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Cite this: Chem. Commun., 2014, 50, 6771 Received 6th March 2014, Accepted 3rd April 2014
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Water-dispersible silicon dots as a peroxidase mimetic for the highly-sensitive colorimetric detection of glucose† Qiong Chen, Meiling Liu, Jiangna Zhao, Xue Peng, Xiaojiao Chen, Naxiu Mi, Bangda Yin, Haitao Li, Youyu Zhang* and Shouzhuo Yao
DOI: 10.1039/c4cc01703j www.rsc.org/chemcomm
We demonstrate that photoluminescent Si-dots exhibit intrinsic peroxidase-like activity, and can catalyze the oxidization of 3,30 ,5,50 tetramethylbenzidine (TMB) by H2O2, and produce a color change. This strategy can be used to detect glucose with high sensitivity and selectivity.
Semiconductor quantum dots (QDs) have an enormous variety of potential applications due to their excellent optical properties. For instance, Li reported a glucose biosensor based on nanocomposite films of CdTe QDs and glucose oxidase (GOx).1 Bahshi and colleagues reported the sensing of glucose by MB+-functionalized CdSe/ZnS QDs.2 Cao demonstrated that a simply assembled complex consisting of CdTe QDs and GOx can be used to sensitively determine glucose based on its effective fluorescence quenching by H2O2.3 However, heavy metal ion-containing nanoparticles may suffer from intrinsic limitations such as potential toxicity and chemical instability.4 Therefore, it is important to develop excellent nanomaterials for fabricating stable biosensors with good biocompatibilities. As one of the most inert, nontoxic, abundant and low-cost nanomaterials, silicon nanomaterials are used in sensors and corrosion protection due to their attractive advantages, including excellent optical, electronic, mechanical properties and surface tailorability.5 Compared to heavy metal ion-containing quantum dots, Si-dots are biocompatible, inexpensive and chemically stable. In recent years, many reports have been focused on the synthesis of Si-dots by physical, physicochemical, chemical, and electrochemical etching of bulk Si.4,6 The reported Si-dots have always been modified by grafting a water-soluble material on to the particle surface4 and the modified Si-dots may be excellent candidates for biological imaging applications.7 Recently, our research group reported a new way to obtain label-free Si-dots and applied them in fabricating several biosensors.4,8 Based on their unique optical properties, glucose4 and Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China. E-mail: [email protected]; Fax: +86 73188872531; Tel: +86 75188865515 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4cc01703j
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pesticide8 biosensors were developed. In order to expand the application of Si-dots, it is extremely important to explore their unknown advantages and develop new applications of Si-dots. In the present work, we demonstrate that Si-dots have an intrinsic peroxidase mimetic catalytic ability for the first time. Based on the peroxidase mimetics of Si-dots, and the selective catalytic oxidation of glucose by glucose oxidase,9 naked eye glucose colorimetric analysis was carried out, which is shown in Fig. 1. This new type of biosensor does not require complex modification or enzyme immobilization. This offers a simple, sensitive and selective colorimetric method for glucose determination in serum. The Si-dots were successfully synthesized by the phosphomolybdic acid (POM)-assisted electrochemical etching of bulk Si (see the detailed synthesis in the ESI†). The morphology and optical properties of the as-prepared Si-dots were characterized and the results are shown in Fig. S1 (ESI†). The transmission electron microscope (TEM) (Fig. S1A, ESI†) image and the histogram of particle size distribution (inset) indicate that the Si-dots mostly appear as spherical dots with a high monodispersity, and with diameters of 4–25 nm. The average size was about 12 nm in diameter. Electric diffraction (ED) (Fig. S1B, ESI†) and high resolution transmission electron microscopy (HRTEM) (Fig. S1C, ESI†)
Fig. 1 Schematic illustration of the colorimetric detection of glucose using glucose oxidase (GOx) and Si-dots.
