Evaluation of novel Fmoc-tripeptide based hydrogels as immobilization supports for electrochemical biosensors Giovanni Fusco a,b , Laura Chronopoulou a , Luciano Galantini a , Andrea Zerillo b , Zulkarnain M. Rasik c , Riccarda Antiochia b , Gabriele Favero b , Andrea D'Annibale a , Cleofe Palocci a , Franco Mazzei b, ⁎ a Department of Chemistry, University of Rome La Sapienza, Italy b Department of Chemistry and Drug Technologies, University of Rome La Sapienza, Italy c Department of Chemistry, Sepuluh Nopember Institute of Technology, Surabaya, Indonesia abstract article info Article history: Received 9 August 2017 Received in revised form 6 October 2017 Accepted 6 October 2017 Available online 07 October 2017 The immobilization procedure is one of the key factors affecting the electron transfer based biosensors perfor- mance. It must assure both the enzyme retention activity and an efficient communication between the redox center of the enzyme and the electrode surface. In this regard, hydrogels are attractive materials due to their three-dimensional hydrophilic networks and their high-water concentration, promoting the biomolecule long- term stability and providing a suitable scaffold for trapping. To this aim, we have synthesized a gelling tripeptide Fluorenylmethyloxycarbonyl-triphenylalanine (FmocPhe 3 ) produced starting from Fmoc-phenylalanine and diphenylalanine by the catalytic activity of Lipase in water and characterized its performance as a new immobi- lization support for biosensors. For this purpose, we have evaluated the main bioelectrochemical properties of Trametes versicolor Laccase (TvL) immobilized within the hydrogel and a hydrogel-nanocomposite structure in the presence of gold nanoparticles (AuNPs). Either the Fmoc and the benzyl rings inside the tripeptide structure contributed to the hydrogel network formation by π-π stacking interactions, which promoted the physical en- trapment of Laccase as well as the interaction with the carbon-based electrode surface. Furthermore, the π-elec- trons flow throughout the tripeptide based hydrogel allowed a good electron transfer between the immobilized enzyme and the electrode. The obtained results showed a significative increase in the hydrogel nanocomposite- based graphite biosensor performance with respect to those obtained with the hydrogel-based graphite biosen- sor. The experimental results, in terms of analytical performance and costs, assessed the possible use of FmocPhe 3 based hydrogels in the development of electrochemical biosensors. © 2017 Elsevier B.V. All rights reserved. Keywords: Hydrogel Gelling tripeptides Gold nanoparticles Laccase Electrochemical biosensors 1. Introduction The main aspect to be accurately evaluated in electron transfer- based biosensors assembly is the immobilization of the electrochemi- cally active proteins on the electrode surface. An ideal immobilization procedure should: a) assure an optimal protein loading; b) retain the native structure and bio-electrocatalytic properties of the immobilized protein; c) allow a site oriented protein immobilization, to reach an ef- ficient communication with the electrode surface [1–4]. For these rea- sons, the optimization of protein immobilization procedures is essential to increase biosensors performance. In the last years, several immobilization techniques have been inves- tigated in order to develop electrochemical biosensors [5–10]. In partic- ular, hydrogels represent an ideal enzyme immobilization support [11– 15]. In fact, hydrogels polymeric structure is characterized by the pres- ence of a cross-linked network, swollen with water, isolating the immobilized biomolecules and reproducing the microenvironment found in biological media. Gelling oligopeptides have been also employed in this regard [16]. They are biocompatible, biodegradable and can be easily tailored for different applications. Hydrogel formation can be controlled by operating on chemical, physical and biological pa- rameters. Gelling oligopeptides are able to lead to the formation of dif- ferent secondary structures, such as alpha-helices and beta-sheets [17]. Moreover, a number of dipeptides linked to fluorenylmethoxycarbonyl (Fmoc) spontaneously form fibrous hydrogels under physiological conditions. The structural and physical properties of these gels are dictated by the amino acidic sequence of the peptide building blocks. The self-assembly is driven by hydrogen bonding and π–π attractive interactions between Fmoc π-electrons [18]. Furthermore, the synthesis of Fmoc-oligopeptides can be triggered in an aqueous phase by employing an enzymatic reaction, avoiding the use of organic solvents that could affect their immobilization behavior for enzymatic proteins [19]. We recently reported the use of lipolytic en- zymes for the preparation of stable FmocPhe 3 hydrogels [20] which were successfully employed as controlled delivery systems for selected Microchemical Journal 137 (2018) 105–110 ⁎ Corresponding author. E-mail address: franco.mazzei@uniroma1.it (F. Mazzei). https://doi.org/10.1016/j.microc.2017.10.002 0026-265X/© 2017 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc