Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Development a novel approach of chemiluminescent probe array Xiaohua Li, Zhujun Zhang ⇑, Liang Tao, Yongbo Li, Yunyun Li Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel CL probe array assay was first
developed. It gives rapid and high throughput
detection. It breaks traditional development
view in solid phase supports.
a r t i c l e
i n f o
Article history: Received 14 July 2013 Received in revised form 1 October 2013 Accepted 5 October 2013 Available online 12 October 2013 Keywords: Chemiluminescent probe array Immobilization High-throughput Glucose Co3O4–SiO2 mesoporous nanocomposite material
a b s t r a c t A new chemiluminescent (CL) probe array assay approach was first developed. The new CL probe array was based on Co3O4–SiO2 mesoporous nanocomposite material, which not only has an excellent catalytic effect on the luminol–H2O2 CL reaction in alkaline medium but also can be used for the immobilization of enzymes. As a model, the novel bifunctional CL probe array has been applied to the high-throughput determination of glucose in human. The linear range of the glucose concentration was 3–90 lM and the detection limit was 0.36 lM. It breaks traditional development view in solid phase supports and provides new insights into the application of mesoporous material. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme [1–3]. Many different carriers have been studied for the immobilization of enzymes. They can be classified as inorganic and organic according to their chemical composition. Organic carriers include cellulose, dextran, agar, agarose, chitin, alginate, collagen, albumin, carbon, polystyrene, polyacrylatepolymethacrylates, polyamides, polyacrylamide, vinyl, and allyl-polymers [4,5]. Inorganic carriers ⇑ Corresponding author. Tel.: +86 29 85308184; fax: +86 29 85307774. E-mail address: [email protected] (Z. Zhang). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.012
include bentonite, glass, silica, metals, and metal oxides [6,7]. Inorganic carriers often display good mechanical properties, thermal stability, and resistance against microbial attack and organic solvents. However, non-porous materials like metal and metal oxides only have small binding surfaces. Mesoporous material is one of the most promising carriers for enzyme immobilization. The unique properties of mesoporous materials were huge surface area and restricted pore nanospaces. Furthermore, mesoporous materials offer nanostructure for enzymes that can be finely tuned through controlling their pore structure, transport and microenvironment [8–10]. Recently, some research groups have immobilized enzymes on mesoporous silica which showed improvement on enzyme stability, catalytic activity, products specificity, and
68
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71
resistance to extreme environmental conditions [11–13]. However, all of the present carriers only play one role of supporter. Nanotechnology has provided new opportunities in many fields. One of the most important applications of nanoparticles is catalysis, where the large surface area per unit volume of nanosized catalysts enhances reactions. Recently, much attention has been extended to the CL of nanomaterial systems, to improve the sensitivity and the stability [14]. Our group has demonstrated that Co3O4 nanoparticles could enhance the luminol–H2O2 CL system [15–17]. Jia et al. reported a two-step method for the preparation of a Co3O4–SiO2 mesoporous nanocomposite material that exhibits very high activity for CO oxidation even at a temperature as low as 76 °C [18,19]. Recently, bifunctionalized nanoparticles have gained great interest in the biomedical applications [20]. Bifunctional nanoparticles integrate two formerly distinct functionalities into a single entity with superior and sometimes unprecedented properties such as detection by multiple imaging modalities, or detection and therapy. In this work, we report a bifunctional Co3O4–SiO2 mesoporous nanomaterials entrapped function enzymes inside the nanochannel by a simple synthesis operation. The Co3O4–SiO2 mesoporous nanomaterials are well dispersed with a diameter of 20–30 nm. Glucose oxidase (GOx) was chosen as a model enzyme to prepare the enzyme reactor. Meanwhile, the Co3O4–SiO2 mesoporous nanomaterials exhibited higher specific catalytic effect on the luminol–H2O2 CL reaction in alkaline medium owing to its high specific surface area and mesoporous channels. The combination of excellent luminescent properties and enzyme reactor suggest a great promise in the application of these bifunctional nanoparticles in catalysis chemiluminescence and in biomedicine such as determination of glucose in serum (Scheme 1). To the best of our knowledge, this is the first example of the use of nanomaterial to plays two important roles of catalyzer of CL reaction and supporter. The new CL probe array has been successfully applied to highthroughput determination of glucose in human serum. Experimental Materials and methods 3-Aminophthalhydrazide (luminol) and glucose oxidase (GOx) were purchased from Sigma–aldrich (USA). Sodium hydroxide solution (NaOH), activated carbon, d-Glucose, Co (NO3)2, tetraethoxysilane (TEOS), and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Millipore Milli-Q (18 MX cm) water was used in all experiments. All reagents were used without further purification. The 96-well plates were provided by JET BIOFIL products.