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patterns of the Si-dots indicate that these nanoparticles exhibit single crystalline structures. To confirm the presence of a hydrogen terminated surface (H–Si), optical properties of the Si-dots, Fourier-transform infrared spectroscopy (FTIR), UV-vis and fluorescence spectra (FL) measurements were performed. The FTIR spectrum (Fig. S1D, ESI†) shows a strong stretching vibration of Si–H bonds at around 900 cm1 and the stretching vibration of coupled H–Si–Si–H bonds at 2100 cm1.4 The UV-vis absorption (red) and photoluminescence (black) spectra of the Si-dots in aqueous solution are presented in Fig. S1E (ESI†). The absorbance at about 290 nm is due to the Si-dots.10 It has been reported that the PL properties (e.g., wavelength) of H-terminated Si-dots are sensitively dependent on the dot size.11 The emission spectrum of the Si-dots solution has a narrow band ranging from 400 to 500 nm, which further illustrates that the sizes of the Si-dots are uniform.11 The PL quantum yield of the as-prepared Si-dots was up to B9.4% according to the Williams method, which is the same as in the literature.4 All of the results show that Si-dots with a hydrogen terminated surface were successfully synthesized by the phosphomolybdic acid (POM)-assisted electrochemical etching of bulk Si. As shown in Fig. S2A and B (ESI†), the PL intensities of the Si-dots were not affected by the temperature or the pH, suggesting that the Si-dots were stable when they were incubated under a wide range of pH (2–9) and temperatures (20–90 1C) for 2 h. To prove the feasibility of the strategy, the catalytic oxidation of the peroxidase substrate TMB was tested in the presence of the Si-dots and H2O2. Fig. 2 shows the UV-vis absorption spectra for the different test solutions. Fig. 2 (inset) and Fig. S1E (ESI†) show that the Si-dots and the TMB didn’t both have absorptions at 370 nm and 652 nm. That is to say, the absorption peaks at 370 nm and 652 nm reflect the existence of the oxidative product of TMB. Compared with the absorption spectrum of the mixture of the Si-dots and the TMB solution (curve c), weak absorptions at 370 nm and 652 nm were observed in the case of TMB and H2O2 (curve b), which indicated that the weak oxidation of TMB by H2O2 had occurred. A much stronger absorbance was observed in the presence of the Si-dots in a TMB and H2O2 solution (curve a), indicating that
Fig. 2 The effects of the Si-dots in various reaction systems. (a) TMB, Si-dots and H2O2, (b) TMB, and H2O2, (c) Si-dots and TMB. Reaction conditions: the solutions were incubated in 0.2 mM NaAc–HAc buffer (pH 4.0) at 30 1C for 10 min. Inset shows the color changes of the different samples.
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the reaction of TMB with H2O2 was greatly accelerated by the Si-dots. Accordingly, a noticeable color change of the solution also was observed in the presence of the Si-dots in the oxidation system of TMB and H2O2 (see the inset in Fig. 2). To further characterize the peroxidase-like activity of the Si-dots, several typical peroxidase substrates, such as 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and o-phenylenediamine (OPD) were used as replacements for the TMB. (Fig. S3, ESI†). All of the results confirmed that the as-prepared Si-dots exhibited an intrinsic peroxidase-like activity which was similar to that of horseradish peroxidase (HRP) (eqn (1)). Si-dots H2 O2 þ TMB ! H2 O þ oxidizedTMB
(1)
It is well known that enzymatic activity is dependent upon the substrate and the reaction conditions. Therefore the catalytic activity of the Si-dots may be dependent on pH, temperature and H2O2 concentration. As shown in Fig. S4 (ESI†), the UV-vis absorbance at 652 nm increased in intensity with pH in the range of 3.0–4.0 and temperature in the range of 25–40 1C, and then declined on increasing the pH and temperature further. The experimental data imply that the Si-dots reached their highest catalytic activity at the vicinity of pH 4.0 and 40 1C. The effects of H2O2 concentration on the catalytic activity of the Si-dots were also investigated. The intensity of the absorption at 652 nm sharply increased with increasing H2O2 concentration from 10 mM to 200 mM, and then it gradually slows down when the concentration is beyond 300 mM. It is suggested that the reaction of H2O2 with TMB catalyzed