Ratiometric Fluorescent Probe for Alkaline Phosphatase Based on

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Ratiometric Fluorescent Probe for Alkaline Phosphatase Based on Betaine-Modified Polyethylenimine via Excimer/Monomer Conversion Fangyuan Zheng, Sihua Guo, Fang Zeng,* Jun Li, and Shuizhu Wu* State Key Laboratory of Luminescent Materials and Devices, College of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China S Supporting Information *

ABSTRACT: Alkaline phosphatase (ALP) is an important diagnostic indicator for a number of human diseases since abnormal level of ALP is closely related to a variety of pathological processes; hence, the development of convenient and reliable assay methods for monitoring ALP is of great significance for medical sciences as well as biological diagnostics. Herein, we report the first ratiometric fluorescent sensing system for ALP. This sensing system consists of two components: the betainemodified and positively charged polyethylenimine (PEI) and the negatively charged pyrene derivative containing one ALP-responsive phosphate group (Py-P, an aliphatic phosphate ester). In the absence of ALP, the two-component sensing system shows the excimer’s emission of Py-P, since Py-P molecules complex with the positively charged polyelectrolyte via electrostatic interactions, leading to the formation of pyrene excimers. While in the presence of ALP, the phosphate moieties are cleaved from Py-P molecules due to the enzymatic reaction, thereby destroying the electrostatic interactions; as a result, the system displays the monomer emission of Py-P. This assay system is operable in aqueous media with a very low detection limit of 0.1 U/mL. The system is capable of detecting ALP in such biological fluid as serum, and this strategy may provide a new and effective approach for designing ratiometric sensing systems for detecting other biomolecules. Until now, several elegant fluorescent probes for ALP have been reported.48−52 However, these probes are all based on small molecular compounds and belong to the “turn-on” or “turn-off” fluorescent probes, in which an increase or decrease in a single fluorescence intensity upon the enzyme-catalyzed hydrolysis of a phosphate group is employed as the sensing signal; and this single-fluorescence-intensity sensing signal may be disturbed by various factors such as variations in excitation intensity, emission collection efficiency, and probe concentration. Moreover, for the vast majority of these probes, in order to change the fluorescence of fluorophore, phosphate group was linked directly to the phenolic hydroxyl group to generate phenolic phosphate ester. However, the study by Kawaguchi et al. indicates that the probes based on phenolic phosphate ester are not ALP-specific; these probes are also responsive toward other phosphatases (can be hydrolyzed by other phosphatases such as PTPs and PSPs).53 Thus, it is highly desirable to design an ALP-specific and ratiometric fluorescent probe. On the other hand, it is well-known that polyelectrolytes can easily form aggregates in the presence of substances with opposite charge, due to electrostatic interactions. Hyperbranched polyethylenimine (PEI), which has high cationic charge densities in the polymeric chains, is a polyelectrolyte widely used in biological applications. However, its high

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lkaline phosphatase (ALP), which catalyzes the dephosphorylation process of proteins, nucleic acids, and small molecules, can be found in a variety of tissues (intestine, liver, bone, kidney, and placenta) of nearly all living organisms.1−3 This enzyme has been extensively used as a biomarker in enzyme immunoassays and molecular biology,4 and ALP is also one of the most commonly assayed enzymes in routine clinical practice. Because ALP’s abnormal level is closely related to a variety of pathological processes, ALP is an important diagnostic indicator of many human diseases, such as bone diseases (osteoblastic bone cancer, Paget’s disease, and osteomalacia) and liver diseases (liver cancer, hepatitis, and obstructive jaundice).5−7 Therefore, the development of convenient and reliable assay methods for monitoring ALP activity/level is extremely important and valuable, not only for clinical diagnoses but also for biomedical research. A number of techniques have been successfully utilized to assay the activity of ALP in various samples in the past years, such as colorimetric,8 chromatographic,9 chemiluminescence,10 electrochemical,11 isotope label,12 and surface-enhanced resonance Raman scattering. However, they often require sample processing and/or complex instrumentation; therefore, diagnostic assays with high sensitivity, selectivity, simplicity, and cost-effectiveness are still desired. As a nondestructive method, the fluorescence technique is extremely attractive in this regard due to its simplicity, high sensitivity, and real-time detection.13−41 And this technique has been extensively applied in the study of various enzyme-catalyzed processes.42−47 © 2014 American Chemical Society

Received: July 8, 2014 Accepted: September 11, 2014 Published: September 11, 2014 9873

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cationic charge densities also bring about severe problems,54−56 for example, the membrane-disruptive properties that frequently cause high cytotoxicities, the strong binding with negatively charged proteins which deteriorate serum stability of the polyplexes, and so on. Previous innovative researches by Jiang and co-workers and Ishihara and co-workers indicate that zwitterionic materials, such as those based on betaine or phosphocholine, exhibit excellent biocompatibility via inhibition of protein adsorption due to the electrostatically induced hydration.57−64 Zwitterionic polymers/compounds can endow biocompatibility to various materials due to their excellent resistance to nonspecific protein adsorption. In our previous study, we also used a zwitterionic compoundbetaine compound (N,N-dimethyl(acrylamidopropyl)ammonium propanesulfonate)to modify 25 kDa polyethylenimine (PEI 25K) via the Michael addition reaction.65 The betaine-modified PEI shows good biocompatibility and still has high cationic density, because betaine is a neutral molecule and Michael addition only converts part of primary amines to secondary amines. As for aromatic fluorophores such as pyrene, their fluorescence emission varies as a result of excimer/monomer interconversion. It is known that pyrene monomers (emission at 350−400 nm) form intramolecular excimers (emission at 450−500 nm) due to aggregation,66−69 and the excimer could return to the monomer state via deaggregation, and this feature has been employed to design fluorescent probes. We envision that, by integrating the biocompatible betaine-modified PEI and an ALP-responsive negatively charged pyrene derivative, and by making use of the ALP-induced charge conversion, we could modulate the excimer/monomer emissions of pyrene via aggregation/deaggregation, thereby realize ratiometric sensing for the enzyme ALP. In this study, we designed a novel ratiometric fluorescent probe system for ALP. This probe system consists of two components: the first one is the hyperbranched, betaine-modified and positively charged PEI; the second is the negatively charged pyrene derivative containing an ALP-responsive phosphate group (Py-P, an aliphatic phosphate ester). In the absence of ALP, the twocomponent system shows the excimer emission of Py-P, since Py-P molecules bind along the positively charged polyelectrolyte due to electrostatic interactions, which lead to the formation of excimers. While in the presence of ALP, the phosphate moieties are removed from Py-P molecules due to the enzymatic reaction, and as a result, the system displays the monomer’s emission of Py-P. The schematic illustration for the fluorescent ratiometric detection is shown in Figure 1.

Figure 1. Schematic illustration for the probe system, and its fluorescent response toward alkaline phosphatase (ALP). The two photos were taken under a hand-held UV lamp (365 nm), with the solution (50 μM Py-P and 0.03 mg/mL betaine-modified PEI) in the absence or presence of 30 U/L ALP for 60 min.

