Colorimetric Logic Gate for Pyrophosphate and Pyrophosphatase via

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A Colorimetric Logic Gate for Pyrophosphate and Pyrophosphatase via Regulating the Catalytic Capability of Horseradish Peroxidase Chuanxia Chen, Dan Zhao, Jian Sun, and Xiurong Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10712 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 12, 2016

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ACS Applied Materials & Interfaces

A Colorimetric Logic Gate for Pyrophosphate and Pyrophosphatase via Regulating the Catalytic Capability of Horseradish Peroxidase Chuanxia Chen †‡, Dan Zhao †‡, Jian Sun † and Xiurong Yang *, † †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: Pyrophosphate, Pyrophosphatase, Colorimetric, Horseradish peroxidase, “INH” logic gate.

ABSTRACT: By regulating the catalytic capability of horseradish peroxidase (HRP), an artful colorimetric assay platform for pyrophosphate (PPi) and pyrophosphatase (PPase) was unprecedentedly designed. In this work, Cu(I), generated by reducing Cu(II) in the presence of ascorbate, could inhibit HRP’s catalytic capability of transforming colorless 3,3’,5,5’tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The robust coordination between PPi and Cu(II) is able to discourage the reduction of Cu(II) to Cu(I) effectively, thus restoring the original catalytic capability of HRP and regenerating blue-colored oxTMB. Upon PPase introduction, PPi would be hydrolyzed into orthophosphate, which could release Cu(II) free from the Cu(II)-PPi complex, and thus in turn allows the catalytic capability of HRP to be inhibited by Cu(I). HRP was activated or deactivated to different degrees depending on PPi or

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PPase levels, which could be indicated by using HRP-triggered catalytic system as a signal amplifier, thus paving a way for PPi and PPase sensing. Based on the colorimetric sensor for PPi and PPase, an “INH” logic gate was rationally constructed. With the merits of high sensitivity and selectivity, cost-effectiveness and simplification, our proposed analytical system has also been verified to have potential to be utilized for enzyme inhibitor screening and diagnosis of PPase-related diseases.

INTRODUCTION

Pyrophosphate (PPi), as a type of biologically crucial anions, is the hydrolysis product of adenosine triphosphate under cellular conditions, and it performs important functions in pathological, energetic and metabolic progress.1-2 Inorganic pyrophosphatase (PPase) could specifically catalyze the hydrolysis of inorganic PPi into orthophosphate (Pi), which is an efficient exergonic process, providing thermodynamic energy and phosphate substrates for several biosynthetic reactions and being able to control the PPi level in cells.3-5 Moreover, PPase has been proved to play essential roles in phosphorus metabolism, carbohydrate metabolism and evolutionary

events.6-8

Accordingly,

certain

diseases,

including

colorectal

cancer,

hyperthyroidism and lung adenocarcinomas, are associated with the abnormal expression level of PPase.9 In this context, a simple, sensitive, cost-effective and convenient assay for PPase activity is urgently needed for elucidating the PPase-related biological processes and diseases.

A few conventional and elegant methods have so far been developed and devoted to assay PPase activity, such as practical automated method utilizing robotics10 and chromatography.11 Dismayingly, they are generally limited by the specialized instruments, expensive materials or laborious, time-consuming operations. To get out of these predicaments, optical approaches have


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been widely utilized for bioanalysis because of their distinct merits, such as simplicity, low-cost, rapidity and high sensitivity, and even could be readily monitored by naked eyes without resorting to complicated equipment. Actually, a few organic dye-based colorimetric and fluorescent chemosensors have been exploited for PPase activity detection based on click chemistry,12 or the discrimination between Pi product and PPi substrate directly by these organic probes13-15 or bridged by Cu(II).16 Additionally, the rapid development of nanotechnology has tremendously fueled the growth of analysis and testing technology.17 Some colorimetric18 and fluorescent nanomaterials19-20 have been designed into optical sensors for determination of PPase activity by assessing the enzymatic hydrolysate-induced release of inorganic Cu(II) from the Cu(II)−PPi complex. Synthesis as well as modification and purification of organic probes or nanomaterials is required in these previously reported optical methods, which severely impedes their practical applications. Consequently, simple, flexible and effective methods for PPase detection are still highly desired to keep pace with expectations in future point-of-care testing. Colorimetric assays that employ the chromogenic reactions catalyzed by enzyme or enzyme mimics (nanomaterials or metal ions), could endow detection an excellent ultrasensitivity and ease operation. This is benefited from the intrinsic amplification of signal in an exponential manner induced by enzyme or enzyme mimics,21 as well as the easily achieved signal output with the aid of simple instrument or visually. During these catalyst-mediated chromogenic reactions, colorless substrates, like 3,3’,5,5’-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) or o-phenylenediamine (OPD), are readily oxidized to their colored derivatives by H2O2 under the catalysis of natural peroxidase,22 enzyme mimics23-24 or Fenton-reagent.25-26 What is more, horseradish peroxidase (HRP) is extensively used in