by the Si-dots reached equilibrium within 300 mM. Hence, the catalytic activity was best in 300 mM H2O2 acidic solutions (pH 4.0) at 40 1C. From Fig. S4 (ESI†), a linear relationship in the range of 1 to 200 mM (R2 = 0.956) was obtained, which implies that the Si-dots catalytic system could be used in analytical systems involved with H2O2oxidation. To investigate the kinetic mechanism of the peroxidase-like activity of the Si-dots, apparent steady-state kinetic parameters for the peroxidase-like color changing reaction were determined by changing the concentrations of TMB and H2O2 in this system, respectively. Kinetic experiments were carried out using 10 mL of Si-dots in 0.5 mL of 0.2 M NaAc–HAc buffer solution (pH 4.0) containing 320 mM TMB as the substrate, and the H2O2 concentration was 20 mM. The initial reaction rate was calculated using the A Beer–Lambert Law c ¼ where, c is the substrate concentration, eb A is the absorbance, b is the thickness of the solution. The absorbance data were back-calculated to give concentrations using a molar absorption coefficient, e, of 39 000 M1 cm1 for the TMBderived oxidation products.12 Apparent steady-state reaction rates at different concentrations of the substrate were obtained by calculating the slopes of the absorbance changes with time. In this work, typical Michaelis–Menten curves were obtained for both TMB and H2O2 by monitoring the absorbance changes at 652 nm for 5 min (Fig. S5A and B, ESI†).13 The Michaelis–Menten constant (Km) and maximum initial velocity (Vmax) were obtained from Lineweaver– Burk plots of the double reciprocal of the Michaelis–Menten equation, 1 1 1 ¼ Km =Vmax þ (2) n ½S Km
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Table 1 Comparison of Michaelis–Menten constants (Km) and maximum reaction rates (Vmax) of the oxidation reaction catalyzed by the Si-dots, reported C-dots and HRP
Catalyst
Substance
Km/mM
Vmax/108 M s1
HRP31
TMB H2O2
0.434 3.702
10.00 8.71
Si-dots
TMB H2O2
1.502 0.065
14.72 5.65
where n is the initial velocity, Vmax is the maximal reaction velocity, [S] is the concentration of the substrate, and Km is the Michaelis constant. The results are shown in Fig. S5 (ESI†) and summarized in Table 1. The smaller the value of Km, the stronger the affinity between the enzyme and the substrate, and the more efficient the catalyst. The apparent Km value for the Si-dots with H2O2 as the substrate was lower than that of HRP (Table 1), suggesting that the Si-dots have a higher affinity for H2O2 than HRP. The apparent Km value of the Si-dots with TMB was also larger than that of HRP, suggesting that the Si-dots have a lower affinity for TMB than HRP. To further investigate the catalytic mechanism of the Si-dots in the system, we measured their activity under standard reaction conditions by varying the concentration of TMB at a fixed concentration of H2O2, and vice versa. The double reciprocal plots of the initial velocities against the concentrations of one substrate were obtained over a range of concentrations of the second substrate (Fig. S5C and D, ESI†). The lines were parallel, which is characteristic of a ping–pong mechanism,14 which is also observed in HRPbased systems. The experimental facts indicate that the Si-dots bind and react with the first substrate, then release the first product before reacting with the second substrate, which is similar to HRP and some other nano-materials.12 A possible catalytic mechanism of the Si-dots is presented in Fig. 1. First, it is well known that the as-prepared hydrogen terminated Si-dots were initially catalytically inactive.4 Therefore, the H–Si-dots were partially oxidized to their core–shell structure by H2O2, on which H2O2 molecules were adsorbed, and the H2O2 decomposed into active oxygen species with oxene characteristics.15 They then become electrophilic and are prone to oxidize TMB to TMBOX (Fig. 3). We measured the amperometric responses of the
Fig. 3 (A) UV-vis spectra for the mixed solutions of TMB, Si-dots and glucose incubation solution (pH 7.0 buffer, containing GOx) in 0.2 M NaAc–HAc buffer (pH 4.0) at 40 1C. The TMB concentration was 320 mM and 10 mL Si-dots dispersions were used. Inset: the corresponding images of the colored products. (B) Dependence of the absorbance at 652 nm on the concentration of glucose from 0.017 mM to 5 mM. Inset: the corresponding linear calibration plot.
