A phenol biosensor based on immobilizing tyrosinase to modified core-shell magnetic nanoparticles supported at a carbon paste electrode

Analytica Chimica Acta 533 (2005) 3–9

A phenol biosensor based on immobilizing tyrosinase to modified core-shell magnetic nanoparticles supported at a carbon paste electrode Zhimin Liu, Yanli Liu, Haifeng Yang, Yu Yang, Guoli Shen, Ruqin Yu∗ State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People’s Republic of China Received 6 August 2004; received in revised form 28 October 2004; accepted 28 October 2004 Available online 21 December 2004

Abstract A phenol biosensor was developed based on the immobilization of tyrosinase on the surface of modified magnetic MgFe2 O4 nanoparticles. The tyrosinase was first covalently immobilized to core-shell (MgFe2 O4 –SiO2 ) magnetic nanoparticles, which were modified with amino group on its surface. The resulting magnetic bio-nanoparticles were attached to the surface of carbon paste electrode (CPE) with the help of a permanent magnet. The immobilization matrix provided a good microenvironment for the retaining of the bioactivity of tyrosinase. Phenol was determined by the direct reduction of biocatalytically generated quinone species at −150 mV versus SCE. The resulting phenol biosensor could reach 95% of steady-state current within 20 s and exhibited a high sensitivity of 54.2 ␮A/mM, which resulted from the high tyrosinase loading of the immobilization matrix. The linear range for phenol determination was from 1 × 10−6 to 2.5 × 10−4 M with a detection limit of 6.0 × 10−7 M obtained at a signal-to-noise ratio of 3. The stability and the application of the biosensor were also evaluated. © 2004 Elsevier B.V. All rights reserved. Keywords: Magnetic bio-nanoparticles; Tyrosinase; Biosensor; Phenol

1. Introduction Phenol is an important contaminant in ground water and surface water [1]. Spectrophotometry and chromatography are the commonly used analytical methods [2,3]. However, these methods are used to be complicated in sample pretreatment and unsuitable for in situ monitoring. Many efforts have been made for the simple and effective determination of phenol to solve these problems. Amperometric biosensors based on tyrosinase have proved to be sensitive and convenient tools for this purpose [4–10]. Tyrosinase can catalyse two reactions: ortho-hydroxylation of phenols to catechol and the further oxidation of catechols to ortho-quinones, both in the presence of molecular oxygen. The phenol can be detected via the reduction of the produced quinone at low potential. The key aspect in the construction of this kind biosensor is ∗

Corresponding author. Tel.: +86 731 882 2782; fax: +86 731 882 2782. E-mail address: [email protected] (R. Yu).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.10.077

the immobilization procedure of tyrosinase on the sensor surface. The immobilization methods such as physical adsorption [11], covalent cross-linking [12], incorporation within carbon paste [7], immobilization in polymer films [13], entrapment in cyro-hydrogel [14] and some sol–gel matrices [8,9,15] have been reported. But some of these methods are rather complicated in manipulation and poor in stability and/or weak in retaining the bioactivity of tyrosinase. Searching for a simple and reliable scheme to immobilize tyrosinase is of considerable interest. In recent years, magnetic nanoparticles as special biomolecule immobilizing carriers are becoming the focus of research. Due to its special properties, magnetic nanoparticles have been used in cell separation processes [16], radio-immunoassay [17], biological missile [18] and DNA separation [19]. The successful applications of magnetic nanoparticles in the immobilization of biomolecules have also been reported [20–22]. Tanaka and Matsunaga reported the fully automated chemiluminescence immunoassay of insulin by using antibody–protein

