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Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection
Biosensors and Bioelectronics 36 (2012) 18–21
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection Jingjing Wang a, Dongxue Han b, Xiaohong Wang a,n, Bin Qi a,n, Meisheng Zhao c,n a
Key Lab of Polyoxometalate Science of Ministry of Education, Faculty of Chemistry, Northeast Normal University, Changchun 130024, PR China Changchun Institute of Applied Chemistry Chinese Academy of Sciences, PR China c Eye Hospital of the Second Clinical Hospital of Jilin University, Jilin University, Changchun 130020, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 December 2011 Received in revised form 6 March 2012 Accepted 18 March 2012 Available online 10 April 2012
Polyoxometalates (H3PW12O40, H4SiW12O40 and H3PMo12O40) have been proven to possess intrinsic peroxidase-like activity for the first time, which can catalyze oxidation of the peroxidase substrate 3,30 ,5,50 -tetramethylbenzidine (TMB) by H2O2 to form a blue color in aqueous solution. Among them, H3PW12O40 (PW12) exhibits higher catalytic activity to TMB than natural enzyme HRP and other two POMs. In addition, H3PW12O40/graphene exhibited higher activity than H3PW12O40 in this catalytic oxidation reaction due to the effect of graphene in promoting the electron transfer between the substrate and catalyst. POMs/H2O2/TMB system provides a simple, accurate approach to colorimetric detection for H2O2 or glucose. The colorimetric method based on POMs showed good response toward H2O2 and glucose detection with a linear range from 1.34 10 7 to 6.7 10 5 mol/L and 1 10 7 to 1 10 4 mol/L, respectively. The results showed that it is a simple, cheap, more convenient, highly selective, sensitive, and easy handling colorimetric assay. & 2012 Elsevier B.V. All rights reserved.
Keywords: Polyoxometalates Peroxidase-like Colorimetric detection H2O2 Glucose
1. Introduction Artificial enzyme mimics have attracted much attention because there are many disadvantages in the application of natural enzymes such as expensive caused by preparation, purification and storage, and easily inhibition of their activity (Shoji and Freund, 2001). Peroxidase enzymes could activate H2O2 to perform outstanding oxidizability in nature and have long been targeted by biomimic chemistry (Ellis et al., 2009). At present, a large number of artificial enzymes have been constructed by incorporating a catalytic center into a variety of scaffolds to mimic natural peroxidase enzymes, including nanoparticles (Liu and Tang, 2010), gel (Wulff, 2002) or polymers (Liu and Shen, 2009). Recently, it was reported that Fe3O4 magnetic nanoparticles possessed intrinsic enzyme mimetic activity similar to that found in natural peroxidases (Wei and Wang, 2008; Zhang et al., 2010), which was used to detect H2O2 and thrombin. FeS nanostructure (Sanz et al., 2005), BiFeO3 nanoparticles (AbdRabboh and Meyerhoff, 2007), polymer-coating CeO2 nanoparticles (Peng et al., 2007), bimetallic alloy nanoparticles (He et al., 2010), graphene oxide (Song et al., 2010) and positively charged gold nanoparticles (Jv et al., 2010) were found with peroxidase n
Corresponding authors. Tel.: þ 86 431 88930042; fax: þ 86 431 85099759. E-mail addresses: [email protected], [email protected] (X. Wang), [email protected] (M. Zhao). 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.03.031
activity. Nevertheless, these examples demonstrate the potential as peroxidase mimetics, further efforts to find or develop superior enzyme mimetics with more sensitivity, reusability, and stability are still required to underway and hold significant incentives in the field of bioinorganic chemistry. Polyoxometalates are the well-known metal-oxo-cluster compounds, which are emerging as useful materials for numerous applications, especial in biology (Goozerh and Proust, 1998). POMs exhibit many ideal properties in biological and medical fields, such as being nano-sized, removed by the renal system easily, lower toxicity level, stability in biological conditions, and applied as antitumor agents (Wang et al., 2003), HIV inhibitior (Judd et al., 2001), and antiviral agents (Inoue et al., 2005). Our interest is the application of POMs in colorimetric multiplexed immunoassay. It is well-known that POMs exhibit fast and reversible multi-electron redox processes without any significant structural alteration, which has made them prominent candidates as redox sensors and electrochromic or photochromic materials (Liu and Tang, 2010). Meanwhile, one- or two-electron-transfer oxidation of organic substrates into the corresponding compounds is an important reaction in organic synthesis (Wang et al., 2010) using H2O2 as an oxidant. Therefore, POMs might mimic peroxidase enzymes to oxidize some dyes such as TMB to oxidation form with blue color in colorimetric multiplexed immunoassay. TMB is a dye used as a horseradish peroxidase (HRP) substrate in various bioassays upon the oxidation form of blue. TMB has been used to demonstrate the peroxidase-like
J. Wang et al. / Biosensors and Bioelectronics 36 (2012) 18–21
activities of Fe3O4 nanoparticles (Zhang et al., 2010) and singlewalled carbon nanotube (Song et al., 2010). POMs as colorimetric multiplexed immunoassay agents to detect glucose have not been reported by far. In the present study, we have investigated the peroxidase mimetics of POMs and POM/graphene in detection of glucose to propose a simple and sensitive detection method for glucose in aqueous solutions for the first time. It is known that most of glucose biosensors are based on the oxidation or reduction of enzymatically produced H2O2 (Fu et al., 2011). Therefore, the accurate and rapid determination of H2O2 is practically important due to its application in food, pharmaceutical, clinical, industrial, and environmental analysis (Yao et al., 2006). Jin group has reported that the resulting Prussian blue based nanoelectrode arrays show a low detection limit of 1 mM (Xian et al., 2007). It had been reported recently that graphene/Prussian blue nanocomposite multilayer films were used to detect H2O2 by electrochemical surface plasmon resonance (EC-SPR) spectroscopy with the detection limit up to 1 mM (Mao et al., 2011). The better results were attributed to the promoting effect on Prussian by graphene. To the best of our knowledge, no attempt has been made to fabricate POMs/TMB system or POM on graphene/TMB system to colorimetrically detect H2O2 and glucose.
