Switching Peroxidase-mimic Activity of Protein Stabilized Pt

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Switching Peroxidase-mimic Activity of Protein Stabilized Pt Nanozymes by Sulfide Ions: Substrate Dependence, Mechanism and Detection Yan Liu, Yuanlin Zheng, Ding Ding, and Rong Guo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03430 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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Switching Peroxidase-mimic Activity of Protein Stabilized Pt Nanozymes by Sulfide Ions: Substrate Dependence, Mechanism and Detection Yan Liu,* Yuanlin Zheng, Ding Ding and Rong Guo* School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, Jiangsu, P. R. China ABSTRACT: In the present work, we use β-casein as a model protein to prepare a smart β-casein stabilized Pt nanoparticle (CM-PtNP) with peroxidase mimicking activity, and systematically investigate sulfide-mediated switching effect and mechanism of CM-PtNP nanozyme’s activity. Sulfide-mediated activity switching effect depends heavily on the physicochemical properties of nanozymes and the identity of substrate. On one hand, the binding of sulfide to Pt nanozyme surface leads to the transform from Pt2+ to Pt0, resulting in more active sites and the activity “switching on”; on the other hand, the binding of sulfide ions via Pt-S interaction blocks the active sites, resulting in the activity “switching off”. For substrates 3,3′,5,5′-tetramethylbenzidine (TMB) and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), the two factors play different decisive roles since the interaction of substrate molecules with nanozyme allows their different distributions on nanozyme surfaces. By virtue of this specific response, excellent sulfide colorimetric sensors with different limit of detection were developed based on CM-PtNP with different substrates. This is the first report about a fundamental understanding of how substrates influence the anion-mediated activity switching

effect

by

illuminating

the

nature

of

anion-nanozyme

interaction

and

nanozyme-substrate interaction. This may be useful to rationally predict the environment factors on the activities of the nanozyme and to design an effective signal amplification based on target-induced nanozyme deactivation/activation.

Corresponding Authors

*Fax：+86-514-87311374. Email: [email protected]. [email protected].

1

ACS Paragon Plus Environment

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1. INTRODUCTION Nanomaterial-based artificial enzymes (nanozymes) have been attracting increasing attention in the past few years, since they exhibit several advantages such as low cost, stability against denaturing, and tunability in catalytic activities.1-5 Very recently, chemists have reported the environment factors such as metal ions and small organic molecules existing in biological and environmental systems, are able to switch the enzyme like activity of nanozyme effectively.6-11 Based on this, a facile target-induced nanozyme deactivation/activation has proven to be an effective signal amplification method for fabricating colorimetric sensor. Among these researches, Hg2+ induced nanozyme deactivation/activation has been widely used to a signal amplifier to detect Hg2+ concentrations.12-14 Although Huang and co-workers have recently discovered that some metal ions, such as Hg2+ and Ag+ could greatly enhance the catalytic activity of citrate-capped PtNPs,12,13 it is also found that Hg2+ and Ag+ can inhibit effectively the enzyme activity of protein-capped noble metal nanozyme.15,16 These researches suggest that the same target-induced activity switching effect depends on both the physiochemical properties of the nanozyme core and the surface modifiers. Despite the significant advances of the nanozyme based signal amplification method, its performance is still limited to some extent due to the limited functionality of the used nanozyme and the incomplete understanding of the environmental factors mediated switching mechanism. In addition to the composition, crystallinity, and structure of nanozyme core, the catalytic performance of nanozymes is closely associated with the intrinsic properties of the surface modifier.17-19 Rational selection of modifiers can increase binding affinity and accelerate the catalyzed reaction by bringing substrates into proximity with the nanozyme’s active sites. Proteins have been extensively explored to not only synthesize nanocrystals with small size and excellent monodispersity but also significantly increase the stabilization and biocompatibility of the system.20-22 More importantly, it introduces highly specific or multiple functionalities onto these hybrid nanoparticles based nanozyme for further multifunction application.23,24 Since amino acid composition and sequence as well as mesoscale degree of molecular order play important roles in protein assistant the synthesized process, it is still a great challenge to design and construct multifunctional nanozymes with extremely high enzyme like activity by using proteins. In addition, the rational prediction of the environment factors on these nanozyme’s activity based on deep insights into the activity switching mechanism would be advantageous for 2


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improving its signal amplification performance. Anions (such as S2-, F−, Cl−, Br−, and I−) exist widely in biological, food and environmental areas,25-27 but scarce information is available on anions mediated switching effect and mechanism of nanozyme’s activity. The presence of sulfide in natural water and wastewater derived from various manufacturing or industrial processes has being attracted great attention in recent years. As a traditional environmental pollutant, excessive existence of sulfide can cause serious environmental problems and pose hazards to living systems. This inspires us to carry out a deep research on how sulfide ions influence the structure, stability and activity of nanozymes, and to fabricate the corresponding sensor based on the sulfide-mediated activity switching. Besides its practical functions, this serves as an effective tool to quantitatively investigate the physiochemical processes and sensing mechanisms for nanozyme activity switching by environmental factors. Platinum (Pt) nanomaterials have received increasing attention since they have glamorous features such as small size, excellent catalytic performance, and good biocompatibility.28-31 Recently, Pt nanomaterials have been reported to possess enzyme like activities,31-33 but application of the enzyme-like activities of the Pt nanozymes in biological and environmental areas remains largely unexplored. Thus, controlling the size and structures of Pt nanozyme to achieve superior enzymatic activities and multiple functions is challenging. In the present work, we use β-Casein as a model protein to prepare a smart β-Casein /PtNP nanozyme