supernatant was collected as soon as possible and storied at 2− 8 °C for use). All solvents (pyridine, acetic anhydride, methanol, diethyl ether, acetone, and acetonitrile) were analytical grade. The water used throughout the experiments was the triple-distilled water which was further treated by ionexchange columns and then by a Milli-Q water purification system. Synthesis of Py-P. 1-Pyrenylmethanol (244 mg, 1.05 mmol), phosphoric acid (crystalline 98 mg, 1 mmol), and pyridine (395 mg, 5 mmol) were dissolved in acetonitrile (8 mL). Then, N,N-diisopropylethylamine (258 mg, 2 mmol) was added dropwise into the solution. The mixture was stirred for 5 min. Finally, acetic anhydride (204 mg, 2 mmol) was slowly added. The mixture was stirred at 90 °C for 24 h. After being cooled to room temperature, acetonitrile was evaporated and the mixture was extracted by dichloromethane (3 × 30 mL) and pure water. The aqueous layer was collected and removed by rotary evaporator. The remaining solid was dissolved in methanol (2 mL), and then an excessive amount of diethyl ether was poured into the solution. The mixture was put in centrifuge tube and centrifuged to get a precipitate. The precipitate was washed by diethyl ether and acetone several times to get a white solid (170 mg, yield 55%). 1H NMR (δ ppm, in D2O, Supporting Information Figure S1): 8.48−8.53 (d, 1H), 8.15−8.23 (m, 5H), 8.05−8.10 (s, 2H), 7.98−8.03 (t, 1H), 5.59−5.67 (d, 2H). 31P NMR (δ ppm, in D2O, Supporting Information Figure S1): −22.69. ESI-MS (Supporting Information Figure S1): m/z 310.08 [M − H]−. Synthesis of N,N-Dimethyl(acrylamidopropyl) Ammonium Propanesulfonate (Betaine Compound, DMAAPS). DMAAPS was synthesized by the ring-opening reaction of 1,3propane sultone with N,N-dimethylaminopropylacrylamide (DMAPAA) in anhydrous acetone at ambient temperature. DMAPAA (4.0 g, 25.6 mmol) and acetone (15 mL) were charged into a 50 mL dried round-bottom flask. 1,3-Propanesultone (3.0 g, 24.6 mmol) solution in 15 mL of anhydrous acetone was added dropwise into the flask in 0.5 h, and stirring was proceeded for 16 h at the same temperature. The resulting white precipitate was filtered, washed with acetone, and dried under vacuum, yielding a white powder (6.29 g, yield 92%). 1H NMR (δ ppm, in D2O, Supporting Information Figure S2): 2.07 (m, 2H), 2.22 (m, 2H), 2.99 (t, J = 8 Hz, 2H), 3.12(m, 6H), 3.40 (m, 4H), 3.49 (t, J = 8 Hz, 2H), 5.80 (d, J = 12.0 Hz,

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EXPERIMENTAL SECTION Reagents and Materials. N-[3-(Dimethylamino)propyl] acrylamide (DMAPAA, 98%) and 1,3-propane sultone were purchased from TCI Shanghai. 1-Pyrenemethanol was obtained from J&K Chemical Ltd. Phosphoric acid crystalline, polyethylenimine branched (average Mw ∼25 000 by LS, average Mn ∼10 000 by GPC) and ALP (from bovine intestinal mucosa) were purchased from Sigma-Aldrich. N,N-Diisopropylethylamine (DIPEA) and tris(hydroxymethyl)aminomethane (Tris base) were purchased from Aladdin Reagents. Alkaline phosphatase enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shanghai Enzyme-linked Biotechnology Co. Ltd. Human serum was supplied by The Sixth Affiliated Hospital, Sun Yat-sen University (first, the blood was centrifuged for 20 min at 3000 rpm; then the 9874

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bind along the polymer chain, thereby causing the formation of excimer forms. From Figure 2, it can also be seen that, when the concentration of betaine-modified PEI is increased to 0.03 mg/mL, the excimer emission intensity reaches the maximum; further increase in the PEI concentration will lead to the simultaneous decrease of both the monomer and excimer emissions, which is probably caused by the concentrationinduced quenching. Hence, in the following experiments, we adopt the concentrations of 50 μM Py-P and 0.03 mg/mL betaine-modified PEI to prepare the probe system. Fluorescent Response of the Probe System toward ALP. To investigate the probe system’s fluorescent response toward ALP, we measured the fluorescent spectra of the system (50 μM Py-P and 0.03 mg/mL betaine-modified PEI) in the absence or presence of ALP. The fluorescence spectra of the system were periodically recorded during incubation of the assay system with ALP (20 U/mL) at 37 °C in Tris−HCl buffer (pH 8.0, 10 mM), as shown in Figure 3A.

