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Real-Time Colorimetric Assay of Inorganic Pyrophosphatase Activity Based on Reversibly Competitive Coordination of Cu2+ between Cysteine and Pyrophosphate Ion Jingjing Deng, Qin Jiang, Yuexiang Wang, Lifen Yang, Ping Yu,* 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, Beijing 100190, China S Supporting Information *

ABSTRACT: In this study we demonstrate a new colorimetric method for real-time pyrophosphatase (PPase) activity assay based on reversible tuning of the dispersion/aggregation states of gold nanoparticles (Au-NPs) by controlling the coordination of Cu2+ between cysteine and pyrophosphate ion (PPi) with PPase. The addition of Cu2+ to the cysteine-stabilized Au-NP dispersion results in the aggregation of Au-NPs, while the further addition of PPi to this aggregation turns the aggregated Au-NPs into their dispersed state because of the higher coordination reactivity between Cu2+ and PPi than that between Cu2+ and cysteine. The subsequent addition of PPase to the PPi-triggered dispersed Au-NPs restores the aggregation state of Au-NPs because PPase catalyzes the hydrolysis of PPi into orthophosphate and thus consumes PPi in the reaction system. In this study, we utilize this reversibility of the change between the aggregation/dispersion states of AuNPs for real-time colorimetric monitoring of PPase activity by continuously measuring the ratio of absorbance at the wavelength of 650 nm (A650) to that at 522 nm (A522) in the time-dependent UV−vis spectra of Au-NP dispersions containing different activities of PPase. To calculate the kinetics of the PPase-catalyzed hydrolysis of PPi, the A650/A522 values are converted into PPi concentrations to obtain the time-dependent changes of PPi concentrations in the dispersions containing different activities of PPase. The initial reaction rates (v0) are thus achieved from the time-dependent logarithm of PPi concentrations with the presence of different PPase activities. Under the experimental conditions employed here, the v0 values are linear with the PPase activity within a range from 0.025 to 0.4 U with a detection limit down to 0.010 U (S/N = 3). Moreover, the colorimetric method developed here is also employed for PPase inhibitor evaluation. This study offers a simple yet effective method for realtime PPase activity assay.

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designability of the surface chemistry of Au-NPs and the technical simplicity, high sensitivity, and selectivity of the asformed colorimetric methods. To date, colorimetric methods have been widely used for detecting various kinds of species such as nucleic acids, proteins, saccharides, and metal ions.5 Recent attempts have well extended the application of this kind of method to enzyme activity assay; unfortunately, most of the methods reported so far serving this purpose are based on the color change of Au-NPs induced by the products produced from the enzymatic reactions, instead of the substrate to be consumed during reaction.6 This is understandable since the change between the dispersion/aggregation states of Au-NPs is usually irreversible, especially for cases in which the aggregation of Au-NPs is caused by non-cross-linking interactions such as salt effects. While real-time enzyme activity assay based on the substrate change is more direct and specific and thus highly expected, the irreversibility of the change between the

eveloping technically simple yet effective methods for real-time enzyme activity assay is of critical importance both for reaction screening in metabolic pathways and for the early diagnosis of diseases.1 Inorganic pyrophosphatase (PPase) is one kind of enzyme that specifically catalyzes the hydrolysis of inorganic pyrophosphate ion (PPi) into orthophosphate (Pi), providing a thermodynamic pull for biosynthetic reactions.2 To date, PPase has been demonstrated to be directly relevant to phosphorus metabolism, carbohydrate metabolism, and evolutionary events.3 In this context, a method for simple and real-time PPase activity assay is very essential for understanding the PPase-related biological processes. While several elegant methods have so far been developed for PPase activity assay,4 these methods are limited by the expensive materials, sophisticated instruments, or their time-consuming experimental procedures. As a consequence, an effective yet technically simple method for real-time PPase activity assay is still highly desired. Benefiting from the unique optical properties of gold nanoparticles (Au-NPs), Au-NP-based colorimetric assays have recently attracted increasing attention due to the © 2013 American Chemical Society

Received: August 9, 2013 Accepted: September 9, 2013 Published: September 9, 2013 9409

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Scheme 1. Schematic Illustrations of the Mechanisms for (A) Reversible Competitive Coordination Chemistry Regulated by PPase, (B) Colorimetric Determination of PPase Activity and the Inhibition Efficiency of NaF, and (C) the Catalytic Reaction of PPase

in Scheme 1 B. To the best of our knowledge, this is the first example of real-time PPase activity assay with a simple Au-NPbased colorimetric method. This study not only provides a new colorimetric principle for real-time assay of PPase enzyme activity but also paves an avenue to the development of novel colorimetric methods based on rational design of the surface chemistry of Au-NPs through reversibly competitive coordination chemistry.

aggregation/dispersion states of Au-NPs triggered by enzyme substrate unfortunately invalidates colorimetric methods for real-time enzyme activity assay.7 To overcome this problem, we have recently developed a colorimetric method for PPi assay, the substrate for PPasecatalyzed hydrolysis reaction.8 This method was based on the competitive coordination of Cu2+ between cysteine and PPi, in which PPi could competitively coordinate with Cu2+ in the presence of cysteine, resulting in the dispersion of Cu2+triggered aggregated Au-NPs.8 Herein, we interestingly find that the competitive coordination of Cu2+ between cysteine and PPi could be reversibly tuned by changing the concentration of PPi in the Au-NP dispersion with PPase, as shown in Scheme 1 A and described below. As reported in our early study,8 the addition of Cu2+ into the Au-NP dispersion containing cysteine essentially triggers the aggregation of Au-NPs. Upon the addition of PPi, the aggregated Au-NPs tend to solubilize into the aqueous solution to form a PPi-triggered dispersion of AuNPs (Scheme 1 A) because of the stronger binding ability between PPi and Cu2+ than that between cysteine and Cu2+.9 On the other hand, further addition of PPase into this dispersion results in the reaggregation of Au-NPs due to the hydrolysis of PPi into Pi under the catalysis of PPase. To summarize, the presence of PPi causes the Cu2+-triggered aggregated Au-NPs to remain in a dispersion state, while the addition of PPase (i.e., the consumption of PPi) causes the PPidispersed Au-NPs to tend to reaggregate. The degree of reaggregation of Au-NPs is directly related to the consumption of PPi (i.e, PPase activity), and thus, this principle could be used for real-time monitoring of the PPase activity through the change in the UV−vis spectrum of the Au-NP dispersion. The reversibility of this method essentially enables the system to real-time monitor PPase activity in aqueous media and, furthermore, to evaluate the inhibitor of PPase, as displayed

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EXPERIMENTAL SECTION

Chemicals and Reagents. Chloroauric acid (HAuCl4· 3H2O), trisodium citrate, cupric sulfate anhydrous, and sodium pyrophosphate were purchased from Beijing Chemical Reagent Co. (Beijing, China). L-Cysteine was obtained from Kanto Chemical Corp. (Japan). Baker’s yeast inorganic PPase (EC 3.6.1.1) was purchased from Sigma. One unit of Baker’s yeast PPase liberates 1.0 μmol of inorganic orthophosphate/min at pH 7.2 and 25 °C. All other chemicals were at least analytical grade reagents and used without further purification. Aqueous solutions were prepared with Milli-Q water (18.2 MΩ·cm−1). Unless otherwise pointed out, all experiments were carried out at room temperature. Synthesis of Au-NPs. Au-NPs with a diameter of about 13.5 nm were synthesized as reported before.10 Briefly, trisodium citrate (10 mL, 38.8 mM) was added to a boiling solution of HAuCl4 (100 mL, 1 mM), and the resulting solution was kept continuously boiling for another 30 min to give a wine red mixture. The mixture was cooled to room temperature and then filtrated through a Millipore syringe (0.45 μm) to remove the precipitate, and the filtrate was stored in a refrigerator at 4 °C for use. The size of the synthetic nanoparticles was about 13.5 nm as confirmed by the TEM image and UV−vis spectroscopy (TU-1900 spectrophotometer, Beijing Purkinje General Instrument Co. Ltd., China) with an 9410

