Poly-L-lysine-modified with ferrocene to obtain a redox polymer for mediated glucose biosensor application
Introduction
Redox polymers have been well accepted for application in biosensors, energy conversion, bioelectrocatalysis and storage technologies. The importance of this kind of material in the development of biosensors resides in their function as an immobilization matrix for enzymes because they favour the mediated electron transfer (MET) process, which allows their reuse in other applications [1], [2].
The importance of MET in the development of second-generation biosensors is focused on preventing inconveniences with oxygen species observed in first-generation biosensors, since it facilitates the transport of electrons from the enzyme to the electrode surface because some redox enzymes contain catalytic sites deep in the protein structure, such as glucose oxidase (GOx) [3]. This type of based-electron transfer includes those that use reversible redox molecules with a potential gradient for electron shuttling where redox polymers are included [4], [5]. Redox polymers have been investigated since the past 70 years, but they gained importance in 1989 when Degani and Héller published the transfer of electrons with the enzyme glucose oxidase and its application in an amperometric glucose biosensor [6]. The design of novel redox polymers allows the high efficiency of bioelectrodes to increase biocatalytic activity, electron transfer kinetics, buffer changes in pH, thermostability, crosslinking capabilities, biocompatibility and control of the degree of swelling or solvation.
The first generation of redox polymer used, for instance, Os-complexes modified with polyvinyl imidazoles and later copolymers composed of methacrylates, acrylates, or acrylamides [7], using the enzyme glucose oxidase [8] and later incorporating ferrocene in polymers such as linear polyethyleneimine (LPEI) published by the Minteer group [9], as well as catechol-modified polypeptide [10]. Ferrocene mediators have been the most used due to some advantages, such as insensitivity to O2, pH independence, stability in their oxidation/reduction states (Fe(II)/Fe(III) for Fc/Fc+), good interaction with enzymes and reversible electron transfer kinetics at low redox potentials [11]. In addition, to achieve faster electron transfer, photochemical stability, low biotoxicity or decreasing electrochemical potential to follow a reaction, ferrocene derivatives have been used as mediators for the detection of hydrazine [12], glucose [4], p-synephrine [13], epinephrine [14] and dopamine [15], among others. In energy matter, for instance, in redox batteries [16] or fuel cells [17]. In medical research, ferrocene byproducts have been evaluated for potential application as drug candidates for cancer therapy [18]. Furthermore, ferrocene derivatives are attractive due to their electrochemical and catalytic properties [19]. In this context, they can be dissolved in the support electrolyte or trapped with other reagents, such as polymers, to be deposited on surfaces [20] to confer other properties, such as control of the reactive oxygen species. In this context, ferrocene-based polymer redox (FBPR) was used for the first time in 1984 with GOx, and its application has increased over time [21]. Mainly, polypyrene-derived polymers [22] and polythiophenes [23] have been reported because they are conductive polymers that can improve the electrical communication between enzymes and electrode surfaces. On the other hand, cyclodextrins have been used for their electrochemical stability and complex formation that facilitate their solubility in aqueous systems and provide a controllable environment around the dendritic core. Therefore, cyclodextrins are good candidates to stabilize molecules for GOx immobilization using ferrocene as a mediator [24], [25], [26]. Other polymers already reported as polyethyleneimine have been used for being rich in primary amino groups, flexible and hydrophobic [27], [28], [29]. Additionally, polyvinyl has hydrophilic properties that allow good interaction with enzymes, even though its biocompatibility is questionable [30]. Chitosan is a biocompatible biopolymer with adhesion capacity and water permeability, but its low electron transfer efficiency even when ferrocene is added limits its application [31], [32]. In this sense, poly-L-lysine is a cationic biopolymer with the potential to be used as a ferrocene-based polymer redox (FBPR) because it is rich in amino groups, biodegradability, and not antigenicity [33], which enables its usefulness in biomedical applications, drug release or the food industry [34] and could allow the adhesion of proteins as crosslinker agents in biosensors due to its electrical charge and intrinsic properties [35], [36].
In the present investigation, poly-L-lysin [37] was modified by a very simple method, with the anchor of carboxylate ferrocene (Fc) through its activation with the mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy-succinimide (NHS) and the covalent bond with the amino group of PLL to obtain the redox polymer Fc-PLL. The stoichiometric relationship between Fc/NHS/EDC was standardized, and its characterization of the redox polymer was performed by FT-IR and cyclic voltammetry. Fc-PLL was used with glucose oxidase enzyme for evaluation as a glucose biosensor by amperometry, obtaining a stable bioelectrode with good electrocatalytic properties and sensibility.
Section snippets
Reagent and chemicals
All the chemicals used in the experiments were analytical reagents with high purity. Ferrocene-carboxylic acid (97%), N-Hydroxy-succinimide (98%), N-(3-Dimethylaminopropyl)-N’-ethyl-carbodiimide-hydrochloride (98%), Poly-L-lysine solution 0.1% (w/v) in H2O were obtained from Sigma-Aldrich and used without further modification. Glucose oxidase (GOx) enzyme from Aspergillus Niger (100,000–120,000 U/g) type X-S, D-(+)-glucose ACS reagent, were purchased from Sigma-Aldrich. Ethylene glycol
Results and discussion
The modification of carboxylate Fc (FcCOOH) with PLL was analysed by FTIR (IR-Affinity-1 FTIR SHIMADZU). The spectrum of FcCOOH (Fig. 1-B) shows characteristic peaks similar to those reported by the National Institute of Standards and other works [41], [42]. νC-H 2600 cm−1 and 3000 cm−1 correspond to OH-acid stretching, νC = O and νC-O at 1649 cm−1 and 1286 cm−1, while a peak at 1459 cm−1 corresponding to aromatic bond C = C and at 509 cm−1 describes νFe-C.
While the FTIR for poly-L-lysine (Fig.
Conclusions
The synthesis of the Fc-PLL redox polymer was carried out in an effortless way by activating the carboxyl group of Fc-COOH with a mixture of EDS/NHS under mild reaction conditions. This modification was characterized by FTIR analysis. Electrochemical evaluation by cyclic voltammetry showed that the formal potential of 0.19 V (vs. Ag/AgCl) was more cathodic, conserving the carbonyl group of the imide of the ferrocene chain, and the Fc-PLL was soluble in water, facilitating its preparation and
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J. Ledesma-Garcia reports financial support was provided by Mexican Council for Science and Technology.
Acknowledgements
The authors acknowledge the Mexican Council for Science and Technology (CONACYT) for the financial support through the project Ciencia de Frontera 2019 grant No. 845132.
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