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chemical analysis27-31 due to its exceptional performances including superior stability, rapid response, remarkable catalytic efficiency, low cost and splendid biocompatibility. In this work, we proposed an artful colorimetric assay platform for PPi and PPase activity by regulating the catalytic capability of HRP. The sensing principle is based on the robust coordination of PPi with Cu(II), the hydrolysis of PPi catalyzed by PPase, the reduction of Cu(II) to Cu(I) by ascorbate and the Cu(I)-mediated catalytic capability of HRP, which could in rotation initiate the HRP-catalyzed chromogenic reaction of TMB to act as an amplifier system. The analysis is readily achieved by measuring absorbance change or observing color variation. Furthermore, an ‘INH’ logic gate is rationally developed rooted in the restoration and inhibition of HRP’s catalytic capability induced by PPi and PPase. EXPERIMENTAL SECTION Chemicals and apparatus. Horseradish peroxidase was purchased from Shanghai Sangon Biotech. Co., Ltd. (China). Pyrophosphatase and sodium ascorbate were obtained from SigmaAldrich (America). Pyrophosphate and CuSO4 were bought from Beijing Chemical Reagent Co. (China). TMB solution was purchased from eBioscience, Inc (America). All the other reagents were analytical grade, and used as received. Clinical human serum samples were gifted from the Second Hospital of Jilin University (China). The absorbance was collected from an EL 808 ultra microplate reader (Bio-Tec instrument) using 96-well plate. UV-vis spectra were surveyed on a Cary 50 UV-vis spectrophotometer (Varian). The X-ray photoelectron spectroscopy (XPS) samples were analyzed by an ESCALAB MK II spectrometer (VG Scientific) with Al Kα radiation as the X-ray source. Peak position


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were internally referenced to the C1s peak (284.6 eV). The pH values of buffer solutions were adjusted and measured using a pH-10 pH meter (Sartorius). Detection of PPi. Briefly, 25 µL of 14 µM CuSO4, 25 µL of PPi with different concentrations, 5 µL of 100 µM sodium ascorbate and 35 µL of 5.75 mU/mL HRP were gradually added into each well of a 96-well plate that containing 50 µL Tris-HCl buffer (50 mM, pH 7.0) and 30 µL H2O. After incubated at 37°C for 30 min, 30 µL of TMB was introduced for color development. Then 50 µL of 2 M H2SO4 was used to stop the enzymatic reaction. Optical densities of resultant solutions in each well were recorded at 450 nm using the microplate reader. Detection of PPase. Into a 96-well plate, each well containing 50 µL Tris-HCl buffer (50 mM, pH 7.0) and 20 µL Mg2+ (100 µM) was sequentially loaded with 25 µL of 30 µM PPi and 10 µL of PPase with various concentrations, and then incubated at 37°C for 30 min. 25 µL of 14 µM CuSO4, 5 µL of 100 µM sodium ascorbate and 35 µL of 5.75 mU/mL HRP were gradually added. After incubated at 37°C for 30 min, 30 µL TMB was introduced for color development. Then 50 µL of 2 M H2SO4 was used to stop the enzymatic reaction. Optical densities of resultant solutions in each well were recorded at 450 nm using the microplate reader. PPase Inhibition Assay. First, 30 µL NaF with various concentrations were initially added into 10 µL of 25 mU/mL PPase solutions, and incubated at 25°C for 15 min. Then, the mixture solution consisting of 50 µL Tris-HCl buffer (50 mM, pH 7.0), 20 µL Mg2+ (100 µM) and 25 µL of 30 µM PPi was sequentially added into these NaF-treated PPase solutions, and incubated at 37°C for 30 min. Subsequently, 25 µL of 14 µM CuSO4, 5 µL of 100 µM sodium ascorbate and 35 µL of 5.75 mU/mL HRP were gradually added. After incubated at 37°C for 30 min, 30 µL TMB was introduced for color development. Finally, 20 µL of 5 M H2SO4 was used to stop the