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absorption peak at around 522 nm. A cysteine-stabilized Au-NP dispersion was prepared by adding an aqueous solution of cysteine (70 μL, 25 μM) to a dispersion of Au-NPs (270 μL, 5 nM), and the resulting mixture was then incubated in a 25 °C water bath for 30 min. PPase Activity Assay. Colorimetric PPase activity assay was performed under the following procedures. A 5 μL volume of PPase with different activities ranging from 0.025 to 1 U was added to the aqueous dispersion consisting of Au-NPs (270 μL, 5 nM), cysteine (70 μL, 25 μM), PPi (60 μL, 2 mM), and Cu2+ (60 μL, 4 mM). Immediately after the addition of PPase to the cysteine-stabilized Au-NP dispersions containing PPi and Cu2+, the UV−vis absorption spectra of the dispersions were consecutively recorded every 2 min at 25 °C. After 30 min, the dispersions were photographed with a digital camera (Canon IXUS 951S, Japan). Inhibitor Efficiency Evaluation. For investigating the inhibition efficiency of NaF on PPase, we initially added PPase (5 μL, 0.2 U) to 60 μL of aqueous solutions of NaF with different concentrations (100 nM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM) in a 25 °C water bath. After 15 min, the NaF-treated PPase was added to the aqueous dispersion consisting of Au-NPs (270 μL, 5 nM), cysteine (70 μL, 25 μM), PPi (60 μL, 2 mM), and Cu2+ (60 μL, 4 mM). After 30 min, the dispersions were photographed with a digital camera (Canon IXUS 951S, Japan) and UV−vis spectroscopic measurements were obtained.

to this dispersion essentially triggers the aggregation of Au-NPs through the coordination between Cu2+ and cysteine, resulting in a color change from wine red to blue and the production of a new absorption peak at 650 nm (Figure 1 B, vial 2, blue curve) (Figure 1 A, step 1). Further addition of PPi (60 μL, 38.3 mM) to the dispersion of Cu2+-triggered aggregated Au-NPs enables the change of the dispersion color from blue (vial 2, blue curve) to dark red (vial 3, red curve) (Figure 1 A, step 2) because of the stronger coordination between PPi and Cu2+ than that between cysteine and Cu2+, consistent with our earlier study.8 On the other hand, the addition of PPase (5 μL, 6 U) to the cysteine-stabilized Au-NP dispersion containing Cu2+ (60 μL, 4 mM) and PPi (60 μL, 38.3 mM) obviously leads to the change in the color of the Au-NP dispersion from dark red to blue (Figure 1 B, vial 4, green curve) (Figure 1 A, step 3), essentially demonstrating the reversibility of the present reaction system. Such reversibility could consequently be used for real-time PPase activity assay, as demonstrated later. To investigate the utility of the reversibility described above for real-time PPase activity assay, the interference from the products produced from the PPase-based biocatalytic reaction was studied. Although the direct product of PPase-catalyzed hydrolysis of PPi is PO43−, the protonation of PO43− is always present in the aqueous solution at physiological pH.11 Therefore, all the phosphate-related anions (Pi), including PO43−, HPO42−, and H2PO4−, were investigated in this study. As shown in Figure 2 B, the addition of Cu2+ essentially results in the aggregation of Au-NPs due to the coordination between cysteine and Cu2+ (Figure 2 B, vial 5, blue curve). However, the presence of PPi essentially prevents the aggregation of Au-NPs caused by Cu2+, resulting in a red-colored dispersion (Figure 2 B, vial 1, black curve). Different from this, the addition of PO43−, HPO42−, or H2PO4− does not prevent this aggregation (Figure 2 B, vial 2, red curve; vial 3, green curve; vial 4, pink curve), essentially demonstrating these products do not interfere with the PPase activity assay because of their weaker coordination capability with Cu2+, as compared with that of cysteine. The large difference in the capability between PPi and other phosphate anions to compete with cysteine in the coordination with Cu2+ could also be evidently seen from the A522/A650 values in Figure 2 C. This feature actually forms the basis for the development of our technically simple yet effective colorimetric method for real-time PPase activity assay, as demonstrated below. Figure 3 A depicts the typical time-dependent UV−vis spectra of a Au-NP dispersion containing cysteine, PPi, Cu2+, and PPase. The spectra were consecutively recorded every 2 min at 25 °C immediately after addition of PPase to the aqueous dispersion consisting of Au-NPs, cysteine, PPi, and Cu2+. After 30 min, the dispersions were photographed, as shown in Figure 3 A. For comparison, the time-dependent UV−vis spectra of the Au-NP dispersion containing cysteine, PPi, and Cu2+ without the addition of PPase were also consecutively recorded (Figure 3 B). As can be seen from Figure 3 A, the addition of PPase clearly results in a gradual decrease in A522 and a gradual increase in A650 at the same time as a function of time. Accordingly, the color of the dispersion also changes from the initial wine red (vial 1) to the final violet blue (vial 2) (Figure 3 A, inset). Contrarily, both the spectrum and the color of the Au-NP dispersion containing only cysteine, PPi, and Cu2+ (i.e., no PPase) remain unchanged (Figure 3 B). Moreover, the sole addition of PPase to the Au-NP dispersion containing cysteine (i.e., no PPi) does not lead to a change of