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A–bacterial magnetic particle complexes [20]. Wang and He reported the application of core-shell magnetic nanoparticles in biomedicine [21]. In this paper, a novel immobilization method for tyrosinase based on magnetic nanoparticles for constructing amperometric biosensor will be reported. The prepared magnetic MgFe2 O4 nanoparticles were first coated with SiO2 to form core-shell (MgFe2 O4 –SiO2 ) magnetic nanoparticles then modified with 3-aminopropyltriethoxysilane (APTES), leaving primary amine groups on the surface. Glutaraldehyde was subsequently used, one aldehyde group reacted with the amine groups, the other could form an imine linkage with the primary amine group on proteins. Thus tyrosinase would be attached onto the surface of core-shell magnetic nanoparticles via glutaraldehyde, forming magnetic bio-nanoparticles. Finally the magnetic bionanoparticles were immobilized on the surface of carbon paste electrode in the presence of magnetic field. The biosensor prepared with the aforementioned immobilization procedure has been successfully used for the detection of phenol. Comparing with the conventional immobilization methods, the procedure with core-shell (MgFe2 O4 –SiO2 ) magnetic nanoparticles shows advantages of high sensitivity, simplicity in manipulation and low cost.

2. Experimental 2.1. Reagents Tyrosinase (EC 1.14.18.1, 2870 U mg−1 , from mushroom), tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) were purchased from Sigma–Aldrich. The glutaraldehyde (25% water solution) was obtained from Shanghai Biochemical Reagents (Shanghai, China). Ammonium hydroxide (30 wt.%) and polyethylene glycol (PEG, 5 kDa) were the products of Tiantai Fine Chemicals (Tianjin, China). All other chemicals were of analytical grade and were used as-received. Doubly distilled water was used throughout the experiments. The supporting electrolyte was 0.05 M phosphate buffer solution (PBS), prepared with KH2 PO4 and Na2 HPO4 . 2.2. Preparation of core-shell (MgFe2 O4 –SiO2 ) magnetic nanoparticles surface modified with amine groups 2.2.1. Synthesis of magnetic MgFe2 O4 core In a typical procedure, MgSO4 , Fe(NO3 )3 ·9H2 O, NaOH, and NaCl were mixed (molar ratio 1:2:8:10) and ground together in an agate mortar for about 30 min. The reaction started readily during the mixing process, accompanied by release of heat. As the reaction proceeded, the mixture became mushy and underwent gradual changes in color from colorless to light red (∼1 min) and finally brown (∼10 min). The mixture was then placed in a quartz crucible, inserted into a quartz tube, annealed at 700 ◦ C for 1 h, and subsequently

cooled to room temperature. Samples were collected, washed several times with water, and dried at 120 ◦ C overnight in a drying oven. 2.2.2. Silica coating on the magnetic core The silica coating on the magnetic MgFe2 O4 core was prepared according to the literature [23]. Briefly, 0.015 g of MgFe2 O4 was added to 100 ml of 2-propanol and sonicated for about 30 min. Under continuous stirring, 3.0 g of PEG, 5.2 ml of water, 9.0 ml of ammonia solution (30 wt.%) and 0.1 ml of TEOS were consecutively added into the above suspension, the reaction was allowed to continue for 24 h under stirring. After the reaction was completed, the products were collected through centrifugation at 5000 rpm, followed by washing with water and ethanol several times, and then dried at 80 ◦ C overnight in an oven. The core-shell (MgFe2 O4 –SiO2 ) magnetic nanoparticles were obtained. 2.2.3. Surface modification of the core-shell magnetic nanoparticles About 1.0 g of core-shell magnetic nanoparticles were suspended in 10 ml of 95% ethanol/H2 O solution and sonicated for 10 min, then 0.5 ml of APTES was added, the reaction was allowed to proceed for 8 h under stirring. Then the precipitates were washed with water and ethanol several times in an ultrasonic bath. The core-shell magnetic nanoparticles with amine groups on its surface were prepared. 2.3. The preparation of the magnetic bio-nanoparticles The conjugation procedure of magnetic bio-nanoparticles is shown in Fig. 1. The preparation of the magnetic nanoparticles was realized by using glutaraldehyde as cross-linking reagent. Under the agitation, 0.5 g of amine group modified core-shell magnetic nanoparticles was added to 5 ml of 2.5% glutaraldehyde, the reaction mixture was kept at room temperature for 3 h. Then the mixture was centrifugated and the precipitates were washed with PBS three times. After adding 5 ml of PBS, 1.0 mg of tyrosinase was added, the mixture was agitated for 12 h at 4 ◦ C. Excess tyrosinase was removed by washing with PBS, the formed magnetic bio-nanoparticles were stored at 4 ◦ C in a refrigerator. 2.4. Fabrication of the enzyme electrode A solid carbon paste electrode (CPE) was prepared according to the procedure reported elsewhere [24]. The paraffin (400 mg) was melted at 60 ◦ C and mixed with graphite powder (500 mg). The mixture was blended thoroughly to obtain a homogeneous paste. A PVC tube (8 mm i.d. and 10 mm depth) was first filled with a magnet (8 mm i.d. and 2 mm depth, producing an inhomogeneous magnetic field, 0.2 T at the surface), then the homogeneous paste was squeezed into the PVC tube holder, the tube was left to harden for a day. The PVC nut was turned to extrude ∼1 mm thick outer paste

