Abstract
A new electrochemical biosensor was constructed for the glucose detection. Iron oxide nanoparticles were prepared using co-precipitation method. Polyvinyl alcohol-Fe3O4 nanocomposite was fabricated by dispersing synthesized nanoparticles in polyvinyl alcohol (PVA) solution. Glucose oxidase (GOx) was immobilized on PVA-Fe3O4 nanocomposite via physical adsorption. The mixture of PVA, Fe3O4 nanoparticles and GOx was drop cast on a tin (Sn) electrode surface (GOx/PVA-Fe3O4/Sn). The Fe3O4 nanoparticles were characterized by X-ray diffraction (XRD). Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FESEM) techniques were used to characterize the PVA-Fe3O4 and GOx/PVA-Fe3O4 nanocomposites. The electrochemical performance of the modified biosensor was investigated using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Presence of Fe3O4 nanoparticles in PVA matrix enhanced the electron transfer between enzyme and electrode surface and the immobilized GOx showed excellent catalytic characteristic toward glucose. The GOx/PVA-Fe3O4/Sn bioelectrode could measure glucose in the linear range from 1 to 30 mM with a sensitivity of 9.36 ''A mM-1 and exhibited a lower detection limit of 8 ''M at a signal-to-noise ratio of 3. The value of Michaelis-Menten constant (KM) was calculated as 1.42 mM. The modified biosensor also has good anti-interfering ability during the glucose detection, fast response (10 s), good reproducibility and satisfactory stability. The results demonstrated that the GOx/PVA-Fe3O4/Sn bioelectrode is promising in biosensor construction.
1. Introduction
The accurate detection of glucose concentration is vital in various fields such as biology, biochemistry, clinical chemistry and food analysis. For this aim, glucose biosensor is widely used for determination of glucose in blood and food [1, 2]. Between several detection methods, electrochemical biosensors have attracted considerable attention due to their excellent selectivity, simplicity and low cost. Electrochemical techniques, particularly amperometric biosensors based on glucose oxidase (GOx) immobilization, which can catalyze the oxidation of glucose, have been widely used in glucose sensing [3-5].
Different types of nanomaterials with various size, shapes, physical and chemical properties owing to advances in synthetic methodologies are being extensively used in biosensors. These materials exhibit significant benefits due to their small size, large surface-to-volume ratio, optical characteristics and high catalytic properties compared to macroscale materials. Because of their adsorption ability, they are useful for the immobilization of biomolecules such as GOx [6-9]. Moreover, the capability of nanoparticles in electron transfer improvement between electrode and active site of the enzyme make them suitable for applying in enzymatic biosensors applications [6]. Wide variety of metal nanoparticles including gold (Au) [10, 11], silver (Ag) [12], palladium (Pd) [13], platinum (Pt) [14, 15] and metal oxide [16] nanoparticles have been extensively used to construct different sensors, due to their unique features [17]. In recent years, Fe3O4 magnetic nanoparticles have attracted a lot of attention in many fields such as biotechnology, pharmacy, cell separation and drug delivery, Owing to their interesting properties including biocompatibility, low toxicity, strong supermagnetism, catalytic activity and the ease of preparation process. These magnetic nanoparticles are appropriate for immobilization of different biomolecules such as enzymes. Therefore, it would be promising to use them in biosensor applications [18-22]. It must be mentioned that, by modifying these nanoparticles, using conductive polymers and biopolymers, can be overcome the problems of aggregation and rapid degradation [6].
A number of polymers have been used to stabilize the biomolecules in the design of biosensors. Among them, polyvinyl alcohol (PVA) has been widely utilized as a matrix for entrapment of GOx due to its biocompatibility, non-toxicity, water solubility, good chemical and thermal stability. PVA has large numbers of hydroxyl groups, which provides a biocompatible microenvironment [23, 24].