Instruments The Synergy™2 Multi-Mode Microplate Reader with the lownoise photomultiplier tubes (PMT) detector was used for the detection of luminescence. An external dispense module pumped fluid from the supply bottles to the two injectors located inside the instrument. Online monitoring of each probe can be computercontrolled via BioTek’s Gen5 PC software. The transmission electron microscopy (TEM) images were obtained with JEM-2100 TEM (Hitachi, Japan). Scanning electron microscopy (SEM) images were obtained with Quanta 200 SEM (FEI, Japan). All of the nanoparticles were centrifuged on an HC-3018R centrifuge (Anhui, China). The CL spectrum was obtained using the modified Hitachi F4600 spectrofluorimeter combined with a flow injection system. The surface area, pore size, and pore volume were determined by N2 adsorption–desorption isotherms obtained at 77.156 K on a Micromeritics ASAP 2020M. Preparation of Co3O4–SiO2 mesoporous nanocomposite material entrapped function enzymes inside the nanochannel synthesised The bifunctional Co3O4–SiO2 mesoporous nanomaterials entrapped function enzymes inside the nanochannel synthesised referring to the procedure reported by Jia et al. with some change [19]. The 20 g of activated carbon was impregnated under magnetic stirring with 5.4 mL of TEOS diluted with 8 mL of ethanol. The solution was completely absorbed by the activated carbon in 5 min under continuous stirring. Then the composite material was transferred into a muffle oven and annealed at 350 °C for 30 min in air. The above C–SiO2 composite material was added to 10.4 mL of 4.0 M Co (NO3)2 solution under magnetic stirring for 30 min. The resulting solid was transferred into a muffle oven and calcined at 550 °C for 90 min in air. Co3O4–SiO2 mesoporous nanocomposite material was obtained. A 2-mL volume of 6 mg mL 1 GOx stock solution dissolved in 5 mM pH 6.5 tris (hydroxymethyl) aminomethane–HCl buffer was added to 15 mg Co3O4–SiO2 mesoporous nanocomposite material. The mixture was stirred for 8 h at 4 °C. The Co3O4–SiO2 mesoporous nanocomposite material-immobilized GOx was separated from solution by centrifugation and washed with distilled water. Measurements of CL probe array The Co3O4–SiO2 mesoporous nanocomposite material-immobilized GOx was well dispersed in 10 mL distilled water, and then was added into each well (100 lL each probe). Glucose solution was added into the each probe (100 lL). The mixture was then incubated at room temperature for 20 min to yield the testing sample solution. A 50 lL volume of luminol solution were injected into
Scheme 1. The measurement principle of the glucose probe array.
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71
69
the each CL probe and readings were acquired. All the experiments were carried out at room temperature. The probe array can be tested simultaneously. Results and discussion Characterization of Co3O4–SiO2 mesoporous nanocomposite material TEM image of Co3O4–SiO2 mesoporous nanocomposite material demonstrated uniform distribution of size between 20 and 30 nm (Fig. 1A). Nitrogen sorption isotherms (Fig. 1B) revealed that the Co3O4–SiO2 nanocomposite is mesoporous with a Brunauer Emmett Teller (BET) surface area of about 523.500 m2 g 1 and a pore volume of 0.33 cm3 g 1. Comparison of catalytic activity of Co3O4–SiO2 mesoporous nanocomposite material and Co3O4 nanoparticles The comparison catalysis effect of typical metal ions and nanoparticals, like Co(II), Cr(III), Cu(II), Co3O4, CeO2, CuO and horseradish peroxidase (HRP) with that of Co3O4–SiO2 mesoporous nanomaterials for H2O2 (1 10 6 M) was performed on the Synergy™2 Multi-Mode Microplate Reader (Fig. 2). The results showed that Co3O4–SiO2 mesoporous nanocomposite material exhibited higher specific catalytic effect on the luminol–H2O2 CL reaction in alkaline medium. The performances of CL probe array A batch CL system was used for the kinetic study of the CL reaction. Fig. 3A shows the kinetic curve of the probe. A CL emission which lasted less than 2 s was observed when 50 lL (5 10 5 M) luminol was injected into the probe. Fig. 3B shows that the CL
Fig. 2. (a) Co3O4–SiO2 mesoporous nanocomposite material; (b) Co3O4 nanoparticles; (c) CeO2 nanoparticles; (d) CuO nanoparticles; (e) Co (II); (f) Cr (III); (g) Cu (II); (h) HRP. Experimental conditions: pH 12.0, luminol 5 10 5 M, 1 10 6 M H2O2, catalyst concentration: 1 10 3 g L 1.