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Si-dots modified glassy carbon electrode (Si-dots/GCE). The reduction current increased sharply to reach a steady-state value for the Si-dots/GCE with the addition of H2O2 (Fig. S6, ESI†), which clearly demonstrated that the Si-dots exhibited an electrocatalytic activity towards H2O2 reduction, which may promote electron transfer between the electron acceptor (H2O2) and the electron donator (the underlying electrode). The stability of the Si-dots and the robustness of the peroxidase activity in wide pH and temperature ranges is crucial for extending their applications. Fig. S7 (ESI†) shows that the catalytic activity of the Si-dots does not change after they were incubated at a wide range of pH (2–9) and temperatures at 20–90 1C for 2 h, which makes them suitable for expanding their applications in biomedicine and environmental fields. The nature of the peroxidase-like activity of the Si-dot nanostructures may originate from their catalytic ability to reduce H2O2. According to the research mentioned above, H2O2 can oxidise the TMB in the presence of peroxidase. With H2O2 as a product of the catalytic oxidation of glucose, glucose could then be detected based on the following reaction. GOx
D-glucose þ O2 þ H2 O ! H2 O2 þ gluconic acid
(3)
Considering that the assay conditions, such as the enzymatic factor, temperature, pH value and incubation time, have a significant effect on the detection of glucose, the experimental conditions, such as, pH, incubation temperature and time were optimized (Fig. S8, ESI†). The optimum temperature, time and pH were 35 1C, 30 min and 7, respectively. Under the optimized conditions, a linear relationship was obtained between the absorbance and the glucose concentration in the range of 0.17 to 200 mmol L1 (R2 = 0.986), with a detection limit of 0.05 mmol L1 (Fig. 3). This detection method based on Si-dots gave a lower detection limit than the method using HRP and other nanoparticles as catalysts (Table S1, ESI†). To investigate the selectivity for glucose detection, control experiments were performed using fructose, lactose, and maltose. The results are shown in Fig. S9 (ESI†). Even when the concentrations of the control samples were 10 times larger than that of glucose, no obvious response was observed, indicating that the colorimetric detection method can be used to detect glucose selectively. The excellent specificity and high sensitivity of the sensor suggested that the developed method could be directly applied for detecting glucose in real samples. Therefore, we detected glucose in serum samples of patients with diabetes provided by Hunan Normal University Hospital. Fig. S10 (ESI†) shows the UV-vis spectra and the inset shows that the experiment performed showed good colorimetric differentiation. According to the calibration curve, the concentration of glucose in blood was 10.2 mM (110.8 mg dL1). A blood glucose test is positive when the amount of glucose is more than 100 mg dL1. This colorimetric method is therefore applicable for determining glucose in blood samples, suggesting that our system was successful in the detection of glucose in real samples. In summary, we firstly reported that our Si-dots possess an intrinsic peroxidase-like activity. On the basis of our research,
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we provide a simple, inexpensive, highly sensitive and selective colorimetric assay to detect glucose in serum. As a novel nanoperoxidase mimetic, the Si-dots show several advantages over HRP, such as stability, dispersibility and high catalytic efficiency. Therefore, we expect that these Si-dots have great potential for applications in biotechnology. This work was supported by the National Natural Science Foundation of China (21375037, 21275037, 21305042), the Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and the Doctoral Fund of Ministry of Education of China (No. 20134306110006).
Notes and references 1 X. Li, Y. Zhou, Z. Zheng, X. Yue, Z. Dai, S. Liu and Z. Tang, Langmuir, 2009, 25, 6580–6586. 2 L. Bahshi, R. Freeman, R. Gill and I. Willner, Small, 2009, 5, 676–680. 3 L. Cao, J. Ye, L. Tong and B. Tang, Chem. – Eur. J., 2008, 14, 9633–9640. 4 Y. Yi, J. Deng, Y. Zhang, H. Li and S. Yao, Chem. Commun., 2013, 49, 612–614. 5 S. Song, Y. Qin, Y. He, Q. Huang, C. Fan and H. Y. Chen, Chem. Soc. Rev., 2010, 39, 4234–4243.
