Fe(III)-based coordination polymer nanoparticles: peroxidase-like catalytic activity and their application to hydrogen peroxide and glucose detection

Abstract

Fe(III)-based coordination polymer nanoparticles (FeCPNPs) have been prepared for the first time by simple mixing of ferric chloride and sodium hexametaphosphate (SHAM) aqueous solutions at room temperature. It was found that such FeCPNPs possess peroxidase-like activity capable of catalyzing the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) by H₂O₂ turning the solution blue in color. Based on these findings, a simple, sensitive and selective colorimetric assay to detect H₂O₂ was developed, with the linear range and detection limit estimated to be from 1 μM to 50 μM (r = 0.997) and 0.4 μM, respectively. The application of this colorimetric assay to glucose detection both in buffer solution and diluted serum has also been demonstrated successfully. This glucose sensor exhibits excellent performance with a linear range from 2 μM to 20 μM (r = 0.985) and a detection limit of about 1 μM.

---

1. Introduction

During the past decades, considerable attention has been paid on the artificial enzyme mimics¹ because natural enzymes suffer from some serious disadvantages, such as easy denaturation by environmental changes, digestion by proteases, expensive preparation and purification.^2,3 Indeed, numerous enzyme mimetics such as hemin, hematin, cyclodextrin, porphyrin, etc. have been successfully developed.⁴ Among them, peroxidase enzymes have caused widespread interest among researchers due to their great potential in diagnostic detection of H₂O₂ and glucose.⁵

They are important for minimizing diabetic complications to maintain blood glucose concentrations within the normal physiological range⁶ because of the great importance of sugar sensing in human blood and their potential use in fuel cell applications.⁷ Up to now, a number of glucose sensors have been developed.⁸ Among them, horseradish peroxidase (HRP) has been widely used to fabricate sensors for detection of the products from reactions catalyzed by glucose oxidase (GOx).⁹ In comparison with HRP, nanomaterials have created widespread interest in their use as high-efficiency catalysts due to their large surface-to-volume ratio as well as unique properties such as low-cost, easy obtainability, more stability to biodegradation and less vulnerability to denaturation. Yan and co-workers first reported that Fe₃O₄ nanoparticles possess peroxidase-like activity similar to HRP.¹⁰ Up to now, however, only very limited nanostructures including Fe₃O₄ nanoparticles,¹¹polymer coated CeO₂ nanoparticles,¹²BiFeO₃ nanoparticles,¹³single-walled carbon nanotubes,¹⁴graphene oxide,¹⁵ FeS nanostructures,¹⁶ positively-charged gold nanoparticles¹⁷ and carboxyl functionalized mesoporous polymers¹⁸ have been successfully used in this assay. Among these nanomaterials, iron-containing materials exhibit excellent performance in colorimetric detection of H₂O₂.^11,13,16,19 However, most of these materials need a complicated fabrication process. For example, the fabrication of Fe₃O₄ nanoparticles needs several steps; temperature condition is strict in the preparation of BiFeO₃ nanoparticles; the purification of single-walled carbon nanotubes is time-consuming; N₂ atmosphere protection is necessary in the formation of nanostructured FeS. Therefore, easy-to-prepare material is a strong need in the assay of colorimetric detection of H₂O₂. To the best of our knowledge, the use of Fe(III)-based coordination polymer nanoparticles (FeCPNPs) for colorimetric H₂O₂ detection has not been reported so far. In this paper, we demonstrate for the first time that FeCPNPs can be rapidly prepared by simple mixing of aqueous ferric iron and sodium hexametaphosphate solutions at room temperature. It suggests that the resultant FeCPNPs possess peroxidase-like activity capable of catalyzing oxidation of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂ to develop a blue color in aqueous solution, leading to a simple method for colorimetric detection of H₂O₂ with a linear detection range from 1 μM to 50 μM (r = 0.997) and a detection limit of 0.4 μM. Knowing that H₂O₂ is the main product of glucose oxidation, glucose can also be subsequently detected. The linear range of glucose detection was 2 μM to 20 μM (r = 0.985) and the detection limit was estimated to be 1 μM.

2. Experimental

2.1 Materials

Na₂HPO₄, FeCl₃·6H₂O, HRP, sodium acetate (NaAc), SHAM, glucose, maltose, fructose and lactose were purchased from Beijing Chemical Corp. Acetic acid, TMB, glucose and H₂O₂ (30 wt%) were purchased from Aladin Ltd. (Shanghai, China). GOx was purchased from Aldrich Chemical Company. All chemicals were used as-received without further purification. The water used throughout all experiments was purified through a Millipore system.