probe array exhibited high uniformity. The relative standard deviation (RSD) for inter-assay precision was 4.8% (11). The amount of GOx immobilized in Co3O4–SiO2 mesoporous nanocomposite material The amount of GOx immobilized in Co3O4–SiO2 mesoporous nanocomposite material was measured by adding 15 mg Co3O4– SiO2 mesoporous nanocomposite material to 2 mL 6 mg mL 1 GOx solution and the mixture was stirred for 8 h at 4 °C. The supernatant was separated from the solid material by centrifugation and the amount of immobilized enzyme was calculated from the difference in the absorbance of the supernatant at 450 nm before and after addition of the support (Fig. 4). The results showed that Co3O4–SiO2 mesoporous nanocomposite material shows a greater adsorption capacity of GOx than Co3O4 nanoparticle. Co3O4–SiO2 mesoporous nanocomposite material provides a biocompatible environment for GOx to keep its bioactivity by virtue of their biochemical inertness and relative mechanical strength. Measurements of CL probe array The determination of glucose was studied under the optimum conditions using the prepared CL probe array. It was found that the response signals were proportional to the concentrations of glucose. The linear calibration plot was DI = 6070.9C 16287C (where I is the CL intensity and C is the concentration of glucose, R2 = 0.9981) (Fig. 5). The linear calibration range is from 3 10 6 to 9 10 5 M with detection limit (LOD, 3r) of 3.6 10 7 M. As compared with the analytical methods previously published in literatures [21], the proposed method shows high sensitivity for glucose analysis. The novel CL probe array exhibits good reproducibility. The intra-assay precision of the analytical method was calculated by analyzing 10 6 M glucose (n = 11). The RSD for the intra-assay precision was 4.2%. Anti-interference study of the developed CL probe array
Fig. 1. (A) TEM image of Co3O4–SiO2 mesoporous nanocomposite material. (B) Nitrogen sorption isotherms of the as-prepared Co3O4–SiO2 composite catalyst measured at 77.156 K. The BJH pore distribution curve based on the adsorption isotherm is also shown as an inset.
The potentially interfering substances in serum were tested by analyzing a standard solution of glucose (5.0 10 6 M). The tolerable limit of an interfering species was taken as a relative error less than 5%. It showed that 20-fold leucine, lysine, creatinine, glutamic acid, aspara, gine, serine, carbamide peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide do not interfere with the determination. The interference of protein in human serum could be ignored when human serum was ultrafiltered, and the present
70
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71
200000 150000 100000
B
A
Max V
t at Max V
CL Intensity (a.u.)
250000
-52274400000.000 mRLU/m
300000
50000 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
Time (s) Fig. 3. (A) The l kinetic curve of the probe. (B) The uniformity of CL probe array. Experimental conditions: pH 12.0, luminol 5 10
5
M, 5 10
5
M glucose.
Table 1 Analysis results of glucose in human serum. Sample Sample Sample Sample Sample Sample
1 2 3 4 5
Proposed method (mM)
o-Toluidine method (mM)
3.98 ± 0.16 5.56 ± 0.21 4.88 ± 0.31 5.79 ± 0.28 4.21 ± 0.15
4.12 5.73 5.01 5.92 4.18
sample solutions. The results matched well with those obtained with the o-toluidine method are shown in Table 1. Conclusions Fig. 4. UV–vis spectra of glucose oxidase (GOD) solution (pH 6.5): (a) 6.0 mg mL 1 GOD solution; (b) After adsorption to Co3O4 nanoparticle; (c) After adsorption to Co3O4–SiO2 mesoporous nanocomposite material.
In this work, we developed a novel bifunctional CL probe array for the determination of glucose. When compared with other common catalysts, the Co3O4–SiO2 mesoporous nanocomposite material exhibited higher catalytic activity toward luminol–H2O2 CL reaction. Co3O4–SiO2 mesoporous nanocomposite material exhibited very high catalytic activity towards luminol–H2O2 CL reaction in alkaline medium and provided confined nanospace that could stabilize catalytic centers and enzymes. The novel CL probe array has been successfully applied to the determination of glucose in human serum. Our work reveals that the mesoporous materials will be a promising platform as enzyme mimetics and biocatalyst carriers. Acknowledgments
Fig. 5. Calibration graph for glucose. Experimental parameters: pH 12.0, 5 10 luminol.