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ChemComm 6 (a) A. Shiohara, S. Hanada, S. Prabakar, K. Fujioka, T. H. Lim, K. Yamamoto, P. Northcote and R. D. Tilley, J. Am. Chem. Soc., 2010, 132, 248–253; (b) Z. H. Kang, C. H. A. Tsang, Z. D. Zhang, M. L. Zhang, N.-B. Wong, J. Antonio Zapien, Y. Y. Shan and S.-T. Lee, J. Am. Chem. Soc., 2007, 129, 5326–5327; (c) V. Kumar, Nanosilicon, Elsevier Ltd, 2007; (d) P. K. Sudeep, Z. Page and T. Emrick, Chem. Commun., 2008, 6126–6127. 7 (a) F. Erogbogbo, K.-T. Yong, I. Roy, G. Xu, P. N. Prasad and M. T. Swihart, ACS Nano, 2008, 2, 873–878; (b) F. Erogbogbo, K.-T. Yong, I. Roy, R. Hu, W.-C. Law, W. Zhao, H. Ding, F. Wu, R. Kumar, M. T. Swihart and P. N. Prasad, ACS Nano, 2010, 5, 413–423. 8 Y. Yi, G. Zhu, C. Liu, Y. Huang, Y. Zhang, H. Li, J. Zhao and S. Yao, Anal. Chem., 2013, 85, 11464–11470. 9 (a) D. Chen, H. Feng and J. Li, Chem. Rev., 2012, 112, 6027–6053; (b) Q. Zhang, S. Wu, L. Zhang, J. Lu, F. Verproot, Y. Liu, Z. Xing, J. Li and X.-M. Song, Biosens. Bioelectron., 2011, 26, 2632–2637. 10 Z. F. Li and E. Ruckenstein, Nano Lett., 2004, 4, 1463–1467. 11 Z. Kang, Y. Liu, C. H. A. Tsang, D. D. D. Ma, X. Fan, N.-B. Wong and S.-T. Lee, Adv. Mater., 2009, 21, 661–664. 12 W. Shi, Q. Wang, Y. Long, Z. Cheng, S. Chen, H. Zheng and Y. Huang, Chem. Commun., 2011, 47, 6695–6697. 13 L. Su, J. Feng, X. Zhou, C. Ren, H. Li and X. Chen, Anal. Chem., 2012, 84, 5753–5758. 14 L. Z. Gao, J. Zhuang, L. Nie, J. B. Zhang, Y. Zhang, N. Gu, T. H. Wang, J. Feng, D. L. Yang, S. Perrett and X. Y. Yan, Nat. Nanotechnol., 2007, 2, 577–583. 15 Z. Kang, C. H. A. Tsang, N.-B. Wong, Z. Zhang and S.-T. Lee, J. Am. Chem. Soc., 2007, 129, 12090–12091.
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Cite this: Chem. Commun., 2014, 50, 6771 Received 6th March 2014, Accepted 3rd April 2014
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Water-dispersible silicon dots as a peroxidase mimetic for the highly-sensitive colorimetric detection of glucose† Qiong Chen, Meiling Liu, Jiangna Zhao, Xue Peng, Xiaojiao Chen, Naxiu Mi, Bangda Yin, Haitao Li, Youyu Zhang* and Shouzhuo Yao
DOI: 10.1039/c4cc01703j www.rsc.org/chemcomm
We demonstrate that photoluminescent Si-dots exhibit intrinsic peroxidase-like activity, and can catalyze the oxidization of 3,30 ,5,50 tetramethylbenzidine (TMB) by H2O2, and produce a color change. This strategy can be used to detect glucose with high sensitivity and selectivity.
Semiconductor quantum dots (QDs) have an enormous variety of potential applications due to their excellent optical properties. For instance, Li reported a glucose biosensor based on nanocomposite films of CdTe QDs and glucose oxidase (GOx).1 Bahshi and colleagues reported the sensing of glucose by MB+-functionalized CdSe/ZnS QDs.2 Cao demonstrated that a simply assembled complex consisting of CdTe QDs and GOx can be used to sensitively determine glucose based on its effective fluorescence quenching by H2O2.3 However, heavy metal ion-containing nanoparticles may suffer from intrinsic limitations such as potential toxicity and chemical instability.4 Therefore, it is important to develop excellent nanomaterials for fabricating stable biosensors with good biocompatibilities. As one of the most inert, nontoxic, abundant and low-cost nanomaterials, silicon nanomaterials are used in sensors and corrosion protection due to their attractive advantages, including excellent optical, electronic, mechanical properties and surface tailorability.5 Compared to heavy metal ion-containing quantum dots, Si-dots are biocompatible, inexpensive and chemically stable. In recent years, many reports have been focused on the synthesis of Si-dots by physical, physicochemical, chemical, and electrochemical etching of bulk Si.4,6 The reported Si-dots have always been modified by grafting a water-soluble material on to the particle surface4 and the modified Si-dots may be excellent candidates for biological imaging applications.7 Recently, our research group reported a new way to obtain label-free Si-dots and applied them in fabricating several biosensors.4,8 Based on their unique optical properties, glucose4 and Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China. E-mail: [email protected]; Fax: +86 73188872531; Tel: +86 75188865515 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4cc01703j
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pesticide8 biosensors were developed. In order to expand the application of Si-dots, it is extremely important to explore their unknown advantages and develop new applications of Si-dots. In the present work, we demonstrate that Si-dots have an intrinsic peroxidase mimetic catalytic ability for the first time. Based on the peroxidase mimetics of Si-dots, and the selective catalytic oxidation of glucose by glucose oxidase,9 naked eye glucose colorimetric analysis was carried out, which is shown in Fig. 1. This new type of biosensor does not require complex modification or enzyme immobilization. This offers a simple, sensitive and selective colorimetric method for glucose determination in serum. The Si-dots were successfully synthesized by the phosphomolybdic acid (POM)-assisted electrochemical etching of bulk Si (see the detailed synthesis in the ESI†). The morphology and optical properties of the as-prepared Si-dots were characterized and the results are shown in Fig. S1 (ESI†). The transmission electron microscope (TEM) (Fig. S1A, ESI†) image and the histogram of particle size distribution (inset) indicate that the Si-dots mostly appear as spherical dots with a high monodispersity, and with diameters of 4–25 nm. The average size was about 12 nm in diameter. Electric diffraction (ED) (Fig. S1B, ESI†) and high resolution transmission electron microscopy (HRTEM) (Fig. S1C, ESI†)
Fig. 1 Schematic illustration of the colorimetric detection of glucose using glucose oxidase (GOx) and Si-dots.
