form coumarin@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 both GMP (10 mM) and 7-amino-4-methyl coumarin (0.0286 mM) to form a precipitate. The resulting precipitate was then centrifuged and washed with water several times until no fluorescence of 7-amino-4-methyl coumarin was observed from the supernatant of the coumarin@Tb-GMP (0.784 mg) suspension (Figure S1). Ratiometric ALP Activity Assay. A ratiometric fluorescent ALP activity assay was performed under the following procedures. A total of 2 μL of ALP with activities ranging from 0.025 to 2.0 U was added into the mixture (1 mL, pH 8.0) containing 50 mM Tris-HCl, 1 mM MgCl2, and 0.784 mg coumarin@Tb-GMP. Immediately after the addition of ALP, the fluorescent spectrum of each of the mixtures was consecutively recorded every 1 min at 37 °C, and after 20 min, the mixtures were used for photographing with a digital camera (Canon IXUS 951S, Japan) under a 365 nm UV lamp. Fluorescent spectra were recorded on a HITACHI F-4600 fluorescence spectrophotometer (Hitachi, Japan). For ratiometric detection, the excitation wavelength of the courmarin@ Tb-GMP nanoparticles was optimized to be 315 nm (Figure S2), and the ratio of fluorescence intensity at 552 nm (F552) for the Tb-GMP network to that at 450 nm for coumarin (F450) was employed as the signal readout for the ALP activity. ALP Inhibitor Efficiency Evaluation. To investigate the application of the ratiometric fluorescent assay for evaluating the inhibition efficiency of Na3VO4 toward ALP, we initially added 2 μL of aqueous solution of Na3VO4 with different concentrations (0 μM, 100 μM, 200 μM, 500 μM, 1 mM, 2 mM, 5 mM, 10 mM, and 20 mM) into the aqueous solution of ALP (2 μL, 0.2 U) in a 37 °C water bath. After 20 min, the Na3VO4-treated ALP was added into the mixture (1 mL, pH 8.0) containing 50 mM Tris-HCl, 1 mM MgCl2, and 0.784 mg coumarin@Tb-GMP. The resulting mixtures were allowed to stand by for 20 min and then were used for photographing and fluorescent measurements.

molecule to be encapsulated into the Tb-GMP ICP network to form the coumarin@Tb-GMP nanoparticles, as shown in Scheme 1. In aqueous media containing no ALP, the coumarin@Tb-GMP nanoparticles are water-stable and emit green light. Upon the presence of ALP, the phosphate group in the GMP ligand is cleaved, resulting in the destruction of the Tb-GMP network and the release of encapsulated coumarin dye into solution. This process eventually results in the decrease in the fluorescence intensity emitted from the TbGMP network itself and the increase in the fluorescence intensity emitted from coumarin, which provides a straightforward basis for the ratiometric assay for ALP. To the best of our knowledge, this is the first study on the real-time ratiometric ALP activity assay with stimulus responsive infinite coordination polymer nanoparticles. With the help of a fluorescent lamp (365 nm), this method can even possess on-site visible features, which could be further developed for simple clinical applications. This study not only provides a new mechanism for the real-time assay of ALP activity but also paves an avenue to the development of ratiometric methods based on rational design of the structures of infinite coordination polymers for practical applications.

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EXPERIMENTAL SECTION Chemicals and Solutions. Guanine monophosphate (GMP) was purchased from Sigma-Aldrich. Terbium nitrate hexahydrate (Tb(NO 3 ) 3 ·6H 2 O), sodium orthovanadate (Na3VO4), and 7-amino-4-methyl coumarin were all purchased from Aladdin (Shanghai, China). ALP (CIAP, Calf intestine alkaline phosphatase) was purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China). All the chemicals were of at least analytical grade and used without further purification. The reaction buffer for ALP was 50 mM Tris-HCl (pH 8.0) containing 1 mM MgCl2. Note, one unit of ALP was defined as the amount of enzyme that catalyzes the hydrolysis of 1 μmol of 4-nitrophenyl phosphate per minute at pH 9.8 and 37 °C. Milli-Q water (Millipore, Bedford, MA) was used in the study. Unless stated otherwise, the experiments were carried out at room temperature. Synthesis of Tb-GMP Network and Coumarin@TbGMP Nanoparticles. Synthesis of the Tb-GMP ICP network was performed as reported previously.40 Briefly, Tb-GMP ICP was synthesized by mixing an aqueous solution of Tb(NO3)3· B