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X-ray diffraction (XRD) was obtained with Rigaku D/max 2550 X-ray Diffractometer.

3. Results and discussions 3.1. Characterization of the magnetic nanoparticles

Fig. 1. Schematic diagram for preparation of magnetic bio-nanoparticles.

layer to achieve enough magnetic field on the electrode surface. The CPE was polished with ultra-fine emery paper until a smooth surface was obtained, the electrode surface was cleaned with doubly distilled water. The fabrication of the enzyme electrode was accomplished by adding 30 ␮l of magnetic bio-nanoparticles in PBS onto the CPE surface. After 30 min, the surface was washed with PBS. In the presence of magnetic field, the magnetic bionanoparticles were firmly immobilized on the electrode surface. When not in use, the enzyme electrode was stored in a dry state at 4 ◦ C.

The purity of the as-prepared crystals (Section 2.2.1) was examined using powder X-ray diffraction. The XRD pattern of the final powders presented in Fig. 2 only shows peaks corresponding to MgFe2 O4 (JCPDS card No. 17-0464). This confirmed that the synthesis method was feasible and complete. The morphology and structure of the synthesized products are observed through TEM (Fig. 3(a)). The resulting MgFe2 O4 powder consisted of particles of 15–30 nm, which were well dispersed and were seen to be nearly spherical with irregular polyhedrons. PEG, a dispersion reagent, was added to the suspension of MgFe2 O4 nanoparticles, the hydrolysis of TEOS initiated when ammonia was added as a catalyst. After the reaction was completed, the coating of silica on the surface of magnetic MgFe2 O4 nanoparticles was formed, producing core-shell (MgFe2 O4 –SiO2 ) magnetic nanoparticles. Fig. 3(b) shows the TEM image of MgFe2 O4 nanoparticles after they were coated with shells of ∼10 nm thickness. At this stage, the core-shell magnetic nanoparticles still exhibited the original morphology of MgFe2 O4 core but with an enlarged size due to coated shell structure. The core-shell nanoparticles were nearly spherical and the particle diameter was ∼120 nm. The results also showed that the coating thickness of the shell could be changed when some experimental parameters (e.g. the water/TEOS molar ratio, the amount of ammonia) varied. However, proper shell thickness was favourable to keep the high magnetic-field intensity and the stability of the magnetic nanoparticles. The coating of silica on the magnetic nanoparticles facilitated the dispersion of nanoparticles. Also the presence of OH on the

2.5. Measurements Cyclic voltammetric and amperometric experiments were performed using a PAR 273 potentiostat/galvanostat and model 270 software (EG & G Princeton Applied Research, Princeton, NJ, USA). A conventional three-electrode system was employed with the enzyme electrode as working electrode, a Pt foil as auxiliary electrode, and a saturated calomel electrode (SCE) as the reference against which all potentials were measured. Cyclic voltammetric measurements were done in an unstirred electrochemical cell. Amperometric experiments were carried out in a stirred cell by applying a potential of −150 mV to the working electrode and aliquots of phenol standard solution were successively added to the cell. Current–time curves were recorded after a steady-state current was achieved. Transmission electron micrographs (TEM) of magnetic nanoparticles were obtained with a Hitach-800 Transmission electron microscope (Hitachi, Japan).

Fig. 2. XRD image of magnetic MgFe2 O4 nanoparticles.