with

peroxidase

mimicking

activity,

and

systematically

investigate

the

sulfide-mediated switching effect and mechanism of the peroxidase-mimic activity. The sulfide induced activity switching effect depends heavily on the physicochemical properties of nanozymes and the identity of substrate. By virtue of this specific response, a sulfide colorimetric sensor with excellent sensitivity and selectivity based on CM-PtNP was developed. To the best of our knowledge, this is the first systematic report about anions mediated switching activity of nanozymes with different substrates, which has provided new mechanistic insights into substrates dependent anions-mediated activity switching by illuminating the nature of anion-nanozyme interaction and nanozyme-substrate interaction. This investigation is of particular significance for mechanistic understanding and technological applications of peroxidase nanomimetics for biotechnology, environmental chemistry, and medicine. 3


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2. EXPERIMENTAL SECTION 2.1. Chemicals and Materials. β-Casein was purchased from Sigma (>99%). Hydrogen tetrachloroaurate tetrahydrate (H2PtCl6·6H2O,

99%),

3,3′,5,5′-tetramethylbenzidine

(TMB)

and

2,2′-azinobis

(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma. All other reagents used were of analytical grade from Beijing Company, and ultrapure Millipore water (18.2 MΩ) was used as the solvent. All glassware was washed with aqua regia (HCl: HNO3 in 3:1 ratio by volume), and rinsed with ethanol and ultrapure water. β-Casein powders were dispersed in water under stirring at 50 ◦C, and the dispersions were stored at 4◦C overnight to allow complete hydration. 2.2. Synthesis of β-casein Functionalized PtNPs. Pt nanoparticles were prepared first by mixing 0.4 mL 3 mM H2PtCl6 solution with 40 µL of β-casein aqueous solution with a desired concentration in 2.96mL10mM PBS buffer (pH=7). After stirring 90 min at 45℃, 0.6 mL of ice cold 10 mM NaBH4 was added under stirring. After the complete reaction (5 h), the solution was dialysed with water for 24 h to remove excess β-casein. 2.3. β-casein-PtNPs as Peroxidase Mimetics. To investigate the peroxidase-like activity of the as-prepared β-casein-PtNPs, the catalytic oxidation of the peroxidase substrate TMB in the presence of H2O2 was tested. In a typical experiment, 30 µL of 8.0 mM TMB, 10 µL of the β-casein-PtNPs stock solution, and 60 µL of 4 M H2O2 were added into 2.9 mL of 0.2 M pH 4.0 acetate buffer at 20 °C. The solution was then transferred for UV-vis scanning after incubating for 15 minutes. 2.4. Microcalorimetry. Heats of dilution were measured using a VP-ITC titration microcalorimeter from MicroCal Inc., Northampton, MA at (25 ± 0.1) ℃. Experiments were carried out titrating concentrated sulfide first into buffer and then into β-casein-PtNPs solution. In a typical experiment, β-casein-PtNP solution was placed in the 1.438 cm3 sample cell of the calorimeter and sulfide ion solution was loaded into the injection syringe. Sulfide ion solution was titrated into the sample cell as a sequence of 25 injections of 10 µL aliquots. The duration of each injection was 10 s, and the time delay (to allow equilibration) between successive injections was 240 s. The contents of the sample cell were stirred throughout the experiment at 307 rpm to ensure thorough mixing. Raw data were obtained as a plot of heating rate (µcal s-1) against time (min). These raw data were then integrated to obtain a plot of observed enthalpy change per mole of injected 4


