BSA-stabilized Pt nanozyme for peroxidase mimetics and its application on colorimetric detection of mercury(II) ions

Biosensors and Bioelectronics 66 (2015) 251–258

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BSA-stabilized Pt nanozyme for peroxidase mimetics and its application on colorimetric detection of mercury(II) ions Wei Li a, Bin Chen a, Haixiang Zhang a, Yanhua Sun b, Jun Wang b, Jinli Zhang a, Yan Fu a,n a Key Laboratory for Green Chemical Technology MOE, Key Laboratory of Systems Bioengineering MOE, Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, PR China b Department of Chemistry and Biology, School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 28 August 2014 Received in revised form 25 October 2014 Accepted 18 November 2014 Available online 20 November 2014

Bovine serum albumin (BSA) is chosen as the nucleation templates to synthesize Pt-based peroxidase nanomimetics with the average diameter of 2.0 nm. The efficient Pt nanozymes consist of 57% Pt0 and 43% Pt2 þ , which possess highly peroxidase-like activity with the Km values of 0.119 mM and 41.8 mM toward 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), respectively. Interestingly, Hg2 þ is able to down-regulate the enzymatic activity of Pt nanoparticles, mainly through the interactions between Hg2 þ and Pt0. It is the first report to explore a colorimetric Hg2 þ sensing system on the basis of peroxidase mimicking activities of Pt nanoparticles. One of our most intriguing results is that BSA-stabilized Pt nanozymes demonstrate the ability to sense Hg2 þ ions in aqueous solution without significant interference from other metal ions. The Hg2 þ detection limit of 7.2 nM is achieved with a linear response range of 0–120 nM, and the developed sensing system is potentially applicable for quantitative determination of Hg2 þ in drinking water. & 2014 Elsevier B.V. All rights reserved.

Keywords: Protein Platinum Nanoparticle Nanozyme Hg2 þ detection

1. Introduction Mercury is well known as one of the most toxic metals, and accumulation of mercury in human body can cause strong damage to central nervous system (Kim et al., 2012). Consequently, great efforts have been devoted to develop colorimetric, fluorescent and electrochemical Hg2 þ sensors based on the selective interactions of mercury(II) with small organic molecules, synthetic polymers, DNAzymes, proteins, as well as metal nanoclusters (Hu et al., 2010; Lee et al., 2007; Li et al., 2008; Liu and Lu, 2007; Liu et al., 2010; Ono and Togashi, 2004; Sener et al., 2014; Wei et al., 2010; Xue et al., 2008). Mercury ions have been reported to be deposited on the surfaces of Au gold nanoparticles through Hg–Au amalgamation to switch their peroxidase-like activities (Long et al., 2011). However, Au3 þ , Pt4 þ and Pb2 þ ions can also be deposited on the bimetallic Pt–Au nanoparticles and cause interference in Hg2 þ -sensing system (Tseng et al., 2012). Lien et al. investigated the effects of different metal ions on the peroxidase-like activities of gold nanoparticles, and developed a series of fluorescent sensing systems adopting aurophilic/metallophilic interactions. When Bi3 þ and Hg2 þ coexisted, strong Hg–Au amalgamation contributed to a large decrease in the peroxidase-like activity of Bi3 þ – n

Corresponding author. Fax: þ 86 22 27890643. E-mail address: [email protected] (Y. Fu).

http://dx.doi.org/10.1016/j.bios.2014.11.032 0956-5663/& 2014 Elsevier B.V. All rights reserved.

Au NPs, resulting in the limit of 1.2 nM for Hg2 þ detection (Lien et al., 2014). Therefore, sensitive and selective detection of Hg2 þ ions can be achieved through modulating enzymatic activity of metal nanomaterials. Platinum nanomaterials have been reported to demonstrate four kinds of enzyme-like activity including superoxide dismutase, catalase, oxidase and peroxidase, therefore, many efforts are devoted to control the particle sizes and the nanostructures of platinum to mimic enzymatic activities by using dendrimer, DNA and protein (Borodko et al., 2011; Knecht et al., 2008; Tiwari et al., 2013; Wang et al., 2013). For example, apoferritin-encapsulated Pt nanoparticles consisting of 74% Pt0 and 26% Pt2 þ had the Km of 0.22 mM toward TMB and 187.25 mM toward H2O2 (Fan et al., 2011). Moreover, i-motif DNA has been used as the nucleation template to synthesize Pt nanozymes with the average size of 2.9 nm, which exhibited 8-times higher affinity to TMB and 10times lower affinity to H2O2, compared to native horseradish peroxidase (Fu et al., 2014; Gao et al., 2007). Although Pt nanoclusters/nanoparticles possess highly peroxidase-like activities, they have not been utilized in the colorimetric detection of toxic metal ions. Therefore, we are motivated to investigate the effects of metal ions on the peroxidase-like activity of Pt nanoparticles, as well as to explore rapid, sensitive and selective Hg2 þ -sensing system. BSA is an abundant, multifunctional and biocompatible biomacromolecule, and it provides multiple binding sites for