2. Material and methods 2.1. Chemicals and materials The purchased chemicals (analytical grade) were used without further purification. 3,30 ,5,50 -tetramethylbenzidine (TMB) was purchased from j&k chemica, Beijing. Na3WO4, H3PO4, HCl, and 30% H2O2 etc. were purchased from Beijing Chemical Reagent Company (Beijing, China). Glucose oxidase (GOx) was purchased from Sigma-Aldrich (Milwaukee, WI). Other reagents and chemicals were at least analytical reagent grade. The water used throughout all experiments was purified. H3PW12O40 (PW12), H3PMo12O40 (PMo12) and H4SiW12O40 (SiW12) were synthesized according to a previous method (Rocchiccioli-Deltcheff et al., 1983; Ding et al., 2005; Shi et al., 2005). 2.2. Instrument Absorption spectra were recorded on a Cary 500 UV–vis-NIR spectrophotometer. 2.3. Preparation of the PW12/grapheme The PW12/graphene was prepared according to the Ref. (Zhou and Han, 2010) and characterized by UV–vis (Fig. S1).
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To examine the influence of incubation temperature on the PW12 activity, catalytic reactions incubated in different temperature water baths from 20 to 60 1C were investigated, under conditions identical to those used for Test I. As control experiments, 50 mL of water was used instead of 50 mL of PW12. 2.5. Glucose detection using GOx and PW12 Glucose detection was tested as follows: (referred to as Test II) (a) 20 mL of 20 mg/mL GOx and 200 mL of glucose of different concentrations in phosphate buffered saline (PBS, pH 7.0) were incubated at 37 1C for 30 min. (b) 40 mL of 800 mM TMB and 20 mL H3PW12O40 in 250 mL buffer solution (pH 3.0) were added into the above 280 mL glucose reaction solution; (c) the mixed solution (pH 3.326 ) was incubated at room temperature for 15 min; (d) the mixed solution was used to perform the adsorption spectroscopy measurement.
3. Results and discussion 3.1. The peroxidase activity of POMs The POMs were used to catalyze the oxidation of a peroxidase substrate TMB by H2O2 to the oxidized colored product (Eq. (1)), which provides a colorimetric detection of H2O2. Combination of the catalytic reaction of glucose with glucose oxidase as catalyst (GOx) (Eq. (2)) and the POMs catalytic reaction (Eq. (1)), a colorimetric method (Scheme 1) for glucose detection could be developed. This method exhibited sensitive and selective response toward glucose detection. POMs
H2 O2 þTMB!2H2 Oþ oxidized TMB GOx
O2 þglucose!H2 O2 þgluconic acid
ð1Þ ð2Þ
In order to evaluate the performance of POMs as peroxidase mimetics, three POMs had been chosen to study their catalytic activity in detecting H2O2 (Fig. S2), including H3PW12O40 (PW12), H4SiW12O40 (SiW12), and H3PMo12O40 (PMo12). The results show that PW12, SiW12 and PMo12 exhibited different catalytic activity in oxidation of TMB by H2O2. It can be seen that without any catalyst, TMB could not be oxidized to oxidative form with blue color. This is consistent to Yan’s previous study (Gao et al., 2007). Using POMs such as PW12 and SiW12, the oxidation of TMB could occur by H2O2. The absorption spectra indicated that the presence of PW12 and SiW12 gave a 740%, 730% response when compared with the one in the absence of the PW12 or SiW12. However, using PMo12 as a catalyst could catalyze both O2 and H2O2 to oxidize TMB efficiently, while the absorption spectrum with H2O2 is similar to that without H2O2. It is supposed that PMo12 has
2.4. H2O2 detection using PW12 as peroxidase mimetics To investigate the peroxidase-like activity of POMs, the catalytic oxidation of the peroxidase substrate TMB in the presence of H2O2 was designed. Experiments were carried out as follows (referred to as Test I): (a) 10 mM H3PW12O40 in a reaction volume of 1000 mL buffer solution (pH 3.0) with 800 mM TMB as substrate, and H2O2 concentration was 10 mM; (b) the mixed solution was incubated at room temperature (25 1C) for 10 min; (c) 500 mL of the resulting reaction solution was diluted with 500 mL water and then used for adsorption spectroscopy measurement. To examine the influence of reaction buffer pH on the PW12 activity, the acetate buffer solutions from pH 2.0 to 9.0 were investigated, under conditions identical to those used for Test I.
