Fluorometric Method for Inorganic Pyrophosphatase Activity Detection

Article pubs.acs.org/ac

Fluorometric Method for Inorganic Pyrophosphatase Activity Detection and Inhibitor Screening Based on Click Chemistry Kefeng Xu, Zhonghui Chen, Ling Zhou, Ou Zheng, Xiaoping Wu, Longhua Guo, Bin Qiu, Zhenyu Lin,* and Guonan Chen Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University, Fuzhou, Fujian 350116, China ABSTRACT: A fluorometric method for pyrophosphatase (PPase) activity detection was developed based on click chemistry. Cu(II) can coordinate with pyrophosphate (PPi), the addition of pyrophosphatase (PPase) into the above system can destroy the coordinate compound because PPase catalyzes the hydrolysis of PPi into inorganic phosphate and produces free Cu(II), and free Cu(II) can be reduced by sodium ascorbate (SA) to form Cu(I), which in turn initiates the ligating reaction between nonfluorescent 3-azidocoumarins and terminal alkynes to produce a highly fluorescent triazole complex, based on which, a simple and sensitive turn on fluorometric method for PPase can be developed. The fluorescence intensity of the system has a linear relationship with the logarithm of the PPase concentration in the range of 0.5 and 10 mU with a detection limit down to 0.2 mU (S/N = 3). This method is cost-effective and convenient without any labels or complicated operations. The proposed system was applied to screen the potential PPase inhibitor with high efficiency. The proposed method can be applied to diagnosis of PPase-related diseases.

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of the dispersion/aggregation states of gold nanoparticles (AuNPs) by controlling the coordination of Cu(II) between cysteine and PPi with PPase.15 On the basis of the competition assay approach and fluorescent 11-mercaptoundecanoic acidcapped AuNCs with special optical properties, Sun et al. established a convenient, sensitive and real-time assay of PPase activity.16 The Cu(I) catalyzed alkyne−azide cycloaddition reaction (CuAAC) is one of the most famous click chemistry reactions,17 which owns the characteristics of high efficiency, mild conditions and inertness to surrounding biological milieu. Many sensors with high sensitivity and selectivity based on CuAAC have been developed, such as colorimetric assay18 and electrochemical sensors.19 In early studies, fluorescence biosensors based on CuAAC for Cu(II) in a serum sample,20 DNA sequence21 and histidine22 were developed because a highly fluorescent triazole complex can be produced through ligating the nonfluorescent 3-azidocoumarins and terminal alkynes.23 It has been reported that the strong interaction between PPi and Cu(II) can prevent the reduction of Cu(II) to Cu(I) by ascorbate,24 which in turn inhibit the click reaction. The presence of PPase can hydrolysis the PPi and hinder the

yrophosphatase (EC 3.6.1.1), or inorganic pyrophosphatase (PPase), is a hydrolase enzyme that can specifically turn pyrophosphate (P2O74−, PPi) into inorganic phosphate. The degradation of PPi by PPase is a high energy discharge reaction, which provides a thermodynamic pull for biosynthetic reactions.1,2 Early studies showed that PPase has direct relation with phosphorus metabolism, carbohydrate metabolism and evolutionary events.3,4 Therefore, PPase is crucial to the energy anabolism taking place in all living organisms and the depletion of PPase would lead to cell death.5 Little attention has been paid to develop a sensitive and convenient determination method for PPase, though the structure and mechanism of PPases has been extensively studied.6−10 The radio labeling technique is the most frequently used method for PPase detection.11,12 Because radio labeled materials are costly and need expensive counting equipment and, more importantly, there exists a great potential hazard to humans, the application of this technique is limited. To address this drawback, some other methods were explored for PPases activity determination. For example, based on the PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate, Eriksson et al. established a real-time bioluminescence method for PPase determination.13 On the basis of the fact that the hydrogen-ion concentration will decrease during the PPi hydrolysis reaction and results in small pH changes, a rapid and sensitive method for PPase mensuration was developed.14 Recently, Deng et al. proposed a colorimetric method for PPase activity measuring based on reversible tuning © 2014 American Chemical Society