5
M
This work was supported financially by the Fundamental Research Funds for the Central Universities (Program No. GK 20091004) and National Natural Science Foundation of China (No. 30872371). References
method can be directly applied to the determination of glucose in human serum. Analytical applications The proposed method was utilized for the determination of glucose in human serum. The serum samples were pretreated by ultrafiltration to remove protein before glucose assay. The samples were first analyzed with the o-toluidine method (630 nm) [22]. The samples were then reassayed with the proposed CL probe array. The samples were diluted with distilled water to yield test
[1] E. Severac, O. Galy, F. Turon, C.A. Pantel, J.S. Condoret, P. Monsan, A. Marty, Enzyme Microb. Technol. 48 (2011) 61–70. [2] G. Michael, Evolving new types of enzymes, Curr. Biol. 23 (2013) R214–R217. [3] J.F. Ma, L.H. Zhang, Z. Liang, W.B. Zhang, Y.K. Zhang, Anal. Chim. Acta 632 (2009) 1–8. [4] G.M. Rios, M.P. Belleville, D. Paolucci, J. Sanchez, J. Membr. Sci. 242 (2004) 189– 196. [5] H.J. Kim, Y. Sum, S.H. Lee, Jung-A. Kim, H.S. Kim, J. Mol. Catal. B: Enzym. 83 (2012) 8–15. [6] J.N. Talbert, J.M. Goddard, Colloids Surf. B Biointerfaces 93 (2012) 8–19. [7] S.A. Ansari, Q. Husain, Biotech. Adv. 30 (2012) 512–523. [8] J. Kim, H. Jia, C.W. Lee, S.W. Chung, J.H. Kwak, Y. Shin, A. Doh-nalkova, B.G. Kim, P. Wang, J.W. Grate, Enzyme Microb. Technol. 39 (2006) 474–480. [9] Z. Zhou, M. Hartmann, Top Catal 55 (2012) 1081–1100. [10] B. Rodriguez-Colinas, M. Alcalde, A.O. Ballesteros, F.J. Plou, Adv. Mater. 23 (2011) 5275–5282.
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71 [11] C.H. Lee, T.S. Lin, C.Y. Mou, Nano Today 4 (2009) 165–179. [12] J.M. Thomas, R. Raja, Acc. Chem. Res. 41 (2008) 708–720. [13] A. Takimoto, T. Shiomi, K. Ino, T. Tsunoda, A. Kawai, F. Mizukami, K. Sakaguchi, Microporous Mesoporous Mater. 116 (2008) 601–606. [14] A. Ambrosi, M. Castaneda, A. Killard, M. Smyth, S. Alegret, A. Merkoci, Anal. Chem. 79 (2007) 5232–5240. [15] X.H. Li, Z.J. Zhang, L. Tao, Analyst 138 (2013) 1596–1600. [16] J.X. Xie, Y.M. Huang, Anal. Methods 3 (2011) 1149–1155. [17] X.H. Li, Z.J. Zhang, L. Tao, M. Gao, Spectrochim. Acta A 107 (2013) 311–316.
71
[18] S. Matsuura, R. Ishii, T. Itoh, T. Hanaoka, S. Hamakawa, T. Tsunoda, F. Mizukami, Mesoporous Mater. 127 (2010) 61–66. [19] J.J. Jia, M. Schwickardi, C. Weidenthaler, W. Schmidt, S. Korhonen, B.M. Weckhuysen, F. Schüth, J. Am. Chem. Soc. 133 (2011) 11279–11288. [20] M. Wang, C. Wang, K.L. Young, L. Hao, M. Medved, T. Rajh, H.C. Fry, L. Zhu, G.S. Karczmar, C. Watson, J.S. Jiang, N.M. Markovic, Chem. Mater. 24 (2012) 2423– 2425. [21] W. Chen, L. Hong, A.L. Liu, J.Q. Liu, X.H. Lin, X.H. Xia, Talanta 99 (2012) 643– 648. [22] F.F.J. Charles, R. Robert, J.P. Anthony, Clin. Chim. Acta. 43 (1973) 105–111.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Development a novel approach of chemiluminescent probe array Xiaohua Li, Zhujun Zhang ⇑, Liang Tao, Yongbo Li, Yunyun Li Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel CL probe array assay was first
developed. It gives rapid and high throughput
detection. It breaks traditional development
view in solid phase supports.