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enzymatic reaction. Optical densities of resultant solutions in each well were recorded at 450 nm using the microplate reader. RESULT AND DISCUSSIONS The design and establishment of the sensing system. The working principle of the designed method for determinating the PPi and PPase activity is schematically shown in Scheme 1. As is well-known, native HRP could catalyze the oxidization of colorless TMB to produce a high colorimetric signal due to its intrinsic enzymatic activity. Whereas, the enzymatic activity of HPR could be efficiently hampered by Cu(I) due to the interaction of Cu(I) with amino acid (including phenylalanine, tyrosine and tryptophan) residues in HRP.32 As Cu(I) is not stable enough in solution, in-situ generation of Cu(I) is an imperative. Evoked by this, ascorbate is chosen to obtain Cu(I) by reducing Cu(II). The copper-regulated HRP's enzymatic activity could be developed for analyzing the PPi and PPase activity. Theoretically, PPi could strongly coordinate with Cu(II) to form a Cu(P2O7)26- complex,18,

20, 33

which would hinder the

transformation of Cu(II) into Cu(I) induced by ascorbate, thus restoring the enzymatic activity of HPR. After incubated with PPase, PPi is hydrolyzed into orthophosphate which could not coordinate with Cu(II). Therefore, the free Cu(II) could be reduced to Cu(I) and then hampers the enzymatic ability of HPR again. By using the HRP-catalyzed chromogenic reaction of TMB as a signal amplifier, the colorimetric signal varies depending on the levels of target. A simple, sensitive and cost-effective colorimetric assay platform for selective determination of PPi and PPase activity was thus proposed.


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Scheme 1. Illustration of the biosensing strategy for the detection of PPi and PPase activity via regulating the catalytic capability of HRP. The feasibility of the proposed sensing protocol was primarily evaluated. TMB solution was colorless in the absence of HRP (Figure 1a), while oxidized to generate its blue product (oxTMB) under the catalysis of HRP (Figure 1b). The blue derivative changed into a yellow one after H2SO4 was added, exhibiting an absorption band around 450 nm (Figure S1, ESI). Redox reaction of Cu(II) with sodium ascorbate help to in situ generate Cu(I), which faded the color of oxTMB solution and weakened the absorbance by hampering the catalytic capability of HRP (Figure 1e and Figure S1, ESI). However, no obvious colorimetric change was observed when individual Cu(II) or sodium ascorbate was introduced (Figure 1c, d), undoubtedly indicating a much lower inhibiting ability of Cu(II) or sodium ascorbate alone for HRP compared with their mixture. However, if Cu(II) was preferentially mixed with PPi, it lost the opportunity to be reduced to Cu(I) by sodium ascorbate. So the catalytic capability of HRP was barely affected, and the oxTMB remained its native high colorimetric signal (Figure 1f). After incubated with PPase, Cu(II) was in free and could be reduced to Cu(I) due to the hydrolysis of PPi, which in turn inhibited HRP’s activity and thus bleached the color of solution, with a decrease in absorbance (Figure 1g and Figure S1, ESI). The aforementioned results confirmed the method


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was feasible. To make the sensing principle more reliable, the effects of individual Pi, PPi and PPase on the catalytic capability of HRP in the absence of Cu(II) were taken into consideration. No distinguishable alteration in the absorbance signal of the sole HRP-TMB without Cu(II) was observed in the presence of individual Pi, PPi or PPase (Figure S2, ESI), implying the lack of direct interactions between HRP and these substances. This result further confirmed that only the interaction of HRP with Cu(II)-ascorbate could lead to massive colorimetric signal change. The subsequent PPi or PPase activity sensing was accomplished by means of interacting with Cu(II) directly or indirectly.