Scheme 1. Schematic illustration of colorimetric detection of glucose by using glucose oxidase (GOx) and POM-catalyzed reactions.
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J. Wang et al. / Biosensors and Bioelectronics 36 (2012) 18–21
intrinsic higher catalytic oxidative activity and can facilitate the fast oxidation of TMB only in the present of O2 but no need of H2O2. Thus, PMo12 is useless for detecting H2O2 and glucose. Therefore, PW12 was selected as the catalyst in our detecting system from the above comparison because of easy preparation and more stability than SiW12. In addition, the catalytic activity of simple acid, such as HCl and H2SO4, had been checked in our detecting system (Fig. S3). It can be seen that the catalytic activity of these two mineral acids is lower than that of PW12. Moreover, the simple acid is corrosive and is not beneficial to environment. So the simple acid has been replaced by more novel and beneficial environmental catalysts. 3.2. PW12 as peroxidase mimetics and its use in H2O2 detection According to the above discussion, PW12 can catalyze the oxidation of TMB by H2O2 to obtain the TMB oxidized form with blue color (Fig. S4), which shows that PW12 has intrinsic peroxidaselike catalytic activity. The catalytic reaction can be detected by monitoring TMB absorbance change at 652 nm (Gao et al., 2007). It is known that the pH and temperature are the important parameters for the H2O2 detection. The pH-dependent response curve is showed in Fig. S5. The catalytic oxidation of TMB with H2O2 catalyzed by PW12 was much faster in acidic solutions than in neutral or basic solutions. The catalytic activity at pH 3 was highest. Thus, the pH 3.0 buffer solution was taken as the optimal reaction solution to get a high catalytic activity. The reaction temperature-dependent response curves were shown in Fig. S6. DA had been used to evaluate the catalytic activity of PW12 under different temperatures, where DA¼A(PW12, 652 nm) -A(blank, 652 nm). The large DA is, the higher catalytic activity is. A highest value could be obtained at 25 1C (about room temperature) in Fig.S6b. Thus, 25 1C was taken as the optimal reaction temperature. The system discussed above could be used to detect H2O2 because the catalytic activity of the PW12 is H2O2 concentration dependent (Eq. (1)), the method could be developed for H2O2 detection under the optimal conditions (25 1C, pH 3.0 buffer). Fig. 1 shows a H2O2 concentration as low as 1.34 10 7 mol/L could be detected with a linear range from 1.34 10 7 to 6.7 10 5 mol/L. In the results reported by Wu group (Wang et al., 2011), it can be seen that POMs [(CH3)4N]2.5H7.5[Eu(GeW11O39)(H2O)2]2 4.5H2O (Ge-POMs) gave the
Fig. 1. Linear calibration plot for H2O2. The error bars represent the standard deviation of three measurements.
detection limits with the H2O2 concentration in a linear range from 0 to 0.135 mM with a detection limit of 0.16 mM for the discoloration and from 0 to 0.15 mM with a detection limit of 0.68 mM for the luminescence recovery based on S/N¼3, respectively. Compared to Wu’s result, our process is much more convenient and sensitive, and the stability of PW12 is higher than that of Ge-POMs. In addition, the preparation of Ge-POMs needs more procedures than that of PW12. It is well known that the result of luminescence method is often influenced by the environmental conditions such as the kind of solution, dissolvent and temperature. For further analysis of the catalytic mechanism, the apparent steady-state kinetic parameters for PW12 were determined. Maximum initial velocity (Vmax) and Michaelis–Menten constant (Km) were obtained using Lineweaver–Burk plot (Gao et al., 2007; Peng et al., 2007). Comparison of the kinetic parameters of PW12, Fe3O4 MNPs (magnetic nanoparticles), and HRP was given in Table S1. The Km and Vmax of PW12 with TMB are 0.11 mM and 4.31 10 7 M S 1 at pH 3, respectively. This result confirms that the PW12 behaved as oxidase-like mimetic. The apparent Km value of PW12 with H2O2 as the substrate was significantly higher than that for HRP, consistent with the observation that a higher H2O2 concentration was required to achieve maximal activity for PW12. The apparent Km value of the PW12 with TMB as the substrate was about two point five and four times lower than HRP and Fe3O4. From the Table S1, it can be concluded that the PW12 have a higher activity than HRP and Fe3O4 MNPs. This also indicates that, similar to HRP, PW12 binds and reacts with the first substrate, then releases the first product before reacting with the second substrate. The catalytic effect is related to PW12 electronic structures and their interactions with H2O2 and TMB. Neither H2O2 nor PW12 alone can oxidize TMB efficiently (Fig. S2a). This shows that the interactions between PW12, H2O2, and TMB are important for the catalytic reaction.