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transition metal ions (Bal et al., 1998; Belatik et al., 2012; Duff and Kumar, 2009). So far, BSA has been recognized as an efficient template to stabilize fluorescent/catalytic metal nanoclusters and direct oriented growth of metal nanocrystals (Goswami et al., 2011; Guo and Irudayaraj, 2011; Huang et al., 2011; Lee et al., 2013; Wang et al., 2011; Xie et al., 2007, 2009). In this study, natural BSA is chosen as the nucleation template to synthesize Pt nanoparticles using K2PtCl4 as the precursor. By using the characterizations of CD, UV–vis, TEM and XPS, it is demonstrated that the size distribution and the peroxidase-like activity of Pt nanoparticles are greatly associated with the reducing agent and the pH value used in the synthesis process. Importantly, Hg2 þ is able to down-regulate the enzymatic activity of Pt nanoparticles. It is the first report to explore a colorimetric Hg2 þ sensing system on the basis of peroxidase mimicking activities of Pt nanoparticles. BSA-stabilized Pt nanozymes demonstrate the ability to sense Hg2 þ ions in aqueous solution without significant interference from other metal ions.

2.1. Chemicals BSA (purity 497%), dimethylamine borane (DMAB) and NaCl were purchased from Sigma-Aldrich. K2PtCl4, CuCl2  2H2O, Pb(CH3COO)2  3H2O, Hg(ClO4)2  3H2O, Na2PdCl4 and HAuCl4  3H2O were purchased from Alfa Aesar. H2O2 (30 wt%) was purchased from J&K Scientific, and TMB was purchased from Heowns. Other reagents such as sodium borohydride (NaBH4), CoCl2  6H2O, MnCl2  4H2O, CdCl2  5/2H2O, ZnCl2, MgCl2  6H2O, CaCl2, FeCl3  6H2O, Ni(NO3)2  6H2O, Na2HPO4 and NaH2PO4, were purchased from Tianjin Kermel Chemical Reagent Company. All the chemical reagents except BSA were of the purity higher than 99.9%, and used without further treatment. The water used in all experiments was distilled for three times. 2.2. Preparation of Pt nanoparticles Pt nanoparticles were synthesized through the reduction of K2PtCl4 by DMAB or NaBH4 in the presence of BSA. Firstly, 10 μM BSA dissolved in 10 mM PBS buffer (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, respectively) were firstly incubated with K2PtCl4 at the [K2PtCl4]/[BSA] ratio of 30. After incubation for 2 h at 25 °C, freshly prepared DMAB or NaBH4 aqueous solution was added to the mixture at the [reductant]/[precursor] ratio 5, to start the reduction reaction at 25 °C. The obtained BSA–Pt can be used as the nanozyme after 5 h reaction. 2.3. Reaction kinetics Time-dependent absorption was monitored at 652 nm using UV–vis spectroscopy at 25 °C. In order to detect the peroxidaselike activities of Pt nanoparticles, the concentration of TMB and H2O2 was fixed at 0.125 mM and 125 mM, respectively, in PBS buffer at pH 4.0. Then certain amount of as-prepared BSA–Pt solution was added into the working solution to initiate the reaction. All the experiments were repeated thrice for reproducibility. The initial velocities (v) were calculated according to Eqs. (1) and (2):

υ=

A 652 εL

Vmax[S] Km + [S]

(3)

where v was the initial velocity of the reaction, Vmax was the maximal rate of reaction, [S] was the substrate concentration, and Km was the Michaelis–Menten constant. Km and Vmax were obtained by the Lineweaver–Burk plot method according to Eq. (4):

