A novel immobilization matrix for the biosensing of phenol: self assembled monolayers of calixarenes Phenolün biyosensörle bir immobilizasyon

Aim: The development of calixarene based phenol biosensor. Methods: This study describes the application of a calixarene derivative, 5,17-diamino-25,27-bis(3-thiol-1-oxypropane)-26,28-dihydroxycalix[4]arene (HS-Calix-NH 2 ) which has both amino and thiol functionalities, in the practical surface modifications for biomolecule binding. The structure of HS-Calix-NH 2 allows easy inter-action with Au surface and one-step biomolecule immobilization. Self-assembled monolayers (SAMs) of p-amino-functionalized mercaptoalkylcalixarene (HS-Calix-NH 2 ) were formed onto the Au electrode. Then, Laccase (Lac) enzyme was immobilized onto the modified surface by crosslinking with glutaraldehyde (GA). Resulted electrode (HS-Calix-NH 2 /Lac) was used for the electrochemical analysis of phenolic compounds at − 50 mV. Results: The linearity was observed in the range of 0.1–100 μ M and 1.0–100 μ M for catechol and phenol, respectively. The potential use of the biosensor was investigated for phenol analysis in artificial samples which simulate the industrial waste water, which is highly acidic and composed of concentrated salt, without needing any sample pre-treatment step. Conclusion: The prepared Lac biosensor has a potential for rapid, selective and easy detection of phenolic con-taminations in samples.

The formation of SAMs using calixarenes was tested first time in our previous study calixarenes containing hydroxyl (to prepare glucose oxidase biosensor) [10] and thiol and in this study calixarenes containing amine and thiol as a functional group for the covalent immobilization of enzymes (to prepare laccase biosensor). Here, we describe the application of a functional calixarene derivative; 5,17-diamino-25,27-bis(3-thiol-1-oxypropane)-26,28-dihydroxycalix [4]arene (HS-Calix-NH 2 ) for the practical, one-step immobilization of biomolecules onto the Au surfaces that is very promising in biosensor applications. In order to construct phenolic biosensor, Lac was covalently immobilized onto the surface of calixarene modified gold surfaces, namely HS-Calix-NH 2 which was successfully synthesized, characterized and applied in dye-sensitized solar cells in our previous work [12]. Formation of SAMs on the electrode was performed via bonds between gold and thiol groups of calixarenes, thus free amino groups of HS-Calix-NH 2 remained on exterior side of the surface. These amino functionalities of HS-Calix-NH 2 played a key role for the stable immobilization of the enzyme by crosslinking with glutaraldehyde (GA). GA is a proper bifunctional crosslinker which binds biomolecules to the immobilization support through their amino groups, with suitable reaction time, pH value and temperature [13]. Biosensor preparation was performed by mixing of Laccase (Lac) enzyme, bovine serum albumin (BSA) and GA and then, coated onto the HS-Calix-NH 2 -modified Au electrode. During the measurements, Lac is oxidized by molecular oxygen [14] and then, reduced again by the help of phenolic substrates acting as a donor for electrons to regenerate the enzyme, the phenolic substrates are converted to the quinone and/or phenoxy radicals. These oxidized species can be reduced at the electroactive surfaces at potentials below than 0.0 V [15]. After optimization studies, analytical characteristics were investigated and HS-Calix-NH 2 /Lac biosensor was applied for analysis of the phenol amount in artificial wastewater samples.

Materials
Laccase (Lac, EC 1.10.3.2, from Agaricus bisporus, lyophilized powder, 6.8 U per mg solid), GA solution (25%, v/v), dimethyl sulfoxide (DMSO), BSA were purchased from Sigma Aldrich. All other chemicals were analytical grade. TLC analyses were performed on DC Alufolien Kieselgel 60 F254 (Merck). Drying of solvents were carried out by storing them over molecular sieves (Aldrich; 4 Å, 8-12 mesh). All reactions, except noted ones, were conducted under nitrogen atmosphere. All starting supplies used for the synthesis of calixarenes were of standard analytical grade from Merck or Aldrich and used without further purification. To prepare all aqueous solutions, deionized water, passed through a Millipore milli-Q Plus water purification system, was used.

Apparatus
Voltammetric and amperometric measurements were carried out by Palm Sens analyzer (Palm Instruments, Houten, Netherlands). The experiments were performed in a reaction cell (10 mL) at room temperature. A three electrode system consisted of Au (1.6 mm diameter, 99.95% purity; BASI, USA) as the working electrode, a Pt (Metrohm, Switzerland) as the counter electrode and a Ag/AgCl (in 3.0 M KCl, Metrohm, Switzerland) electrode as the reference electrode were used. Surface characterization of the biosensor was carried out on a JEOL5600-LU model scanning electron microscope (SEM). SEM images were taken using an acceleration voltage of 20 kV.