3.3. Glucose detection using GOx and PW12 The glucose detection was performed in two separated steps (Test I and Test II) as mentioned in the experimental section because GOx could be denatured at pH 3.0 buffer solution. When the reaction of Eq. (2) was finished at a pH 7.0 PBS buffer solution, the H2O2 produced in the glucose oxidation reaction by GOx was detected using the PW12 (Eq. (1)). Thus, colorimetric detection of glucose could be realized easily using our method. Fig. 2 shows a glucose concentration as low as 1 10 7 mol/L could be detected with a linear range from 1 10 7 to 10 4 mol/L. Qu group reported that glucose can be detected as low as 1 10 6 mol L 1, and the linear range is from 1 10 6 to 2 10 5 mol L 1 with graphene oxide at pH 4 (Song et al., 2010). In the results by Li group (Jv et al., 2010), it can be seen that positively-charged gold nanoparticles as peroxidiase mimic gave the detection limits of the glucose concentration in a linear range from 1.8 10 5 to 1.1 10 3 mol L 1 with a detection limit of 4 10 6 mol L 1for the discoloration. Compared to the above results, the detection limits of glucose obtained by our group is lower than that obtained using Fe3O4 magnetic nanoparticles (Wei and Wang, 2008), graphene oxide and gold nanoparticles as peroxidase mimetic. The test had been done to demonstrate the selectivity of POM colorimetric assay for glucose. The control experiment (Fig. S7) shows that the absorbance hardly increased for glucose analogs: fructose, lactose, and maltose. The selectivity is shown in Fig. S7. Even when the control sample was used at concentrations as high as 5 mM, the signal remained as low as the background signal. Compared with glucose, the color difference can be distinguished even by the naked eye.
J. Wang et al. / Biosensors and Bioelectronics 36 (2012) 18–21
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colorimetric assay to detect glucose with a linear range from 1 10 7 to 1 10 4 mol/L in buffer solution. Based on these findings, polyoxometalates showed great potential applications in varieties of simple, robust, cost-effective, and easy-to-make biosensors in the future. Meantime, graphene-functionalized PW12 exhibited better catalytic activity than the pure PW12 for oxidation of TMB with H2O2. This result showed that graphene could promote the electron transfer between the substrate and catalyst in our detecting system.
Acknowledgments This work was supported by the National Natural Science Foundation of China (20871026, 51078066). Supported by the analysis and testing foundation of Northeast Normal University and the major projects of Jilin Provincial Science and Technology Department.
Appendix A. Supporting information Fig. 2. Linear calibration plot for glucose. The error bars represent the standard deviation of three measurements.
Since glucose oxidase has high affinity to oxidized glucose (Wei and Wang, 2008; Gill et al., 2008; Sanz et al., 2005; Kurita et al., 2004; Pang et al., 2009; Abd-Rabboh and Meyerhoff, 2007; Cosnier et al., 2000; Abe et al., 2008). According to the calibration curve, the concentration of glucose in the normal blood sample is 4.89 mM. The general range of blood glucose concentration in healthy and diabetic persons is about 3–8 mM and 9–40 mM, respectively (Xu et al., 2007). When the blood glucose concentration is deviant, we can detect it easily using this system. Therefore, this colorimetric method is applicable to real samples to determine glucose concentration. 3.4. The peroxidase mimics by PW12/graphene It is known that graphene could promote the electron transfer between the substrate and catalysts. Therefore, we prepared PW12/graphene to evaluate this hypothesis. To examine the influence of different catalyst on oxidation of substance TMB with H2O2 as oxidant, the two group tests were devised with (1) PW12 and (2) PW12/graphene as catalyst respectively under conditions identical to those used for Test I. The color degree of picture 2 is darker than that of picture 1 (Fig. S8) and the absorbance of group 1 (A¼0.236) is lower than 2 (A¼ 0.260). These results show that PW12/graphene could facilitate the oxidation of substance TMB with H2O2 as oxidant comparing to PW12 as peroxidase mimics. 4. Conclusion In summary, the catalytic oxidation of peroxidase substrate TMB with H2O2 using the PW12 was realized. The results indicate that PW12 possesses intrinsic peroxidase-like activity and its catalysis is strongly dependent on pH, temperature and H2O2 concentration, similar to horseradish peroxidase (HRP). PW12 as a mimic peroxidase shows some advantages (such as easy preparation, low-cost, stability, more convenience, easy handling and sensitivity) over natural enzymes. The colorimetric method showed good response toward H2O2 detection with a linear range from 1.34 10 7 to 6.7 10 5 mol/L. More importantly, we design and develop a simple, cheap, highly selective and sensitive
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.03.031.