Km 1 1 = + v Vmax[S] Vmax

(4)

The detection was carried out in 3 mL PBS buffer at pH 4.0 containing 0.125 mM TMB, 125 mM H2O2, 900 nM BSA–Pt nanoparticles (calculated from Pt precursor) and different concentrations of Hg2 þ ions. Firstly, 9 μL BSA–Pt solutions (Pt concentration of 300 μM) were added into 1.5 mL PBS buffer. Then certain volumes of Hg(ClO4)2 stock solution were added into the mixtures to reach the final concentration of 0–1.2 μM (final concentrations in 3 mL reaction system), and the above mixtures were diluted to 2.93 mL using three-distilled water. Finally, 30 μL of TMB stock solution and 38.2 μL of H2O2 stock solution were added successively to initiate the reactions. After incubation for 20 min at 25 °C, the A652 signals were recorded by UV–vis spectroscopy. To investigate the effects of other metal ions including Na þ , Mg2 þ , Ca2 þ , Mn2 þ , Ni2 þ , Zn2 þ , Co2 þ , Cu2 þ , Pd2 þ , Cd2 þ , Fe3 þ and Au3 þ , 9 μL BSA–Pt solutions were added into 1.5 mL PBS buffer at pH 4.0. Certain volumes of stock solutions of different metal ions were added into the above mixtures to reach the final concentrations of 10 μM (concentration in 3 mL reaction system). Then the above mixtures were diluted to 2.93 mL using threedistilled water. Finally, 30 μL of TMB stock solution and 38.2 μL of H2O2 stock solution were added successively to initiate the reactions. After incubation for 20 min at 25 °C, the A652 signals were recorded by UV–vis spectroscopy. 2.5. Detection of Hg2 þ in real water samples Tap water was collected from our laboratory in Tianjin University (Tianjin, China). The as-prepared water samples were spiked with Hg2 þ at 60 nM and 96 nM. The sensing experiments were carried out in 3 mL reaction systems (pH 4.0) containing 0.125 mM TMB, 125 mM H2O2, 900 nM BSA–Pt nanoparticles (calculated from Pt precursor). Firstly, 9 μL as prepared BSA–Pt solution and certain volumes of Hg(ClO4)2 stock solution were added into the real water (final volume was fixed at 2.93 mL), of which the pH value was adjusted to 4.0 by H3PO4. Then 30 μL of TMB solution and 38.2 μL of H2O2 solution was added successively to initiate the reactions. After incubation for 20 min at 25 °C, the A652 signals were recorded by UV–vis spectroscopy. The recoveries were calculated according to Eq. (5):

(1)

Rate of recovery = (C3 − C1)/C2 × 100%

(2)

where C1, C2 and C3 represent the concentrations measured in unfortified samples, fortifications and fortified samples, respectively.

dCp dt

v=

2.4. Detection of Hg2 þ

2. Experimental section

Cp =

where Cp represented the concentration of o  TMB, ε was the extinction coefficient of o  TMB, ε ¼ 3.9  104 M  1 cm  1, L was the optical path length of 1 cm. In order to calculate the enzymatic parameters of Pt nanoparticles, serial solutions of various TMB or H2O2 concentrations were done using buffer at pH 4.0. The kinetic parameters were determined via Michaelis–Menten Eq. (3):

(5)

W. Li et al. / Biosensors and Bioelectronics 66 (2015) 251–258

2.6. Characterizations 2.6.1. UV–vis spectroscopy UV–vis spectra were recorded by Varian Cary300 spectrophotometer at 25 °C using a quartz glass cuvette with 1 cm path length. The reaction kinetics was monitored at the wavelength of 652 nm.

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2.6.5. Inductively coupled plasma mass spectrometry (ICPMS) The concentration of Hg2 þ ions in the tap water was determined by ICPMS (Agilent 7700CE).