2.2 Preparation of FeCPNPs

FeCPNPs were prepared by direct mixing of FeCl₃ and SHAM aqueous solutions. In brief, 60 μL of 1.0 M FeCl₃ was added to 3.94 mL of 6 mM SHAM aqueous solution at room temperature under shaking, leading to a large amount of precipitates. The resulting precipitates thus formed were washed with distilled water and centrifuged twice. Then, the resultant products were redispersed in water for characterization and further use.

2.3 Detection of H₂O₂

For detection of H₂O₂, measurements were carried out by monitoring the absorbance change at 652 nm. In a typical run, 50 μL of FeCPNPs dispersion was added to 800 μL of NaAc buffer solution (pH 4.0), followed by addition of 200 μL of TMB solution (1 mM in ethanol). The concentration of H₂O₂ was 42 mM, unless otherwise stated. The UV-vis spectra were recorded after reaction for 20 min at 45 °C.

2.4 Detection of glucose

Glucose detection was performed according to the following three steps: (1) 100 μL of 1 mg mL⁻¹ GOx and 100 μL of glucose of different concentrations in 200 μL of 10 mM Na₂HPO₄ buffer (pH 7.0) were incubated at 37 °C for 1 h; (2) 200 μL of TMB (1 mM in ethanol) and 200 μL of the FeCPNPs dispersion were added to the above glucose reaction solution; (3) the resulting mixture was incubated at 45 °C for 20 min before measurements.

For glucose determination in serum, the serum sample was first diluted 200-fold for the measurement. In control experiments, 5 mM maltose, 5 mM lactose, and 5 mM fructose were used instead of 1 mM glucose.

2.5 Characterization

Transmission electron microscopy (TEM) measurement was made on a HITACHI H-8100 EM (Hitachi, Tokyo, Japan) with an accelerating voltage of 200 kV. UV-vis spectra were obtained on a UV5800 Spectrophotometer.

3. Results and discussion

The structure of amorphous iron and hexaphosphate has already been studied by Mali's group and an idealized model has been constructed.²⁰ Similarly, the FeCPNPs fabricated in our assay should be in accordance with their model in which each metaphosphate group is bonded to two iron atoms and each iron atom is bonded to three phosphorus atoms and is shared between two molecules. Fig. 1a shows the TEM image of the FeCPNPs thus formed, indicating the formation of nanoparticles with diameters in the range of 20 to 50 nm. The chemical composition of the nanoparticles was further determined by the energy dispersion spectrum (EDS), as shown in Fig. 1b. The peaks of P and Fe were observed, indicating that the nanoparticles are formed from ferric iron and SHAM. The Si and other peaks originate from the ITO substrate. All these observations confirm the successful fabrication of FeCPNPs.

Fig. 1 (a) TEM image and (b) the corresponding EDS of FeCPNPs thus formed.

To examine the peroxidase-like activity of the FeCPNPs, the catalytic oxidation of the peroxidase substrate TMB in the presence of H₂O₂ was tested. The inset in Fig. 2 shows the photographs of TMB solutions under different conditions. It is seen that TMB solutions in the absence and presence of H₂O₂ exhibit no color change, indicating that no oxidation reaction occurs in the absence of FeCPNPs. However, after the addition of FeCPNPs to the solution containing H₂O₂ and TMB, a blue color was observed after incubation. All these observations indicate that FeCPNPs have peroxidase-like catalytic ability and thus can catalyze oxidation of TMB in the presence of H₂O₂.

Fig. 2 UV-vis spectra of TMB solution (black line), TMB–H₂O₂ (red line), TMB–H₂O₂–FeCPNPs (blue line) and FeCPNPs solution (purple line) in pH 4.0 acetate buffer (TMB: 0.1 mM; H₂O₂: 42 mM; FeCPNPs: 2.6 mg mL⁻¹). Inset: typical photographs of three samples to the corresponding lines.

UV-vis spectra of various TMB solutions in acetate buffer (pH 4.0) at 45 °C shown in Fig. 2 further reveal the differences between these samples. It is seen that TMB solutions both in the absence and presence of H₂O₂ exhibit no strong absorption peaks ranging from 330 to 800 nm. However, after addition of FeCPNPs, the TMB–H₂O₂ solution exhibits three strong absorption peaks centered at 372, 456, and 652 nm, respectively, which can be attributed to that FeCPNPs catalyze the oxidation of TMB by H₂O_2. Additionally, the absorbance at 652 nm increased with increasing H₂O₂ concentration (Fig. S1, ESI†). It should be noted that the FeCPNPs sample itself exhibits a weak absorption at 652 nm and thus makes negligible contribution to the whole UV-vis intensity of FeCPNP-involved sample measurements. All these observations further confirm that FeCPNPs possess peroxidase-like activity for oxidation of TMB by H₂O₂.