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Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection Jingjing Wang a, Dongxue Han b, Xiaohong Wang a,n, Bin Qi a,n, Meisheng Zhao c,n a
Key Lab of Polyoxometalate Science of Ministry of Education, Faculty of Chemistry, Northeast Normal University, Changchun 130024, PR China Changchun Institute of Applied Chemistry Chinese Academy of Sciences, PR China c Eye Hospital of the Second Clinical Hospital of Jilin University, Jilin University, Changchun 130020, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 December 2011 Received in revised form 6 March 2012 Accepted 18 March 2012 Available online 10 April 2012
Polyoxometalates (H3PW12O40, H4SiW12O40 and H3PMo12O40) have been proven to possess intrinsic peroxidase-like activity for the first time, which can catalyze oxidation of the peroxidase substrate 3,30 ,5,50 -tetramethylbenzidine (TMB) by H2O2 to form a blue color in aqueous solution. Among them, H3PW12O40 (PW12) exhibits higher catalytic activity to TMB than natural enzyme HRP and other two POMs. In addition, H3PW12O40/graphene exhibited higher activity than H3PW12O40 in this catalytic oxidation reaction due to the effect of graphene in promoting the electron transfer between the substrate and catalyst. POMs/H2O2/TMB system provides a simple, accurate approach to colorimetric detection for H2O2 or glucose. The colorimetric method based on POMs showed good response toward H2O2 and glucose detection with a linear range from 1.34 10 7 to 6.7 10 5 mol/L and 1 10 7 to 1 10 4 mol/L, respectively. The results showed that it is a simple, cheap, more convenient, highly selective, sensitive, and easy handling colorimetric assay. & 2012 Elsevier B.V. All rights reserved.
Keywords: Polyoxometalates Peroxidase-like Colorimetric detection H2O2 Glucose
1. Introduction Artificial enzyme mimics have attracted much attention because there are many disadvantages in the application of natural enzymes such as expensive caused by preparation, purification and storage, and easily inhibition of their activity (Shoji and Freund, 2001). Peroxidase enzymes could activate H2O2 to perform outstanding oxidizability in nature and have long been targeted by biomimic chemistry (Ellis et al., 2009). At present, a large number of artificial enzymes have been constructed by incorporating a catalytic center into a variety of scaffolds to mimic natural peroxidase enzymes, including nanoparticles (Liu and Tang, 2010), gel (Wulff, 2002) or polymers (Liu and Shen, 2009). Recently, it was reported that Fe3O4 magnetic nanoparticles possessed intrinsic enzyme mimetic activity similar to that found in natural peroxidases (Wei and Wang, 2008; Zhang et al., 2010), which was used to detect H2O2 and thrombin. FeS nanostructure (Sanz et al., 2005), BiFeO3 nanoparticles (AbdRabboh and Meyerhoff, 2007), polymer-coating CeO2 nanoparticles (Peng et al., 2007), bimetallic alloy nanoparticles (He et al., 2010), graphene oxide (Song et al., 2010) and positively charged gold nanoparticles (Jv et al., 2010) were found with peroxidase n
Corresponding authors. Tel.: þ 86 431 88930042; fax: þ 86 431 85099759. E-mail addresses: [email protected], [email protected] (X. Wang), [email protected] (M. Zhao). 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.03.031
activity. Nevertheless, these examples demonstrate the potential as peroxidase mimetics, further efforts to find or develop superior enzyme mimetics with more sensitivity, reusability, and stability are still required to underway and hold significant incentives in the field of bioinorganic chemistry. Polyoxometalates are the well-known metal-oxo-cluster compounds, which are emerging as useful materials for numerous applications, especial in biology (Goozerh and Proust, 1998). POMs exhibit many ideal properties in biological and medical fields, such as being nano-sized, removed by the renal system easily, lower toxicity level, stability in biological conditions, and applied as antitumor agents (Wang et al., 2003), HIV inhibitior (Judd et al., 2001), and antiviral agents (Inoue et al., 2005). Our interest is the application of POMs in colorimetric multiplexed immunoassay. It is well-known that POMs exhibit fast and reversible multi-electron redox processes without any significant structural alteration, which has made them prominent candidates as redox sensors and electrochromic or photochromic materials (Liu and Tang, 2010). Meanwhile, one- or two-electron-transfer oxidation of organic substrates into the corresponding compounds is an important reaction in organic synthesis (Wang et al., 2010) using H2O2 as an oxidant. Therefore, POMs might mimic peroxidase enzymes to oxidize some dyes such as TMB to oxidation form with blue color in colorimetric multiplexed immunoassay. TMB is a dye used as a horseradish peroxidase (HRP) substrate in various bioassays upon the oxidation form of blue. TMB has been used to demonstrate the peroxidase-like
J. Wang et al. / Biosensors and Bioelectronics 36 (2012) 18–21
activities of Fe3O4 nanoparticles (Zhang et al., 2010) and singlewalled carbon nanotube (Song et al., 2010). POMs as colorimetric multiplexed immunoassay agents to detect glucose have not been reported by far. In the present study, we have investigated the peroxidase mimetics of POMs and POM/graphene in detection of glucose to propose a simple and sensitive detection method for glucose in aqueous solutions for the first time. It is known that most of glucose biosensors are based on the oxidation or reduction of enzymatically produced H2O2 (Fu et al., 2011). Therefore, the accurate and rapid determination of H2O2 is practically important due to its application in food, pharmaceutical, clinical, industrial, and environmental analysis (Yao et al., 2006). Jin group has reported that the resulting Prussian blue based nanoelectrode arrays show a low detection limit of 1 mM (Xian et al., 2007). It had been reported recently that graphene/Prussian blue nanocomposite multilayer films were used to detect H2O2 by electrochemical surface plasmon resonance (EC-SPR) spectroscopy with the detection limit up to 1 mM (Mao et al., 2011). The better results were attributed to the promoting effect on Prussian by graphene. To the best of our knowledge, no attempt has been made to fabricate POMs/TMB system or POM on graphene/TMB system to colorimetrically detect H2O2 and glucose.