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sulfide (△Hobs, kJ mol-1) against sulfide concentration (mM). Control experiments included the titration of sulfide into buffer, buffer into nanoparticles, and buffer into buffer. The last two controls resulted in small and equal enthalpy changes for each successive injection of buffer and, therefore, will not be further considered in the data analysis. 3. RESULTS AND DISCUSSION 3.1. Characterization of Pt NCs. Pt nanoparticles were synthesized using sodium borohydride as a reducing agent in the presence of β-casein. The reduction of platinum ions ([PtCl6]2-) can be confirmed by UV/vis spectroscopy shown in Figure 1A. The absorption peaks of platinum ions at 260 nm was disappeared as the metallic Pt nanoparticles were produced, and the prepared β-casein-PtNP (CM-PtNP) obtained appears a light brown color (inset in Figure 1A). The transmission electron microscopy (TEM) image in Figure 1B indicates that CM-PtNP is sphere-like shape and the average size of CM-PtNP is calculated to be about 3.8 nm, as judged from image analysis of 200 individual particles (Figure 1B). According to the high-resolution TEM (HRTEM) image of Pt nanoparticles (the inset in Figure 1B), the measured lattice fringe spacing is 0.23 nm, which is consistent with the (111) interplane spacing of face centered-cubic Pt NPs. Figure 1C shows the FT-IR spectra of native β-casein and β-casein stabilized PtNPs. For β-casein, the appearance of a band at 1645 cm-1 indicates an unordered structure of β-casein. However, the predominant band centering at 1638 cm-1 indicates that the extended chain of β-casein after binding to PtNPs. Furthermore, casein consists of a highly repetitive sequence of amino acids of aspartic acid (Asp) and glutamic acid (Glu) (17% Asp and 23% of all residues in casein), and the carboxylic group provides the potential for platinum complexing. As expected, the band at 1447 and 1398 cm−1 due to COO− of Asp and Glu residues changes much in the CM stabilized PtNPs (Figure 1C), which indicates that Asp and Glu residues play important roles in the stabilization of PtNPs. Furthermore, the appearance of the strong peak at 1158 and 1085 cm−1 contributed by –CH3 groups of the hydrophobic amino acids residues such as leucine, valine and phenylalanine residues, indicate that the extended chain of casein. X-ray photoelectron spectroscopy (XPS) data revealed that Pt, carbon, nitrogen and oxygen are present at the surface of PtNPs, indicating that the PtNPs are capped by protein (Figure S1). As shown in Figure 1D, the binding energy of the electron on the Pt 4f7/2 orbital of CM-PtNP can 5


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be deconstructed into Pt2+ and Pt0 components with the binding energies of 72.2 eV (63.74%) and 71.0 eV (36.26%), respectively. Furthermore, the main peak (Pt2+ 4f7/2) of PtNPs (72.2 eV) shifts toward a lower binding energy (ca. 1.0 eV) relative to that of Pt2+ (73.2 eV), suggesting the electrons transfer from the carboxylic group of the proteins to the Pt surface. Thus, the existence of Pt2+ species with high content might be attributed to the coordination between Pt nanoparticles and electron-rich groups of casein such as -COO- groups. The obtained CM-PtNPs are found to be stable over 6 months and do not show any signs of aggregation at 4 ◦C. Here, β-casein can stabilize PtNPs by both the electrostatic effect and the steric effect. 3.2. β-Casein-PtNPs as peroxidase mimetics. The peroxidase-like behavior of the synthesized CM-PtNPs was first examined by using 3,3’,5,5’-tetramethylbenzidine (TMB) as a chromogenic substrate. As shown in Figure 2A, CM-Pt NPs can catalyze the oxidation of TMB by H2O2 to produce the typical blue color reaction very fast. The maximum absorbance of the reaction mixture was 652 nm, which originated from the oxidation of TMB. The control experiments showed that the absorbance of the CM-PtNPs -TMB-H2O2 system was much higher than that of the TMB-H2O2 system, and the CM-PtNPs -TMB system had no absorbance at 652 nm. This indicates that both CM-PtNPs and H2O2 are needed for the reaction, which is similar to horseradish peroxidase (HRP). Accordingly, the catalytic activity of CM-PtNPs also relies on pH and temperature variation, and the optimal pH and temperature are pH 4.0 and 20 ℃, respectively (Figure S2). As shown in Figure S2A, the enzyme activity of CM-PtNPs only changes a little between 10 to 30 ℃, which indicates that CM-PtNPs can be used more freely regardless of the room temperature. The steady-state kinetics investigated by the initial rate method was adopted to confirm the kinetic parameters for better assessing the peroxidase activity of CM-PtNPs. In a certain range of substrate concentration, typical Michaelis–Menten curves were obtained for both TMB and H2O2 (Figure S3). Michaelis–Menten constant (Km) and maximum initial velocity (Vmax) were obtained using Lineweaver–Burk plot, and the results are shown in Table S1. As listed in Table S1, the small apparent Km value of CM-PtNPs with both TMB (0.052 mM) and H2O2 (63.86 mM) as substrates indicates that CM-PtNPs has a high affinity for both TMB and H2O2. This may be related to the surface coated casein and the small sized PtNP. Notably, in addition to a high content of acid amino acid residues, such as aspartate and glutamate residues, β-casein contains 6