1H), 6.19−6.52 (m, 2H). ESI-MS (Supporting Information Figure S2): m/z 278.9 [M + H]+. Synthesis of Betaine-Modified PEI. In a typical run, to a reaction flask containing 25 kDa PEI (430 mg, 0.0172 mmol) in 10 mL of methanol under a nitrogen atmosphere, DMAAPS (278 mg, 1 mmol) was added. The reaction mixture was stirred at 45 °C for 48 h and then dialyzed (MW cutoff: 3500) for 3 days against distilled water. Afterward, water was removed by rotary evaporator and dried under vacuum, yielding a yellow semisolid (610 mg, yield 86%). 1H NMR (δ ppm, in D2O, Supporting Information Figure S3) (the graft ratio calculated based on 1H NMR spectrum is 34.4%). Alkaline Phosphatase ELISA Kit Assay. Detection of ALP using the ELISA kit assay was conducted according to the instructions of the ELISA kit, which involves 11 steps. Measurements. 1H NMR and 31P spectra were recorded on a Bruker Avance 600 MHz NMR spectrometer. Mass spectra were obtained through a Bruker Esquire HCT Plus mass spectrometer. UV−vis spectra were recorded on a Hitachi U-3010 UV−vis spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer.

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RESULTS AND DISCUSSION Preparation of the Probe System. To prepare the probe system, the negatively charged pyrene derivative (Py-P) and the betaine-modified PEI were synthesized and characterized (Supporting Information Figures S1−S4). The probe system was then prepared by dissolving the two components (Py-P and the betaine-modified PEI) in the buffer solution. Since the sensing mechanism is based on the conversion of the excimer emission to the monomer emission, the suitable ratio of Py-P to the betaine-modified PEI will ensure optimal performance of the probe system. In order to determine an appropriate ratio for the betaine-modified PEI and Py-P, we measured the emission spectra of Py-P (50 μM) in the presence of varied amounts of the betaine-modified PEI, as shown in Figure 2. It is

Figure 3. (A) Time course of fluorescence spectra of the assay system (50 μM Py-P and 0.03 mg/mL betaine-modified PEI) in the presence of ALP. The measurement was conducted with ALP (20 U/L) in Tris−HCl buffer (pH 8.0, 10 mM) at 37 °C. (B) Plot of the fluorescence intensity ratio of the assay system (50 μM Py-P and 0.03 mg/mL betaine-modified PEI) in Tris−HCl buffer (pH 8.0, 10 mM) at 37 °C vs reaction time in the presence of different amounts of ALP (n = 3). Excitation wavelength: 350 nm.

Figure 2. Fluorescence spectra of Py-P (50 μM) in the presence of different amounts of betaine-modified PEI in Tris−HCl buffer (pH 8.0, 10 mM). Excitation wavelength: 350 nm.

In the absence of ALP, the monomer emission of the system is relatively weak and excimer emission is very strong due to the π−π stacking of Py-P around the polymer chains of the betainemodified PEI in Tris−HCl buffer, which is caused by the electrostatic interactions between the negatively charged Py-P and the positively charged betaine-modified PEI. Upon addition of ALP, the hydrolysis of phosphate ester group catalyzed by ALP converts the negatively charged Py-P into the electroneutral Py−OH; as more and more Py-P molecules are turned

clear that, in the absence of the betaine-modified PEI, Py-P solution only displays its monomer emission at around 378 nm. As the amount of betaine-modified PEI is increased, the excimer emission of Py-P gradually increases, at the same time its monomer emission decreases gradually. We suppose the reason is that, as the amount of betaine-modified PEI is increased, more of the negatively charged Py-P molecules could 9875

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10%).72 This is sensitive enough for determining ALP levels in biological assays, since the normal range of serum ALP in adults is about 46−190 U/L.73 To further confirm that the variation of the excimer/ monomer emissions of the sensing system is indeed induced by ALP, we investigated the fluorescence changes of the sensing system in the presence of ALP (50 U/L) upon addition of varied amounts of sodium orthovanadate (Na3VO4), which is a well-known ALP inhibitor.74 Figure 5 and Supporting

into Py−OH, accordingly electrostatic interactions between the pyrene derivative and polymer decrease and finally do not exist any longer; correspondingly the excimer emission decreases and monomer emission increases gradually. The kinetic activity of the enzymatic reaction was determined by measuring the time course experiments of the system at four different concentrations of ALP, as shown in Figure 3B. Next, we measured the fluorescence response of the probe toward ALP at varied concentrations (reaction at 37 °C for 60 min in Tris−HCl buffer (pH 8.0)), as shown in Figure 4. It can

