Activity of glucose oxidase functionalized onto magnetic nanoparticles
© Kouassi et al; licensee BioMed Central Ltd. 2005
Received: 21 December 2004
Accepted: 11 March 2005
Published: 11 March 2005
Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties.
Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy.
The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles
Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications
The immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials or devices . Enzyme immobilization for example, is a desired biological procedure because of the possible application of immobilized enzymes in continuous operations, product purification, and catalyst recycling . Furthermore, immobilization provides many advantages such as enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth .
In the last decade, nanosize materials have been widely used as support for this purpose. Among these materials, magnetic nanoparticles are very popular when used in conjunction with biological materials including proteins, peptides, enzymes [4–9] antibodies and nucleic acids , because of their unique properties [4–9]. The ability to tract magnetically labeled entities or target organelles using magnetic force offer the opportunity to conduct biological operations with increased specificity.
Magnetite (Fe3O4) are biocompatible superparamagnetic materials that have low toxicity and strong magnetic properties . They have been widely used for in vivo examination including magnetic resonance imaging, contrast enhancement, tissue specific release of therapeutic agents, hyperthermia [10, 11], magnetic field assisted radionucleide therapy , as well as in vitro binding of proteins and enzymes [4–8].
Magnetite nanoparticles have been used as support material for binding of enzymes including yeast alcohol dehydrogenase  and lipase  directly via carbodiimide activation. This method brought about considerable promise because of its simplicity and efficiency. Recently, γ-Fe2O3magnetic nanoparticles were used for binding Candida rugosa lipase after acetylation of thiophene functionalized nanoparticles, or through nitroso-derivative formed on the surface of the particles by reacting nitroso tetrafluoroborate in methylene chloride. Both methods and more effectively lipase immobilized on acetylated nanoparticles exhibited long term stability. Glucose oxidase (GOX, β-D-glucose oxygen 1-oxidoreductase, EC 22.214.171.124) is a homodimer flavoprotein containing two active sites per molecule [12, 13]. It catalyses the oxidation of β-D-glucose to gluconic acid, concomitant with the reduction of oxygen to hydrogen peroxide. Glucose oxidase has been used to test various types of enzyme immobilization, and is the most commonly studied in the construction of biosensors for glucose assay development [12, 14, 15]. A more recent study  examined the activity of cholesterol oxidase activity using carbodiimide activation.
Here, we report the stability and enzymatic activity of glucose oxidase immobilized onto Fe3O4 magnetic nanoparticles using two binding methods, the direct binding via carbodiimide activation of amino functionalized particles and binding to thiophene-functionalized acetylated nanoparticles. A comparison of the stability and activity of glucose oxidase immobilized to magnetite using different protocols will lay the foundation for magnetic immunoassays. The size and structure of the nanoparticles were characterized using Transmission electron microscopy (TEM) and Fourier Transform Infrared (FTIR) spectroscopy, respectively. The stability, activity, and kinetic behavior of bound glucose oxidase were also examined.
Materials and methods
Glucose oxidase (specific activity 200 units/mg protein) from Aspergillus niger was purchased from VWR international (Pittsburgh, USA). Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide, sodium hydroxide, acetic anhydride, glucose, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 % and iron (III) chloride hexahydrate 99%, were obtained from Sigma-Aldrich St Louis (USA). 11-bromoundecanoic acid was obtained from TCI America Portland, USA. The Biorad Protein Assay Reagent Concentrate was purchased from Biorad Laboratories (Hercules, CA). Thiophene-2-thiolate was obtained from Alfa Aesar MA, USA. Iodine was obtained from Mallinckrodt Kentucky, USA, and acetonitrile was obtained from EMD Chemicals (New Jersey, USA). Sodium phosphate monohydrate and potassium phosphate dihydrate were acquired from EM Science (New Jersey, USA). Sodium carbonate was obtained from Orion Research Inc. (Beverly, USA). Magnetic nanoparticles (Fe3O4) were prepared by chemical co-precipitation of Fe2+ and Fe3+ ions in a solution of ammonium hydroxide (magnetic nanoparticles I or Fe3O4 I), or sodium hydroxide (magnetic nanoparticles II or Fe3O4 II) followed by a treatment under hydrothermal conditions [4, 5]. Iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25°C) by adding NH4OH solution (30%) or NaOH 3 M at a pH 10. The precipitates were heated at 80°C for 35 min under continuous mixing and washed 4 times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the supernatant using a magnetic separator of strength greater than 20 megaoersted (MOe). The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction.