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several phosphoserine residues. Although the net charge of β-casein is positive and the zeta potential of CM-PtNP is about 0.4 mV (the isoelectric point of casein being 4.6), there still exist electrostatic attractions between TMB and carboxylate and phosphate groups at pH 4.0. Notably, a rational selection of protein concentration during the synthesizing process is vital to obtain CM-PtNPs with high enzyme activity. The influence of protein concentration on the enzyme activity of CM-PtNPs was investigated (Figure S4). As shown in Figure S4, the enzyme activity of the CM-PtNPs decreases with the increases of casein concentration. A reasonable explanation for this unique behavior is that the coated protein is affecting the proximity of substrates to the nanozyme core, leading to differences in the enzyme activity of nanozyme, which is consistent with our recent studies.24 To further investigate the peroxidase-like activity of CM-PtNPs, the peroxidase-like behavior of CM-PtNPs was also examined by using 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a chromogenic substrate. TMB and ABTS were chosen because they have opposite charge characteristics. TMB contains two amino groups, which likely results in stronger affinity to a negatively charged nanoparticle surface. In contrast to TMB, ABTS containing two sulfo groups, is a negatively charged chromogenic substrate, so there exist electrostatic repulsion between ABTS and β-casein with high content of acidic amino acid residues. As shown in Figure 2B, CM-PtNPs show weak activity towards ABTS, and an oxidized colored product is almost not formed with an absorbance maximum at 420 nm. Thus, CM-PtNPs show significantly lower catalytic activity with ABTS as substrates. Earlier research has demonstrated that the electrostatic action between nanozymes and the substrate matters much in the affinity of substrates towards nanzymes,8,12 which is consistent with our results here. More importantly, caseins can be thought of as amphiphilic block copolymers consisting of blocks with high levels of hydrophobic or hydrophilic amino acid residues.34,35 Therefore, in addition to the electrostatic attraction, there may exists hydrophobic interaction between protein modified PtNPs and the substrates like TMB and ABTS. To confirm this, we study the effect of density of modifier using CM-PtNP (3.8 nm) with the same core size of nanozyme. The surface density of modifier is controlled by incubating CM-PtNP (3.8 nm) in protein solution of different concentrations. Unexpectedly, the peroxidase-like activity of CM-PtNPs exhibits different changing tendency for ABTS and TMB with the increase of protein density (Figure S5). The 7


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activity decreases for TMB and increase for ABTS with the increase of protein density. With the increase of protein density, the hydrophobic interaction between CM-PtNP nanozyme and substrates matters much, resulting in more substrates binding to the nanozyme surfaces and higher activity. On the other hand, more coated protein molecules inhibit the proximity of substrates to the nanozyme core, leading to the lower enzyme activity of nanozyme. Here, the former factor plays a more important role for nanozyme with ABTS as substrate, and hence an enhanced activity with the increase of protein density. On the contrary, the latter factor plays a crucial role for nanozyme with TMB as substrate, and hence a reduced activity with the increase of protein density. The results here further confirm that the electrostatic attraction mainly leads to the binding of TMB to CM-PtNPs, and the hydrophobic interaction mainly results in the binding of ABTS to CM-PtNPs. Understandably, the interaction of substrate molecules with nanozyme may allow their different distributions on nanozyme surfaces. The different binding sites of the two substrates on CM-PtNPs can be demonstrated by the pyrene fluorescence spectra. Pyrene is one of the most popular fluorescent probes of microenvironmental polarity.35,36 Any change in the microenvironmental polarity can be followed by studying the ratio of I1/I3 for pyrene, so pyrene is further used to detect the microenvironment of CM-PtNP. The intensity ratio I1/I3 has a value of 1.78 in CM-PtNP solution, which is similar to the value 1.77 when pyrene is dissolved in the pure water. This indicates the highly polar environment of pyrene molecules in contact with casein due to very low concentration of protein (0.01 mg/mL). As shown in Figure 2B, the addition of ABTS makes the I1/I3 value decreases from 1.71 to 1.07, and the addition of TMB makes the I1/I3 value decreases from 1.71 to 1.52 (Figure 2B). Due to the electrostatic attraction, TMB molecules tend to bind to CM-PtNPs near the negatively charged N-terminal of β-casein, resulting in small change in I1/I3 value. On the contrary, ABTS molecules prefer to bind to nanozyme surface enriched in Pt2+ species via the hydrophobic interaction near the hydrophobic C-terminal. Since pyrene is easy to be located at the hydrophobic microdomain, the binding of ABTS leads to the more obvious change in the microenvironment around pyrene. Notably, the catalytic activity of metallic NPs and clusters is highly related to the valence (oxidation) states of the metal complexes/ions formed on the Pt NP surfaces. Some earlier studies indicated that metallic Pt0 species play crucial roles in the activation of hydrogen peroxide and the contribution 8