Figure 5. Intensity ratio of the assay system in the presence of 50 U/L ALP upon addition of different amounts of ALP inhibitor (Na3VO4) in Tris−HCl buffer (pH 8.0, 10 mM) at 37 °C for 60 min. Final concentrations: 50 μM Py-P and 0.03 mg/mL betaine-modified PEI (n = 3). Excitation wavelength: 350 nm.

Information Figure S7 show that, with the increasing Na3VO4 concentration, the fluorescence intensity ratio (I378/I488) gradually decreases, and when the Na3VO4 concentration reaches about 50 μM, the ratio (I378/I488) levels off. In addition, in order to test the impact of the vanadate on the betaine/PEIPy-P complex, the fluorescent response of the betaine/PEI-PyP complex with different concentrations of sodium orthovanadate in the absence of ALP was measured in Tris−HCl buffer (pH 8.0, 10 mM) at 37 °C for 60 min (Supporting Information Figure S8). It is obvious that sodium orthovanadate basically has no effects on the structure of the betaine/PEI-Py-P complex. These results indicate that ALP can indeed induce the fluorescence changes of the sensing system. Mechanism for ALP Detection. On the basis of the fluorescent response of the sensing system toward ALP, the detection mechanism is proposed as follows: upon addition of ALP into the assay system, the dephosphorylation catalyzed by ALP transforms Py-P into Py−OH, which do not have negative charge, and Py−OH cannot complex with betaine-modified PEI; hence, excimer emission decreases gradually and eventually no longer exists, while monomer emission increases. In order to confirm the detection mechanism, electrospray mass spectrometry (ESI-MS) data of Py-P before and after addition of ALP were measured. The results are shown in Figure 6. The peak at m/z 310.08 corresponds to the negative molecular ion of Py-P. After 30 U/L ALP was added and incubated for 1 h at 37 °C, the peak at m/z 213.98 for the positive molecular ion can be observed, and this corresponds to 1-pyrenylmethanol (namely, Py−OH. Benzylalcohol easily becomes benzyl carbocation in ESI-MS mode). At the same time, the peak at m/z 309.79 for the negative molecular ion of Py-P can be observed as well; these results indicate that our substrate Py-P molecules are not completely hydrolyzed at this ALP concentration, and only part of Py-P molecules are

Figure 4. (A) Fluorescence spectra of the assay system (50 μM Py-P and 0.03 mg/mL betaine-modified PEI) in the presence of different amounts of ALP in Tris−HCl buffer (pH 8.0, 10 mM) at 37 °C for 60 min. (B) The intensity ratio as a function of the ALP level. Excitation wavelength: 350 nm. Data represent mean ± SD from three independent experiments.

be seen that the fluorescence intensity ratio increases steadily with the increasing ALP level. According to the literaturereported model for ratiometric method,70,71 the calibration equation for the probe system herein has been determined as [ALP] = (30.73)[(R − Rmin)/(Rmax − R)], where Kd is the dissociation constant, R is the fluorescence ratio at both wavelengths, namely, I378/I488, Rmin is the minimum ratio value, Rmax is the maximum ratio value, β is a scaling factor, which represents the fluorescence ratio for the denominator of R in zero and saturating [ALP], and the related relationship curve is shown in Supporting Information Figure S5. By using the calibration equation, the background fluorescence caused by various factors such as variations in excitation intensity, emission collection efficiency, and probe concentration can be canceled out. The fluorescence of the probe solution also changes gradually from blue-green to blue-violet upon addition of ALP. Moreover, the detection limit is determined to be 0.1 U/L (Supporting Information Figure S6), and the quantification limit is determined as 0.5 U/L (with RSD less than 9876

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fluorescence assay for ALP detection. Since there is relatively high concentration of ALP existing in human serum (46−190 U/L for adults), the serum samples were diluted 20-fold for the measurement; hence, with the detection limit of 0.1 U/L and the calibration equation (Supporting Information Figure S5), this probe can be used for detecting ALP levels in biological diagnostics. Compared with other reported fluorescent probes for ALP (as shown in Supporting Information Table S2), the probe system herein can realize ALP detection in the fluorescent ratiometric mode, while currently fluorescent probes for ALP function in either turn-on or turn-off mode; furthermore, the probe system can detect ALP in human serum samples and could serve as a convenient straightforward one-step fluorescent assay for ALP.