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Figure 1. (A) SEM image of the coumarin@Tb-GMP ICP nanoparticles. Inset, photograph of the dispersion of coumarin@Tb-GMP ICP nanoparticles (0.784 mg) in 1 mL of Tris-HCl (50 mM, pH 8.0) excited by a 365 nm UV lamp. (B) SEM image of Tb-GMP ICP nanoparticles. Inset, photograph of the dispersion of Tb-GMP ICP nanoparticles (0.784 mg) in 1 mL of Tris-HCl (50 mM, pH 8.0) excited by a 365 nm UV lamp. (C) Fluorescence emission spectra of Tb-GMP ICP nanoparticles (0.784 mg; black curve), coumarin@Tb-GMP ICP nanoparticles (0.784 mg; blue curve), and coumarin (red curve) in 1 mL of Tris-HCl (50 mM, pH 8.0). λex = 315 nm.

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RESULTS AND DISCUSSION Mechanistic Investigation into Ratiometric Fluorescent ALP Assay. To prepare the fluorescence probe for the ratiometric assay of ALP activity, one of the commonly used fluorescent dyes, 7-amino-4-methyl coumarin (coumarin), was carefully selected (Figure S3) as the guest molecule to form coumarin@Tb-GMP nanoparticles by taking advantage of the adaptive inclusion properties of the supramolecular network of Tb-GMP ICP, as demonstrated previously.40,41 The morphology of as-formed Tb-GMP ICP nanoparticles and the coumarin@Tb-GMP nanoparticles was then analyzed by scanning electron microscopy (SEM), as shown in Figure 1A,B. The coumarin@Tb-GMP nanoparticles were colloidal spheres with diameters ranging from 40 to 50 nm (Figure 1A), which was almost in the same range as that of the Tb-GMP block network (Figure 1B). The encapsulation of coumarin does not largely change the structure or the morphology of the Tb-GMP network. As displayed in Figure 1C (black curve), the Tb-GMP network itself emits green luminescence at 498, 552, 594, and 628 nm owing to the energy transfer from the guanine base to the emissive D4 state of the Tb3+ ion, which was promoted by the coordination of O6 and N7 moieties to the Tb3+ ion during the self-assembling process.40,42−45 To verify the encapsulation of coumarin into the Tb-GMP network, we measured the broadband fluorescence spectra of the as-formed coumarin@Tb-GMP nanoparticles upon UV excitation and found that the fluorescence emission spectrum of the coumarin@Tb-GMP dispersion (blue curve) shows the contributions from the emission from both coumarin dye (450 nm, red curve) and the Tb-GMP network (498, 552, 594, and 628 nm, black curve; Figure 1C). Accordingly, the coumarin@Tb-GMP nanoparticles emit greenish-blue fluorescence (Figure 1A, inset), rather than green fluorescence as emitted by the pure Tb-GMP network (Figure 1B, inset), when being excited by a 365 nm UV lamp. These results demonstrate the successful encapsulation of coumarin into the Tb-GMP network to form the coumarin@Tb-GMP nanoparticles that were used as the fluorescent probe for the ratiometric ALP activity assay, as demonstrated below. As displayed in the Figure 2 inset, the addition of ALP into a Tris-HCl buffer dispersion containing coumarin@Tb-GMP nanoparticles leads to dissociation of the white precipitate (vials 1 and 2), accompanied by a fluorescent color change of the

Figure 2. Fluorescent spectra of the coumarin@Tb-GMP ICP nanoparticles (0.784 mg) dispersed in 1 mL of Tris-HCl (50 mM, pH 8.0) before (black curve) and after (red curve) the addition of ALP (2 μL, 2.0 U; λex = 315 nm). Inset: Photographs of the coumarin@TbGMP ICP nanoparticles (0.784 mg) dispersed in 1 mL of Tris-HCl (50 mM, pH 8.0) before (vial 1) and after (vial 2) the addition of ALP (2 μL, 2.0 U). Photographs of vial 1 (vial 3) and vial 2 (vial 4) when the dispersions were excited by a 365 nm UV lamp.