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surface moiety on silica surface [27]. The resulting core-shell magnetic nanoparticles were characterized by high surfaceto-volume ratio, strong magnetism and good dispersion. 3.2. Cyclic voltammetric characterization Fig. 4 shows the cyclic voltammograms of the enzyme electrode in air-saturated 0.05 M PBS (pH 7.0). In the absence of phenol (Fig. 4(a)), only low background current was observed. Upon addition of phenol to the buffer solution (Fig. 4(b)), the cyclic voltammogram gave a reduction current with the shape of catalytic wave, which was due to the reduction of quinone species liberated from the enzyme reaction catalysed by the tyrosinase on the electrode surface [28]. 3.3. Optimization of experimental variables The pH dependence of the enzyme electrode was studied between 4.5 and 8.0 in 0.05 M PBS in the presence of 25 ␮M phenol. As shown in Fig. 5, the optimum response current was achieved in the pH range 6.0–7.0. This pH range is in agreement with the optimum pH of 5–8 reported for free tyrosinase [29]. In order to obtain maximum sensitivity, pH 7.0 was chosen in subsequent experiments. The effect of applied potential on the amperometric signal and background current of the biosensor was investigated over the potential range −300 to 0 mV. As can be seen in Fig. 6, the highest signal-to-background current was obtained at −150 mV. When the applied potential was more negative than −150 mV, a higher signal current was achieved, however, the background current also increased. Therefore, an applied potential of −150 mV was selected for the amper-

Fig. 3. TEM images of: (a) magnetic MgFe2 O4 core (enlarged 600,000 times) and (b) magnetic core-shell (MgFe2 O4 –SiO2 ) nanoparticles (enlarged 80,000 times).

shell surface was helpful to complete the modification of the core-shell magnetic nanoparticles [25]. It should be noted that PEG, which acted as a steric stabilizer, was very useful in the preparation of core-shell nanoparticles. According to previous literature data, PEG is thought to be anchored onto the surface of silica particles and extend from the particle surface to contribute a steric stabilization function [26]. Thus the high degree of aggregation between nanoparticles was prevented by the repulsion force and solvation layer of the PEG

Fig. 4. Cyclic voltammograms of the enzyme electrode at a scan rate of 100 mV s−1 in 0.05 M air-saturated PBS (pH 7.0) without (a) and with (b) 0.4 mM phenol.

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Fig. 7. Current–time response curve of the enzyme electrode on increasing the phenol concentration in 25 ␮M steps in 0.05 M PBS (pH 7.0) at an applied potential of −150 mV vs. SCE. Fig. 5. Effect of pH on the enzyme electrode response studied by amperometric method for 25 ␮M phenol in 0.05 M PBS. Operating potential −150 mV vs. SCE.

ometric measurements. Moreover, the low applied potential was favourable to minimize the interferences from the coexisting electroactive species. 3.4. Electrode response characteristics Fig. 7 displays a typical current–time plot of the biosensor under the optimized experimental conditions after the addition of successive aliquots of phenol to the PBS under stirring. A steady-state baseline current was reached quickly, the sensor responded rapidly to the concentration increments

Fig. 6. Current–potential curves of the biosensor examined by amperometric method under conditions (a) air-saturated 0.05 M PBS (pH 7.0) under stirring; (b) 25 ␮M phenol solution using the same PBS as in the first solution.

of the substrate and 95% steady-state current was obtained within 20 s. The response rate was comparable to that of the grafting copolymer titanium oxide sol–gel membrane [30] and was much faster than that observed with the pure silica sol–gel based biosensor (∼60 s) [8]. The fast response was attributed to the rapid diffusion of substrate from bulk solution to enzyme. Fig. 8 shows the dependence of the reduction current on the concentration of phenol. The response to phenol is linear (r = 0.998) in the concentration range from 1 × 10−6 to 2.5 × 10−4 M. The detection limit was 6.0 × 10−7 M at a signal-to-noise of 3. The resulting biosensor exhibited a sensitivity of 54.2 ␮A/mM, which was higher than those of cyrohydrogel based enzyme electrode, sol–gel silicate/Nafion composite electrode and TiO2 sol–gel/grafting copolymer based tyrosinase biosensor [14,10,30]. The high sensitivity of

Fig. 8. Steady-state calibration plot of the enzyme electrode for phenol in PBS (pH 7.0) at an applied potential of −150 mV vs. SCE.