2. Material and methods 2.1. Chemicals and materials The purchased chemicals (analytical grade) were used without further purification. 3,30 ,5,50 -tetramethylbenzidine (TMB) was purchased from j&k chemica, Beijing. Na3WO4, H3PO4, HCl, and 30% H2O2 etc. were purchased from Beijing Chemical Reagent Company (Beijing, China). Glucose oxidase (GOx) was purchased from Sigma-Aldrich (Milwaukee, WI). Other reagents and chemicals were at least analytical reagent grade. The water used throughout all experiments was purified. H3PW12O40 (PW12), H3PMo12O40 (PMo12) and H4SiW12O40 (SiW12) were synthesized according to a previous method (Rocchiccioli-Deltcheff et al., 1983; Ding et al., 2005; Shi et al., 2005). 2.2. Instrument Absorption spectra were recorded on a Cary 500 UV–vis-NIR spectrophotometer. 2.3. Preparation of the PW12/grapheme The PW12/graphene was prepared according to the Ref. (Zhou and Han, 2010) and characterized by UV–vis (Fig. S1).
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To examine the influence of incubation temperature on the PW12 activity, catalytic reactions incubated in different temperature water baths from 20 to 60 1C were investigated, under conditions identical to those used for Test I. As control experiments, 50 mL of water was used instead of 50 mL of PW12. 2.5. Glucose detection using GOx and PW12 Glucose detection was tested as follows: (referred to as Test II) (a) 20 mL of 20 mg/mL GOx and 200 mL of glucose of different concentrations in phosphate buffered saline (PBS, pH 7.0) were incubated at 37 1C for 30 min. (b) 40 mL of 800 mM TMB and 20 mL H3PW12O40 in 250 mL buffer solution (pH 3.0) were added into the above 280 mL glucose reaction solution; (c) the mixed solution (pH 3.326 ) was incubated at room temperature for 15 min; (d) the mixed solution was used to perform the adsorption spectroscopy measurement.
3. Results and discussion 3.1. The peroxidase activity of POMs The POMs were used to catalyze the oxidation of a peroxidase substrate TMB by H2O2 to the oxidized colored product (Eq. (1)), which provides a colorimetric detection of H2O2. Combination of the catalytic reaction of glucose with glucose oxidase as catalyst (GOx) (Eq. (2)) and the POMs catalytic reaction (Eq. (1)), a colorimetric method (Scheme 1) for glucose detection could be developed. This method exhibited sensitive and selective response toward glucose detection. POMs
H2 O2 þTMB!2H2 Oþ oxidized TMB GOx
O2 þglucose!H2 O2 þgluconic acid
ð1Þ ð2Þ
In order to evaluate the performance of POMs as peroxidase mimetics, three POMs had been chosen to study their catalytic activity in detecting H2O2 (Fig. S2), including H3PW12O40 (PW12), H4SiW12O40 (SiW12), and H3PMo12O40 (PMo12). The results show that PW12, SiW12 and PMo12 exhibited different catalytic activity in oxidation of TMB by H2O2. It can be seen that without any catalyst, TMB could not be oxidized to oxidative form with blue color. This is consistent to Yan’s previous study (Gao et al., 2007). Using POMs such as PW12 and SiW12, the oxidation of TMB could occur by H2O2. The absorption spectra indicated that the presence of PW12 and SiW12 gave a 740%, 730% response when compared with the one in the absence of the PW12 or SiW12. However, using PMo12 as a catalyst could catalyze both O2 and H2O2 to oxidize TMB efficiently, while the absorption spectrum with H2O2 is similar to that without H2O2. It is supposed that PMo12 has
2.4. H2O2 detection using PW12 as peroxidase mimetics To investigate the peroxidase-like activity of POMs, the catalytic oxidation of the peroxidase substrate TMB in the presence of H2O2 was designed. Experiments were carried out as follows (referred to as Test I): (a) 10 mM H3PW12O40 in a reaction volume of 1000 mL buffer solution (pH 3.0) with 800 mM TMB as substrate, and H2O2 concentration was 10 mM; (b) the mixed solution was incubated at room temperature (25 1C) for 10 min; (c) 500 mL of the resulting reaction solution was diluted with 500 mL water and then used for adsorption spectroscopy measurement. To examine the influence of reaction buffer pH on the PW12 activity, the acetate buffer solutions from pH 2.0 to 9.0 were investigated, under conditions identical to those used for Test I.