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patterns of the Si-dots indicate that these nanoparticles exhibit single crystalline structures. To confirm the presence of a hydrogen terminated surface (H–Si), optical properties of the Si-dots, Fourier-transform infrared spectroscopy (FTIR), UV-vis and fluorescence spectra (FL) measurements were performed. The FTIR spectrum (Fig. S1D, ESI†) shows a strong stretching vibration of Si–H bonds at around 900 cm1 and the stretching vibration of coupled H–Si–Si–H bonds at 2100 cm1.4 The UV-vis absorption (red) and photoluminescence (black) spectra of the Si-dots in aqueous solution are presented in Fig. S1E (ESI†). The absorbance at about 290 nm is due to the Si-dots.10 It has been reported that the PL properties (e.g., wavelength) of H-terminated Si-dots are sensitively dependent on the dot size.11 The emission spectrum of the Si-dots solution has a narrow band ranging from 400 to 500 nm, which further illustrates that the sizes of the Si-dots are uniform.11 The PL quantum yield of the as-prepared Si-dots was up to B9.4% according to the Williams method, which is the same as in the literature.4 All of the results show that Si-dots with a hydrogen terminated surface were successfully synthesized by the phosphomolybdic acid (POM)-assisted electrochemical etching of bulk Si. As shown in Fig. S2A and B (ESI†), the PL intensities of the Si-dots were not affected by the temperature or the pH, suggesting that the Si-dots were stable when they were incubated under a wide range of pH (2–9) and temperatures (20–90 1C) for 2 h. To prove the feasibility of the strategy, the catalytic oxidation of the peroxidase substrate TMB was tested in the presence of the Si-dots and H2O2. Fig. 2 shows the UV-vis absorption spectra for the different test solutions. Fig. 2 (inset) and Fig. S1E (ESI†) show that the Si-dots and the TMB didn’t both have absorptions at 370 nm and 652 nm. That is to say, the absorption peaks at 370 nm and 652 nm reflect the existence of the oxidative product of TMB. Compared with the absorption spectrum of the mixture of the Si-dots and the TMB solution (curve c), weak absorptions at 370 nm and 652 nm were observed in the case of TMB and H2O2 (curve b), which indicated that the weak oxidation of TMB by H2O2 had occurred. A much stronger absorbance was observed in the presence of the Si-dots in a TMB and H2O2 solution (curve a), indicating that
Fig. 2 The effects of the Si-dots in various reaction systems. (a) TMB, Si-dots and H2O2, (b) TMB, and H2O2, (c) Si-dots and TMB. Reaction conditions: the solutions were incubated in 0.2 mM NaAc–HAc buffer (pH 4.0) at 30 1C for 10 min. Inset shows the color changes of the different samples.