a r t i c l e
i n f o
Article history: Received 14 July 2013 Received in revised form 1 October 2013 Accepted 5 October 2013 Available online 12 October 2013 Keywords: Chemiluminescent probe array Immobilization High-throughput Glucose Co3O4–SiO2 mesoporous nanocomposite material
a b s t r a c t A new chemiluminescent (CL) probe array assay approach was first developed. The new CL probe array was based on Co3O4–SiO2 mesoporous nanocomposite material, which not only has an excellent catalytic effect on the luminol–H2O2 CL reaction in alkaline medium but also can be used for the immobilization of enzymes. As a model, the novel bifunctional CL probe array has been applied to the high-throughput determination of glucose in human. The linear range of the glucose concentration was 3–90 lM and the detection limit was 0.36 lM. It breaks traditional development view in solid phase supports and provides new insights into the application of mesoporous material. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme [1–3]. Many different carriers have been studied for the immobilization of enzymes. They can be classified as inorganic and organic according to their chemical composition. Organic carriers include cellulose, dextran, agar, agarose, chitin, alginate, collagen, albumin, carbon, polystyrene, polyacrylatepolymethacrylates, polyamides, polyacrylamide, vinyl, and allyl-polymers [4,5]. Inorganic carriers ⇑ Corresponding author. Tel.: +86 29 85308184; fax: +86 29 85307774. E-mail address: [email protected] (Z. Zhang). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.012
include bentonite, glass, silica, metals, and metal oxides [6,7]. Inorganic carriers often display good mechanical properties, thermal stability, and resistance against microbial attack and organic solvents. However, non-porous materials like metal and metal oxides only have small binding surfaces. Mesoporous material is one of the most promising carriers for enzyme immobilization. The unique properties of mesoporous materials were huge surface area and restricted pore nanospaces. Furthermore, mesoporous materials offer nanostructure for enzymes that can be finely tuned through controlling their pore structure, transport and microenvironment [8–10]. Recently, some research groups have immobilized enzymes on mesoporous silica which showed improvement on enzyme stability, catalytic activity, products specificity, and
68
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71
resistance to extreme environmental conditions [11–13]. However, all of the present carriers only play one role of supporter. Nanotechnology has provided new opportunities in many fields. One of the most important applications of nanoparticles is catalysis, where the large surface area per unit volume of nanosized catalysts enhances reactions. Recently, much attention has been extended to the CL of nanomaterial systems, to improve the sensitivity and the stability [14]. Our group has demonstrated that Co3O4 nanoparticles could enhance the luminol–H2O2 CL system [15–17]. Jia et al. reported a two-step method for the preparation of a Co3O4–SiO2 mesoporous nanocomposite material that exhibits very high activity for CO oxidation even at a temperature as low as 76 °C [18,19]. Recently, bifunctionalized nanoparticles have gained great interest in the biomedical applications [20]. Bifunctional nanoparticles integrate two formerly distinct functionalities into a single entity with superior and sometimes unprecedented properties such as detection by multiple imaging modalities, or detection and therapy. In this work, we report a bifunctional Co3O4–SiO2 mesoporous nanomaterials entrapped function enzymes inside the nanochannel by a simple synthesis operation. The Co3O4–SiO2 mesoporous nanomaterials are well dispersed with a diameter of 20–30 nm. Glucose oxidase (GOx) was chosen as a model enzyme to prepare the enzyme reactor. Meanwhile, the Co3O4–SiO2 mesoporous nanomaterials exhibited higher specific catalytic effect on the luminol–H2O2 CL reaction in alkaline medium owing to its high specific surface area and mesoporous channels. The combination of excellent luminescent properties and enzyme reactor suggest a great promise in the application of these bifunctional nanoparticles in catalysis chemiluminescence and in biomedicine such as determination of glucose in serum (Scheme 1). To the best of our knowledge, this is the first example of the use of nanomaterial to plays two important roles of catalyzer of CL reaction and supporter. The new CL probe array has been successfully applied to highthroughput determination of glucose in human serum. Experimental Materials and methods 3-Aminophthalhydrazide (luminol) and glucose oxidase (GOx) were purchased from Sigma–aldrich (USA). Sodium hydroxide solution (NaOH), activated carbon, d-Glucose, Co (NO3)2, tetraethoxysilane (TEOS), and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Millipore Milli-Q (18 MX cm) water was used in all experiments. All reagents were used without further purification. The 96-well plates were provided by JET BIOFIL products.