3. Results and discussion 3.1. BSA-templated synthesis of Pt nanoparticles

2.6.2. Circular dichroism spectroscopy (CD) CD spectra were obtained with a Jasco J-810 spectropolarimeter at 25 °C using a quartz glass cuvette with 1 mm path length. All the CD spectra were measured from 350 nm to 190 nm at a scan speed of 100 nm/min, and each CD spectrum was an average of three scans. 2.6.3. Transmission Electron Microscopy (TEM) Electron microscopy was performed on JEM-2010FEF equipment (JEOL, Japan). Specimens were prepared by dropping 10 μL of as-prepared BSA–Pt solution on a carbon-coated grid, and after incubation for 5 min excess liquid was removed with filter paper. 2.6.4. X-ray Photoelectron Spectroscopy (XPS) XPS measurements were carried out on the PHI5000 Versaprobe. The whole preparation process of specimen was conducted under nitrogen gas atmosphere. 30 mL of as-prepared BSA–Pt solution was dropped onto a clean silicon wafer (6 mm  6 mm), which was rinsed by water, chloroform and ethanol before being used. After the wafer dried, another 30 mL solution was dropped and this operation was repeated thrice.

Time-dependent UV–vis spectroscopy was performed to monitor the interactions between BSA and K2PtCl4, as well as the reducing process of Pt nanoparticles. UV–vis spectrum of BSA shows a maximum absorption at 280 nm, and the solution of K2PtCl4 and DMAB exhibits no apparent absorption at longer wavelength than 280 nm. After adding K2PtCl4 precursor to BSA at the molar ratio of 30, the peak at 280 nm as well as the absorption at longer wavelength gradually increases in the initial 2 h (Fig. 1a). Seidel et al. reported that the binding of PtCl42 − to λ-DNA resulted in a similar increase in longer wavelength absorbance (Seidel et al., 2004). At pH 4.0, BSA molecules exhibit an elongated shape (roughly 4 nm  12.9 nm), of which some disulfides become accessible to coupling with metal. Therefore, the observed changes in the UV spectra may be attributed to the coordination between PtCl42 − and electron-rich groups of BSA such as –SH, –C¼O, –NH2, etc. Upon addition of DMAB, a significant increase in absorbance at longer wavelength occurs within the initial 30 min (Fig. 1b). No absorption band at 290–300 nm or 347–375 nm is detected, suggesting that binuclear or tetranuclear clusters cannot be found under these conditions (Borodko et al., 2011). CD spectrum of BSA exhibits two characteristic bands at 208 nm and 222 nm, respectively (Fig. 1c). Addition of Pt2 þ ions

Fig. 1. Time-dependent UV–vis spectra of the mixture of 10 μM BSA and 300 μM Pt2 þ (a) and BSA–Pt, (b) after reduction by DMAB at pH 4.0 ([DMAB]/[Pt2 þ ]¼ 5), with 10 min as an interval, (c) CD spectra of 1 μM BSA, BSA–Pt2 þ ([Pt2 þ ]/[BSA]¼30:1, after incubation for 2 h) and BSA–Pt (after incubation with DMAB for 2 h, [DMAB]/[Pt2 þ ] ¼5), respectively, and (d) UV–vis absorption-time course curves of TMB–H2O2 reaction system (pH 4.0) catalyzed by BSA, BSA–Pt2 þ and BSA–Pt reduced by DMAB at pH 4.0 ([DMAB]/[Pt2 þ ] ¼ 5, [TMB]¼ 125 μM, [H2O2] ¼125 mM, [Pt]¼ 900 nM).

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results in a slight increase in the intensity of these two negative peaks without band shift, which suggests that BSA almost maintains its conformation. After adding DMAB ([DMAB]/[K2PtCl4]¼ 5), a blue-shift of 2 nm is detected for the peak at 208 nm, indicating that BSA undergoes a slight conformational change in the reduction process. In order to test the peroxidase mimicking activity, we choose TMB and H2O2 as the peroxidase substrates, and all the reaction kinetics are monitored at 652 nm corresponding to oxidized TMB. Among BSA, BSA–Pt2 þ and BSA–Pt, only the reduced BSA–Pt possesses peroxidase-like activity (Fig. 1d). Concentration of H2O2 greatly influences the reaction kinetics (Fig. S1). Addition of H2O2 (125 mM) into the reaction system results in an equilibrium time of approximate 15 min. Although both the BSA–Pt2 þ interaction and the Pt2 þ reduction lead to increasing absorbance at longer wavelength, it is reasonable to conclude that Pt2 þ can be reduced to Pt0 by DMAB combining with the results of UV, CD and reaction kinetics. Moreover, after precipitation of BSA molecules induced by saturated (NH4)2SO4 solution, neither BSA nor Pt can be detected in the supernatant, demonstrating the complexation of BSA and Pt.