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the reaction of TMB with H2O2 was greatly accelerated by the Si-dots. Accordingly, a noticeable color change of the solution also was observed in the presence of the Si-dots in the oxidation system of TMB and H2O2 (see the inset in Fig. 2). To further characterize the peroxidase-like activity of the Si-dots, several typical peroxidase substrates, such as 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and o-phenylenediamine (OPD) were used as replacements for the TMB. (Fig. S3, ESI†). All of the results confirmed that the as-prepared Si-dots exhibited an intrinsic peroxidase-like activity which was similar to that of horseradish peroxidase (HRP) (eqn (1)). Si-dots H2 O2 þ TMB ! H2 O þ oxidizedTMB
(1)
It is well known that enzymatic activity is dependent upon the substrate and the reaction conditions. Therefore the catalytic activity of the Si-dots may be dependent on pH, temperature and H2O2 concentration. As shown in Fig. S4 (ESI†), the UV-vis absorbance at 652 nm increased in intensity with pH in the range of 3.0–4.0 and temperature in the range of 25–40 1C, and then declined on increasing the pH and temperature further. The experimental data imply that the Si-dots reached their highest catalytic activity at the vicinity of pH 4.0 and 40 1C. The effects of H2O2 concentration on the catalytic activity of the Si-dots were also investigated. The intensity of the absorption at 652 nm sharply increased with increasing H2O2 concentration from 10 mM to 200 mM, and then it gradually slows down when the concentration is beyond 300 mM. It is suggested that the reaction of H2O2 with TMB catalyzed by the Si-dots reached equilibrium within 300 mM. Hence, the catalytic activity was best in 300 mM H2O2 acidic solutions (pH 4.0) at 40 1C. From Fig. S4 (ESI†), a linear relationship in the range of 1 to 200 mM (R2 = 0.956) was obtained, which implies that the Si-dots catalytic system could be used in analytical systems involved with H2O2oxidation. To investigate the kinetic mechanism of the peroxidase-like activity of the Si-dots, apparent steady-state kinetic parameters for the peroxidase-like color changing reaction were determined by changing the concentrations of TMB and H2O2 in this system, respectively. Kinetic experiments were carried out using 10 mL of Si-dots in 0.5 mL of 0.2 M NaAc–HAc buffer solution (pH 4.0) containing 320 mM TMB as the substrate, and the H2O2 concentration was 20 mM. The initial reaction rate was calculated using the A Beer–Lambert Law c ¼ where, c is the substrate concentration, eb A is the absorbance, b is the thickness of the solution. The absorbance data were back-calculated to give concentrations using a molar absorption coefficient, e, of 39 000 M1 cm1 for the TMBderived oxidation products.12 Apparent steady-state reaction rates at different concentrations of the substrate were obtained by calculating the slopes of the absorbance changes with time. In this work, typical Michaelis–Menten curves were obtained for both TMB and H2O2 by monitoring the absorbance changes at 652 nm for 5 min (Fig. S5A and B, ESI†).13 The Michaelis–Menten constant (Km) and maximum initial velocity (Vmax) were obtained from Lineweaver– Burk plots of the double reciprocal of the Michaelis–Menten equation, 1 1 1 ¼ Km =Vmax þ (2) n ½S Km
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Table 1 Comparison of Michaelis–Menten constants (Km) and maximum reaction rates (Vmax) of the oxidation reaction catalyzed by the Si-dots, reported C-dots and HRP
Catalyst
Substance
Km/mM
Vmax/108 M s1
HRP31
TMB H2O2
0.434 3.702
10.00 8.71
Si-dots
TMB H2O2
1.502 0.065
14.72 5.65
where n is the initial velocity, Vmax is the maximal reaction velocity, [S] is the concentration of the substrate, and Km is the Michaelis constant. The results are shown in Fig. S5 (ESI†) and summarized in Table 1. The smaller the value of Km, the stronger the affinity between the enzyme and the substrate, and the more efficient the catalyst. The apparent Km value for the Si-dots with H2O2 as the substrate was lower than that of HRP (Table 1), suggesting that the Si-dots have a higher affinity for H2O2 than HRP. The apparent Km value of the Si-dots with TMB was also larger than that of HRP, suggesting that the Si-dots have a lower affinity for TMB than HRP. To further investigate the catalytic mechanism of the Si-dots in the system, we measured their activity under standard reaction conditions by varying the concentration of TMB at a fixed concentration of H2O2, and vice versa. The double reciprocal plots of the initial velocities against the concentrations of one substrate were obtained over a range of concentrations of the second substrate (Fig. S5C and D, ESI†). The lines were parallel, which is characteristic of a ping–pong mechanism,14 which is also observed in HRPbased systems. The experimental facts indicate that the Si-dots bind and react with the first substrate, then release the first product before reacting with the second substrate, which is similar to HRP and some other nano-materials.12 A possible catalytic mechanism of the Si-dots is presented in Fig. 1. First, it is well known that the as-prepared hydrogen terminated Si-dots were initially catalytically inactive.4 Therefore, the H–Si-dots were partially oxidized to their core–shell structure by H2O2, on which H2O2 molecules were adsorbed, and the H2O2 decomposed into active oxygen species with oxene characteristics.15 They then become electrophilic and are prone to oxidize TMB to TMBOX (Fig. 3). We measured the amperometric responses of the
Fig. 3 (A) UV-vis spectra for the mixed solutions of TMB, Si-dots and glucose incubation solution (pH 7.0 buffer, containing GOx) in 0.2 M NaAc–HAc buffer (pH 4.0) at 40 1C. The TMB concentration was 320 mM and 10 mL Si-dots dispersions were used. Inset: the corresponding images of the colored products. (B) Dependence of the absorbance at 652 nm on the concentration of glucose from 0.017 mM to 5 mM. Inset: the corresponding linear calibration plot.