Xin et al. have studied screen-printed carbon electrode modified by Fe3O4-Au magnetic nanoparticles coated horseradish peroxidase and graphene sheets-nafion film based hydrogen peroxide biosensor [25]. Godarzi et al. have prepared carbon nanotubes decorated by Fe3O4 nanoparticles for highly stable and selective non-enzymatic glucose biosensor [26]. Yang et al. have described core-shell Fe3O4-enzyme-polypyrrole nanoparticles for glucose biosensor using potentiometric technique [27]. Tan et al. have prepared PVA/ZnO nanorods composite films for hydrogen peroxide biosensor [28]. Lad et al. have fabricated PVA-silica hybrid films for encapsulation of GOx based glucose biosensor using electrochemical technique [29]. Fang et al. have designed glassy carbon electrode modified by PVA-multiwalled carbon nanotubes'Pt nanoparticles hybrids for non-enzymatic hydrogen peroxide sensor [30].
In this paper, a novel glucose biosensor is presented. It is fabricated by dispersing synthesized Fe3O4 nanoparticles in PVA solution and GOx is immobilized by physical adsorption in PVA-Fe3O4 nanocomposite, drop cast on a tin (Sn) electrode surface (GOx/PVA-Fe3O4/Sn). The characterization and electrochemical performance of the modified biosensor were investigated.
2. Materials and methods
2.1. Reagents
Ferrous chloride (FeCl2) and ferric chloride (FeCl3) were products of Sigma'Aldrich. Hydrochloric acid (HCl) was purchased from Scharlau. Sodium hydroxide (NaOH) was bought from Merck. These materials have been used for the synthesis of Fe3O4 nanoparticles. PVA (87'89% hydrolyzed, average MW = 72000), D(+)-glucose and GOx (Type VII-S, 250000 U g'1) were obtained from Sigma-Aldrich. Disodium phosphate (Na2HPO4) and monopotassium phosphate (KH2PO4) were used for preparation of phosphate buffer solution (PBS). All chemicals were used without further purification. Distilled water was used for preparation of all aqueous solution.
2.2. Preparation of Fe3O4 nanoparticles
Fe3O4 nanoparticles have been synthesized by earlier protocol reported co-precipitation method [31]. The aqueous solution of 0.85 mL of HCl and 25 mL of deoxygenated water was prepared. 5.2 g of FeCl3 and 2.0 g of FeCl2 were dissolved in solution with vigorous stirring. The mixed solution of iron salts was added drop-wise into 250 mL of 1.5 M NaOH under vigorous stirring at room temperature. The stirring continued for 30 minutes. During the procedure nitrogen gas was bubbling through solution and the pH value was maintained at about 11-12. The black precipitate was separated by centrifugation and washed three times with deoxygenated water to remove impurities. To neutralize anionic charge on nanoparticles, 500 mL of 0.01 M HCl then added into the mixture with stirring.
2.3. Construction of GOx/PVA-Fe3O4/Sn electrode
A PVA (3%) solution was prepared by dissolving 3 g of PVA in 100 mL distilled water under magnetic stirring at 85''C for 2 hours. The solution of glucose oxidase (4 mg mL-1) was prepared in phosphate buffer. 100 ''L Fe3O4 nanoparticles (10 mg mL-1) was dispersed in 500 ''L PVA solution by vigorous stirring at room temperature. As a result, viscous solution consisting of PVA and dispersed Fe3O4 nanoparticles was obtained. Resulting solution and 250 ''L of prepared GOx solution was slowly mixed for 1 minute. Subsequently, 10 ''L of this mixture was drop cast on the surface of the Sn electrode and then dried in air at room temperature to fabricate GOx/PVA-Fe3O4/Sn electrode.
2.4. Apparatus and measurements
X-ray diffraction (XRD) (Co-K'', Philips) was used to investigate the structure and crystal size of Fe3O4 nanoparticles. The PVA-Fe3O4 and its interaction with GOx were characterized by Fourier transform infrared (FTIR) spectrophotometer (Nicolet, model NEXUS 670). Field emission scanning electron microscopy (FESEM) (Tescan, model Mira 3-XMU) was used to study the surface morphology of nanocomposite films. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed on an Autolab Potentiostat/Galvanostat. All electrochemical measurements were carried out at room temperature in phosphate buffer (pH 7) solution, using three-electrode system with a Sn electrode as working electrode, a Pt wire as auxiliary electrode and an Ag/AgCl electrode as reference.