Magnetic nanoparticles I (50 mg) produced, using a solution of ammonium hydroxide were added to 1 mL of phosphate buffer (0.05 M. pH 7.4). After adding 1 mL of carbodiimide solution (0.02 g/mL) in phosphate buffer (0.05 M. pH 7.4), the mixture was sonicated for 15 min. Following the carbodiimide activation, 2 mL of glucose oxidase (1000 units /mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath. The magnetic nanoparticles were separated from the mixture using a magnetic separator. The precipitates containing Fe3O4 nanoparticles I and Fe3O4 bound glucose oxidase (GOX-Fe3O4 I) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles .
A second functionalization protocol using a modification of the strategy adopted to immobilize Candida rugosa lipase on the γ-Fe2O3 was implemented (Magnetic nanoparticles II). Briefly, 1.5 g Fe3O4 was added to 5 g of 11-bromoundecanoic acid dissolved in 15 mL of ethanol. 11-bromoundecanoic acid was covalently linked to the nanoparticles surfaces by heating the mixture with microwave irradiation for 10 min. Functionalization of the particle was achieved through nucleophilic substitution with the 2-thiophene thiolate. In practice, 2-thiophene thiolate (7 mL) was added to the mixture containing the particles and heated in microwave for 5 minutes. The mixture was washed with ethanol and transferred in a round bottom flask. Acetic anhydride (4 mL) and 34.6 mL of iodine (0.01N) were successively added to the particles and agitated. The mixture was heated for 1 h under reflux condition . The particles were washed several times with water, once with 10% sodium carbonate solution and finally with ethanol. The acetylated particles were reacted directly with the enzyme covalently linked to the particles via C = N bond .
A = (C i - C s )* V (1)
Where A is the amount of bound enzyme, C i and C s are the concentration of the enzyme initially added for attachment and in the supernatant, respectively (mg/mL) and V is the volume of the reaction medium (mL). The size of Fe3O4 magnetic nanoparticles, GOX-Fe3O4 I and GOX-Fe3O4 II were characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL) and structure by FTIR spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared as follows: a drop of magnetic nanoparticles was dispersed in nanopure water. The resulting solution was sonicated for 4 min to obtain better particle dispersion. A drop of the dispersed solution was then deposited onto a copper grid and dried overnight at room temperature. The binding of GOX onto the magnetic nanoparticles was investigated using FTIR spectroscopy. Samples for FTIR analysis were prepared in phosphate buffer pH 7.4. The activity of bound GOX was determined by measuring the initial rate of formation of hydrogen peroxide at a given temperature following the formation of a red quinoneimine dye. The principle of enzymatic determination of the activity of glucose oxidase is described as follows: Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide. Phenol + 4-AAP, in the presence of peroxidase (POX), produces a quinoneimine dye that is measured at 500 nm using a Beckman Du Spectrometer to provide an absorbance that is proportional to the concentration of glucose in the sample. The reaction is described as follows:
The activity of glucose oxidase was measured as follows. An assay mixture was prepared by mixing 500 U of horseradish peroxidase, 0.015 mmol of 4-aminoantipyrine (4-AAP), 0.025 mmol of phenol and 5 mmol of glucose in 50 mL of phosphate buffer solution (0.05 M. pH 7.4) to result in a glucose concentration of 0.1 M. To start the enzymatic reaction, 2 mL of the assay solution was added to 15 mL centrifuge test tubes containing GOX-Fe3O4 and mixed by vortex. A solution of free GOX of the same molar concentration was used to evaluate the activity of the free enzyme for comparison. The solution was incubated at various temperatures (37–80°C) at specific intervals of time (30 min) and the supernatant was separated from GOX-Fe3O4 using a magnetic separator. 10 μL aliquots of the supernatant were then taken for determining the concentration of hydrogen peroxide following the procedure by Trinder . The activity of the enzyme can be calculated using the following equation:
Where ABS sample denotes the absorbance of the sample, ABS Std is the absorbance of the standard solution, and C the concentration of glucose in the sample.
