Immobilization of Tyrosinase on ( 3-Aminopropyl ) triethoxysilane-Functionalized Carbon Felt-Based Flow-Through Detectors for Electrochemical Detection of Phenolic Compounds

Tyrosinase (TYR) was covalently immobilized onto amino-functionalized carbon felt (CF) surface via glutaraldehyde (GA). Prior to the TYR-immobilization, primary amino group was introduced to the CF surface by treatment with 3-aminopropyltriethoxysilane (APTES). The resulting TYR-immobilized CF was used as a working electrode unit of an electrochemical flow-through detector for monoand di-phenolic compounds (i.e., catechol, p-cresol, phenol and p-chlorophenol). Additionally, flow injection peaks based on electroreduction of the enzymatically produced o-quinone species were detected at −0.05 V vs. Ag/AgCl. The resulting TYR/GA/APTES/CF biosensor responded well to all compounds tested with limits of detection range from 7.5 to 35 nmol−1 (based on three times S/N ratio). Moreover, such modified electrode exhibits good stability and reproducibility for catechol. No serious degradation of the peak current was found over 30 consecutive injections.


Introduction
A group of chemicals known collectively as endocrine disrupting compounds (EDCs) are suspected of interfering with the normal function of the endocrine system causing adverse effects in humans and environment.These include a range of synthetic oestrogens, pesticides, plasticizers, and phenolics. 1With the increasing concern over health and environmental issues, there is a great necessity to detect the environmental pollution such as phenolic compounds.3][4][5] Many of them are very toxic, showing harmful effects on plants, animals, and human health.The development of bioselective detection units for phenols has increased rapidly in recent years.
Analytical methods such as chromatography, [6][7][8][9] chemiluminescence, 10 capillary zone electrophoresis, [11][12][13] and spectrophotometric methods 14 are currently employed to determine phenols.However, time-consuming and low sensitivities limit their applications in situ.Therefore, there is an interest in developing simple, sensitive, and effective analytical techniques for their determination.5][26] Based on these activities, a number of TYR-based electrochemical biosensors have been proposed for the determination of mono-and di-phenolic compounds.In particular, the development of highly sensitive biosensors for chlorophenol compounds is an important topic, because chlorophenol compounds are extremely toxic contaminants in ground and surface water.Various immobilization approaches of enzyme such as physical adsorption, covalent binding, encapsulation, entrapment and cross-linking have been proposed.Among them, covalent binding has the advantage that the enzyme is generally strongly immobilized on the surface and unlikely to detach from the surface during repeated use.
Carbon felt (CF) is a microelectrode ensemble of micro-carbon fibre (cca.7 μm in diameter), and possesses a random three-dimensional structure.The CF has high surface area (≈0.1-10 m 2 g −1 ), and shows high conductivity and excellent electrolytic efficiency.Furthermore, the porous structure of CF causes very low diffusion barrier against the solution flow.0][31] On account of these CF characteristics, the novel chemical immobilization strategy of TYR onto the CF surface has been established.
In this study, the primary amino group (-NH 2 ) was induced onto the CF surface by using APTES.The GA was then covalently immobilized onto APTES-modified CF surface via -NH 2 of APTES.After that, the TYR was also covalently bonded to CF surface through another aldehyde group of GA.The resulting TYR/GA/APTES/CF biosensor was used as a working electrode unit of biocatalytic enzymatic flow-through detector.The characteristics of the modified surfaces were investigated using field emission scanning electron microscopy (FESEM).The activities of the immobilized TYR toward phenolic compounds were evaluated.Meanwhile, parameters such as GA concentration, applied potential, enzyme immobilization time, and electrolyte pH were discussed and optimized.Operational stability and storage stability were also investigated.
Flow injection analysis (FIA) system is composed of a double plunger pump (DMX 2000T, SNK) with a six-way injection valve (SVM-6M2, SNK, 200 μl injection loop) and CFbased electrochemical flow-through detection. 16All FIA experiments were measured at room temperature.Air-saturated phosphate buffer (0.1 mol l −1 , pH=7.0) was used as a carrier.Before the measurements, the carrier solution was flowed at flow rate of 3.0 ml min −1 for 1000 s under the applied potential of −0.05 V vs. Ag/AgCl to remove weakly adsorbed TYR from the CF surface and to reduce the background current.Then, 200 μl of standard solutions of phenolic compounds were injected, and the cathodic peak currents based on the electroreduction of o-quinone species produced by TYR reaction.