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RESULTS AND DISCUSSION As displayed in Figure 1 B, the cysteine-stabilized Au-NP dispersion was red in color and exhibited a strong absorption at 522 nm (Figure 1 B, vial 1, black curve). The addition of Cu2+

Figure 1. (A) Schematic illustration of reversibly competitive coordination of Cu2+ between surface-confined cysteine and PPi regulated by PPase. (B) UV−vis spectra and photographs (inset) of the mixtures prepared by separate addition of cysteine (70 μL, 25 μM) (vial 1, black curve), cysteine (70 μL, 25 μM) + Cu2+ (60 μL, 4 mM) (vial 2, blue curve), cysteine (70 μL, 25 μM) + Cu2+ (60 μL, 4 mM) + PPi (60 μL, 38.3 mM) (vial 3, red curve), or cysteine (70 μL, 25 μM) + Cu2+ (60 μL, 4 mM) + PPi (60 μL, 38.3 mM) + PPase (5 μL, 6 U) (vial 4, green curve) to citrate-stabilized Au-NPs (270 μL, 5 nM). 9411

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Figure 2. (A) Schematic illustration of the competitive coordination between cysteine and substrate (PPi) or products (Pi) with Cu2+. (B) UV−vis spectra and photographs (inset) of the mixtures prepared by separate addition of cysteine (70 μL, 25 μM) + pure water (60 μL) + Cu2+ (60 μL, 4 mM) (vial 5, blue curve), cysteine (70 μL, 25 μM) + PPi (60 μL, 2 mM) + Cu2+ (60 μL, 4 mM) (vial 1, black curve), cysteine (70 μL, 25 μM) + PO43− (60 μL, 2 mM) + Cu2+ (60 μL, 4 mM) (vial 2, red curve), cysteine (70 μL, 25 μM) + HPO42− (60 μL, 2 mM) + Cu2+ (60 μL, 4 mM) (vial 3, green curve), or cysteine (70 μL, 25 μM) + H2PO4− (60 μL, 2 mM) + Cu2+ (60 μL, 4 mM) (vial 4, pink curve) to citrate-stabilized Au-NPs (270 μL, 5 nM). The final concentration of these anions in the resulting Au-NP dispersions (600 μL) was 200 μM. (C) Relative signal intensity of A522/A650 for different anions as indicated in the figure. The A522/A650 values were calculated from the data from (B). Error bars show the standard deviations (n = 3).