Biosensor preparation
Smoothing and polishing of the electrode surface were made using a piece of cloth with various sized alumina powder (Gamma, 0.05; 0.1; 0.3; 1.0 mm), rinsed with pure water and placed in an ultrasonic bath to remove any substances from the surface. Afterwards, the electrode was cleaned and polished as follows: initially, the electrode was immersed into the ethanol/deionized water mixture (1 : 1 v/v), then it was put in an ultrasound bath type sonicator (Elma Schmidbauer GmbH, Singen, Germany) for 5 min. After washing with Millipore water, the cyclic voltammetry (CV, voltage range between 0.5 and 1.5 V with a scan rate of 50 mV/s) was applied in 0.5 M H 2 SO 4 until reproducible voltammetric responses were obtained. The electrode was then washed with deionized water. For the surface modification, initially, 0.25 mg HS-Calix-NH 2 was dissolved in 0.025 mL of DMSO and adjust to 0.25 mL with PBS (NaCl 8.0 g·L − 1 , KCl 0.2 g·L − 1 , Na 2 HPO 4 1.44 g·L − 1 , KH 2 PO 4 0.24 g·L − 1 and pH 7.4), this solution was dropped on the electrode and allowed to stand 30 min at room temperature. During the incubation time SAMs of p-aminofunctionalized mercaptoalkylcalixarene (HS-Calix-NH 2 ) were formed onto the Au electrode. The electrode surface was washed with PBS to remove non-bound molecules. For the enzyme immobilization, 1.0 mg Lac was dissolved in 0.05 M sodium phosphate buffer (0.005 mL, pH 6.0), the mixture of 2.5 μL of 2.5% of GA and 2.5 μL of BSA (1.0 mg·mL − 1 ) solution was prepared in PBS and spread over the surface of HS-Calix-NH 2 modified gold electrodes, dried for 1 h at ambient conditions. The resulted surface was shown in Scheme 1.

Measurements
Electrochemical measurements for the phenolic substrates (phenol and catechol) were carried out at − 50 mV in 50 mM sodium phosphate buffer (pH 6.0) [16]. When the phenolic compounds are added into the working buffer, immobilized Lac on the surface of working electrode is oxidized by molecular oxygen. After that it was reduced again by phenolic compounds, acting as electron donors for the enzyme regeneration. Then, the phenolic compounds are oxidized to quinone, phenoxy radicals, or both, and subsequently, these oxidized species can be reduced at the working electrode [17]. After characterization, the performance of HS-Calix-NH 2 /Lac biosensor was tested by using artificial waste water sample with highly acidic nature (50 g/L NaCl and 100 g/L phenol in 1.0 M HCl). The sample was diluted with working buffer and added into the cell as a substrate. The phenol calibration curve was used for the calculation of phenol concentration in the sample.

