Appl Biochem Biotechnol DOI 10.1007/s12010-013-0625-0

Artificial Enzyme Mimics for Catalysis and Double Natural Enzyme Co-immobilization Xiaohua Li & Zhujun Zhang & Yongbo Li

Received: 16 July 2013 / Accepted: 30 October 2013 # Springer Science+Business Media New York 2013

Abstract This work presents a new chemiluminescent (CL) probe array assay. The new type CL probe array is based on enzyme mimics of Co3O4–SiO2 mesoporous nanocomposite material, which not only have an excellent catalytic effect on the luminol–H2O2 CL reaction in an alkaline medium but also can be used for the immobilization of enzymes. The linear range of the lactose concentration is 3.0×10−7 to 1.0×10−5 g mL−1 and the detection limit is 6.9×10−8 g mL−1. β-Galactosidase and glucose oxidase were selected as a model for enzyme assays to demonstrate the applicability of Co3O4–SiO2 mesoporous nanocomposite material in multienzyme immobilization. The novel bifunctional CL probe array has been successfully applied to the determination of lactose in milk. Keywords Chemiluminescentprobe array . Co3O4 − SiO2 mesoporousnanocomposite material . Immobilization . Lactose

Introduction Natural enzymes are natural protein molecules that act as highly efficient catalysts in biochemical reactions under mild conditions. However, most natural enzymes are not stable and exhibit an inherently low durability to harsh reaction conditions and require expensive preparation [1–3]. In recent years, enzyme immobilization techniques and artificial enzyme mimetics have been studied to overcome this drawback [4, 5]. Nanomaterials possess quantum size effect, volume effect, surface effect, and macroscopic quantum tunneling effect compared with traditional solid material [6, 7]. Yan et al. first reported that Fe3O4 nanoparticles exhibit an intrinsic enzyme mimetic activity similar to natural peroxides. Compared with natural enzymes, the nanoparticles as enzyme mimetics have advantages including high stability, resistance to high concentrations of substrate, lowcost of preparation, and are easy to store and treat [8–10]. Our group has reported that Co3O4 nanoparticles exhibited greater catalytic activity compared with natural HRP, CuO nanoparticles, α-Fe2O3 nanorods, NiO nanoparticles, and other common catalysts [11]. Nanoparticles could potentially replace the use of enzymes in sensors. However, at present, it is not possible X. Li : Z. Zhang (*) : Y. Li School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, China e-mail: [email protected]

Appl Biochem Biotechnol

that all natural enzymes can be replaced with enzyme mimetics. In many cases, several natural enzymes and enzyme mimetics can work together in a specific order. Generally, immobilized natural enzymes typically have greater thermal and operational stability than the soluble form of the enzyme [12–15]. Recently, mesoporous silica was studied as a host material for immobilized enzymes because of its large internal surface area and tunable pores, which showed improvement on enzyme stability, catalytic activity, product specificity, and resistance to extreme environmental conditions. The novel supports enable high enzyme loading and activity retention compared with traditional non-porous materials like silica, metal, and metal oxides that only have small binding surfaces [16, 17]. Mesoporous material is one of the most promising supports for enzyme immobilization. However, most present carriers only play one role of supporter. In this work, we made full use of the unique properties of Co3O4–SiO2 mesoporous nanocomposite material. Firstly, Co3O4–SiO2 mesoporous nanocomposite material as peroxidase mimetics was first found to greatly enhance the chemiluminescent (CL) of luminol– H2O2 system. Co3O4–SiO2 mesoporous nanocomposite material exhibited greater catalytic activity compared with Co3O4 nanoparticles owing to its high specific surface area and mesoporous channels. Secondly, Co3O4–SiO2 mesoporous nanocomposite material was first used for the immobilization of the two types of natural enzymes. Co3O4–SiO2 mesoporous nanocomposite material provides confined nanospace that could stabilize catalytic centers. They enable the ability of the natural enzymes to bind its outer surface and inner surface and catalyze the desired reaction. In the CL system, Co3O4–SiO2 nanocomposite material plays two important roles of catalyzer and supporter. The measurement principle was based on the following reactions: βgalactosidase lactose þ H2 O  → β  D  galactose þ β  D  glucose glucose oxidase β  D  glucose þ O2  → D  glucono  1 ; 5  lactone þ H2 O2

Co3 O4 −SiO2 nanocomposite material luminol þ H2 O2 þ OH −  → 3  aminophthalate þ hν ð425 nmÞ

Microarrays allow multiple parallel detection in a single experiment. The CL method combined with the microarray technique is a promising approach. Based on these, we designed a novel CL probe array (Scheme 1).