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to their catalytic activities.37 Here, in addition to the low affinity of ABTS to CM-PtNPs, ABTS binding to the nanozyme surface enriched in Pt2+ species may also leads to the lower catalytic activity (Figure 5a). 3.3. Sulfide ion Mediated Activity Switching Effect and Mechanism of CM-PtNP Nanozyme. The metal–sulfur bond plays an important role in various areas such as medical, catalysis, electronics, and material science. Thus, the sulfide ions may influence the enzymatic activity of CM-PtNP. To verify this hypothesis, activity assays were conducted to assess the influence of sulfide ions first using TMB as a substrate. The studies were carried out by preincubating CM-PtNP and S2-, and samples without S2- were considered as controls. Here, CM-PtNP activity was suppressed to about 50 % with S2- at a concentration of 2.0 µM (Figure 3A), which indicates that S2- can effectively switch off the enzyme activity of CM-PtNP nanozyme at very low ion concentration. To further demonstrate the activity switching effect by sulfide ions, the influence of sulfide ion on the peroxidase-like of CM-PtNPs were examined by using ABTS as a chromogenic substrate (Figure 3B). Interestingly, different from the activity “switching off” for CM-PtNPs with TMB as substrate, sulfide ions exhibit activity “switching on” for CM-PtNPs with ABTS as substrate. In order to get insight into sulfide ion mediated activity switching mechanism of CM-PtNP, we used TEM, FTIR, fluorescence spectra and XPS measurements. TEM images shows that the size of PtNPs remains almost unchanged after the addition of sulfide ion (Figure 4A). Thus, sulfide-mediated activity switching is not due to the size change of CM-PtNPs. In the FT-IR spectra (Figure 4B), the predominant band centering at 1638 cm-1 restores to 1645 cm-1, which indicates that the extended chain recovers to the unordered structure of β-casein. Furthermore, the peaks at 1158 and 1085 cm−1 contributed by –CH3 groups of the hydrophobic amino acids residues also change after the addition of sulfide ions. This indicates that the binding of sulfide ion to PtNPs makes some amino acid residues release from PtNP surfaces and lead to the change of the protein chain. Furthermore, the microenvironment polarity of pyrene in CM-PtNP decreases to 1.71 after the addition of sulfide ions (Figure 4D), indicating that pyrene molecules are located at a less hydrophilic domain. This also indicates the structure change of protein molecule after the addition of sulfide ions. 9


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XPS data shows that the addition of sulfide ions increases the content of metallic Pt0, e.g., the fraction of Pt2+ and Pt0 is determined as 55.12% and 44.88 % (Figure 4C), which is due to the reduction of Pt2+ by sulfide ions. Since the reduced Pt0 species in the Pt nanozyme mainly contribute to their catalytic activities, the increase in Pt0 species may leads to the activity “switching on”. Accordingly, there is another factor at work. That is, sulfide ions bound on CM-PtNP surfaces via Pt-S bond blocks the active sites of CM-PtNP nanozyme, resulting in activity “switching off”. As shown in the above section, the interaction of substrate molecules with nanozyme allows their different distributions on nanozyme surfaces. TMB molecules tend to bind to CM-PtNPs near the negatively charged N-terminal of β-casein, and ABTS molecules prefer to bind to nanozyme surface enriched in Pt2+ species near the hydrophobic C-terminal. Here, for TMB, the latter “blocking” factor plays a more crucial role and leads to the activity “switching off” by sulfide ions. For ABTS, the former factor matters much and leads to the activity “switching on” (Figure 5b). Notably, it is found that the activity switching efficiency for the two substrates differs much in different sulfide concentration. Figure 3C shows the effect of sulfide ion concentration on the peroxidase-like activity of CM-PtNPs with different substrates. As shown, sulfide-mediated activity switching efficiency (slope) with ABTS as substrate is much stronger that that with TMB as substrate at very low sulfide concentration. Upon the addition of sulfide ions, the added ions prefer to bind to nanozyme surfaces enriched in Pt2+ species. At the same time, ABTS molecules are easy to bind to nanozyme surface near Pt2+ species. Thus, the factor that the reduced Pt0 species results in activity “switching on” plays a crucial role, so sulfide-mediated switching efficiency is stronger for nanozyme with ABTS as substrates. Blocking the active sites of CM-PtNP nanozyme due to the binding of sulfide ions gradually becomes more and more important, resulting in the efficiency difference gradually decreases with the further increase of sulfide concentration. It is worth mentioning that, for ABTS substrate, sulfide ions will “switch off” the activity of CM-PtNPs with the sulfide concentration increasing above 2 µM (Figure S6). This is due to the crucial role of the blocking factor at higher ion concentration (Figure S7). To further investigate S2−-mediated activity switching mechanism of PtNP nanozyme, we substituted casein in the PtNP nanozyme system with bovine serum albumin (BSA) and 10