Received: October 22, 2014 Accepted: December 8, 2014 Published: December 8, 2014 816

dx.doi.org/10.1021/ac503958r | Anal. Chem. 2015, 87, 816−820

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Scheme 1. Structure and Sensing Mechanism of Fluorescent Sensor for PPase

volume of PPase with a different final concentration ranging from 0.5 to 40 mU was added to the aqueous dispersion consisting of HEPES buffer (168 μL, 10 mM, pH 7.2) and PPi (12.5 μL, 2 mM) in a 37 °C water bath. After 60 min, Cu(II) (2 μL, 10 mM) was added and the solution was incubated for 15 min. Then propargyl alcohol (5 μL, 10 mM), 3-azido-7hydroxycoumarin (5 μL, 10 mM) and sodium ascorbate (8 μL, 10 mM) were added to reach a final volume of 200 μL. Incubation was carried out in the dark for 15 min to form the fluorescent 1,2,3-triazole compounds through the CuAAC reaction at room temperature, and then data of fluorescence emission spectra were collected.1 The CuAAC reaction was performed under the optimum conditions described in earlier reports.20−22,25 Each point was detected for three times, and the average values were used for quantification.

complexation between Cu(II) and PPi. The free Cu(II) can be reduced to Cu(I) and initiates the CuAAC reaction between 3azidocoumarins and propargyl alkyne, which results in the enhancement of the fluorescence intensity of the system, and the fluorescence intensity enhancement had a relationship with the PPase activity. On the basis of this mechanism, a sensitive fluorometric method for PPase activity can be developed. Furthermore, the proposed system was applied to screen the inhibitor for PPase with high efficiency.

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EXPERIMENTAL SECTION Chemicals and Reagents. Sodium ascorbate (SA), propargyl alcohol, copper sulfate pentahydrate (CuSO4· 5H2O) and other reagents were purchased from Alfa Aesar China Co. Ltd. (Tianjin) and used as received. 4-(2Hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES) was obtained from Shanghai Chemical Reagent Company (Shanghai, China). Sodium pyrophosphate was purchased from Beilian Fine Chemicals Co. Ltd. (Tianjin). Baker’s yeast inorganic PPase (EC3.6.1.1) was purchased from Sigma-Aldrich Co. LLC. One unit of Baker’s yeast PPase can liberate 1.0 μmol of inorganic orthophosphate/min at pH 7.2 and 25 °C. 3-Azide-7hydroxycoumarin was synthesized according to the literature.23 All other chemicals were at least analytical grade reagents and used directly without further purification. Aqueous solutions were prepared with Milli-Q water (18.4 MΩ·cm−1). Unless otherwise mentioned, all experiments were carried out at room temperature. Instruments. Fluorescence measurements were carried out on a 970-CRT fluorescence spectrometer (Hitachi Ltd., Shanghai, China) using a square quartz cuvette that can hold 300 μL of solution. Excitation and emission slits were all set for a 10.0 nm band-pass. The excitation wavelength was set at 395 nm, and the emission spectra were collected from 415 to 600 nm. The fluorescence intensity at 475 nm was used to evaluate the performances of the proposed assay strategy. UV−visible absorption spectra were recorded using a Lambda 750 UV−vis spectrophotometer. All measurements were carried out at room temperature unless stated otherwise. Procedure for PPase Activity Assay. PPase activity assay was performed under the following procedures. A 2.0 μL

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RESULTS AND DICUSSIONS Principle of the Detection System. The protocol of the proposed fluorometric method is shown in Scheme 1. In the absence of PPase, the addition of Cu(II) into PPi solution can make Cu(II) coordinate with PPi and form a Cu(P2O7)26− complex, the strong association between PPi and Cu(II) would hamper the transformation of Cu(II) into Cu(I).15,24,26 After incubation with PPase, PPi is hydrolyzed into orthophosphate and which cannot coordinate with Cu(II), so the added Cu(II) is free in the solution and can be reduced to form Cu(I) by sodium ascorbate. Then Cu(I), in turn, triggers the reaction between the fluorescently inactive compounds of 3-azido-7hydroxycoumarin and propargyl alcohol to produce a highly fluorescent triazole complex. The enhanced fluorescence signal has a relationship with the concentration of PPase, based on which, a sensitive, cost-effective and simple fluorometric method for PPase activity determination can be developed. Simple experiments were carried out to verify our presumption. As shown in Figure 1A, the mixed solution containing 3-azido-7-hydroxycoumarin and propargyl alcohol gives off a weak fluorescence signal (curve a). A strong fluorescence signal can be detected after the addition of Cu(II) and sodium ascorbate into the above-mentioned system (curve b); this means the CuAAC reaction occurred and the fluorescent compounds was formed. While PPi was added 817