Scheme 1 The measurement principle of the lactose probe array

Appl Biochem Biotechnol

Experimental Materials and Instruments Sodium hydroxide solution (NaOH) and activated carbon, lactose, ethanol, Co(NO3)2, and tetraethoxysilane (TEOS) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Carbamide peroxide, tert-butyl hydroperoxide and cumene hydroperoxide were purchased from Aladdin Co. Ltd. 3-Aminophthalhydrazide (luminol), β-galactosidase, and glucose oxidase were purchased from Sigma (St. Louis, MO, USA). Millipore Milli-Q (18 MΩ cm) water was used in all experiments. All reagents were used without further purification. Ultrapure water was used in all experiments. The 96well plates were provided by JET BIOFIL products. A SynergyTM 2 multi-mode microplate reader with a low-noise photomultiplier tube (PMT) detector was used. Transmission electron microscopy (TEM) images were obtained with JEM2100 TEM (Hitachi, Japan). The As-prepared Co3O4–SiO2 Mesoporous Nanocomposite Material as Peroxidase Mimetics The Co3O4–SiO2 nanocomposite material was prepared similarly as described in the literature with some changes [18]. Fifteen grams of activated carbon was impregnated under magnetic stirring with 4 mL of TEOS diluted with 5 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 mL of aqueous Co(NO3)2 solution (4 M) 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. Co-immobilization of Glucose Oxidase and β-Galactosidase in Mesoporous Co3O4–SiO2 Nanocomposite Material A 2-mL volume of glucose oxidase and β-galactosidase solution dissolved in 5 mM pH 7.4 phosphate buffer was added to 12 mg Co3O4–SiO2 mesoporous nanocomposite material. The mixture was stirred for 5 h at 4 °C. The Co3O4–SiO2 mesoporous nanocomposite material-immobilized glucose oxidase, β-galactosidase, and glucose oxidase were separated from the solution by centrifugation and washed with ultrapure water.

Results and Discussion Characterization of Co3O4–SiO2 Nanocomposite Material TEM image of Co3O4–SiO2 mesoporous nanocomposite material demonstrated uniform distribution size of about 30 nm (Fig. 1). Nitrogen sorption isotherms (Fig. 1) revealed that the Co3 O 4–SiO 2 nanocomposite is mesoporous with a Brunauer– Emmett–Teller (BET) surface area of about 523 m2 g−1 and a pore volume of 0.3 cm3 g−1.

Appl Biochem Biotechnol

Fig. 1 a TEM image of Co3O4-SiO2 mesoporous nanocomposite material. b Nitrogen sorption isotherms of the as-prepared mesoporous nanocomposite material measured at 77 K, the BJH pore distribution curve based on the adsorption isotherm is also shown as an inset

The Peroxidase-Like Activity of the Co3O4–SiO2 Mesoporous Nanocomposite Material and Performance of CL Probe Array for Lactose Measurements To examine the peroxidase-like activity of the Co3O4–SiO2 mesoporous nanocomposite material. The effects of Co3O4–SiO2 mesoporous nanocomposite material on the luminol–H2O2 system were tested. Figure 2a shows the kinetic curve of the luminol–H2O2 CL reaction. An ultraweak CL emission which lasted less than 2 s was observed when 50 μL (1×10−7 M) H2O2 was injected into 50 μL (5×10−5 M) luminol (a). Luminol–H2O2 CL intensity was increased after the addition of Co3O4 nanoparticles (b). Luminol–H2O2 CL intensity was increased sharply after the addition of Co3O4–SiO2 mesoporous nanocomposite material due to its high specific surface area and mesoporous channels (c). Figure 2b shows that the CL probe array exhibits high uniformity. The relative standard deviation (RSD) for inter-assay precision was 4.5 % (n=11). Linearity and Detection Limits The chosen conditions for the luminol–H2O2–Co3O4–SiO2 mesoporous nanocomposite material CL system was 5.0×10−5 M luminol in 0.01 M NaOH. We chose 2 mg mL−1 Co3O4–