Enzyme immobilization procedures
The CF sheet [from Nihon Carbon Co.] was cut into 10 mm × 3 mm × 3 mm in size (weight, cca.12-13 mg), and washed with doubly distilled water under ultrasonication for 10 min, dried in vacuum for 1 h.The fabricated procedure is similar as previously reported by the authors of this work. 15Briefly speaking, the CF was dipped into a solution of APTES in toluene, 250 g l −1 .After 1 h incubation at room temperature, the CF was washed with toluene under ultrasonication for 2 min and dried in vacuum for 1 h.The APTES-modified CF was immersed in different concentrations of aqueous GA solution (2 ml) and incubated at room temperature for 15 min.The activated CF surfaces were then washed thoroughly with pure water and the CF was placed in enzyme aqueous solution.The activated GA/APTES-functionalized CF was immersed in 2 ml of TYR buffer solutions.After the incubation at 4 °C for 1 h, the CFs were washed with phosphate buffer (0.1 mol l −1 , pH=7.0) to remove the weakly adsorbed TYR.

Results and discussion
3.1 FESEM measurements of the bare and modified CF Usually, the activity of immobilized enzyme is significantly influenced by the conformation and structure of enzyme on the matrix surface.In order to obtain the interfacial properties of modified CF surfaces, the FESEM measurements were performed to understand the characteristics of the CF surface.Fig. 1 shows the FESEM images of (A) bare CF, and (B) TYR/GA/APTES/CF electrode.Differing from the bare CF (Fig. 1A), a micrometre-sized, island-like structure and/or film-like structure were observed on the TYR/GA/APTES-modified CF surface (Fig. 1B).This is also evidence of the successful modification on the CF surface.3.3 Optimization of the immobilization parameters using catechol

Effect of GA on the peak current responses of catechol
In this study, catechol was used as a model substrate.TYR catalyses two-electron oxidation of o-diphenols to o-quinones, which is called catecholase activity in the presence of molecular oxygen. 33The produced o-quinones can be electrochemically reduced to o-diphenols at a low overpotential. 34The effects of GA volume fraction on the peak currents were examined.To understand the importance of covalent bonding by using GA, experiments were carried out by changing the GA volume fraction from 0 to 25 %, as shown in Fig. 3(A).With the increase in GA concentration, the currents also increased to a maximum at 20 % of GA.
Higher volume fractions of GA (25 %) resulted in almost the same response.This result indicates that the GA is essential for immobilization of TYR by covalent bonding to detect the catechol.Furthermore, the GA immobilization time upon the peak current of catechol (Fig. 3(A) inset) was investigated.The peak current reached a platform with increasing immobilization time from 15 min to 6 h.Then it decreased while immobilization time increased.It can be concluded that the activity of enzyme was damaged by long reaction time with GA, which is consistent with the result of Fig. 3D.

Effect of pH in the electrolyte solution on the peak current responses
The influence of the pH of the electrolyte solution on the peak-current response was investigated for a catechol concentration of 10 μmol l −1 over the pH range from 5 to 9 using 100 mmol l −1 phosphate buffer solution.Fig. 3 (B) shows that the enzymatic activity was dependent on the pH, exhibiting higher activity at pH=6.5.This optimum pH is in good accordance with other biosensors described in the literature. 35Therefore, pH=6.5 phosphate buffer solution was chosen throughout this study.ground current, which is probably due to the reduction of dissolved oxygen in carrier.Thus, −0.05 V vs. Ag/AgCl was selected as an optimum applied potential of the TYR-CF based flow biosensor.

Effect of TYR immobilization time on the peak current responses
The relationship between the adsorption time of TYR and the peak current responses of catechol (10 μmol l −1 ) are depicted in Fig. 3(D).The peak current responses were found to be non-dependent on the adsorption time over the range from 30 min to 24 h.The maximum response of 10 μmol l −1 of catechol is 1 h.It can be considered that the adsorbed TYR molecule that contributes to the signal generation is adsorbed on the GCE surface during the initial stage of net adsorption processes.This result implies that the adsorption process of the electrochemically active TYR-layer is relatively rapid, and the initial adsorption layer mainly contributes to the generation of the current response.Both short and long immobilization times were not preferable for fabricating the TYR/GA/APTES/CF based biosensor.Therefore, one hour was chosen throughout this study.