Biosensing studies
Initially the modified surface was characterized via CV using K 3 Fe(CN) 6 as redox probe. The difference in the voltammograms as a result of surface coating was shown in Figure 1. The oxidation and reduction peaks are obtained at about 0.25 and 0.18 V for bare electrode (peak-to-peak separation of 70 mV), 0.27 and 0.15 V for HS-Calix-NH 2 modified surface (peak-to-peak separation of 120 mV). A drop in the peak current when the Au electrode was coated with HS-Calix-NH 2 was observed due to the rather inefficient electron transfer properties. The surface morphologies of HS-Calix-NH 2 and HS-Calix-NH 2 /Lac modified surfaces were characterized by SEM and the images were shown in Figure 2. Figure 2 gives information about the surface before and after enyzme immobilization. In Figure 2A, the images of the electrode surface after calixarene covering and Figure 2B shows the big difference after covalent immobilization of the Lac. It can be seen that the addition of enzyme and the crosslinking enzyme and calixarene on the surface of Au electrode using GA caused the formation of coverage on the Au surface. The membrane structure was formed after immobilization step that includes crosslinking with the bifunctional cross-linker in the presence of Lac on the electrode surfaces ( Figure 2B).
The effect of pH on the HS-Calix-NH 2 /Lac response was also examined over the range of pH 4.5-7.0 (50 mM sodium acetate and sodium phosphate buffers) by using catechol (250 μM). As shown in Figure 3, lower biosensor response was obtained with lower or higher pHs than 6.0. A further increase or decrease in pH led to decrease in the enzymatic activity. Hence, pH 6.0 is decided as the optimum pH for HS-Calix-NH 2 /Lac biosensor, which is similar to that of previously reported Lac biosensors [18]. In another work, the optimum pH was obtained at 5.5 for the Lac biosensor in which the enzyme was immobilized in the histidine modified montmorillonite matrix on a GCE [19]. It can be said that optimum pH for Lac is varied according to the ionic features of immobilization matrices used for biomolecules.
The effect of HS-Calix-NH 2 amount on the biosensor response was investigated using different amount of (0.5, 1.0 and 2.0 mg·mL − 1 ) HS-Calix-NH 2 during Lac immobilization. The results (Figure 4) indicate that using 1.0 mg/mL HS-Calix-NH 2 showed higher sensor responses (that can be seen from the slope of the linear graph) than 0.5 and 2.0 mg/mL − 1 . And also the biosensor response showed saturation at 2.5 mM of catechol when the biosensor was prepared using 2 mg of calixarene (data not shown). The lower amount of HS-Calix-NH 2 does not have enough functional group to immobilize enzyme. The decrease on the current with the increased amount of calixarene can be attributed that the limit for the diffusion phenol and of oxygen as a co-substrate of enzymatic reaction. Quinones,   which are produced in enzymatic reaction, are reduced at the working electrode and the thick layers of calixarenes produce also diffusion barrier for the oxidized species. As shown in Figure 5, current change was proportional to catechol and phenol concentration in the range from 0.1 to 100 μM and from 1.0 to 100 μM, respectively. When the concentration of catechol or phenol were more than 100 μM, substrate saturation was observed, showing a typical Michaelis-Menten kinetic mechanism. K Mapp and I max values were calculated (using GraphPad program) as 118 μM and 6.41 μA for catechol and 114 μM and 1.95 μA for phenol, respectively.
For the analytical characterization of HS-Calix-NH 2 / Lac electrode, a certain catechol concentration in calibration curve was used for detecting the operational stability, repeatability and limit of detection (LOD). In the repeatability study, optimized HS-Calix-NH 2 /Lac biosensor was tested with the addition of 50 μM catechol consecutively. After the measurements (n = 8), standard deviation and coefficient of variation values were assessed as ± 1.52 and 3.17%, respectively. Furthermore, LOD was estimated via 3S b /m formula where S b is standard deviation of measurements and m is the slope of standard curve, using eight consecutive measurements at 0.1 μM catechol. It was calculated as 0.06 μM. Concomitantly, to test the operational stability of HS-Calix-NH 2 / Lac electrode, 50 μM catechol additions (121 measurements) were accomplished during 42 h and the activity of Lac electrode decreased as 22.7%. The comparison of analytical characteristics of the prepared laccase biosensors were summarized in Table 1. The aim of the preparation of newly designed biosensors is to improve one or more performance characteristics such as linearity for the substrate, operational/storage stability, LOD etc. When compared the HS-Calix-NH 2 /Lac to biosensors in the literature, for catechol better linearity and lower LOD were obtained than Tyr biosensor, which was constructed on 3-mercaptopropionic acid (MPA) SAM on a Au disk electrode [20], peroxidase biosensor, which was designed on poly(glycidylmethacrylate-co-vinyl ferrocene) grafted iron oxide nanoparticles [23]. The Lac/NiNPs/cMWCNTs/ PANI/AuE electrode lost 15% of its initial activity after its 200 uses during the span of four months when stored at  4°C [24], HS-Calix-NH 2 /Lac was used to detect catechol for 121 measurements consecutively. Additionally, the effect of possible interfering substances such as ethanol, ascorbic acid and uric acid was experimented for the novel proposed biofilm layer to provide higher accuracy in the sample application study. To monitor the effect of interfering substances, initially catechol standard (50 μM) was added into the working buffer (50 mM, pH 6.0, phosphate buffer). Subsequently, ascorbic acid, uric acid and ethanol were added to the working buffer under the same conditions, respectively. Moreover, the same catechol amount was introduced into the reaction cell and it was observed that initial and last catechol signals were similar. On the other hand, there was no dramatic change in the current responses when the 10 μM interfering substances were added into the reaction cell as shown in Figure 6.
In the final step, the developed HS-Calix-NH 2 /Lac biosensor system was applied to artificial waste water, which was prepared with 1.0 M HCl solution containing 50 g·L − 1 NaCl and 100 g·L − 1 phenol [26]. Prior to the sample application, a new calibration curve for phenol was carried out and artificial waste water was diluted as 10 times in working buffer and the final solution was added into the reaction cell to detect the phenol in artificial sample. As a consequence, the corresponding results were compared with known concentration and it was found that there was a 101.5% as a recovery (n = 5). The HS-Calix-NH 2 /Lac biosensor showed high stability and an extensive linear range for the detection of phenol, and the strategy can be easily broaden to screen other phenolic compounds.

Conclusions
In conclusion, a functional calixarene derivative 'HS-Calix-NH 2 ' was used to modify the Au electrode via formation of SAMs. Lac was chosen as a model enzyme for the fabrication of phenol biosensors. After optimization of preparation and working conditions, the HS-Calix-NH 2 /Lac biosensor was calibrated for catechol in batch systems. The biosensor was applied for phenol analysis in artificial waste water samples. The obtained data for the HS-Calix-NH 2 /Lac biosensor was verified the practical application without requiring a sample treatment. And it was shown that the prepared Lac biosensor has a potential for rapid, selective and easy detection of phenolic contaminations in samples.