Figure 1. Absorbance intensities at 450 nm and the photographs of the mixtures prepared by separate addition of none (a), HPR (b), HRP + sodium ascorbate (c), HRP + Cu(II) (d), HRP + sodium ascorbate + Cu(II) (e), PPi + Cu(II) + sodium ascorbate + HRP (f) and PPase + PPi + Cu(II)+ sodium ascorbate + HRP (g) to TMB solutions. Error bars represent the standard deviations (n = 3). Construction of an “INH” logic gate for PPi and PPase. Originated from the activated and deactivated catalysis of HRP upon the oxidization of TMB induced by PPi and PPase, an “INH” logic gate was legitimately designed (Figure 2A). The corresponding colorimetric signals, the histograms and the truth table were presented in Figure 2A, Figure 2B and Figure 2C,


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respectively. In this logic gate, PPi and PPase were employed as inputs, and the normalization absorbance at 450 nm (NA450) was acted as output signal. For input, the presence of PPi or PPase was defined as “1” state, and their absence as “0” state. For output, large NA450 value ( > 0.35) and small NA450 value ( < 0.35) were defined as “1” and “0”, individually. We can also utilize the color change as the output by defining the blue color of the system as the “1” state and the nearly colorless solution as the “0” state. With no input (0, 0) or with PPase input (1, 0) alone, the output was “0”. When subjected to PPi (0, 1) alone, the system solution was blue and NA450 increased sharply, giving an output signal of “1”. With the two inputs appeared together (1, 1), the NA450 was extremely weak and the output was returned to “0”, indicating PPase could efficiently inhibit the PPi-activated output signal. Thus, a Boolean logic gate was rationally constructed using PPi and PPase as inputs, and further utilized for PPi and PPase detection.

Figure 2. Schematic diagram and colorimetric signals (A), histograms (B), truth table (C) of the “INH” logic gate. Inset in (A) is the photographs of color change with different inputs. Error bars in (B) represent the standard deviations (n = 3). Detection of PPi. Typically routes to detect PPi have been established by virtue of the coordination of PPi with certain metal ions, mainly Zn(II),34-35 Fe(III)36 and Cu(II).37-38 Among


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these metal ions, Cu(II) is preferred because of its better distinguish ability for PPi from other phosphate-related anions, which could be easily applied to optical sensing via integrating with chromogenic or fluorogenic reactions. In this work, a HRP-participated chromogenic reaction was employed, in which the HRP dealt with signal generation and signal amplification via catalyzing. An extremely high sensitivity thus could be anticipated. Firstly, the coordination between PPi and Cu(II) was evaluated. XPS measurements were performed to survey the change in oxidation state of copper element after binding with PPi and being reduced by sodium ascorbate. The results were shown in Figure S3 (ESI). A characteristic Cu 2p3/2 binding energy peak around 934 eV and a corresponding satellite peak around 942.7 eV were observed for untrammeled Cu(II).39-40 No obvious shift in binding energy was observed for Cu(P2O7)26- complex. However, when reacted with sodium ascorbate, the Cu 2p3/2 binding energy peak was changed to ~932 eV accompanying with the disappearance of the satellite peak around 942.7 eV, indicative of the standpoint that Cu(II) could be reduced to Cu(I) by sodium ascorbate,39 which has been well accepted up to now.12 After adding sodium ascorbate to the inadequate PPi-treated Cu(II), Cu 2p3/2 binding energy peaks around 934 eV and 932 eV appeared simultaneously, indicating the coexistence of Cu(II) and Cu(I). The incomplete reduction of Cu(II) was ascribed to the inhibiting effect of PPi rooted in the robust affinity of PPi to Cu(II). In addition, the coordination of Cu(II) with PPi has proved to debase the copper oxidation−reduction potential significantly,20 which helps in understanding the reason why PPi could inhibit the redox reaction between Cu(II) and sodium ascorbate. It follows that copper element was in its monovalent or/and divalent form depending on whether or how much sodium ascorbate or/and PPi were added, thus launching the colorimetric assay for PPi.


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Figure 3. (A) Absorbance change with the increase of PPi concentration from 20 to 6000 nM. Inset shows the corresponding color change. (B) Calibration curve and linear range of this assay for PPi detection. (C) and (D) individually represent the response of absorbance with different inorganic anions solely or coexisting with PPi. Insets are the color changes of the solution with typical anions. PPi is 3 µM, and other anions are 20 µM. Error bars represent the standard deviations (n = 3). As depicted in Figure S4 A (ESI), nearly 1400 nM Cu(II) could weaken the catalytic capability of HRP to the fullest (nearly 80%). The absorbance of the sensing system for PPi has negligible alteration when Cu(II) was below 1400 nM (Figure S4 B, ESI), implying that an identical sensitivity was achieved in the 5-1400 nM concentration range. Further increasing the Cu(II) concentration would not be in favor of the highly sensitive quantification of PPi and then PPase. Additionally, a wider dynamic range could be achieved with higher level of Cu(II). For the sake of a good sensitivity and a wide dynamic range, 1400 nM Cu(II) was used for the following experiments. The absorbance/sensitivity increased progressively with the increase of the HRP concentration, and tended to be constant when HRP exceeded 0.805 mU/mL (Figure S4 C, ESI). Thus, 0.805 mU/mL was used as the optimal concentration for HRP. To obtain the