Analytical characteristics of the present biosensor
After optimization of the fabrication parameters above, the analytical properties of TYR/GA/APTES/CF biosensor were subsequently evaluated.Fig. 4 depicts the calibration curves of (a) catechol, (b) p-cresol, (c) 4-CP, and (d) phenol obtained by flow injection analysis, which plots the cathodic peak current vs. catechol concentration, obtained under certain conditions (applied potential, −0.05 V; carrier flow rate, 3.25 ml min −1 ; carrier, pH=7.0).The magnitude of the peak-current response by this TYR-based flow-biosensor was linear in the concentration range between 1.0 to 30 μmol l −1 with a detection limit of 0.008 μmol l −1 , based on the peak current signal-to-noise of 3. Table 1 summarizes the performance characteristics of the modified biosensor.The TYR/GA/APTES/CF-based biosensor has the ability to detect low concentration of analytes.Judging from the calibration plots, the fabricated biosensor is useful for detecting not only di-phenolic compounds, but also mono-phenolic compounds, especially 4-CP.Table 2 summarizes some characteristics of recent covalent bonding-based phenol sensors compared to our sensor.Overall, the device reported here compares favourably with other reported tyrosinase sensors in terms of the parameters outlined.As compared with them, our sensors have the advantages of lower detection limit, higher sensitivity, and good storage stability.It can be concluded that the TYR/GA/APTES/CF-based biosensor has broad specificity toward both mono-and di-phenolic compounds with good characteristics.

Operational stability of the TYR/GA/ APTES/CF-based flow-through detector
Operational stability is one of the important factors for the practical use of enzyme-based biosensors or as biocatalysts. 36Fig. 5 displays the typical 30 consecutive flow injection peaks for the sensor with the concentration of 10 μmol l −1 catechol.The relative standard deviation (RSD) was 1.85 for 30 successive assays, this is superior to TYR-entrapped carbon paste (RSD = 2.5 %, n = 30). 37 can be seen from Fig. 5 that no serious peak degradation was observed over 30 consecutive injections by using catechol as the substrate.It is known that quinone compounds are highly unstable, and easily polymerize and inactivate the TYR. 38The polymerized product causes the fouling of the TYR-based enzyme electrode surface, lead-  ing to the serious degradation of the response. 39But in this case, the flow-through system prevents the surface fouling caused by the polymerized products.6. Storage stability of the TYR/GA/APTES/ CF-based flow-through detector Storage stability is an important factor for the application of immobilized enzymes, because native enzymes usually quickly lose their activity. 40The relative remaining activity for the determination of catechol over 30 days storage period were checked.The modified electrode maintained 78 % of original activity for catechol after 25 days of storage by checking the activity every 5 days.The results indicate that the TYR/GA/APTES/CF biosensor has good storage characteristics toward the electrochemical detection of catechol.

Conclusions
In this study, GA was used to immobilize TYR onto the APTES-modified CF surface.The TYR/GA/APTES/CF biosensor shows excellent results on sensitivity, operational stability, and storage stability for phenolic compounds.The biosensor exhibited excellent operational stability over 30 injections, and maintained 78 % of the original catecholase activity after 25 days of storage.It is also useful for the continuous monitoring of mono-phenolic compounds in our case.Furthermore, this enzyme immobilization strategy would be useful not only for biosensors, but also for biofuel cells.
List of abbreviations and symbols

Fig. 3 (
Fig.3(C)shows the effect of applied potential on the peak current of 10 μmol l −1 catechol.Cathodic peak current appeared at +0.15 V and increased with change in the potential from +0.15 to −0.05 V, and the maximum value was observed at −0.05 vs. Ag/AgCl.Gradual decrease in peak current in more negative potential region (from −0.1 to −0.2 V) can be attributed to the increased back-

a
Slope of the linear portion of calibration curve b Noise level, 7 nA (S/N = 3)

Table 1 -
Analytical performances of biosensor

Table 2 -
Comparison of the biosensor for the determination of phenolic compounds