Similar to peroxidase, the catalytic activity of FeCPNPs is dependent on pH, temperature and H₂O₂ concentration, the corresponding information is shown in Fig. 3. The results show that the optimal pH and temperature for the detection of H₂O₂ were evaluated to be 4.0 and 45 °C, respectively. Note that FeCPNPs require a higher concentration of H₂O₂ to reach the maximum level of peroxidase-like activity, indicating that the catalytic activity of the FeCPNPs is more stable at high H₂O₂ concentration than HRP. This behavior was very similar to that observed with other NP-based peroxidase mimetics and HRP.^10,15

Fig. 3 Dependence of the FeCPNPs peroxidase-like activity on (a) pH, (b) temperature and (c) H₂O₂ concentration. Experiments were carried out using 50 μL FeCPNPs or 0.5 ng mL⁻¹ HRP in 1 mL acetate buffer (pH 4.0) with 0.1 mM TMB as a substrate. The H₂O₂ concentration was 10 mM at pH 4.0 and 45 °C unless otherwise stated. The maximum point in each curve was set as 100%.

On the basis of the peroxidase-like property of FeCPNPs, a colorimetric method for H₂O₂ and glucose detection was designed. As demonstrated above, the color variation of TMB oxidation catalyzed by FeCPNPs was H₂O₂ concentration-dependent. This indicates that the absorbance change can be used for detection of H₂O₂. The absorbance at 652 nm was proportional to H₂O₂ concentration from 0.001–0.05 mM (Fig. 4a) with a detection limit of 0.4 μM. H₂O₂ is the main product of GOx-catalyzed reaction of glucose oxidation; therefore, colorimetric detection of glucose can also be realized using FeCPNPs instead of the traditionally used HRP. Since GOx would not be stable in pH 4.0 buffer solution, the glucose detection can be performed as follows: first, glucose is catalyzed by GOx to form glucose acid in a pH 7.0 buffer solution and the substrate oxygen is converted to H₂O₂ at the same time;²¹ then, FeCPNPs can catalyze the oxidation of TMB in the presence of H₂O₂ at pH 4.0, which produces a blue color in the solution. The detailed procedure is described in the Experimental section. A typical glucose response curve is shown in Fig. 4b and the inset shows the corresponding calibration plot: the linear range is from 2 to 20 μM and glucose can be detected as low as 1 μM. Moreover, this method can be used for detecting glucose in human serum samples. It is well known that the general range of blood glucose concentration in healthy and diabetic persons is about 3–8 mM and 9–40 mM, respectively.²¹ Therefore, this colorimetric method is applicable to diluted real samples to determine the glucose concentration. According to the calibration curve, the concentration of glucose in the serum sample used in our assay is estimated to be 3.2 mM, the corresponding data are shown in Fig. 5. Control experiments show that the absorbance increased slightly for glucose analogues, including fructose, lactose and maltose, with concentrations 4-fold higher than that of glucose (Fig. 6), indicating that the present sensing system shows high selectivity for glucose due to the high affinity of GOx to oxidized glucose.

Fig. 4 Dependence of the absorbance at 652 nm on the concentration of H₂O₂ (a) and glucose (b) in the range from 1 μM to 1 mM and 2 μM to 1 mM, respectively. The insets show the corresponding linear calibration plots.

Fig. 5 The absorbance at 652 nm for buffer solution and a 200-fold diluted serum sample, error bars represent the standard deviation for three measurements.

Fig. 6 Selectivity analysis for glucose detection by monitoring the relative absorbance. The analyte concentrations were as follows: 5 mM fructose, 5 mM lactose, 5 mM maltose, and 1 mM glucose. Inset: the color change of different solutions.

4. Conclusions

In summary, simple mixing of ferric chloride and sodium hexametaphosphate (SHAM) aqueous solutions at room temperature has been proven to be an effective strategy for rapid preparation of FeCPNPs with peroxidase-like activity. The use of such FeCPNPs for sensitive and selective colorimetric H₂O₂ and glucose detection has been demonstrated successfully. Our present study is important because it provides us a novel peroxidase-like catalyst for colorimetric detection of H₂O₂ and glucose, and therefore, could have potential applications in biotechnology and disease surveillance.

Acknowledgements

This work was supported by National Basic Research Program of China (No. 2011CB935800).