Appl Biochem Biotechnol

Fig. 2 a Luminol–H2O2 (a), luminol–H2O2–Co3O4 nanoparticles (b), and luminol–H2O2–Co3O4–SiO2 mesoporous nanocomposite material (c). Experimental conditions: pH 12.0, luminol 5×10−5 M, Co3O4–SiO2 nanocomposite material 1.0×10−3 g L−1, H2O2 1.0×10−5 M. b The uniformity of CL probe array. Experimental conditions: pH 12.0, 5.0×10−5 M luminol, 1.0×10−5 M lactose

SiO2 nanocomposite material-immobilized β-galactosidase and glucose oxidase. The calibration curve for lactose was obtained under the chosen conditions. It was found that the CL intensity was linear with the lactose concentration. As Fig. 3 shows, the linear range was from 3.0×10−7 to 1.0×10−5 g mL−1, and the regression equation is △I=146.68 C-2969.8 (where I is the CL intensity and C is the concentration of H2O2, R2 =0.9985). The detection limit (LOD, 3σ) was 6.9×10−8 g mL−1. The intra-assay precision of the analytical method was calculated by analyzing 5×10−6 g mL−1 lactose (n=7). The RSD for the intra-assay precision was 4.2 %. Anti-interference Study of the Developed CL Probe Array The potentially interfering substances in serum were tested by analyzing a standard solution of lactose (2.0×10−6 g mL−1). The tolerable limit of an interfering species was taken as a relative error less than 5 %. It showed that 100-fold Ca2+, NO3−, NH4+, and SO42− do not interfere with

Fig. 3 Calibration graph for lactose. Experimental parameters: pH 12.0, 5×10−5 M luminol

Appl Biochem Biotechnol

the determination. Twentyfold lysine, creatinine, glutamic acid, leucine, serine, asparagine, tert-butyl hydroperoxide, carbamide peroxide, and cumene hydroperoxide do not interfere with the determination. Mechanism Discussion Many researchers have proposed that the CL luminescence results from the excited species produced in catalytic oxidation. Some authors have reported that the surface of metal oxide nanoparticles can be oxidized to form an O–O bond. This bond can break to yield active intermediates such as·OH and·O2. Lately, some studies showed that nickel foam-supported Co3O4 nanowire array electrode exhibited better performance for H2O2 electroreduction in terms of both activity and mass transport property [19–21]. The maximum CL emission wavelength was still located at about 425 nm, indicating that the luminophor for the CL system was still the excited-state 3-aminophthalate anions. Co3O4–SiO2 nanocomposite material does not produce a new luminophor of the chemiluminescent reaction. The possible mechanism of luminol–H2O2 with Co3O4–SiO2 mesoporous nanocomposite material as catalyzer can be speculated. The surface of Co3O4–SiO2 mesoporous nanocomposite material can be oxidized to form an O–O bond. This bond can break to yield hydroxyl radicals. Then, the resulting hydroxyl radicals reacted with luminol anion and HO2− to form luminol radicals and superoxide radical anions, followed by a reaction with each other to form the excited-state 3-aminophthalate anions. Analytical Applications To demonstrate the application of the CL probe array based on glucose oxidase and βgalactosidase for lactose analysis. Aliquots of commercial milks were distributed in centrifuge tubes and centrifuged at 20,000 rpm for 30 min. Both tube pellet (insoluble proteins) and supernatant (milk fats) were eliminated. The clarified milk was diluted with ultrapure water to yield the testing sample solution. The response of the sample solution was measured and compared with that of a set of lactose standard solutions. The results are shown in Table 1 which matched with Lane–Eynon method.

Conclusions A novel bifunctional CL probe array for the determination of lactose was established. It offers new insights into the application of nanoparticle materials as a probe array. When compared with Co3O4 nanoparticles, the Co3O4–SiO2 mesoporous nanocomposite material exhibited higher catalytic activity toward luminol–H2O2 CL reaction. The double natural enzymes were also immobilized in Co3O4–SiO2 mesoporous nanocomposite material matrix and investigated Table 1 Analysis results of lactose in milk


Found (mg mL−1)±RSD (%, n=3)

Lane–Eynon method (mg mL−1)