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between PPi and Cu2+, and reached a plateau after 20 min, so 20 min was chosen in further experiments. The effect of the reaction time between PPi and PPase was studied also. As shown in Figure 1D, one can find that the fluorescence intensity at 475 nm increased sharply with the increase of reaction time between PPase and PPi, which tended to be constant after 60 min. To obtain an effective hydrolysis for PPase assay, a slightly longer time was needed to complete the reaction, thus a 60 min hydrolysis time was used. The effect of the temperature on the system was studied also because it can affect the activity of PPase. Figure 1E shows that 37 °C is the suitable temperature. Additionally, the pH values are also important to the enzymatic processes. The results showed that the fluorescence intensity of the system increase first with the increase of the pH value and decrease if the pH is higher than 7.2 (Figure 1F). So, pH 7.2 was chosen as the optimized condition. Performance of the Sensing System. The activity of PPase was tested by the addition of different activity units of PPase into the test solution. Figure 2A reveals the fluorescence

Figure 1. (A) Fluorescence responses of the sensing system under different conditions: (a) before click reaction, (b) after click reaction, (c) after click reaction with PPi, (d) after click reaction with PPi and PPase, (e) before click reaction with PPase. PPi, 250 μM; PPase, 40 mU. (B) Effect of PPi concentration on the fluorescence intensity of the system. Cu(II): 100 μM. (C) Effect of the reaction time between PPi and Cu(II) on the fluorescence intensity of the system. (D) Effect of incubation time between PPase and PPi on the fluorescence intensity of the system. (E) Effect temperature on the fluorescence intensity of the system. (F) Effect of media pH on the fluorescence intensity of the system.

Figure 2. (A) Fluorescence intensity of the system at different PPase concentrations. From a to i: 0, 0.5, 1, 2, 4, 6, 8, 10 and 20 mU. (B) Calibration curve between the fluorescence intensity and the PPase concentrations.

simultaneously, only a little fluorescence signal enhancement was observed (curve c), which indicated the CuAAC reaction was inhibited. A further test was carried out after PPi incubated with PPase first, and the fluorescence of the system was enhanced greatly (curve d). If PPase only was added into the mixed solution of 3-azido-7-hydroxycoumarin and propargyl alcohol, no obvious fluorescence change was detected (curve e). These results suggested that PPase itself caused no effect on the fluorescence of the system, but it can hydrolyze the PPi and cannot hinder the CuAAC reaction. These phenomena confirmed the principle. Optimization of the Experimental Conditions. In our design principle, the strong association between PPi and Cu(II) would hamper the transformation of Cu(II) into Cu(I). If the concentration of PPi is not high enough, the free Cu(II) in the solution can initiate the CuAAC reaction, which results in the high background signal. But if too much PPi is added, the PPase will hydrolysis free PPi first, which will result in the poor sensitivity of the proposed biosensor. Thus, we studied the concentration of PPi and the time dependence of peak intensity after addition of PPi to explore the inhibition ability of PPi on the Cu(II). As shown in Figure 1B, the fluorescence intensity decreased sharply with the increase of PPi concentration; this indicates as more PPi is added, more Cu(II) is chelated and less Cu(I) is produced. And after 125 μM, it reached a plateau, so 125 μM PPi was used in this study. The coordination reaction between PPi and Cu(II) also affected the fluorescence of the system. As shown in Figure 1C, the fluorescence intensity decreased sharply with the enhancement of complexation time