References

1. G. Wulff, Chem. Rev., 2002, 102, 1.
2. R. Breslow, Acc. Chem. Res., 1995, 28, 146.
3. E. Shoji and M. S. J. Freund, J. Am. Chem. Soc., 2001, 123, 3383.
4. (a) Z. Genfa and P. K. Dasgupta, Anal. Chem., 1992, 64, 517; (b) R. P. Bonarlaw and J. K. M. Sanders, J. Am. Chem. Soc., 1995, 117, 259; (c) M. S. Muche and M. W. Gobel, Angew. Chem., Int. Ed. Engl., 1996, 35, 2126; (d) K. Chen and L. Que, Angew. Chem., Int. Ed., 1999, 38, 2227; (e) Z. H. Liu, R. X. Cai, L. Y. Mao, H. P. Huang and W. H. Ma, Analyst, 1999, 124, 173; (f) L. Fruk and C. M. Niemeyer, Angew. Chem., Int. Ed., 2005, 44, 2603.
5. W. C. Ellis, C. T. Tran, M. A. Denardo, A. Fischer, A. D. Ryabov and T. J. Collins, J. Am. Chem. Soc., 2009, 131, 18052.
6. J. Wang, K. S. Carmon, L. A. Luck and I. I. Suni, Electrochem. Solid-State Lett., 2005, 8, H61.
7. (a) J. L. C. Clark, C. Lyons and A. N. Y. Aead, Science, 1962, 102, 29; (b) G. Reach and G. S. Wilson, Anal. Chem., 1992, 64, 381A; (c) A. P. F. Turner, B. Chen and A. P. Sergey, Clin. Chem. (Washington, D. C.), 1999, 45, 1596; (d) N. Mano, F. Mao and A. Heller, J. Am. Chem. Soc., 2003, 125, 6588.
8. (a) E. S. Forzani, H. Q. Zhang, L. A. Nagahara, I. Amlani, R. Tsui and N. Tao, Nano Lett., 2004, 4, 1785; (b) H. Wei and E. Wang, Anal. Chem., 2008, 80, 2250; (c) S. Liu, J. Tian, L. Wang, Y. Luo, W. Lu and X. Sun, Biosens. Bioelectron., 2011, 26, 4491; (d) W. Lu, Y. Luo, G. Chang and X. Sun, Biosens. Bioelectron., 2011, 26, 4791.
9. (a) V. Sanz, S. de Marcos, J. R. Castillo and J. Galban, J. Am. Chem. Soc., 2005, 127, 1038; (b) K. Abe, K. Suzuki and D. Citterio, Anal. Chem., 2008, 80, 6928; (c) P. Pang, L. Zhang, T. Ge, Y. Cai, S. Yao and C. A. Grimes, Sens. Actuators, B, 2009, 136, 310.
10. L. Gao, J. Zhuang, L. Nie, J. Zhang, N. Gu, T. Wang, J. Feng, D. Yang, S. Oerrett and X. Yan, Nat. Nanotechnol., 2007, 2, 577.
11. N. Ding, N. Yan, C. Ren and X. Chen, Anal. Chem., 2010, 82, 5897.
12. A. Asati, S. Santra, C. Kaittanis, S. Nath and J. M. Perez, Angew. Chem., Int. Ed., 2009, 48, 2308.
13. W. Luo, Y. Li, J. Yuan, L. Zhu, Z. Liu, H. Tang and S. Liu, Talanta, 2010, 81, 901.
14. Y. Song, X. Wang, C. Zhao, K. Qu, J. Ren and X. Qu, Chem.–Eur. J., 2010, 16, 3617.
15. Y. Song, K. Qu, C. Zhao, J. Ren and X. Qu, Adv. Mater., 2010, 22, 2206.
16. Z. Dai, S. Liu, J. Bao and H. Ju, Chem.–Eur. J., 2009, 15, 4321.
17. Y. Jv, B. Li and R. Cao, Chem. Commun., 2010, 46, 8017.
18. S. Liu, L. Wang, J. Zhai, Y. Luo and X. Sun, Anal. Methods, 2011, 3, 1475.
19. S. Liu, J. Tian, L. Wang, Y. Luo, G. Chang and X. Sun, Analyst, 2011 10.1039/c1an15654c.
20. G. Mali, M. Sala, I. Arcon, V. Kaucic and J. Kolar, J. Phys. Chem. B, 2006, 110, 23060.
21. Y. Xu, P. E. Pehrsson, L. Chen, R. Zhang and W. Zhao, J. Phys. Chem. C, 2007, 111, 8638.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1cy00360g

---