Instruments The Synergy™2 Multi-Mode Microplate Reader with the lownoise photomultiplier tubes (PMT) detector was used for the detection of luminescence. An external dispense module pumped fluid from the supply bottles to the two injectors located inside the instrument. Online monitoring of each probe can be computercontrolled via BioTek’s Gen5 PC software. The transmission electron microscopy (TEM) images were obtained with JEM-2100 TEM (Hitachi, Japan). Scanning electron microscopy (SEM) images were obtained with Quanta 200 SEM (FEI, Japan). All of the nanoparticles were centrifuged on an HC-3018R centrifuge (Anhui, China). The CL spectrum was obtained using the modified Hitachi F4600 spectrofluorimeter combined with a flow injection system. The surface area, pore size, and pore volume were determined by N2 adsorption–desorption isotherms obtained at 77.156 K on a Micromeritics ASAP 2020M. Preparation of Co3O4–SiO2 mesoporous nanocomposite material entrapped function enzymes inside the nanochannel synthesised The bifunctional Co3O4–SiO2 mesoporous nanomaterials entrapped function enzymes inside the nanochannel synthesised referring to the procedure reported by Jia et al. with some change [19]. The 20 g of activated carbon was impregnated under magnetic stirring with 5.4 mL of TEOS diluted with 8 mL of ethanol. The solution was completely absorbed by the activated carbon in 5 min under continuous stirring. Then the composite material was transferred into a muffle oven and annealed at 350 °C for 30 min in air. The above C–SiO2 composite material was added to 10.4 mL of 4.0 M Co (NO3)2 solution under magnetic stirring for 30 min. The resulting solid was transferred into a muffle oven and calcined at 550 °C for 90 min in air. Co3O4–SiO2 mesoporous nanocomposite material was obtained. A 2-mL volume of 6 mg mL 1 GOx stock solution dissolved in 5 mM pH 6.5 tris (hydroxymethyl) aminomethane–HCl buffer was added to 15 mg Co3O4–SiO2 mesoporous nanocomposite material. The mixture was stirred for 8 h at 4 °C. The Co3O4–SiO2 mesoporous nanocomposite material-immobilized GOx was separated from solution by centrifugation and washed with distilled water. Measurements of CL probe array The Co3O4–SiO2 mesoporous nanocomposite material-immobilized GOx was well dispersed in 10 mL distilled water, and then was added into each well (100 lL each probe). Glucose solution was added into the each probe (100 lL). The mixture was then incubated at room temperature for 20 min to yield the testing sample solution. A 50 lL volume of luminol solution were injected into
Scheme 1. The measurement principle of the glucose probe array.
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71
69
the each CL probe and readings were acquired. All the experiments were carried out at room temperature. The probe array can be tested simultaneously. Results and discussion Characterization of Co3O4–SiO2 mesoporous nanocomposite material TEM image of Co3O4–SiO2 mesoporous nanocomposite material demonstrated uniform distribution of size between 20 and 30 nm (Fig. 1A). Nitrogen sorption isotherms (Fig. 1B) revealed that the Co3O4–SiO2 nanocomposite is mesoporous with a Brunauer Emmett Teller (BET) surface area of about 523.500 m2 g 1 and a pore volume of 0.33 cm3 g 1. Comparison of catalytic activity of Co3O4–SiO2 mesoporous nanocomposite material and Co3O4 nanoparticles The comparison catalysis effect of typical metal ions and nanoparticals, like Co(II), Cr(III), Cu(II), Co3O4, CeO2, CuO and horseradish peroxidase (HRP) with that of Co3O4–SiO2 mesoporous nanomaterials for H2O2 (1 10 6 M) was performed on the Synergy™2 Multi-Mode Microplate Reader (Fig. 2). The results showed that Co3O4–SiO2 mesoporous nanocomposite material exhibited higher specific catalytic effect on the luminol–H2O2 CL reaction in alkaline medium. The performances of CL probe array A batch CL system was used for the kinetic study of the CL reaction. Fig. 3A shows the kinetic curve of the probe. A CL emission which lasted less than 2 s was observed when 50 lL (5 10 5 M) luminol was injected into the probe. Fig. 3B shows that the CL
Fig. 2. (a) Co3O4–SiO2 mesoporous nanocomposite material; (b) Co3O4 nanoparticles; (c) CeO2 nanoparticles; (d) CuO nanoparticles; (e) Co (II); (f) Cr (III); (g) Cu (II); (h) HRP. Experimental conditions: pH 12.0, luminol 5 10 5 M, 1 10 6 M H2O2, catalyst concentration: 1 10 3 g L 1.
