Catechol Biosensor Design Based on Ferrocene- Derivatized 2,5-Dithienyl Pyrrole Copolymer with 3,4- Ethylenedioxythiophene

Novel conducting platforms based on co(polymerization) of 2,5-dithienyl pyrrole-ferrocene and 3,4-ethylenedioxythiophene were employed for the first time in developing a catechol biosensor. The grafting of Fc moiety onto a hybrid thienyl pyrrole monomer for phenolics detection is proposed for the first time herein to enhance the efficiency of electrochemical reduction of quinone and, in turn, improve the stability of the biosensor in a ‘reagentless’ manner. Tyrosinase enzyme was immobilized by cross-linking onto the carbon nanotubes-enriched electrodeposited films, and catechol was determined with a low detection limit of 2.1 μM. Good operational stability (RSD 3.34 %) was observed during 20 consecutive measurements.


Introduction
New and improved methods have been widely researched because of the major relevance of phenolic detection for beneficial aspects in human physiology and toxicity concerns. Alongside classical analytical methods, electrochemical biosensors are highlighted for their attractive features (fast response time, no requirements for sample pre-treatment, possibility for miniaturization, and portability for on-field measurements) [1,2]. At present, enzymatic phenol biosensors are highly relevant for the chemical, food, or pharmaceutical industry [3][4][5][6]. The operating principle is primarily based on Tyrosinase (Tyr) (E.C. 1.14.18.1, monophenol monooxygenase), which oxidizes monophenols and o-diphenols into corresponding o-quinones whilst reducing O2 to water [7][8][9].
Conducting polymers (CPs) possess π-conjugated backbone, which confers good electronic properties [10,11] and can also meet the requirements of biocompatibility [12,13], efficient electron transfer (allowing both electronic and ionic transport) as well as facility in deposition on the desired type of electrode [14]. As some of the most researched materials in biosensing, the versatility in the synthesis of CPs has enabled immobilization of a wide range of biological moieties (enzymes, antibodies, whole cells, DNA, etc. [15]) and can provide a good option for stabilization of the desired bio-element [16,17].
Considerable efforts have focused on immobilizing Tyr onto CP films for biosensing purposes, mainly PANI [18][19][20], PPy [21,22], and PTh. [23]. Yet, the required deposition potential or processability of the final polymer structure can hinder the applicability; thus copolymerization of the monomers with hydrophilic polymers (polyvinyl alcohol [24], poly(ethylene glycol [25]), glycine methacrylate [26], or aminopropyl moieties [27]) has been employed. Functionalization of the monomer units has also been explored; however, insertion of functional groups onto PPy or PTh has proven unlucrative due to steric hindrances that reduce conjugation length and, in turn, to low conductivity [28]. Recently, a new class of conducting polymers (hybrid 2,5-thienylpyrroles) consisting of both pyrrole and thiophene units has been introduced and employed in a variety of fields from optoelectronics to biodevices. They provide a facility in synthesis with stable electrochemical behavior and tremendous potential for tailor-made functionality based on the N-substitution of the pyrrole fragment. The grafting of functional units onto a conducting matrix can be highly favorable in biosensors for maintaining proper conformation and orientation of the biorecognition element while providing accessibility and efficient mass transfer of analyte. The application of such tailor-made conducting structures in biosensor technology, albeit efficient, is still in the early stages of research, most notably employed in glucose detection [29]. Alternatively, poly 3,4ethylenedioxythiophene (PEDOT) has been highlighted for its superior electrochemical features [23] and can enhance the performance of thiophene-pyrrole structures through copolymerization.
The current study proposes the polymer of ferrocene-derivatized 2,5-thienylpyrrole (further to be referred to as P(SNS-Fc)) and its copolymer with EDOT, for the first time, as immobilization supports for Tyrosinase (P(SNS-Fc)/Tyr and P(SNS-Fc-co-EDOT)/Tyr) in the development of catechol biosensors. Multi-wall carbon nanotubes (MWCNTs) were further introduced to enhance electron kinetics. The analytical characterization and stability investigations proved that the proposed concept represents a good alternative to reported analogs based on conducting matrices.

Electrochemical polymerization and preparation of enzyme electrodes.
Synthesis of P(SNS-Fc) was performed as previously reported [30]. Shortly, P(SNS-Fc) films were prepared through potentiodynamic technique on Pt foil electrodes using 0.01 M monomer (SNS-Fc) in ACN/LiClO4 solution. The potential was cycled between 0.0 and 0.9 V with a scan rate of 100 mV/s. A platinum wire and an Ag/Ag + electrode were used as the counter and reference electrodes, respectively, For P(SNS-Fc-co-EDOT), 5 μL of EDOT was introduced into the electrolysis cell, and the copolymer film was prepared by scanning the potential between 0.0 V and 1.0 V at a scan rate of 100 mV/s. The addition of CNTs was performed by drop-casting 10 µL of 1 mg/mL CNTs-ethanol solution on the surface of the polymer-coated electrode. Immobilization of Tyrosinase (~40-100U) was done by cross-linking with different amounts of 1% glutaraldehyde (7.5 -15 µL).

Instrumentation and principle of measurements.
All amperometric measurements were performed with the potentiostat GAMRY Ref 600 (GAMRY Instruments Inc., Pennsylvania, USA) in a three-electrode cell: Pt foil electrode (0.5 cm 2 ) -working electrode, Pt wire -counter electrode, Ag/AgCl (3M KCl) (BASI)reference electrode. Optimized conditions (pH of buffer electrolyte and applied voltage) and controlled magnetic stirring were applied during amperometric measurements. The detection limit was calculated using 3Sb/m criteria, where m is the slope of the calibration curve, and Sb is the standard deviation of the responses at a minimal concentration (n = 10) [31].