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CONCLUSION In summary, we have developed a novel fluorogenic ratiometric sensing system for ALP, which is based on betaine-modified PEI polymer and functions through excimer/monomer conversion of a pyrene derivative. The polymer betainemodified PEI is positively charged, while the pyrene Py-P contains a phosphate group, which is negatively charged and serves as both the substrate for enzymatic reaction and the fluorophore. Due to the electrostatic interactions, Py-P molecules complex with the polymer, leading to the π−π stacking of Py-P molecules and the corresponding excimer emission. In the presence of ALP, the hydrolysis of Py-P catalyzed by ALP transforms the negatively charged Py-P into the electroneutral Py−OH; accordingly, excimer emission decreases, while monomer emission gradually increases. The system can be used for ALP detection in biological fluid like serum. Due to its ease of use, this system could serve as a onestep straightforward fluorescent assay for ALP. This strategy may offer a technically simple approach for designing ratiometric sensing systems for detecting other biomolecules.

Figure 6. ESI-MS data of Py-P before and after addition of ALP. (A) ESI-MS of Py-P in the absence of ALP. (B) ESI-MS of Py-P (50 μM) in the presence of ALP (30 U/L) at 37 °C for 60 min.

converted into Py−OH. The results confirm the detection mechanism. ALP Detection in Biological Fluid Samples. To evaluate the efficacy of this probe in real samples, first the probe’s detection for ALP in Tris−HCl buffer (pH 8.0, 10 mM) was compared with the detection by using the commonly used commercial assay kit, the ALP-ELISA kit (Supporting Information Table S1). The recovery of added known amounts of ALP is more than 97% by this probe, which suggests the feasibility of the present method. Encouraged by the above results, we applied the assay system for detecting the endogenous ALP levels in human serum samples [Table 1,

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Table 1. Endogenous ALP Detection in Human Serum Samples by Using ELISA Kit Assay and This Probe sample no. c

A Bc Cc Dc

ALP amount (U/L) by ELISA kit assaya 80.10 58.14 93.95 56.81

± ± ± ±

3.12 4.17 2.83 5.32

79.71 57.94 93.56 55.66

± ± ± ±

ASSOCIATED CONTENT

S Supporting Information *

ALP amount (U/L) by this probeb

Synthetic routes, 1H NMR spectra, mass spectra, determination of graft ratio, determination of the detection limit, fluorescence spectra, comparison of ALP detection in buffer by using ELISA kit assay and this probe, absorption spectrum, and comparison of the sensing system herein with other reported probes for ALP. This material is available free of charge via the Internet at http://pubs.acs.org.

3.17 2.09 3.63 2.37

a

When ALP level was determined by using the ELISA kit, the serum samples were 2000-fold diluted for measurement; the values herein represent ALP levels in the undiluted serum samples, which are calculated based on the measurements for the diluted samples. bWhen ALP level was determined by using this probe, the serum samples were 20-fold diluted for measurement; the values herein represent ALP levels in the undiluted serum samples, which are calculated based on the measurements for the diluted samples; the data were obtained from the calibration equation (Supporting Information Figure S5). c Mean ± SD were used to describe the data (n = 3).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

by using the calibration equation (Supporting Information Figure S5) as a standard for this probe]. The endogenous ALP levels in the serum samples measured by this probe are close to those measured by using the ELISA kit. However, as for the ELISA kit (the antibody-based colorimetric assay), the detection process requires complex procedures; while for the assay system reported herein, the detection process is very convenient and could serve as a one-step straightforward

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support by the National Key Basic Research Program of China (Project No. 9877

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