probe array exhibited high uniformity. The relative standard deviation (RSD) for inter-assay precision was 4.8% (11). The amount of GOx immobilized in Co3O4–SiO2 mesoporous nanocomposite material The amount of GOx immobilized in Co3O4–SiO2 mesoporous nanocomposite material was measured by adding 15 mg Co3O4– SiO2 mesoporous nanocomposite material to 2 mL 6 mg mL 1 GOx solution and the mixture was stirred for 8 h at 4 °C. The supernatant was separated from the solid material by centrifugation and the amount of immobilized enzyme was calculated from the difference in the absorbance of the supernatant at 450 nm before and after addition of the support (Fig. 4). The results showed that Co3O4–SiO2 mesoporous nanocomposite material shows a greater adsorption capacity of GOx than Co3O4 nanoparticle. Co3O4–SiO2 mesoporous nanocomposite material provides a biocompatible environment for GOx to keep its bioactivity by virtue of their biochemical inertness and relative mechanical strength. Measurements of CL probe array The determination of glucose was studied under the optimum conditions using the prepared CL probe array. It was found that the response signals were proportional to the concentrations of glucose. The linear calibration plot was DI = 6070.9C 16287C (where I is the CL intensity and C is the concentration of glucose, R2 = 0.9981) (Fig. 5). The linear calibration range is from 3 10 6 to 9 10 5 M with detection limit (LOD, 3r) of 3.6 10 7 M. As compared with the analytical methods previously published in literatures [21], the proposed method shows high sensitivity for glucose analysis. The novel CL probe array exhibits good reproducibility. The intra-assay precision of the analytical method was calculated by analyzing 10 6 M glucose (n = 11). The RSD for the intra-assay precision was 4.2%. Anti-interference study of the developed CL probe array
Fig. 1. (A) TEM image of Co3O4–SiO2 mesoporous nanocomposite material. (B) Nitrogen sorption isotherms of the as-prepared Co3O4–SiO2 composite catalyst measured at 77.156 K. The BJH pore distribution curve based on the adsorption isotherm is also shown as an inset.
The potentially interfering substances in serum were tested by analyzing a standard solution of glucose (5.0 10 6 M). The tolerable limit of an interfering species was taken as a relative error less than 5%. It showed that 20-fold leucine, lysine, creatinine, glutamic acid, aspara, gine, serine, carbamide peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide do not interfere with the determination. The interference of protein in human serum could be ignored when human serum was ultrafiltered, and the present
70
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71
200000 150000 100000
B
A
Max V
t at Max V
CL Intensity (a.u.)
250000
-52274400000.000 mRLU/m
300000
50000 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
Time (s) Fig. 3. (A) The l kinetic curve of the probe. (B) The uniformity of CL probe array. Experimental conditions: pH 12.0, luminol 5 10
5
M, 5 10
5
M glucose.
Table 1 Analysis results of glucose in human serum. Sample Sample Sample Sample Sample Sample
1 2 3 4 5
Proposed method (mM)
o-Toluidine method (mM)
3.98 ± 0.16 5.56 ± 0.21 4.88 ± 0.31 5.79 ± 0.28 4.21 ± 0.15
4.12 5.73 5.01 5.92 4.18
sample solutions. The results matched well with those obtained with the o-toluidine method are shown in Table 1. Conclusions Fig. 4. UV–vis spectra of glucose oxidase (GOD) solution (pH 6.5): (a) 6.0 mg mL 1 GOD solution; (b) After adsorption to Co3O4 nanoparticle; (c) After adsorption to Co3O4–SiO2 mesoporous nanocomposite material.
In this work, we developed a novel bifunctional CL probe array for the determination of glucose. When compared with other common catalysts, the Co3O4–SiO2 mesoporous nanocomposite material exhibited higher catalytic activity toward luminol–H2O2 CL reaction. Co3O4–SiO2 mesoporous nanocomposite material exhibited very high catalytic activity towards luminol–H2O2 CL reaction in alkaline medium and provided confined nanospace that could stabilize catalytic centers and enzymes. The novel CL probe array has been successfully applied to the determination of glucose in human serum. Our work reveals that the mesoporous materials will be a promising platform as enzyme mimetics and biocatalyst carriers. Acknowledgments
Fig. 5. Calibration graph for glucose. Experimental parameters: pH 12.0, 5 10 luminol.