Scheme 1. Schematic illustration of colorimetric detection of glucose by using glucose oxidase (GOx) and POM-catalyzed reactions.
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intrinsic higher catalytic oxidative activity and can facilitate the fast oxidation of TMB only in the present of O2 but no need of H2O2. Thus, PMo12 is useless for detecting H2O2 and glucose. Therefore, PW12 was selected as the catalyst in our detecting system from the above comparison because of easy preparation and more stability than SiW12. In addition, the catalytic activity of simple acid, such as HCl and H2SO4, had been checked in our detecting system (Fig. S3). It can be seen that the catalytic activity of these two mineral acids is lower than that of PW12. Moreover, the simple acid is corrosive and is not beneficial to environment. So the simple acid has been replaced by more novel and beneficial environmental catalysts. 3.2. PW12 as peroxidase mimetics and its use in H2O2 detection According to the above discussion, PW12 can catalyze the oxidation of TMB by H2O2 to obtain the TMB oxidized form with blue color (Fig. S4), which shows that PW12 has intrinsic peroxidaselike catalytic activity. The catalytic reaction can be detected by monitoring TMB absorbance change at 652 nm (Gao et al., 2007). It is known that the pH and temperature are the important parameters for the H2O2 detection. The pH-dependent response curve is showed in Fig. S5. The catalytic oxidation of TMB with H2O2 catalyzed by PW12 was much faster in acidic solutions than in neutral or basic solutions. The catalytic activity at pH 3 was highest. Thus, the pH 3.0 buffer solution was taken as the optimal reaction solution to get a high catalytic activity. The reaction temperature-dependent response curves were shown in Fig. S6. DA had been used to evaluate the catalytic activity of PW12 under different temperatures, where DA¼A(PW12, 652 nm) -A(blank, 652 nm). The large DA is, the higher catalytic activity is. A highest value could be obtained at 25 1C (about room temperature) in Fig.S6b. Thus, 25 1C was taken as the optimal reaction temperature. The system discussed above could be used to detect H2O2 because the catalytic activity of the PW12 is H2O2 concentration dependent (Eq. (1)), the method could be developed for H2O2 detection under the optimal conditions (25 1C, pH 3.0 buffer). Fig. 1 shows a H2O2 concentration as low as 1.34 10 7 mol/L could be detected with a linear range from 1.34 10 7 to 6.7 10 5 mol/L. In the results reported by Wu group (Wang et al., 2011), it can be seen that POMs [(CH3)4N]2.5H7.5[Eu(GeW11O39)(H2O)2]2 4.5H2O (Ge-POMs) gave the
Fig. 1. Linear calibration plot for H2O2. The error bars represent the standard deviation of three measurements.
detection limits with the H2O2 concentration in a linear range from 0 to 0.135 mM with a detection limit of 0.16 mM for the discoloration and from 0 to 0.15 mM with a detection limit of 0.68 mM for the luminescence recovery based on S/N¼3, respectively. Compared to Wu’s result, our process is much more convenient and sensitive, and the stability of PW12 is higher than that of Ge-POMs. In addition, the preparation of Ge-POMs needs more procedures than that of PW12. It is well known that the result of luminescence method is often influenced by the environmental conditions such as the kind of solution, dissolvent and temperature. For further analysis of the catalytic mechanism, the apparent steady-state kinetic parameters for PW12 were determined. Maximum initial velocity (Vmax) and Michaelis–Menten constant (Km) were obtained using Lineweaver–Burk plot (Gao et al., 2007; Peng et al., 2007). Comparison of the kinetic parameters of PW12, Fe3O4 MNPs (magnetic nanoparticles), and HRP was given in Table S1. The Km and Vmax of PW12 with TMB are 0.11 mM and 4.31 10 7 M S 1 at pH 3, respectively. This result confirms that the PW12 behaved as oxidase-like mimetic. The apparent Km value of PW12 with H2O2 as the substrate was significantly higher than that for HRP, consistent with the observation that a higher H2O2 concentration was required to achieve maximal activity for PW12. The apparent Km value of the PW12 with TMB as the substrate was about two point five and four times lower than HRP and Fe3O4. From the Table S1, it can be concluded that the PW12 have a higher activity than HRP and Fe3O4 MNPs. This also indicates that, similar to HRP, PW12 binds and reacts with the first substrate, then releases the first product before reacting with the second substrate. The catalytic effect is related to PW12 electronic structures and their interactions with H2O2 and TMB. Neither H2O2 nor PW12 alone can oxidize TMB efficiently (Fig. S2a). This shows that the interactions between PW12, H2O2, and TMB are important for the catalytic reaction.
