Bioconjugates of Glucose Oxidase and Gold Nanorods Based on Electrostatic Interaction with Enhanced Thermostability
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Bioconjugates made up of an enzyme and gold nanorods (GNRs) were fabricated by electrostatic interactions (layer-by-layer method, LBL) between anionic glucose oxidase (GOD) and positively charged GNRs. The assembled processes were monitored by UV–Vis spectra, zeta potential measurements, and transmission electron microscopy. The enzyme activity assays of the obtained bioconjugates display a relatively enhanced thermostability behavior in contrast with that of free enzyme. Free GOD in solution only retains about 22% of its relative activity at 90 °C. Unexpectedly, the immobilized GOD on GNRs still retains about 39.3% activity after the same treatment. This work will be of significance for the biologic enhancement using other kinds of anisotropic nanostructure and suggests a new way of enhancing enzyme thermostability using anisotropic metal nanomaterials.
KeywordsGold nanorods Enzyme thermostability Glucose oxidase Polyelectrolytes
Considerable effort has been devoted to the study of gold nanoparticles with variable size and shape due to their unique geometry-dependent optical, electronic, and catalytic properties in electronics and optics [1, 2, 3, 4, 5], particularly in the fields of biotechnology and nanotechnology [6, 7, 8]. Recently, gold nanorods (GNRs) have attracted interest due to their unusual properties in electronics and optics [9, 10] and especially in bionanotechnology fields involving bioimaging [11, 12], biosensing [13, 14, 15, 16], DNA expression , cancer therapy , etc. For GNRs, two distinct plasmon bands, a transverse mode (~520 nm) and a longitudinal mode (usually >600 nm), can be observed. This unique optical property of GNRs opens up fascinating applications as biologic and chemical sensors. A versatile layer-by-layer approach to the preparation of polyelectrolyte-coated GNRs films has been reported [19, 20], indicating that polyelectrolytes are effective coating reagents for the modification of GNRs.
The immobilization of an enzyme is one of the crucial factors in a range of biologic techniques. Proteins have traditionally been immobilized on to solid surfaces by a variety of techniques including physical adsorption, solvent casting, covalent binding, and electropolymerization . Although enzymes have been immobilized on to the surface of polystyrene latex , gold nanoparticles (GNPs), or silica nanoparticles [23, 24, 25], there are few reports in which anisotropic nanoparticles have been used to conjugate the enzyme. In this work, GNRs were used to prepare a bioconjugate with an enzyme, using GOD as a model enzyme. The thermostability of the GNR/GOD bioconjugates was dramatically enhanced, and even at 90 °C, its relative activity still remained at about 39.3%.
Materials and reagents. GOD (EC 22.214.171.124, 211 U mg−1fromAspergillus niger) and peroxidase from horseradish (HRP, 969.65 U mg−1) were purchased from Fluka.d-(+)-glucose (99%), Chloroauric acid (HAuCl4·3H2O, 99.9%),l-(+)-Ascorbic acid (AA, 99+%), Silver nitrate (AgNO3, 99.9%), and Cetyltrimethylammonium bromide (CTAB, 98%) were purchased from Alfa-Aesar.o-Dianisidine and Sodium borohydride (NaBH4, 98+%) were obtained from Sigma (USA). The polyelectrolytes, poly (sodium-4-styrenesulfonate) (PSS, Mw ~70,000 g/mol), and poly(diallyldimethylammoniumchloride) (PDADMAC, Mw ca. 200,000–350,000 g/mol) were obtained from Aldrich and used without further purification. All chemicals were used as received.
Synthesis of GNRs. GNRs were prepared according to the seed-mediated growth method. Briefly, a seed solution was prepared by mixing 5 mL of CTAB (0.2 M) and 5 mL of HAuCl4(0.5 mM) with 0.6 mL freshly prepared 10 mM ice-cold NaBH4solution. The color of the solution changed from dark yellow to brownish yellow under vigorous stirring, indicating the formation of the seed solution. After 5 h, this seed solution was used for the synthesis of the GNRs. In a flask, 75 mL of 0.2 M CTAB was mixed with 1.5 mL of 4 mM silver nitrate aqueous solution and 75 mL of 1 mM HAuCl4. After gentle mixing of the solution, 1.05 mL 0.10 M AA was added. While continuously stirring this mixture, 180 μL of the seed solution was added to initiate the growth of the GNRs. These GNRs were aged for 24 h to insure full growth.
Polyelectrolyte Coating of GNRs. About 10 mL of as-prepared GNRs was centrifuged twice at 8,000 rpm for 10 min, the supernatant was discarded, and the precipitate was redispersed in 5 mL 1 mM CTAB. Subsequently, it was added dropwise to 5 mL of PSS (2 g L−1, 1 mM NaCl) aqueous solution. After 1 h adsorption time, it was centrifuged twice at 8,000 rpm to remove excess polyelectrolyte and dispersed in 5 mL deionized water. Finally, the PSS-coated GNRs were added dropwise to 5 mL of PDADMAC (2 g L−1, 1 mM NaCl) aqueous solution. After 1 h, it was centrifuged twice at 8,000 rpm to remove excess polyelectrolyte and dispersed in 5 mL of phosphate buffer solution (10 mM, pH 7.0).