3. Results and discussion
3.1. Characterization of Fe3O4 nanoparticles, PVA-Fe3O4 nanocomposite and GOx/PVA-Fe3O4/Sn electrode
Fig. 1 shows the XRD pattern of the synthesized Fe3O4 nanoparticles. The 2'' values of Fe3O4 nanoparticle 21.3'', 34.9'', 41.5'', 50.3'', 62.6'', 67.0'', and 73.8'' correspond to the crystal planes of (111), (220), (311), (400), (422), (511) and (440), respectively, which is quite similar to that reported before for magnetite nanoparticles [32]. The reflection planes are broad, indicating the nanosize of the Fe3O4 nanoparticles. No peaks of impurities are observed in XRD pattern, revealing the high purity of the Fe3O4 nanoparticles. The average crystal size of Fe3O4 nanoparticles calculated using Debye-Scherrer formula is about 3 nm.
The FTIR spectrum Fig. 2a displays the major peaks related to pure PVA. The adsorption band at 3433.98 cm-1 is attributed to the stretching of O-H, 2921.68 cm-1 peak is related to the symmetric stretching of CH2, 1714.24 cm-1 peak is due to carbonyl groups and 1660.66 cm-1 peak is assigned to the symmetric stretching of carboxylate anion (-COO-). The peak at 1444.67 cm-1 is corresponding to the O-H and C-H bending. The band at 1103.16 cm-1 can be attributed to the C-O stretching and O-H bending vibrations [29, 33, 34].
The FTIR spectrum of PVA-Fe3O4 nanocomposite is represented in Fig. 2b. It obviously reveals the characteristic adsorption bands of PVA and Fe3O4 nanoparticles. The adsorption band at 629.44 cm-1 is associated with the Fe-O stretching vibration and torsional vibration in the tetrahedral and octahedral sites [6]. These results indicate that the Fe3O4 nanoparticles are presented in PVA film.
The FTIR spectra of GOx and GOx/PVA-Fe3O4 nanocomposite are shown in Fig. 2c and 2d, respectively. Two major IR adsorption bands related to amide '' and II have been extensively used for characterization of GOx. The amide I adsorption peak appears at 1640.29 cm-1 belonging to the C-O stretching vibration in the protein's backbone. The band at 564.10 cm-1 exhibits the characteristics of N-H bending and C-N stretching of amid II [35]. The FTIR spectrum of the GOx/PVA-Fe3O4 nanocomposite shows characteristic adsorption bands of GOx and PVA-Fe3O4 nanocomposite. Amide I and II IR bands are also observed in the spectrum of GOx/PVA-Fe3O4 nanocomposite. However, the functional groups of GOx and PVA-Fe3O4 nanocomposite overlap, thus the IR peaks shape become broader. Therefore, we could confirm the immobilization of enzyme with its native structure on nanocomposite matrix.
The morphology of PVA/Sn film, PVA-Fe3O4/Sn electrode and GOx/PVA-Fe3O4/Sn bioelectrode were characterized by FESEM (Fig. 3). As shown in Fig. 3a the surface of PVA film seems very smooth, with no cracks or pores. Fig. 3b shows that Fe3O4 nanoparticles are dispersed almost homogenously in the PVA matrix with some aggregation. It can be seen from Fig. 3b the surface of PVA- Fe3O4 nanocomposite is granular. Such granular surface is believed to be very beneficial for the immobilization of biomolecules. Regular globular morphology of GOx/PVA- Fe3O4 nanocomposite is shown in Fig. 3c. It is indicating that the enzyme molecules successfully immobilized on the surface of PVA- Fe3O4 nanocomposite.