Morphology characterization.
The synthesis and electrochemical characterization of the polymer platforms had been previously detailed in our reports [30,32]. In the current study, particular attention has been given to the characterization of the (co)polymer system involving Tyr enzyme. Both polymer platforms were characterized by scanning electron microscopy (SEM) before and after the addition of CNTs and enzymes. The initial polymer films coated the electrode surface homogenously, and the copolymerization with EDOT led to an increase in the granular aspect of the surface ( Figure. 1a,b), which should provide a large surface area, adequate for biomolecule immobilization, as seen in our previous study [32]. Subsequently, drop-casting of CNTs and enzyme immobilization resulted in typical fibrous structures [33], proving homogenous distribution and cross-linking, with increased porosity for the copolymer platform. The morphology proves similar to the one observed when the glucose oxidase enzyme was immobilized on this type of platform for glucose (bio)sensing [32].

Optimum biosensor design parameters.
The electrochemical characterization of the CP platforms has been recently disclosed when employed in glucose detection [32]. As such, herein, only design parameters related to catechol detection have been investigated. Operational parameters (potential, pH) for P(SNS-Fc)/CNTs/Tyr biosensor were optimized at -0.15 V vs. Ag/AgCl and pH 7.5 (Figure. S1a, S1b). Furthermore, optimum enzyme and cross-linker concentrations were established at 60 U and 10µL (Figure. S1c, S1d). The optimization experiment was performed identically for the copolymer platform P(SNS-Fc-co-EDOT)/CNTs/Tyr, showing very similar optimum parameters ( Figure. S2). The results are in accordance with previous literature reports regarding Tyr biosensors based on the electrochemical reduction of quinone as the detection principle [34].

Analytical characterization.
Analytical characterization of both biosensor platforms ( Figure. 2a-d,3a-d) also revealed very similar performance towards catechol detection as current response evolved linearly in the range 0.02 -0.25 mM with the equation of y = -3.41x -0.01 (R2 = 0.996) and y = -2.5x -0.001 (R2 = 0.995) for Pt/P(SNS-Fc)/CNT/Tyr and Pt/P(SNS-Fc-co-EDOT)/CNT/Tyr biosensor, respectively. The LOD value was calculated as 3.9 µM for the homopolymer-based sensor and it improved to 2.1 µM upon copolymerization of the ferrocenetethered SNS monomer with EDOT. Km and Imax parameters were calculated (according to Lineweaver-Burk plots ( Figure. 2d, 3d)) as 0.874 mM and 2.81 µA and 1.287 mM and 3.34 µA for Pt/P(SNS-Fc)/CNT/Tyr and Pt/P(SNS-Fc-co-EDOT)/CNT/Tyr, respectively. The slight difference observed in analytical data can be discussed considering both increased conductivity and decrease in the number of Fc moieties upon copolymerization, both factors counteracting each other, as proven by previous studies [32]. While the copolymerization increases the conductivity of the platform, the number of available Fc units is the more significant factor, a fact proven by publications concerned with the grafting of Fc moieties onto (non)conducting platforms for biosensing purposes [35,36]. On this note, the motivation to employ an Fc-tethered CP platform in this study has been inspired by several reports on 'mediated' Tyr biosensors for phenol detection [37][38][39][40], which show that the electrochemical catechol recycling is amplified by the presence of a redox shuttle in the biosensing matrix. Our so-called 'reagentless mediated system' approach is more efficient since the redox component is not liable to leaking or diffusion barriers. Thus far, a single similar report on catechol detection is based on a hybrid thienyl pyrrole monomer derivatized with nitrophenol group and copolymerized with polypyrrole [41] that was employed as a spectrophotometric method for determination of analyte (Table 1).

Stability investigations.
Additional reasoning for grafting a mediator unit onto a conducting platform for developing a phenolic biosensor is given by the potential of improving the stability of analysis. Commonly, Tyr biosensors are liable to loss of activity due to the inherent instability and short lifetime of Tyr enzyme [42], but also due to electrode fouling by radical intermediates generated from the enzymatic catalysis [43]. In this context, if the electroreduction of the enzymatically generated quinone is mediated, thus, the reversibility is increased; it is thought that the fouling is reduced and, in turn, the stability of the sensor would increase [37]. The inhibition of the enzyme by its substrate (so-called 'suicide inactivation' [44]) may be delayed due to fast redox cycling [38]. Our study testifies to this, as the linear range reported herein for catechol detection (up to 0.25 mM) is slightly extended in comparison with literature (Table 1) data that reports availability for detection up to 0.103 mM [40], 0.12 mM [48]    indicating good operational stability (~ 90% preserved activity, Figure. 4a, 4b) similar to recent reports based on Tyr enzyme [49,50].

Conclusions
The current study aims to provide an alternative to conducting polymer-based matrices for catechol detection by introducing a ferrocene-tethered hybrid polymer type platform suitable for immobilization of Tyr enzyme. The incorporation of CNTs within the polymer P(SNS-Fc) and copolymer P(SNS-Fc-co-EDOT) matrices granted versatile properties for enzyme immobilization. As a result, adequate analytical characteristics were observed, accompanied by good stability given by the ability of the redox unit to facilitate electrochemical recycling of catechol at the electrode surface in a 'reagentless' manner. Thus, the work proposed herein has a twofold impact, common liabilities of mediated biosensor analysis are resolved, and stability issues regarding phenol detection through electrochemical reduction of quinone are improved.

Funding
Akdeniz University Research Funds.