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lysozyme (Lyz) to evaluate the role of modifiers in the responsive process to sulfide. The two proteins are chosen because they have different properties and differ significantly in their binding affinities to PtNPs. Isoelectric point (pI) of BSA and Lyz is about pH 4.7 and 10.8, respectively. Different from casein with no cysteine, BSA has 35 cysteine residues and Lysozyme has 8 cysteine residues. Thus, in addition to –COOH and –NH2 groups, cysteine residues play important roles in the binding of BSA and Lyz on nanozyme surfaces. The effect of sulfide ion on the enzyme activity of BSA-PtNP and Lyz-PtNP nanozyme is shown in Figure 6. Unexpectedly, S2;− has no influence on the activity of the two nanozymes with both TMB and ABTS as substrates. Thus, the intrinsic property of the modifier is a crucial factor that can enable the nanozyme to respond to S2−. For BSA-PtNPs and Lyz-PtNPs, since there exists strong Pt-S bonding, the binding of S2− to PtNP is hindered, and hence no switch of the enzyme activity. To confirm this, FTIR and fluorescence spectra measurements were used. As shown in Figure S8, it can be seen that BSA-PtNP and Lyz-PtNP bound with S2- showed a FTIR spectrum largely resembling BSA-PtNP and Lyz-PtNP. Also, different from CM-PtNP, the ratio I1/I3 changed a little for BSA-PtNP and Lyz-PtNP. Thus, both FTIR and fluorescence results here indicate that the binding of S2− to BSA-PtNP and Lyz-PtNP is not easy to take place due to the strong protein and PtNP interaction. Accordingly, S2- mediated activity switching depends much on the intrinsic property of surface modifier of PtNP nanozyme. 3.4. Detection of S2−based on peroxidase-like activity switching of CM-PtNP Based on the above results, we wish to develop an alternatively sensitive and selective colorimetric detection for sulfide anion. To evaluate the sensitivity of this S2− detecting system, the changes of substrate absorbance were first monitored with injection of different concentrations of S2− under the optimized experimental conditions with TMB as a substrate. As illustrated in Figure 7A, the absorbance at 652 nm (Abs652 nm) decreased gradually when the concentration of S2− is raised from 0 to 10.0 µM, suggesting that the changes of absorbance could be employed for the quantitative sensing of S2−. It is shown that a good linear relationship between the absorbance and S2− concentration over the range of 0.01–2.0 µM can be acquired as demonstrated in Figure 7B. The detection limit of S2− is determined to be 5 nM with a signal-to-noise ratio of 3. Then, detection of sulfide ions based on CM-PtNP with ABTS as substrates was investigated. Expectedly, LOD of the sulfide sensor based on CM-PtNP with 11


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ABTS as substrates is only 0.8 nM since the activity switching efficiency (slope) with ABTS as substrate is much stronger that that with TMB as substrate. The linear range is from 0.001 to 0.2 µM. Thus, excellent sulfide colorimetric sensors with different limit of detection (LOD) and linear range were developed based on CM-PtNP with different substrates. Thus, the sulfide sensor based on CM-PtNP with both TMB and ABTS as substrates is suitable for use in practice. For comparison, the detection limits and linear ranges for sulfide ion detection by different sensors were summarized in Table S2. Relative to the other approaches reported, the present strategy does have high sensitivity and low detection limit. To further investigate the selectivity of this detection system, other ions were tested including F-, Cl-, Cr3-, I-, CO32-, HCO3-, SO42-, SO32-, NO3-, and C2O42- (Figure S9) at a concentration of 10 µM under the same conditions. Figure S7 shows that only sulfide ions can significantly inhibit the peroxidase-like activity of CM-PtNPs. In the absence of S2−, none of the other ions causes a detectable decrease in absorbance. These results clearly confirm that the CM-PtNPs based sulfide ion sensor is highly selective. In our previous work, we demonstrate that I- can inhibit the activity of CM-AuNP effectively due to the strong Au-I interaction at very ion low concentration,38 so the colorimetric recognition of sulfide based on CM-AuNP was greatly interfered by the presence of iodide (Figure S10). However, sulfide can be alternatively detected by the CM-PtNP nanozyme due to it’s specifically recognition of sulfide ions. Isothermal titration micro calorimetry (ITC) was further employed to investigate the thermodynamic behavior associated with the interaction between nanozyme and ions. Figure 8 shows the ITC curves for the concentrated S2- or me- solutions being titrated into CM-PtNP or CM-AuNP solutions against the final concentration of the anionic ions. The titrating ITC data were corrected by subtraction of the buffer control. For CM-PtNP, the higher exothermic Hobs upon the addition of S2- indicates that the strong interaction between CM-PtNP and S2-. However, the almost zero enthalpy change after the addition of I- indicates that there exists very weak interaction between CM-PtNP and I-. On the contrary, for CM-AuNP, the addition of both S2- and I- leads to the high exothermic Hobs, indicating the strong interaction between the two ions and Au nanozymes. Different from CM-AuNP, CM-PtNP exhibits the specifically recognition of sulfide ions was due to the different interaction between anions and nanozymes. Therefore, the intrinsic property of nanozyme’s core is very crucial in practice. The feasibility of the detection method using the present colorimetric sensor based on 12


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CM-PtNP was investigated by detecting S2- in natural water samples (from Slender West Lake in Yangzhou, P. R. China). The water samples was filtered through a 0.8 µm membrane prior to detection. The recovery studies were carried out on real samples. As shown in Table S3, the recovery obtained is 95.9 – 99.5 %, revealing that the colorimetric sensor could be used for the accurate detection of trace sulfide metals in real samples and has important practical application potential. 4. CONCLUSIONS We rationally designed a smart casein/PtNP nanozymes with peroxidase mimicking activity, and investigate sulfide-induced switching of CM-PtNP nanozyme’s activity. More interestingly, sulfide can exhibit different switching effect on the activity of casein/PtNP by using different substrates and fine-tuning the structure of the nanozyme surface. The switching mechanism was discussed based on XPS, TEM, fluorescence spectra and FTIR studies, indicating that S2− “switch off” activity mainly through blocking the active sites and “switch on” activity due to reduction of Pt2+ into Pt0 species. By virtue of this specific response, a sulfide colorimetric sensor based on CM-PtNP was developed, which allows the detection of S2− with a detection limit of 5 nM based on activity switching off using TMB as substrate and with a detection limit of 0.8 nM based on activity switching off using ABTS as substrate. The present work has provided new mechanistic insights into anion-mediated switching activity of nanozymes with different physiochemical properties and substrates, which may be useful to rationally predict the environment factors on the properties of the nanozyme and to design an effective signal amplification based on nanozyme deactivation/activation. This investigation is of particular significance for mechanistic understanding and technological applications of peroxidase nanomimetics for biotechnology, environmental chemistry, and medicine. ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundations of China (21573190), Nature Science Key Basic Research of Jiangsu Province for Higher Education (15KJA150009) and PAPD.