3.2. Peroxidase-like activities of BSA-stabilized Pt nanoparticles To study the effects of particle sizes, we have synthesized Pt nanoparticles using DMAB and NaBH4 as the reducing agents in the pH range of 3.0–8.0. According to TEM images, Pt nanoparticles reduced by DMAB at pH 4.0 and pH 7.0 show the average diameter of 2.00 nm and 2.43 nm, respectively (Fig. 2a and b). Kinetic studies show that Pt nanoparticles reduced at pH 4.0 react faster (v ¼0.11 μM s  1) than those prepared at pH 7.0 (v ¼0.08 μM s  1). Adopting NaBH4 as the reducing agent, Pt nanoparticles exhibit the particle sizes of 4.19 nm at pH 4.0 as well as 2.36 nm at pH 7.0 (Fig. 2c and d). The reaction rates are determined as 0.036 μM s  1 and 0.095 μM s  1, respectively (Fig. S2 and Table S1). Therefore, smaller Pt nanoparticles possess higher peroxidase mimicking activity. Importantly, these Pt nanoparticles retain their catalytic activity even at a 72 h-incubation. We choose the most active Pt nanoparticles with the size of 2 nm (DMAB, pH 4.0) to perform further studies. For analyzing the catalytic mechanism and acquiring the kinetic parameters, steady-state kinetics for the oxidation of TMB in the presence of H2O2 are investigated by BSA-stabilized Pt nanoparticles. Within the suitable range of TMB and H2O2, typical

Fig. 2. TEM images of BSA–Pt reduced by DMAB at pH 4.0 (a) and pH 7.0 (b), as well as reduced by NaBH4 at pH 4.0 (c) and pH 7.0 (d) at the [reductant]/[precursor] ratio of 5.

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255

Fig. 3. Steady-state kinetic assay and catalytic mechanism of BSA–Pt, the initial velocities in the oxidization of TMB in the presence of H2O2 are measured using 900 nM Pt (calculated from Pt precursor) at pH 4.0 at 25 °C: (a) the concentration of H2O2 is fixed at 125 mM and the TMB concentration is varied, (c) the concentration of TMB is fixed at 0.125 mM and the H2O2 concentration is varied, (b) and (d) are the double-reciprocal plots of (a) and (c), respectively.

Michaelis–Menten curves are obtained as shown in Fig. 3. The data are fitted to Lineweaver–Burk equation to acquire the important enzyme kinetic parameters such as Michaelis–Menten constant (Km) and maximal velocity (Vmax). The apparent Km value toward TMB is 0.119 mM while the Km toward H2O2 is 41.8 mM. Although BSA–Pt exhibits 3-times higher affinity to TMB and 10-times lower affinity to H2O2, the Vmax values suggest that BSA–Pt can react faster than native horseradish peroxidase (Table S2). Importantly, BSA–Pt exhibits much higher affinity toward H2O2 and TMB than those of other Pt nanozymes (Higuchi et al., 2008). For example, Pt nanoparticles of 10– 30 nm showed that the Km values were 68.4 mM and 0.217 mM for H2O2 and TMB, respectively (Chen et al., 2013). Irregular-shaped Pt with the average length of 7 nm as well as the width of 2–5 nm along longitudinal axes, exhibited the affinity of 769 mM and 0.12 mM for H2O2 and TMB, respectively (Gao et al., 2013). Combining with previous reports on metal-or metal oxide-based nanozymes (listed in Table S2), Pt nanoparticles stabilized by native BSA are promising nanomimetics for peroxidase. 3.3. Interactions of BSA–Pt with Hg2 þ ions To understand deeply the surface electronic structures of BSAstabilized Pt nanoparticles, the charge states of Pt species are