dispersion from greenish-blue (vial 3) to blue (vial 4). Meanwhile, such a procedure also leads to a change in the fluorescence spectrum of the coumarin@Tb-GMP nanoparticles; the fluorescent intensity at 450 nm, which was ascribed to the emission of coumarin dye, increases (Figure 2, red arrow). However, those at 498, 552, 594, and 628 nm, which were ascribed to the emission of the Tb-GMP ICP nanoparticles, decrease (Figure 2, blue arrows). This phenomenon could be due to the ALP-catalyzed hydrolysis of the phosphate ester group to convert GMP into a guanosine base, leading to the destruction of the Tb-GMP network and thereby the release of the guest molecule (i.e., coumarin) into the solution. The ratios of F552 to F450 (i.e., F552/F450) were closely associated with the degree of the destruction of the coumarin@Tb-GMP nanoparticles caused by ALP and, as a consequence, could be used for the ratiometric fluorescence assay of ALP activity, as demonstrated below. Ratiometric ALP Activity Assay. Figure 3A depicts the typical time-dependent fluorescent spectra of the Tris-HCl buffer containing coumarin@Tb-GMP ICP nanoparticles after the addition of ALP into the buffer. Upon the addition of ALP, the fluorescent spectrum of the buffer dispersion of the C

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Figure 3. (A) Fluorescent spectra of the dispersion prepared by adding ALP (2 μL, 2.0 U) into the dispersion of the coumarin@Tb-GMP ICP nanoparticles (0.784 mg) in 1 mL of Tris-HCl (50 mM, pH 8.0). Fluorescent spectra were recorded every 1 min after the addition of ALP (2 μL, 2.0 U) at 37 °C. Inset: photograph of the dispersion at 20 min after the addition of ALP excited by a 365 UV lamp. (B) Fluorescent spectra of the dispersion containing only coumarin@Tb-GMP ICP nanoparticles (0.784 mg; i.e., without ALP) in 1 mL of Tris-HCl (50 mM, pH 8.0). Fluorescent spectra were also recorded every 1 min at 37 °C. Inset: photograph of the dispersion at 20 min without the addition of ALP excited by a 365 nm UV lamp.

Figure 4. (A) Plots of time-dependent F552/F450 values versus ALP activity (from top to bottom: 0, 0.025, 0.05, 0.1, 0.2, 0.5, 0.75, 1.0, 1.5, and 2.0 U/ mL). (B) Photographs of buffer solutions containing coumarin@Tb-GMP ICP nanoparticles (0.784 mg) at the time point of 20 min after the addition of different activities of ALP (from vial 1 to vial 8:2.0, 1.5, 1.0, 0.75, 0.5, 0.2, 0.1, and 0 U/mL). (C) Plot of initial rate of enzymatic reaction (v0) versus ALP activity. Inset: plot of v0 versus CALP. Error bars indicate standard deviations (n = 3).

from the initial greenish-blue to blue (Figure 3A, inset). In contrast, neither the spectrum nor the fluorescence color of the dispersion of pure coumarin@Tb-GMP ICP nanoparticles changes as a function of time (Figure 3B). These results further suggest that ALP remarkably catalyzes the hydrolysis of GMP, resulting in gradual destruction of the Tb-GMP network nanoparticles. In addition, we found that the decrease of F552 and the increase of F450 become more dramatic when increasing the activity of ALP in the dispersion (Figure S4), demonstrating the validity of the use of coumarin@Tb-GMP ICP

coumarin@Tb-GMP ICP nanoparticles was recorded immediately and every 1 min consecutively at 37 °C. After 20 min, the dispersions were used for photographing under the excitation of a 365 nm UV lamp, as shown in Figure 3A (inset). For comparison, the time-dependent fluorescent spectrum of the Tris-HCl buffer containing only the coumarin@Tb-GMP ICP nanoparticles (i.e., without ALP) was also recorded (Figure 3B). As could be seen from Figure 3A, the addition of ALP clearly results in a decrease in F552 and an increase in F450. Accordingly, the fluorescence color of the dispersion changes D