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REFERENCES (1) Wei, H.; Wang, E. Nanomaterials with Enzyme-like Characteristics (Nanozymes): Next-generation Artificial Enzymes. Chem. Soc. Rev. 2013, 42, 6060-6093. (2) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X. Intrinsic Peroxidase-like Activity of Ferromagnetic Nanoparticles. Nat. Nano. 2007, 2, 577-583. (3) Cheng, H.; Zhang, L.; He, J.; Guo, W.; Zhou, Z.; Zhang, X.; Nie, S.; Wei, H. Integrated Nanozymes with Nanoscale Proximity for in Vivo Neurochemical Monitoring in Living Brains. Anal. Chem. 2016, 88, 5489-5497. (4) Wang, X.; Hua, Y.; Wei, H. Nanozymes in bionanotechnology: from sensing to therapeutics and beyond. Inorg. Chem. Front. 2016, 3, 41-60. (5) Lin, Y.; Ren, J.; Qu, X. Nano-Gold as Artificial Enzymes: Hidden Talents. Adv. Mater. 2014, 26, 4200-4217. (6) Zhang, K.; Hu, X.; Liu, J. Formation of PdPt Alloy Nanodots on Gold Nanorods: Tuning Oxidase-Like Activities via Composition. Langmuir 2011, 27, 2796-2803. (7) Deng, L.; Yang, X.; Jin, J. Exploiting the Higher Specificity of Silver Amalgamation: Selective Detection of Mercury (II) by Forming Ag/Hg Amalgam. Anal. Chem. 2013, 85, 8594-8600. (8) Singh, S.; Mitra, K.; Shukla, A. Brominated Graphene as Mimetic Peroxidase for Sulfide Ion Recognition. Anal. Chem. 2016, 89, 783-791. (9) Cheng, H.; Lin, S.; Muhammad, F.; Lin, Y.; Wei, H. Rationally Modulate the Oxidase-like Activity of Nanoceria for Self Regulated Bioassays. ACS Sens. 2016, 1, 1336-1343. (10) Weerathunge, P.; Ramanathan, R.; Shukla, R.; Sharma, T. K.; Bansal. V.; Aptamer-Controlled Reversible Inhibition of Gold Nanozyme Activity for Pesticide Sensing. Anal. Chem. 2014, 86, 11937-11941. (11) Nie, D.; Shi G.; Yu, Y. Fe3O4 Magnetic Nanoparticles as Peroxidase Mimetics Used in Colorimetric Determination of 2,4-Dinitrotoluene. Chin J Anal Chem. 2016, 44, 179-185. (12) Long, Y. J.; Li, Y. F.; Liu, Y.; Zheng, J. J.; Tang, J. Huang, C. Z. Visual Observation of The Mercury-Stimulated Peroxidase Mimetic Activity of Gold Nanoparticles. Chem. Commun. 2011, 47, 11939-11941. (13) Lien, C. W; Chen, Y. C.; Chang, H. T.; Huang, C. C. Logical Regulation of The Enzyme-Like Activity of Gold Nanoparticles by Using Heavy Metal Ions. Nanoscale 2013, 5, 8227-8234.