Figure 3. (A) UV−vis spectra of the dispersion prepared by adding PPase (5 μL, 1.0 U) to the mixture consisting of Au-NPs (270 μL, 5 nM), cysteine (70 μL, 25 μM), PPi (60 μL, 2 mM), and Cu2+ (60 μL, 4 mM). UV−vis spectra were consecutively recorded every 2 min shortly after the addition of PPase. Inset: Photographs of the dispersion at 0 min (vial 1) and 30 min (vial 2) after the addition of PPase. (B) UV−vis spectra of the dispersion prepared by adding buffer (5 μL, pH 6.5, 1.2 mM MgCl2) solution to the mixture consisting of Au-NPs (270 μL, 5 nM), cysteine (70 μL, 25 μM), PPi (60 μL, 2 mM), and Cu2+ (60 μL, 4 mM). UV−vis spectra were also recorded every 2 min. Inset: Photographs of the dispersion at 0 min (vial 1) and 30 min (vial 2). The final volume of each resulting mixture was adjusted to 600 μL with Milli-Q water.

on the PPase-regulated competitive coordination of Cu2+ between cysteine and PPi with PPase-catalyzed PPi consumption as a parameter. To estimate the kinetics of the PPase-catalyzed PPi consumption, we summarized the time-dependent A650/A522 values from the UV−vis spectra of Au-NPs containing cysteine, PPi, and Cu2+ with the presence of different activities of PPase (data from Figure 3 A and Figure S2, Supporting Information). As demonstrated previously,5,6 the A650/A522 values could be

the UV−vis spectrum of the dispersion (Figure S1, Supporting Information). These results strongly suggest that PPase can remarkably catalyze the hydrolysis of PPi, resulting in the consumption of PPi in the Au-NP dispersion. More importantly, we found that the rates of the decrease of A522 and increase of A650 become more dramatic with increasing activity of PPase in the dispersions (Figure S2, Supporting Information), suggesting the validity of the Au-NP-based colorimetric method for real-time PPase activity assay based 9412

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Figure 4. (A) Kinetic plots of time-dependent A650/A522 values versus those with different activities of PPase present. Digital photographs of Au-NP dispersions at a time point of 30 min after the addition of different activities of PPase are shown on the right. (B) Calibration curve for PPi without the addition of PPase. (C) Time-dependent consumption of PPi by converting A650/A520 values recorded in (A) into the logarithm of the PPi concentration. (D) Initial rate of enzymatic reaction (v0) versus PPase activity. Inset: linear range of v0 versus CPPase. Error bars indicate standard deviations (n = 3).

Figure 5. (A) UV−vis spectra and photographs (inset) of the dispersions prepared by adding NaF-treated PPase (65 μL, 0.2 U) to the mixtures containing Au-NPs (270 μL, 5 nM), cysteine (70 μL, 25 μM), PPi (60 μL, 2 mM), and Cu2+ (60 μL, 4 mM). The UV−vis spectra were recorded 30 min after the addition of NaF-treated PPase. Inset, from vial 1 to vial 7: the NaF concentrations were 500, 100, 50, 10, 5, 1, and 0.1 μM. (B) Kinetic plot of A650/A522 versus the logarithm of the NaF concentration. The final volume of the resulting dispersion was adjusted to 600 μL with Milli-Q water. Error bars indicate standard deviations (n = 3).

4 A, right panel). These results demonstrate that the kinetics of Au-NP aggregation becomes faster when the activity of PPase in the dispersions is increased through the mechanism shown in Scheme 1 A. To obtain the time-dependent changes of the logarithm of the PPi concentration in the dispersions, A650/A522 values were converted into the concentration of PPi in the dispersions according to the linear relationship between A650/

considered as an indicator of the degree of aggregation/ dispersion of Au-NPs. As typically displayed in Figure 4 A, the A650/A522 values increase with increasing PPase activity. Moreover, the color of the aqueous dispersions of the AuNPs containing cysteine, PPi, Cu2+, and PPase gradually changed from wine red to violet blue at the time point of 30 min when the PPase activity increased from 0 to 1.0 U (Figure 9413