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Figure 5. (A) Fluorescent spectra of the dispersions prepared by adding pure water (2 μL), GDH (2 μL, 25 mg/mL), thrombin (2 μL, 10 μM), GOX (2 μL, 5 mg/mL), EXO (2 μL, 1.0 U/mL), or ALP (2 μL, 1.0 U/mL) into 1 mL of Tris-HCl (50 mM, pH 8.0) buffer containing coumarin@ Tb-GMP nanoparticles (0.784 mg), respectively. (B) F450/F552 values and photographs (inset) of the dispersions prepared by adding pure water (2 μL, vial 1), GDH (2 μL, 25 mg/mL, vial 2), thrombin (2 μL, 10 μM, vial 3), GOX (2 μL, 5 mg/mL, vial 4), EXO (2 μL, 1.0 U/mL, vial 5), or ALP (2 μL, 1.0 U/mL, vial 6) into 1 mL of Tris-HCl (50 mM, pH 8.0) buffer containing coumarin@Tb-GMP nanoparticles (0.784 mg), respectively.

Figure 6. (A) Fluorescent spectra of the dispersions prepared by adding Na3VO4-treated ALP (0.2 U) into 1 mL of Tris-HCl (50 mM, pH 8.0) buffer containing coumarin@Tb-GMP nanoparticles (0.784 mg). The fluorescent spectra were recorded 20 min after the addition of Na3VO4-treated ALP. Red and blue arrows indicate decreasing Na3VO4 concentrations. (B) Photographs of the above dispersions. The Na3VO4 concentrations were (from left to right) 0 μM (vial 1), 100 μM (vial 2), 200 μM (vial 3), 500 μM (vial 4), 1 mM (vial 5), 2 mM (vial 6), 5 mM (vial 7), 10 mM (vial 8), and 20 mM (vial 9). (C) Kinetic plot of F450/F552 versus the logarithm of Na3VO4 concentration. Error bars indicate standard deviations (n = 3).

higher hydrolysis degrees of GMP. Moreover, the fluorescence color of the buffered dispersion containing coumarin@TbGMP ICP nanoparticles observed at the time point of 20 min gradually changed from greenish-blue to blue, when the ALP activity was increased from 0 to 2.0 U/mL (Figure 4B). These results demonstrate that the dissociation degree of the coumarin@Tb-GMP ICP nanoparticles increases with increasing the activity of ALP in the dispersion through the mechanism shown in Scheme 1. The initial reaction rates

nanoparticles as the probe for the ratiometric fluorescent assay of ALP activity. To estimate the kinetics of the ALP-catalyzed GMP hydrolysis, we investigated the initial rates of F552/F450 change from the fluorescent spectra of the dispersion of the coumarin@Tb-GMP ICP nanoparticles in a Tris-HCl buffer with the presence of different activities of ALP (Figure 3A and Figure S4). As typically displayed in Figure 4A, the F552/F450 values decrease with increasing ALP activity, which represents E

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represents the concentration of an inhibitor that is required for 50% inhibition of an enzyme was used as a parameter to evaluate the inhibitor used in this study. As shown in Figure 6C, a sigmoidal profile was obtained from the plot of the F450/F552 values versus the logarithm of Na3VO4 concentration. The IC50 value of 0.2 U ALP was thus calculated to be 2.082 mM, which was consistent with the values reported previously.15,21 These results essentially demonstrate that the ratiometric fluorescence assay developed in this study with the coumarin@Tb-GMP ICP nanoparticles as the probe could also be used to evaluate the inhibitor efficiency.