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(14) Peng, C. F.; Pan, N.; Xie, Z. J.; Wu, L. L. Highly Sensitive and Selective Colorimetric Detection of Hg2+ Based on The Separation of Hg2+ and Formation of Catalytic DNA–Gold Nanoparticles. Anal Meth 2016, 8, 1021-1025. (15) Lien, C. W; Tseng, Y. T.; Huang, C. C.; Chang, H. T. Logic Control of Enzyme-Like Gold Nanoparticles for Selective Detection of Lead and Mercury Ions. Analytical Chemistry 2014 , 86, 2065-2072. (16) Liu, Y.; Xiang, Y.; Ding, D. Structural Effects of Amphiphilic Protein/Gold Nanoparticle Hybrid Based Nanozyme on Peroxidase-Like Activity and Silver-Mediated Inhibition. RSC Adv. 2016, 6, 112435-112444. (17) Hu, Y.; Cheng, H.; Zhao, X.; Wu, J.; Muhammad, F,; Lin, S.; He, J.; Zhou, L.; Zhang, C.; Deng, Y.; Wang, P.; Zhou, Z.; Nie, S.; Wei, H. Surface-Enhanced Raman Scattering Active Gold Nanoparticles with Enzyme-Mimicking Activities for Measuring Glucose and Lactate in Living Tissues. ACS Nano 2017, 11, 5558-5566. (18) Liu, Y.; Ding, D.; Zhen, Y. Amino Acid-Mediated ‘Turn-Off/Turn-On’Nanozyme Activity of Gold Nanoclusters for Sensitive and Selective Detection of Copper Ions and Histidine. Biosens. Bioelectron. 2017, 92, 140-146. (19) Yu, F.; Huang, Y.; Cole, A. J.; Yang, V. C. The Artificial Peroxidase Activity of Magnetic Iron Oxide Nanoparticles and Its Application to Glucose Detection. Biomaterials 2009, 30, 4716-4722. (20) Fan, J.; Yin, J. J.; Ning, B. Direct Evidence for Catalase and Peroxidase Activities of Ferritin–Platinum Nanoparticles. Biomaterials 2011, 32, 1611-1618. (21) Yu, C. J.; Chen, T. H.; Jiang, J. Y. Lysozyme-Directed Synthesis of Platinum Nanoclusters as a Mimic Oxidase. Nanoscale 2014, 6, 9618-9624. (22) Chang, Y.; Zhang, Z. J.; Hao, W.; Yang, J. BSA-Stabilized Au Clusters as Peroxidase Mimetic for Colorimetric Detection of Ag+. Sens. Actuators, B 2016, 232, 692-697. (23) Liu, Y.; Yuan, M.; Qiao, L.; Guo, R. An Efficient Colorimetric Biosensor for Glucose Based on Peroxidase-Like Protein-Fe3O4 and Glucose Oxidase Nanocomposites. Biosens. Bioelectron. 2014, 52, 391-399. (24) Wang, G. L.; Jin, L. Y.; Dong, Y. M.; Wu, X. M.; Li, Z. J. Intrinsic Enzyme Mimicking Activity of Gold Nanoclusters upon Visible Light Triggering and Its Application for Colorimetric Trypsin Detection. Biosens. Bioelectron. 2015, 64, 523-529. (25) Katsu, T.; Mori, Y.; Matsuka, N. Potentiometric Flow Injection Determination of Serum Bromide in Patients with Epilepsy. Journal of pharmaceutical and biomedical analysis 1997, 15, 1829-1832. 15


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Figure 1. UV-vis spectra (A) and TEM image (B) of CM-PtNPs obtained in 0.01 mg/ml β-casein. Inset in B: the size distribution histogram of CM-PtNPs. (C) FT-IR spectra of β-casein (down) and CM-PtNPs (up). (D) XPS spectrum showing the binding energy of Pt 4f in CM-PtNPs.

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1.0

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CM-Pt

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Figure 2. (A) The absorbance at 652 nm (left) and at 415 nm (right) of different system with TMB and ABTS as substrates. (a) substrate-CM-PtNP-H2O2, (b) substrate-CM, (c) substrate-CM-PtNP and (d) substrate-H2O2. (B) The effect of TMB and ABTS on the I1/I3 value of pyrene in CM-PtNPs solution.

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Figure 3. Absorption spectra of the TMB–H2O2 (A) and ABTS–H2O2 (B) mixed solution in the presence of CM-PtNPs before (a) and after (b) the addition of 2 µM S2- in a pH 4.0 HAc-NaAc buffer at 20 °C. (C) The effect of sulfide ion concentration on│A-A0│/A0 with different substrates at low concentration.

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Figure 4. (A) TEM images of CM-PtNP with S2-. (B) FTIR spectra of CM-PtNP without (down) and with S2- (up). (C) XPS spectrum showing the binding energy of Pt 4f in CM-PtNPs with S2-. (D) Fluorescence spectra of pyrene in CM-PtNP solution without (a) and with S2- (b).

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Figure 5. Schematic illustration of the peroxidase-like activity of CM-PtNPs (a) and sulfide mediated activity switching (b) with TMB and ABTS as substrates.

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Lys-Pt NPs

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Figure 6. The relative activity of Pt nanozyme with different modifiers in the absence and in the presence of 2.0 µM S2-.

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[S ]/ µ M

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Figure 7. UV-vis absorption spectra of the TMB–H2O2 (A) and ABTS-H2O2 (C) mixed solution in the presence of CM-PtNPs before and after the addition of S2- with different concentrations. Plots of the absorbance at 652 nm (B) and 415 nm (D) with the concentration of S2-, and the inset being the corresponding calibration curve

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-

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Figure 8. ITC curves of S2- and I- titration into CM-PtNP (A) and CM-AuNP (B) solution.

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Table of Contents

Sulfide-mediated switching the peroxidase-like activity of protein-functionalized platinum nanoparticle nanozyme by using different substrates.

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Graphic Abstract

Sulfide-mediated switching the peroxidase-like activity of protein-functionalized platinum nanoparticle nanozyme by using different substrates.


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Switching Peroxidase-mimic Activity of Protein Stabilized Pt

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