3.3. Glucose detection using GOx and PW12 The glucose detection was performed in two separated steps (Test I and Test II) as mentioned in the experimental section because GOx could be denatured at pH 3.0 buffer solution. When the reaction of Eq. (2) was finished at a pH 7.0 PBS buffer solution, the H2O2 produced in the glucose oxidation reaction by GOx was detected using the PW12 (Eq. (1)). Thus, colorimetric detection of glucose could be realized easily using our method. Fig. 2 shows a glucose concentration as low as 1 10 7 mol/L could be detected with a linear range from 1 10 7 to 10 4 mol/L. Qu group reported that glucose can be detected as low as 1 10 6 mol L 1, and the linear range is from 1 10 6 to 2 10 5 mol L 1 with graphene oxide at pH 4 (Song et al., 2010). In the results by Li group (Jv et al., 2010), it can be seen that positively-charged gold nanoparticles as peroxidiase mimic gave the detection limits of the glucose concentration in a linear range from 1.8 10 5 to 1.1 10 3 mol L 1 with a detection limit of 4 10 6 mol L 1for the discoloration. Compared to the above results, the detection limits of glucose obtained by our group is lower than that obtained using Fe3O4 magnetic nanoparticles (Wei and Wang, 2008), graphene oxide and gold nanoparticles as peroxidase mimetic. The test had been done to demonstrate the selectivity of POM colorimetric assay for glucose. The control experiment (Fig. S7) shows that the absorbance hardly increased for glucose analogs: fructose, lactose, and maltose. The selectivity is shown in Fig. S7. Even when the control sample was used at concentrations as high as 5 mM, the signal remained as low as the background signal. Compared with glucose, the color difference can be distinguished even by the naked eye.
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colorimetric assay to detect glucose with a linear range from 1 10 7 to 1 10 4 mol/L in buffer solution. Based on these findings, polyoxometalates showed great potential applications in varieties of simple, robust, cost-effective, and easy-to-make biosensors in the future. Meantime, graphene-functionalized PW12 exhibited better catalytic activity than the pure PW12 for oxidation of TMB with H2O2. This result showed that graphene could promote the electron transfer between the substrate and catalyst in our detecting system.
Acknowledgments This work was supported by the National Natural Science Foundation of China (20871026, 51078066). Supported by the analysis and testing foundation of Northeast Normal University and the major projects of Jilin Provincial Science and Technology Department.
Appendix A. Supporting information Fig. 2. Linear calibration plot for glucose. The error bars represent the standard deviation of three measurements.
Since glucose oxidase has high affinity to oxidized glucose (Wei and Wang, 2008; Gill et al., 2008; Sanz et al., 2005; Kurita et al., 2004; Pang et al., 2009; Abd-Rabboh and Meyerhoff, 2007; Cosnier et al., 2000; Abe et al., 2008). According to the calibration curve, the concentration of glucose in the normal blood sample is 4.89 mM. The general range of blood glucose concentration in healthy and diabetic persons is about 3–8 mM and 9–40 mM, respectively (Xu et al., 2007). When the blood glucose concentration is deviant, we can detect it easily using this system. Therefore, this colorimetric method is applicable to real samples to determine glucose concentration. 3.4. The peroxidase mimics by PW12/graphene It is known that graphene could promote the electron transfer between the substrate and catalysts. Therefore, we prepared PW12/graphene to evaluate this hypothesis. To examine the influence of different catalyst on oxidation of substance TMB with H2O2 as oxidant, the two group tests were devised with (1) PW12 and (2) PW12/graphene as catalyst respectively under conditions identical to those used for Test I. The color degree of picture 2 is darker than that of picture 1 (Fig. S8) and the absorbance of group 1 (A¼0.236) is lower than 2 (A¼ 0.260). These results show that PW12/graphene could facilitate the oxidation of substance TMB with H2O2 as oxidant comparing to PW12 as peroxidase mimics. 4. Conclusion In summary, the catalytic oxidation of peroxidase substrate TMB with H2O2 using the PW12 was realized. The results indicate that PW12 possesses intrinsic peroxidase-like activity and its catalysis is strongly dependent on pH, temperature and H2O2 concentration, similar to horseradish peroxidase (HRP). PW12 as a mimic peroxidase shows some advantages (such as easy preparation, low-cost, stability, more convenience, easy handling and sensitivity) over natural enzymes. The colorimetric method showed good response toward H2O2 detection with a linear range from 1.34 10 7 to 6.7 10 5 mol/L. More importantly, we design and develop a simple, cheap, highly selective and sensitive
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.03.031.
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