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A522 and the logarithm of the PPi concentration (A650/A522 = 1.013 − 0.2798 log(Cppi/μM), R = 0.9979) (Figure 4 B). The initial reaction rates (v0) were then obtained by calculating the slopes of the initial part of the kinetic curves of different PPase activities (Figure 4 C). Under the experimental conditions employed here, the v0 values were linear with the PPase activity within the range from 0.025 to 0.4 U (Figure 4 D, inset) (v0 = 0.003 + 0.116(CPPase/U), R = 0.9929). The linear relationship also indicates that the reaction was kinetically controlled by PPase and thus could be used for the PPase activity assay. The detection limit, based on a signal-to-noise ratio of 3, was calculated to be 0.010 U. These results substantially validate our colorimetric method for real-time PPase activity assay. Having demonstrated the validity of our colorimetric method, we next investigated the application of this method to evaluating the enzyme inhibitor efficiency. It has been reported that sodium fluoride (NaF) could inhibit the activity of PPase through the formation of the [F − −PPase] intermediate, resulting in an instant decrease in enzyme activity.12 For the control experiment, we first studied whether NaF could induce the aggregation of the cysteine-stabilized AuNPs. We found that the sole addition of NaF to the Au-NP dispersion does not lead to any change either in color or in the UV−vis spectrum (Figure S3, Supporting Information), essentially demonstrating that NaF itself could not result in the aggregation of the cysteine-stabilized Au-NPs. This feature well validates the colorimetric method developed in this study for evaluating the inhibitor efficiency of NaF. To demonstrate this validity further, PPase (0.2 U) was preincubated with 60 μL of aqueous solutions of NaF with different concentrations (i.e., 500, 100, 50, 10, 5, 1, and 0.1 μM) for 15 min at room temperature. Then different concentrations of NaF-treated PPase were respectively added to the Au-NP dispersions (460 μL) containing cysteine, PPi, and Cu2+. After incubation for an additional 30 min, the resulting mixtures were subjected to the UV−vis measurements. As displayed in Figure 5 A, with decreasing NaF concentration from 500 to 0.1 μM, the color of the Au-NP dispersions gradually changed from wine red to purple (inset, from vial 1 to vial 7), corresponding to the decrease in A522 and the increase in A650. It has been reported that PPase activity could be inhibited by micromolar fluoride.12a,c Herein, we found that 500 μM NaF could remarkably inhibit the activity of 0.2 U of PPase since there was no obvious change in either the UV−vis spectra or the color of the dispersions (Figure 5 A, vial 1, black curve). Moreover, IC50 (the half maximal inhibitory concentration) represents the concentration of an inhibitor that is required for 50% inhibition of an enzyme, which could be used as a parameter to evaluate the inhibitors. As shown in Figure 5 B, a sigmoidal profile was obtained by plotting the A650/A522 ratio vs the logarithm of the NaF concentration. The IC50 value of 0.2 U of PPase was calculated to be about 7.076 ± 1.754 μM, which was consistent with the values reported previously.12a,c These results essentially demonstrate that our colorimetric method could also be used to evaluate the inhibitor efficiency.

PPi consumption possesses a near real-time analytical feature. The excellent analytical properties of the colorimetric method demonstrated here substantially enable its application in PPase activity assay and PPase inhibitor efficiency evaluation. This study not only offers a simple yet effective method for real-time PPase activity assay but also opens a new way to the development of colorimetric methods based on rational design of surface chemistry of Au-NPs through reversibly competitive coordination chemistry.

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Authors

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is financially supported by the National Natural Science Foundation of China (Grants 21321003, 20935005, 21127901, 21210007, and 91213305 to L.M. and 21322503 and 91132708 to P.Y.), the National Basic Research Program of China (973 programs, Grants 2010CB33502 and 2013CB933704), and The Chinese Academy of Sciences (Grant KJCX2-YW-W25).

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REFERENCES

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CONCLUSIONS In summary, we have for the first time demonstrated a simple and yet effective colorimetric method for real-time PPase activity assay based on PPase-regulated reversibly competitive coordination of Cu2+ between cysteine and PPi. In addition to the good linearity and low detection limit, the colorimetric method for PPase activity assay based on the PPase-catalyzed 9414

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Analytical Chemistry

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dx.doi.org/10.1021/ac402524e | Anal. Chem. 2013, 85, 9409−9415