1 2

49.6±2.5 39.0±3.2

47.9 38.1










Appl Biochem Biotechnol

as a probe array. The CL probe array has been successfully applied to the determination of lactose in milk. It will provide a new promising platform that enzyme mimetics act as double biocatalysts carriers. Acknowledgments 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 1. Dutton, P. L., & Moser, C. C. (2011). Engineering enzymes. Faraday Discussions, 148, 443–448. 2. Michael, G. (2013). Evolving new types of enzymes. Current Biology, 23, R214–R217. 3. Xie, J. X., Zhang, X. D., Wang, H., Zheng, H. Z., & Huang, Y. M. (2012). Analytical and environmental applications of nanoparticles as enzyme mimetics. Trends in Analytical Chemistry, 39, 114–129. 4. Quin, M. B., & Schmidt-Dannert, C. (2011). Engineering of biocatalysts: from evolution to creation. Catalysis, 1, 1017–1021. 5. Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M., & Fernandez-Lafuente, R. (2007). Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology, 40, 1451–1463. 6. Papageorgiou, D. G., & Bakoglidis, K. (2012). Use of nanomaterials for the improvement of various industrial and biomedical applications. Journal of Environmental Protection and Ecology, 13, 593–602. 7. Chaturvedi, S., & Dave, P. N. (2013). Design process for nanomaterials. Journal of Materials Science, 48, 3605–3622. 8. Pryor, S. W., & Nahar, N. (2010). Deficiency of cellulase activity measurements for enzyme evaluation. Applied Biochemistry and Biotechnology, 162, 1737–1750. 9. Nanda, V., & Koder, R. L. (2010). Designing artificial enzymes by intuition and computation. Nature Chemistry, 2, 15–24. 10. Miletić, N., Nastasović, A., & Loos, K. (2012). Immobilization of biocatalysts for enzymatic polymerizations: possibilities, advantages, applications. Bioresource Technology, 115, 126–135. 11. Li, X. H., Zhang, Z. J., Tao, L., & Gao, M. (2013). Sensitive and selective chemiluminescence assay for hydrogen peroxide in exhaled breath condensate using nanoparticle-based catalysis. Spectrochimica Acta A, 107, 311–316. 12. Hwang, E. T., & Gu, M. B. (2013). Enzyme stabilization by nano/microsized hybrid materials. Engineering in Life Science, 13, 49–61. 13. Werz, D. B. (2012). Chemical synthesis of carbohydrates and their surface immobilization: a brief introduction. Methods in Molecular Biology, 808, 13–29. 14. Lee, C. H., Lin, T. S., & Mou, C. Y. (2009). Mesoporous materials for encapsulating enzymes. Nano Today, 4, 165–179. 15. Pedro, H. A. L., Alfaia, A. J., Marques, J., Vila-Real, H. J., Calado, A., & Ribeiro, M. H. L. (2007). Design of an immobilized enzyme system for naringin hydrolysis at high-pressure. Enzyme Microb. Techno, l40, 442–446. 16. Zhou, Z., & Hartmann, M. (2012). Recent progress in biocatalysis with enzymes immobilized on mesoporous hosts. Topics in Catalysis, 55, 1081–1100. 17. Xie, T., Wang, A., Huang, L. F., Li, H. F., Chen, Z. M., & Wang, Q. Y. (2009). Recent advance in the support and technology used in enzyme immobilization. African Journal of Biotechnology, 8, 4724–4733. 18. Jia, J. J., Schwickardi, M., Weidenthaler, C., Schmidt, W., Korhonen, S., Weckhuysen, B. M., & Schüth, F. (2011). Co3O4–SiO2 nanocomposite: a very active catalyst for CO oxidation with unusual catalytic behavior. Journal of the American Chemical Society, 133, 11279–11288. 19. Lin, C., Ritter, J. A., & Popov, B. N. (1998). Characterization of sol-gel derived cobalt oxide xerogels as electrochemical capacitors. Journal of the Electrochemical Society, 145, 4097–4103. 20. Barbero, C., Planes, G. A., & Miras, M. C. (2001). Redox coupled ion exchange in cobalt oxide films. Electrochemistry Communications, 3, 113–116. 21. Gao, Y. Y., Chen, S. L., Cao, D. X., Wang, G. L., & Yin, J. L. (2010). Electrochemical capacitance of Co3O4 nanowire arrays supported on nickel foam. Journal of Power Sources, 195, 1757–1760.

Artificial enzyme mimics for catalysis and double natural enzyme co-immobilization.

This work presents a new chemiluminescent (CL) probe array assay. The new type CL probe array is based on enzyme mimics of Co3O4-SiO2 mesoporous nanoc...
310KB Sizes 0 Downloads 0 Views