investigated by XPS. As shown in Fig. 4, the binding energy of the electron on the Pt 4f 7/2 orbital of BSA–Pt can be deconstructed into Pt2 þ and Pt0 components with the binding energies of 72.9 eV ( 43%) and 71.3 eV (  57%), respectively (Fan et al., 2011). The existence of Pt2 þ species might be attributed to the coordination between Pt nanoparticles and electron-rich groups of BSA such as –SH, –C ¼O, –NH2, etc. For a sensing system, the ability of a single Pt nanoparticle to convert a multitude of substrate molecules into colored products can make the Pt-based nanozyme an intrinsic signal amplifier (Scrimin and Prins, 2011). The most commonly metallophilic interactions are observed for d10 configuration such as Hg2 þ , Au þ , Pt0, etc. (Doerrer, 2010). Previous reports have shown that metallophilic Hg2 þ –Au þ interactions can effectively quench the fluorescence of Au nanoclusters as well as down-regulate their peroxidase-like activity (Xie et al., 2010; Zhu et al., 2013). This inspired us to investigate the interactions between Hg2 þ and Pt nanoparticles. Interestingly, addition of Hg2 þ decreases the content of metallic Pt0, e.g., the fraction of Pt2 þ and Pt0 is determined as 66% and 34% in the presence of excess Hg2 þ (Fig. 4). Moreover, no apparent aggregation can be observed for BSA–Pt incubated with Hg2 þ (Fig. S3). The reduction of Hg2 þ by Pt0 seems contradictory as considering conventional electrochemical potentials. It has been noted

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Fig. 4. Pt 4f XPS spectra of BSA–Pt ([Pt precursor] ¼ 300 μM) before and after incubated with 300 μM Hg2 þ : black, red, magenta and cyan lines represent the raw curve, the fitted curve, the Pt2 þ and the Pt0 components' curves, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

that the electrochemical potentials of small nanoparticles are lower than their bulk values (Mallick et al., 2001). Krishnadas et al. reported that Cu2 þ can be reduced by Au of the AuAg clusters, which was also an example of galvanic reduction-induced metallophilic interactions (Krishnadas et al., 2014). Yu et al. reported that Hg2 þ induced fluorescence quenching of lysozyme-stabilized Pt4 clusters through metallophilic interactions between Hg(II) (4f145d10) and Pt(0) (4f145d10) (Yu et al., 2014). According to the proposed mechanism for peroxidase-like Pt nanoparticles, metallic Pt0 of BSA–Pt participates in the activation of substrate H2O2 to release ∙OH (Fu et al., 2014). Therefore, Hg2 þ ions potentially regulate the peroxidase-like activity of BSA–Pt through metallophilic interactions between Hg2 þ and Pt nanoparticles. 3.4. Colorimetric detection of Hg2 þ in aqueous solution We test the potential application of BSA-stabilized Pt nanoparticles on the Hg2 þ detection. As the reaction temperature increasing from 20 to 30 °C, the final A652 collected at 20 min only increases by 1.5%. Therefore, temperature variations in the range of 20–30 °C have little impact on the final colorimetric signal. The sensing system contains 900 nM Pt, 0.125 mM TMB and 125 mM H2O2. The operational temperature and the reaction time are fixed at 25 °C and 20 min, respectively. Increasing the concentration of Hg2 þ ions ([Hg2þ ]) results in a color progression from dark blue to colorless (Fig. 5a), which results from the Hg2 þ -induced decrease of active Pt0 species. The A652 signal gradually decreases with the [Hg2 þ ] ranging from 0 to 1200 nM (Fig. 5b). Notably, there exists a good linear relationship between the A652 and the [Hg2 þ ] in the concentration range of 0–120 nM, with the calibration curve of A652 ¼1.25998 0.00422[Hg2 þ ]. The detection limit defined as 3s/slope is determined to be 7.2 nM. The repeatability expressed as the RSD is 2% (n¼ 3). Compared to the LOD, the response time and the operational temperature in other sensing methods (as listed Table 1), BSA-stabilized Pt nanozyme is an efficient candidate in the development of Hg2 þ sensing approaches. This detection system is sensitive enough to detect Hg2 þ in drinking water, which has the maximum allowable limits of 10 nM and 30 nM defined by the US Environmental Protection Agency and the World Health Organization. Selectivity of this protocol is evaluated by various metal ions involving Na þ , Mg2 þ , Ca2 þ , Mn2 þ , Ni2 þ , Zn2 þ , Co2 þ , Cu2 þ , Pd2 þ ,