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calibration curve, PPi samples with contents ranging from 20 to 6000 nM were prepared. The solution color and measured absorbance at 450 nm increased by degrees as increasing PPi concentration from 20 to 3000 nM, and tended to be constant when the PPi concentration exceeded 3000 nM (Figure 3A and Figure S5, ESI). The absorbance was linearly correlated with Log(PPi) in the concentration range of 20-3000 nM (Figure 3B). The limit of detection (LOD) was calculated to be 3.1 nM based on 3σ/s, which was superior to most reported organic dyebased colorimetric sensors41-44 and AuNP-based colorimetric probes.37, 45-46 Recoveries of PPi in several water samples and serum samples were studied. Satisfactory recoveries between 96% and 109.3% were obtained, which was acceptable (Table S1, ESI). In addition, we examined the selectivity of this method for PPi detection. Inorganic orthophosphate anions, including PO43-, HPO42- and H2PO4- (20 µM) were found to have no conflict with PPi detection no matter they were solely added or coexisted with PPi (Figure 3C and D). This could be ascribed to their weaker coordination capability with Cu(II) compared with that of PPi, by which the reduction of Cu(II) is hardly blocked. The discernibility for PPi among these orthophosphate anions is crucial for the later detection of PPase as orthophosphate is generated during the hydrolysis of PPi catalyzed by PPase. Other anions, including F-, Cl-, Br-, I-, S2-, SO42-, SO32-, SCN-, CO32-, C2O42-, NO3- and NO2-, could not obviously disturb the recognition of PPi too (Figure 3C and D). Moreover, other phosphate-related anions, such as ATP, ADP or AMP (3000 nM) were also taken into account. The results in Figure S6 (ESI) depicted that the presence of ATP or ADP had a little disturbance on PPi detection either in the absence or presence of PPi (1000 nM), and the influence from AMP was negligible. This could be ascribed to the rather strong binding affinity of ATP or ADP to Cu(II), although it was much


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weaker than that between PPi and Cu(II). Therefore, our proposed assay is somewhat limited when being applied to real samples with high levels of these potential interfering biomolecules. Detection of PPase. The above discussions have manifested that PPi could sensitively and selectively initiate the HRP-triggered chromogenic reaction via hampering the transformation of Cu(II) to Cu(I), thus affording the possibility to detect PPase with good performance. As the sensitivity improvement of PPase assay relies on the concentration decline of used substrate PPi, 3000 nM PPi was used for this PPase assay. Then, the reaction time between PPi and PPase was studied for PPase detection, the one between Cu(II)-ascorbate and HRP was also investigated, both of which were set to 30 min (Figure S7, ESI).

Figure 4. (A) Absorbance and color changes with the increase of PPase concentration from 0.02 to 10 mU/mL. Inset is the calibration curve and linear range of this PPase sensor. (B) represents the response of absorbance with different proteins and enzymes, and the corresponding photographs. Error bars shown the standard deviations (n = 3). The absorbance at 450 nm was systematically decreased (Figure 4A and Figure S8, ESI) and linear to Log(PPase) in the concentration range of 0.02-6.0 mU/mL (Inset of Figure 4A), accompanied by the visually gradual color fading. The LOD based on 3σ/s was estimated to be 0.012 mU/mL, which was at least 10 times more sensitive than that reported for most