(v0) were then obtained by calculating the slopes of the initial parts of the kinetic curves obtained with different ALP activities (Figure 4C). Under the experimental conditions employed here, the v0 values were linear against the ALP activity within the activity range from 0.025 U/mL to 0.2 U/mL (Figure 4C, inset; v0 = 0.0296 +1.2506 CALP/U, R = 0.9688), which indicates that the reaction is kinetically controlled by ALP and thus could be used for the ALP activity assay. This result also demonstrates that the use of stimulus responsive coumarin@ Tb-GMP ICP enables a ratiometric real-time assay for ALP activity within 20 min even at very low ALP activity. More importantly, due to the use of double signal response as the signal readout, the fluorescent method developed here is reliable since the background fluorescence caused by various factors such as variations in excitation intensity or emission collection efficiency could be well avoided. The detection limit of our ratiometric method was calculated to be 0.010 U/mL (signal-to-noise ratio of 3), which was 1−2 orders of magnitude lower than some of the existing ratiometric methods.15,21,37 The quick response and high sensitivity substantially validate our real-time ratiometric fluorescent method for ALP activity assay. To investigate the selectivity of our ratiometric method, control experiments were conducted using glucose dehydrogenase (GDH), thrombin, glucose oxidase (GOX) and exonuclease (EXO). As shown in Figure 5, the addition of pure water (2 μL), GDH (2 μL, 25 mg/mL), thrombin (2 μL, 10 μM), GOX (2 μL, 5 mg/mL), and EXO (2 μL, 1.0 U/mL) into 1 mL of Tris-HCl (50 mM, pH 8.0) buffer containing 0.784 mg of coumarin@Tb-GMP ICP nanoparticles did not lead to a significant change in the fluorescence color or the fluorescence spectra (Figure 5B, vials 1−5). In contrast, upon the addition of ALP (2 μL, 1.0 U/mL), the fluorescence of the coumarin@TbGMP dispersion clearly changed from greenish-blue to blue (vial 1, vial 6), along with a decrease in F552 and an increase in F450 from the spectra (Figure 5A). These results substantially suggest that these enzymes/proteins (except ALP) did not interfere with the ALP assay with the method developed in this study. Application to ALP Inhibitor Efficiency Evaluation. Having demonstrated the validity of our ratiometric fluorescent assay, we next investigated the possibility of applying the assay for evaluating the enzyme inhibitor efficiency. Sodium orthovanadate (Na3VO4), an inhibitor for ALP,46 was used in our proof-of-concept experiments. To demonstrate this validity, ALP (2 μL, 0.2 U) was preincubated with the aqueous solutions of Na3VO4 with different concentrations for 20 min at room temperature. Then, the Na3VO4-treated ALP solutions were added into the dispersion of the coumarin@Tb-GMP ICP nanoparticles in 1 mL of Tris-HCl (50 mM, pH 8.0) buffer, respectively. After incubating for an additional 20 min, the resulting mixtures were subjected to fluorescent measurements. When the activity of ALP was inhibited by Na3VO4, GMP could not be dephosphorylated by ALP any more, and as a consequence, the greenish-blue fluorescence of the coumarin@ Tb-GMP ICP nanoparticle remained unchanged. As displayed in Figure 6A,B, with decreasing the Na3VO4 concentration from 20 mM (vial 9) to 0 μM (vial 1), the fluorescence color of the coumarin@Tb-GMP ICP gradually changed from greenish-blue to blue-green, accompanied by the decrease in F552 and the increase in F450. Note that, under the conditions employed here, Na3VO4 itself does not cause obvious change in the fluorescence of the coumarin@Tb-GMP nanoparticles (Figure S5). The half maximal inhibitory concentration (IC50) which

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CONCLUSIONS In summary, we have for the first time demonstrated a simple and yet effective ratiometric fluorescent method for a real-time ALP activity assay based on stimulus responsive coumarin@TbGMP ICP nanoparticles. The use of double signal response as the signal readout enables the method developed here to be relatively robust, sensitive, and real-time. The excellent analytical properties of this method substantially enable its application in ALP activity assay and ALP inhibitor efficiency evaluation. This study not only offers a real-time ratiometric ALP activity assay method but also opens a new way to the development of ratiometric fluorescent methods based on a stimulus response of novel infinite coordination polymer nanostructures for practical applications.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +86-10-62559373. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is financially supported by the NSF of China (Grant no. 21435007, 91413117, 21321003, 21210007, 91213305 for L. M. and 21475138, 21322503 for P.Y.), the National Basic Research Program of China (973 programs, 2013CB933704), and The Chinese Academy of Sciences.

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DOI: 10.1021/ac504773n Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ac504773n Anal. Chem. XXXX, XXX, XXX−XXX