Preparation of GOD/GNRs Bioconjugates. The combination of GOD and GNRs was achieved using electrostatic interaction. In detail, the above 1.5 mL cationic PDADMAC-coated GNRs was centrifuged at 8,000 rpm for 10 min, the supernatant was discarded, and the precipitate was incubated with 1.5 mL GOD (1 mg mL−1) dissolved in phosphate buffer (10 mM, pH 7.0) for about 1 h at 30 °C. The resultant mixture was centrifuged to discard free GOD and washed by phosphate buffer containing Tween 20. The target GOD/GNR bioconjugates were finally dispersed under ultrasonication in 1.5 mL phosphate buffer solution (10 mM, pH 7.0) and stored at 4 °C.
Typically, 2.5 mL of a 0.33 mMo-dianisidine solution in 0.1 M buffer, 0.3 mL 5 g L−1glucose solution, and 0.1 mL 0.02% HRP were mixed as substrate. Then, 10 μL of the free GOD solution (1 mg mL−1) was added into the mixture, and the absorption of the mixture was recorded immediately and for the next 4 min. For the GOD/GNR system, the measuring procedures were the same as those for free GOD in solution except for the use of 10 μL GNRs@PSS@PDADMAC@GOD (GOD/GNRs bioconjugates) and 10 μL GNRs@PSS@PDADMAC instead of 10 μL of free GOD, respectively.
Control experiment. 1 mL PDADMAC (2 g L−1, 1 mM NaCl) aqueous solution was mixed with 1 mL GOD (2 mg mL−1) in a phosphate buffer (10 mM, pH 7.0) for about 1 h at 30 °C. The resulting concentration of GOD in the PDADMAC/GOD bioconjugates is 1 mg mL−1. Then, an enzyme activity assay was performed.
Apparatus and measurements. UV–Vis absorption spectra were obtained using a UV-2550 spectrophotometer with temperature controller (S-1700, Shimadzu, Japan). Zeta potentials and size distributions were measured on a Zetasizer nano 90 and Zetasizer 3000HSA (Malvern, England), respectively. TEM was performed with a JEOL-JEM-1011 electron microscope under 100 kV accelerating voltage. Formvar-coated copper grids (200 meshes) were used as the support carrier.
Results and Discussion
The practical application of immobilized enzymes depends on their stability under various conditions, e.g., temperature. In this case, the activity and thermostability of GOD was examined for the GOD/GNRs. The optimum catalytic activity for free GOD was observed at pH 7.0 , and the isoelectric point of GOD is 4.2. Cationic PDADMAC-coated GNRs were, therefore, incubated with GOD in phosphate buffer (10 mM, pH 7.0), in which the GOD was negatively charged.
In order to address the influence of GNRs on the thermostability of GOD, the free GOD in solution and GOD immobilized on to the GNRs were exposed to a defined temperature for 15 min, and the enzyme activity assays  were immediately performed. In this work, the relative activity is defined as follows. For the same concentration of glucose, the UV–Vis absorbance of GOD/GNRs bioconjugates or free GOD at 460 nm, after the reaction with glucose, are represented at different temperatures as Ai,460, except Amax at 40 °C, which was regarded as 100% activity. The relative activities of GOD at different temperatures were obtained from the ratio Ai,460/Amax. In order to eliminate the influence of the absorbance of GNRs on the values of Ai,460, the relative activities of the GOD immobilized on GNRs were obtained from (Ai,460 − A·Ai,0,460)/(Amax − Ai,0,460), where Ai,0,460 represents the absorbance of GNRs@PSS@PDADMAC at 460 nm. In order to make the relative activities of free GOD and the GOD immobilized on GNRs comparable, the amount of GOD used to prepare the GOD/GNRs was the same as that of the free GOD used to measure the enzyme assays. Each set of experiments was carried out in triplicate to confirm the reproducibility of the system.
In summary, we have demonstrated that GOD can be successfully adsorbed on to polyelectrolyte-coated GNRs via electrostatic interactions. According to enzymatic catalysis examination, the GOD/GNRs bioconjugates have extraordinary stability at high temperature in contrast not only with the free enzyme in solution but also previously reported GOD systems with other nanoparticles. Therefore, GNRs can be expected to be a promising matrix for the immobilization of other kinds of enzymes and proteins with greatly enhanced stability for biosensor applications. The present results will be of significance for the biologic enhancement effects using other kinds of anisotropic nanostructures. Although the mechanism by which GNRs dramatically enhance the enzyme thermostability of GOD is still an open question, with further experiments to understand the detailed effect of GNRs on GOD being pursued, the present work has already suggested a new way of enhancing enzyme stability and will be of significance in designing new kinds of enzyme-based nanoreactors for biosensors and biocatalytic reactors using other kinds of anisotropic nanostructures.
This study was supported by Development Program of Science and Technology of Beijing Municipal Education Commission (KM200810028010) and Capital Normal University.
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