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colorimetric and fluorescent assays listed in Table S2 (ESI). The color difference in the absence and presence of various PPase levels was readily distinguishable by naked eyes under room light, which permitted the visual detection of PPase qualitatively and semi-quantitatively without any equipment. In this regard, when the PPase concentration was over 0.02 mU/mL, an obvious color bleaching could be differentiated from the initial solution with naked eyes. The selectivity was also evaluated. Some proteins or enzymes, including bovine serum albumin (BSA), human serum albumin (HSA), glucose oxidase (GOx), trypsin and lysozyme were performed as control. The results persuasively showed that none of these representative interferents, except PPase, could evoke conspicuous colorimetric signal (Figure 4B) and disturb the analysis of PPase activity. Hence, the interference from others could be ignored, manifesting a satisfactory selectivity for PPase detection. This could be ascribed to the highly specific hydrolysis of PPi catalyzed by PPase. Other species are unable to destroy the strong Cu(II)-PPi complex, also the reduction reaction of Cu(II) cannot proceed, and thus no Cu(I) could be generated to inhibit the catalytic capability of HRP. The practicability of this sensing platform was demonstrated by detecting PPase in diluted serum samples (1%). The colorimetric response results were similar with those of the standard system, and a calibration curve could be achieved in the PPase concentration range of 0.1-3.0 mU/mL (Figure S9, ESI). With the calibration curve in serum, four serum samples spiked with different concentrations of PPase were then analyzed. Satisfactory recoveries between 92.7% and 104.0% were obtained (Table S3, ESI), indicating the potential of this method for applications of detecting PPase activity in complicated real samples.


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Inhibition of PPase activity. We also investigated the possibility of the proposed assay for evaluating the PPase inhibition by using an acknowledged PPase inhibitor (NaF) as a model. As displayed in Figure 5 and Figure S10 (ESI), as increasing NaF concentrations from 0 to 200 µM, PPase activity (1.0 mU/mL) was gradually inhibited, corresponding to a gradual decrease in absorbance. The IC50, the inhibitor concentration required for 50% inhibition of the enzyme activity, was figured out to be approximately 5.24 µM. The value was comparable to those previously reported,20,

47

proving our strategy was suitable for screening inhibitor as well as

evaluating the inhibition efficiency.

Figure 5. The inhibition effect of NaF on PPase activity. Error bars represent the corresponding standard deviations (n = 3). To exclude the disturbance from NaF-induced inhibition of HRP activity, we studied how NaF exhibited inhibition effect on the HRP-catalyzed TMB oxidization. As shown in Figure S11 (ESI), as NaF exceeded 0.15 mM, the HRP activity was inhibited gradually with the increase of NaF concentrations. Whereas, the inhibition effect of NaF in the 0.01-0.15 mM concentration range on the HRP activity was negligible, thereby no correction for NaF-induced inhibition of HRP activity was needed. It might be attributed to the fact that at neutral pH the active site of


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HRP is not accessible for inhibitors (NaF, in this work) as it is located in the inner hydrophobic part of the molecule.30, 48 CONCLUSIONS In summary, a colorimetric sensing platform for PPi and PPase with high sensitivity and selectivity was ingeniously designed and established by progressively modulating the catalytic capability of HRP. The robust affinity of PPi with Cu(II), the PPase-catalyzed hydrolysis of PPi, the reduction of Cu(II) to Cu(I) by ascorbate and the Cu(I)-mediated catalytic capability of HRP in rotation initiate the HRP-catalyzed oxidization of TMB, facilitating the colorimetric detection of PPi and PPase by observing color variation or measuring absorbance change. The system possesses some fantastic features: (a) Distinguishing PPi from Pi not only enables selective detection of PPi, but also makes the following analysis of PPase possible. (b) The specificity of enzyme reactions results in an excellent selectivity for PPase detection. (c) The signal amplification derived from catalysis of commercially available HRP endows this method high sensitivity. (d) Simple visual observation. The linear ranges for PPi and PPase activity detections were 20-3000 nM and 0.02-6.0 mU/mL, respectively. The LODs were 3.1 nM and 0.012 mU/mL, for PPi and PPase, separately. Additionally, an “INH” logic gate was successfully devised on the basis of activated and deactivated catalysis of HRP upon the oxidization of TMB induced by PPi and PPase. The proposed analytical system has also been verified to have potential to be utilized for enzyme inhibitor screening and diagnosis of PPase-related diseases. ASSOCIATED CONTENT Supporting Information


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The UV-vis spectra; control experiments; XPS spectra; effect of Cu(II) and HRP concentrations; PPi detection in real samples; interferences from ATP, ADP, AMP; incubation time; PPase detection in serum samples; comparison with reported methods; inhibition effect of NaF. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Fax: +86 431 85689278. E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21435005, 21627808) and the Development Project of Science and Technology of Jilin Province (No. 20150520011JH). REFERENCES (1)

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Table of Contents Graphic and Synopsis


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Colorimetric Logic Gate for Pyrophosphate and Pyrophosphatase via

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