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Si-dots modified glassy carbon electrode (Si-dots/GCE). The reduction current increased sharply to reach a steady-state value for the Si-dots/GCE with the addition of H2O2 (Fig. S6, ESI†), which clearly demonstrated that the Si-dots exhibited an electrocatalytic activity towards H2O2 reduction, which may promote electron transfer between the electron acceptor (H2O2) and the electron donator (the underlying electrode). The stability of the Si-dots and the robustness of the peroxidase activity in wide pH and temperature ranges is crucial for extending their applications. Fig. S7 (ESI†) shows that the catalytic activity of the Si-dots does not change after they were incubated at a wide range of pH (2–9) and temperatures at 20–90 1C for 2 h, which makes them suitable for expanding their applications in biomedicine and environmental fields. The nature of the peroxidase-like activity of the Si-dot nanostructures may originate from their catalytic ability to reduce H2O2. According to the research mentioned above, H2O2 can oxidise the TMB in the presence of peroxidase. With H2O2 as a product of the catalytic oxidation of glucose, glucose could then be detected based on the following reaction. GOx
D-glucose þ O2 þ H2 O ! H2 O2 þ gluconic acid
(3)
Considering that the assay conditions, such as the enzymatic factor, temperature, pH value and incubation time, have a significant effect on the detection of glucose, the experimental conditions, such as, pH, incubation temperature and time were optimized (Fig. S8, ESI†). The optimum temperature, time and pH were 35 1C, 30 min and 7, respectively. Under the optimized conditions, a linear relationship was obtained between the absorbance and the glucose concentration in the range of 0.17 to 200 mmol L1 (R2 = 0.986), with a detection limit of 0.05 mmol L1 (Fig. 3). This detection method based on Si-dots gave a lower detection limit than the method using HRP and other nanoparticles as catalysts (Table S1, ESI†). To investigate the selectivity for glucose detection, control experiments were performed using fructose, lactose, and maltose. The results are shown in Fig. S9 (ESI†). Even when the concentrations of the control samples were 10 times larger than that of glucose, no obvious response was observed, indicating that the colorimetric detection method can be used to detect glucose selectively. The excellent specificity and high sensitivity of the sensor suggested that the developed method could be directly applied for detecting glucose in real samples. Therefore, we detected glucose in serum samples of patients with diabetes provided by Hunan Normal University Hospital. Fig. S10 (ESI†) shows the UV-vis spectra and the inset shows that the experiment performed showed good colorimetric differentiation. According to the calibration curve, the concentration of glucose in blood was 10.2 mM (110.8 mg dL1). A blood glucose test is positive when the amount of glucose is more than 100 mg dL1. This colorimetric method is therefore applicable for determining glucose in blood samples, suggesting that our system was successful in the detection of glucose in real samples. In summary, we firstly reported that our Si-dots possess an intrinsic peroxidase-like activity. On the basis of our research,
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we provide a simple, inexpensive, highly sensitive and selective colorimetric assay to detect glucose in serum. As a novel nanoperoxidase mimetic, the Si-dots show several advantages over HRP, such as stability, dispersibility and high catalytic efficiency. Therefore, we expect that these Si-dots have great potential for applications in biotechnology. This work was supported by the National Natural Science Foundation of China (21375037, 21275037, 21305042), the Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and the Doctoral Fund of Ministry of Education of China (No. 20134306110006).
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