5
M
This work was supported financially by the Fundamental Research Funds for the Central Universities (Program No. GK 20091004) and National Natural Science Foundation of China (No. 30872371). References
method can be directly applied to the determination of glucose in human serum. Analytical applications The proposed method was utilized for the determination of glucose in human serum. The serum samples were pretreated by ultrafiltration to remove protein before glucose assay. The samples were first analyzed with the o-toluidine method (630 nm) [22]. The samples were then reassayed with the proposed CL probe array. The samples were diluted with distilled water to yield test
[1] E. Severac, O. Galy, F. Turon, C.A. Pantel, J.S. Condoret, P. Monsan, A. Marty, Enzyme Microb. Technol. 48 (2011) 61–70. [2] G. Michael, Evolving new types of enzymes, Curr. Biol. 23 (2013) R214–R217. [3] J.F. Ma, L.H. Zhang, Z. Liang, W.B. Zhang, Y.K. Zhang, Anal. Chim. Acta 632 (2009) 1–8. [4] G.M. Rios, M.P. Belleville, D. Paolucci, J. Sanchez, J. Membr. Sci. 242 (2004) 189– 196. [5] H.J. Kim, Y. Sum, S.H. Lee, Jung-A. Kim, H.S. Kim, J. Mol. Catal. B: Enzym. 83 (2012) 8–15. [6] J.N. Talbert, J.M. Goddard, Colloids Surf. B Biointerfaces 93 (2012) 8–19. [7] S.A. Ansari, Q. Husain, Biotech. Adv. 30 (2012) 512–523. [8] J. Kim, H. Jia, C.W. Lee, S.W. Chung, J.H. Kwak, Y. Shin, A. Doh-nalkova, B.G. Kim, P. Wang, J.W. Grate, Enzyme Microb. Technol. 39 (2006) 474–480. [9] Z. Zhou, M. Hartmann, Top Catal 55 (2012) 1081–1100. [10] B. Rodriguez-Colinas, M. Alcalde, A.O. Ballesteros, F.J. Plou, Adv. Mater. 23 (2011) 5275–5282.
X. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 67–71 [11] C.H. Lee, T.S. Lin, C.Y. Mou, Nano Today 4 (2009) 165–179. [12] J.M. Thomas, R. Raja, Acc. Chem. Res. 41 (2008) 708–720. [13] A. Takimoto, T. Shiomi, K. Ino, T. Tsunoda, A. Kawai, F. Mizukami, K. Sakaguchi, Microporous Mesoporous Mater. 116 (2008) 601–606. [14] A. Ambrosi, M. Castaneda, A. Killard, M. Smyth, S. Alegret, A. Merkoci, Anal. Chem. 79 (2007) 5232–5240. [15] X.H. Li, Z.J. Zhang, L. Tao, Analyst 138 (2013) 1596–1600. [16] J.X. Xie, Y.M. Huang, Anal. Methods 3 (2011) 1149–1155. [17] X.H. Li, Z.J. Zhang, L. Tao, M. Gao, Spectrochim. Acta A 107 (2013) 311–316.
71
[18] S. Matsuura, R. Ishii, T. Itoh, T. Hanaoka, S. Hamakawa, T. Tsunoda, F. Mizukami, Mesoporous Mater. 127 (2010) 61–66. [19] J.J. Jia, M. Schwickardi, C. Weidenthaler, W. Schmidt, S. Korhonen, B.M. Weckhuysen, F. Schüth, J. Am. Chem. Soc. 133 (2011) 11279–11288. [20] M. Wang, C. Wang, K.L. Young, L. Hao, M. Medved, T. Rajh, H.C. Fry, L. Zhu, G.S. Karczmar, C. Watson, J.S. Jiang, N.M. Markovic, Chem. Mater. 24 (2012) 2423– 2425. [21] W. Chen, L. Hong, A.L. Liu, J.Q. Liu, X.H. Lin, X.H. Xia, Talanta 99 (2012) 643– 648. [22] F.F.J. Charles, R. Robert, J.P. Anthony, Clin. Chim. Acta. 43 (1973) 105–111.