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the biosensor could be attributed to the favourable microenvironment of the proposed immobilization matrix, which has a general affinity for the tyrosinase loading and favourable for the stabilization of its biological activity to a large extent. 3.5. Reproducibility and stability The repeatability of the current response of one enzyme electrode to 25 ␮M phenol was examined. The relative standard deviation (RSD) was 2.6% for seven successive assays. The electrode-to-electrode reproducibility was determined from the response to 25 ␮M phenol at five different enzyme electrodes, an acceptable reproducibility was obtained with a variation coefficient of 4.8%. The enzyme electrode was stored in a dry state at 4 ◦ C in a refrigerator when not in use. The stability of the biosensor was investigated by measuring the enzyme electrode response with 25 ␮M phenol every 3–4 days. The response current of the biosensor decreased to 92% after storing 10 days, while 85% of the original response retained after 20 days. The enzyme electrode retained 70% of its original response after 1 month testing. The relatively good stability of the enzyme electrode could be attributed to the biocompatibility between core-shell nanoparticles and tyrosinase.

Table 1 Recovery of the tyrosinase biosensor Phenol concentration (␮M) Added

Founda

1.05 5.92 9.24 11.02 15.14 19.27

1.10 5.84 9.30 10.28 14.80 19.64

a

Recovery (%)

104.7 98.6 100.6 93.2 97.7 101.9

An average of three measurements.

Table 2 Phenol concentration in real samples determined by the tyrosinase biosensor and the spectrophotometric method Samplesa

1 2c 3 a b c

Phenol concentration (␮M)b Tyrosinase biosensor

Spectrophotometric

2.23 16.7 52.9

2.33 15.7 52.1

Relative error (%)

95.7 106.3 101.5

Samples were appropriately diluted by pH 7.0, 0.05 M PBS. The values were the average values from three measurements. Relative standard deviation of 2.8% for eight repetitive assays.

3.8. Regeneration of the biosensor 3.6. Interference studies To evaluate the selectivity of the biosensor, the influence of some possible interfering substances were examined in PBS solution (pH 7.0) containing 10 ␮M phenol in the presence of l-cysteine, l-aspartic acid, and l-glutamic acid, each in concentration 50 times of that of phenol, 2000 times of acetaminophenol, 250 times of carbonate, 1000 times of chloride, sulfate, bromide and iodide each. The results showed that these substances did not cause observable interference in the determination of phenol. However, the presence of ascorbic acid and sulfide caused interference (for 50 times of ascorbic acid and sulfide each, the current decreased to 55 and 48% of the original response, respectively), which may be due to ascorbic acid and sulfide could reduce orthoquinone. 3.7. Analytical application To evaluate the accuracy of the proposed biosensor, some assays were made on standard phenol samples. Phenol concentration was determined by the standard addition method and the results were presented in Table 1. It can be seen that the results were satisfactory. Also the determination of phenol in real samples (industrial waste water) were carried out, the results were compared with those obtained by spectrophotometric method, as shown in Table 2, the two methods displayed a good correlation. Therefore, the biosensor provides a possible and simple method for determining the phenol with good precision and accuracy.

The regeneration procedure of this type of biosensor was performed by turning the nut to extrude a 0.1 mm thick outer paste layer and then polishing with an alumina paper wetted with water to produce a smooth shiny surface. The addition of magnetic bio-nanoparticles onto the CPE surface was followed and carried out as described in Section 2.4.

4. Conclusion In this paper, a novel method for the immobilization of tyrosinase on the surface of the modified core-shell magnetic nanoparticles is proposed. Because of the modification of amine groups on the surface of magnetic core-shell nanoparticles, the tyrosinase was covalently immobilized onto the magnetic nanoparticles and then the magnetic nanoparticles were attached to the CPE surface with the help of a permanent magnet. The as-prepared biosensor showed relatively fast response and high sensitivity for phenol measurement. In addition, the preparation of the biosensor was easy as compared with that of silica sol–gel/grafting copolymer based tyrosinase biosensor [5].

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 20375012, 29975006 and 20075006), the Foundation for Ph.D. Thesis Research (No.

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20010532008), and the Foundation of Science Commission of Hunan Province.

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