The effect of temperature on the free GOX, GOX-Fe3O4 I and GOX-Fe3O4 II was estimated by determining the concentration of glucose in the sample at various temperatures. A solution of the assay mixture was added to the various centrifuge test tubes containing bound or free enzymes. The samples were stored in a water bath at specific temperatures (37, 50, 60, 70 and 80°C) and the absorbance was monitored at fixed time intervals to determine the glucose content. The effect of pH on GOX was monitored by measuring the initial rate of glucose oxidation by glucose oxidase in different phosphate and carbonate buffer solutions of pH (5–10) at 25°C.
The thermal stability of free GOX, GOX-Fe3O4 I, and GOX-Fe3O4 II were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (37–80°C) in phosphate buffer (0.05 M, pH 7.4) for 30 min. Aliquots of the reacting solutions were taken at periodic intervals (every 30 min for 6 h) and assayed for enzymatic activity as described above. The first-order inactivation rate constant, k was calculated from the equation:
ln A = ln A0 - kt (5)
where A0 is the initial activity, A is the activity after time t (min) and k is the reaction constant.
The storage stability was examined by measuring the change in the concentration of glucose at room temperature at different time intervals (4 days). Test tubes with samples of GOX-Fe3O4 I, GOX-Fe3O4 II, or free GOX solutions were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 33 days. Thereafter, 3 mL of the assay solution was added, and the residual activity of GOX was assayed.
The kinetic parameters of free GOX, GOX-Fe3O4 I and GOX-Fe3O4 II, Km and Vmax were determined by measuring the initial rates of glucose oxidation (0.2–1 mM) by glucose oxidase (0.25 mg/mL) in phosphate buffer (pH 7.4) at 25°C.
Results and discussion
Summary of binding efficiencies of enzyme-functionalized systems (n = 9).
Bound enzymes (units/mg nanoparticles)
38.4 ± 0.8
27.6 ± 0.6
Kinetic parameters of free GOX, GOX-Fe3O4 I, and GOX-Fe3O4 II determined from the double reciprocal plots.
Vmax (μmol/min mL)
Inactivation rate constants (k) of the free-GOX, GOX-Fe3O4 I, and GOX-Fe3O4II at various temperatures.
Free-GOX k (min-1)
GOX-Fe3O4 I k (min-1)
GOX-Fe3O4 II k (min-1)
Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides using two different base solutions. GOX was bound to the particles by direct attachment via carbodiimide activation and chemically via covalent attachment onto thiophene acetylated magnetic nanoparticles. Confirmation of the binding was demonstrated by FTIR spectroscopy and the sizes of the particles were characterized by TEM. The direct binding of GOX via carbodiimide activation was more effective and resulted in binding efficiency in the range between 94–100% while the binding efficiency was only between 66–72% for the GOX-Fe3O4 II complex. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when the complex was subjected to thermal and pH variations. This study shows that binding onto magnetic nanoparticles can allow the enzyme to acquire the conformational and structural arrangement for a better activity and stability, and suggests that binding of enzyme onto magnetic nanoparticles via carbodiimide activation was efficient for creating bioconjugates for a variety of applications in health and food safety.
The authors acknowledge the USDA challenge grant program for partial funding of this research. Dr Chen Yu is acknowledged for his help in TEM image acquisition.
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