spectra of the sensing system at different PPase concentrations. The fluorescence intensity enhanced sharply with the increasing of PPase concentration in the range of 0.5−8 mU, and increased slowly in the range of 8−20 mU. Figure 2B depicts the relationship between the fluorescence intensity and PPase concentrations. In addition, a linear correlation (the linear equation is I = 404.6 + 333log CPPase, CPPase is the PPase concentration and I is the fluorescence intensity, R = 0.9961) existed between the fluorescence intensity and logarithm of the PPase concentration CPPase in the range of 0.5−10 mU under the optimal conditions. The limit of detection (LOD) was calculated according to the definition of 3σb/s, where σb is the standard deviation of the blank samples, s presents the slope.26,27 In this study, the fluorescence signal of the blank sample was measured 10 times, and the standard deviation (σb) was calculated. Finally, a result of 0.2 mU for the LOD was obtained. This detection limit is lower than that those of the early reported real time assay based on the fluorescent gold nanoclusters (1 mU),16 another real time assay (8 mU)13 and colorimetric assay (10 mU).15 Interference Study. Several proteins (such as lysozyme, human serum albumin (HSA), glucose oxidase (GOx), exonuclease I and exonuclease III) were used as representative interferents to investigate the selectivity of the proposed biosensor. The concentration of interferents was settled as (GOx 0.22 U, lysozyme 0.5 U, HSA 6.0 × 10−5 M, exonuclease 818

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observed in the range from 0.4 to 2.0 μM, which suggests that the proposed sensing system can be applied to monitor inhibitor concentration.

I 0.1 U and exonuclease III 1 U) and the target concentration was 5.6 × 10−7 M (4 mU). As shown in Figure 3, in the

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CONCLUSION The strong interaction between PPi and Cu(II) can effectively hamper the reduction of Cu(II) to Cu(I), which in turn hamper the click reaction between 3-azido-7-hydroxycoumarin and propargyl alcohol to produce highly fluorescence compounds. In the presence of PPase, PPi can undergo hydrolysis into orthophosphate and make Cu(II) free again, and the fluorescence intensity of the system increased significantly. Inspired by this, a simple, fast and sensitive fluorometric method for PPase activity assay based on the click reaction was developed. The method can also be applied to screen the PPase inhibitors efficiently. The assay using PPi substrate may hold potential applications in the diagnosis of PPi or PPase-related diseases.

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Figure 3. Signals of the proposed biosensor at different interferents and targets. Inset is the corresponding photograph of fluorescence emission under UV lamp excitation (365 nm). The concentration of PPase is 4 mU, [GOx] 0.22 U, [lysozyme] 0.48 U, [HSA] 6.0 × 10−5 M, [exo I] 0.1 U, [exo III] 1 U.

AUTHOR INFORMATION

Corresponding Author

*Zhenyu Lin. E-mail: [email protected]. Tel/Fax: 86-59122866135. Address: Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China.

presence of interferents, the fluorescence intensities were very week while the signal of target had a high value and the fluorescence color of the solution obviously brighter in the presence of PPase. The reason is in that these species are unable to destroy the complexation between Cu(II) and PPi, also the CuAAC reaction cannot proceed. These indicate that interference from others can be ignored and the proposed strategy shows excellent selectivity for PPase detection. Screen of Inhibitors for PPase. For demonstrating the potential application of the proposed system to screen the potential enzyme inhibitor, several mineral salts were chosen as models. As shown in Figure 4A, the fluorescence of the system

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program of China (No. 2010CB732403), NSFC for Excellent Youth Scholars of China (21222506), NSFC (21175024, 21375021, 21375019) and program for New Century Excellent Talents in University (NCET-12-0619), Nature Sciences Funding of Fujian Province (2014J06005).

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Figure 4. (A) Effect of different mineral salts on the PPase activity. (B) Linearity of peak intensity with respect to NaF concentrations, respectively. (Inset) Fluorescence spectra of NaF-treated PPase activity, the NaF concentrations from a to f were 0, 0.4, 0.8, 1.2, 1.6 and 2.0 μM.

in the presence of NaF is nearly the same with that of the mixed solution including 3-azidocoumarins and propargyl alkyne, whereas the others are nearly the same with that after CuAAC reaction. These results indicate that NaF is an effective inhibitor for PPase and the other compounds cause no effect on PPase activity. In addition, different concentrations of NaF were added into the sensing system, and the data is summarized in Figure 4B. The fluorescence intensities of the sensing system decreased gradually with the enhancement of the NaF concentrations. Furthermore, a good linear relationship is 819

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dx.doi.org/10.1021/ac503958r | Anal. Chem. 2015, 87, 816−820