Fig. 5. (a) Photograph of the color progression with different concentrations of Hg2 þ in the TMB–H2O2 reaction system (pH 4.0) catalyzed by BSA–Pt at 25 °C, (b) plots of the A652 values with the concentrations of Hg2 þ , inset: linear response to Hg2 þ , (c) the A652 values in the TMB–H2O2 reaction system (pH 4.0) catalyzed by BSA–Pt in the presence of Hg2 þ (1 μM) or other interference ions including Cd2 þ , Pb2 þ , Cu2 þ , Co2 þ , Zn2 þ , Mg2 þ , Fe3 þ , Ni2 þ , Ca2 þ , Na þ , Mn2 þ or Au3 þ (10 μM), respectively. The A0 and A represent the A652 produced by BSA–Pt and BSA–Pt-ion, respectively. All the reaction systems contain 0.125 mM TMB, 125 mM H2O2 and 900 nM Pt, and all the A652 signals are collected at 20 min after initiation. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Cd2 þ , Fe3 þ and Au3 þ (Fig. 5c). In the presence of 10-fold concentration excess compared to [Hg2 þ ], addition of these competing ions into the detecting system individually results in a small fraction of decrease (o 5%) in the final A652. Therefore, BSA-stabilized Pt nanozyme can be considered as a promising candidate with high selectivity towards Hg2 þ against other monovalent,

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Table 1 Comparison of colorimetric platforms for Hg2 þ sensing. System

LOD (nM)

Temperature (°C)

Response time

Reference

G-quadruplex DNAzyme/cysteine T-rich DNA–AuNPs Hemin-G-quadruplex-MBs BSA–Au clusters T-containing-quadruplex DNA BSA–Pt nanozyme

9.2 40 5 3 100 7.2

25 Room temperature 30 40 37 25

24 min 30 min 42 h 25 min 30 min 20 min

Jia et al. (2011) Wang et al. (2008) Huang et al. (2014) Zhu et al. (2013) Li et al. (2009) This work

divalent or multivalent metal ions. Since the valence states of transition metal elements in the nanoparticles are complex, the electrochemical potentials are probably different from their bulk values. Therefore, the selectivity of BSA–Pt toward Hg2 þ against other ions also needs further investigations. To validate that BSA-stabilized Pt nanoparticles are applicable for real water, spiked-recovery experiments with different Hg2 þ concentration are performed by using the tap water from our laboratory and commercial purified water. The recovery rates are calculated as 114% for 60 nM and 103% for 96 nM Hg2 þ spiked in the tap water, while they are measured as 104% for 60 nM and 94% for 96 nM Hg2 þ spiked in the purified water (Table S3). ICPMS has not detected the presence of Hg2 þ in the tap water. Therefore, the real sample was prepared by adding 80 nM Hg2 þ into the tap water. The concentration of 110.9 and 117.4 nM are determined by using the colorimetric method and ICPMS, respectively. Therefore, results obtained by these two methods show no significant difference. The proposed method in this study is applicable for practical analysis of Hg2 þ in real samples. These experiments in real samples demonstrate that the developed sensing system can be potentially applicable for quantitative determination of Hg2 þ in drinking water samples. This proposed method possesses several advantages: the whole process of synthesizing BSA–Pt is ecofriendly, and the chemical reagents are not consuming. In the sensing system, the consumption of BSA–Pt is very little. Compared to BSA–Au clusters (Table S2), Pt nanoparticles exhibit highly peroxidase-like activity at relative low temperature, which is convenient to commonly experimental conditions.

4. Conclusion BSA is chosen as the nucleation template to synthesize Pt-based peroxidase nanomimetics with the average diameter of 2.0 nm. The efficient Pt nanozyme consisting of approximate 57% metallic Pt0 and 43% Pt2 þ , is synthesized through the reduction by DMAB at pH 4.0. These Pt nanoparticles possess highly peroxidase-like activity with the Km values of 0.119 mM and 41.8 mM toward TMB and H2O2 respectively. Interestingly, Hg2 þ is able to down-regulate the enzymatic activity of Pt nanoparticles, mainly through the interactions between Hg2 þ and Pt0. A colorimetric Hg2 þ sensing system is explored on the basis of peroxidase mimicking activities of Pt nanoparticles. One of our most intriguing results is that BSA-stabilized Pt nanozymes demonstrate the ability to sense Hg2 þ ions in aqueous solution without significant interference from other metal ions. The Hg2 þ detection limit of 7.2 nM is achieved with a linear response range of 0–120 nM, and the developed sensing system is potentially applicable for quantitative determination of Hg2 þ in drinking water samples.

Acknowledgments This work was supported by the National Natural Science Foundation of China (21176174 and 21206107), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1161).

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.11.032.

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