Mixed hemi/ad-micelles coated magnetic nanoparticles for the entrapment of hemoglobin at the surface of a screen-printed carbon electrode and its direct electrochemistry and electrocatalysis

Highlights

• A novel disposable H₂O₂ biosensor based on magnetic immobilization of hemoglobin.

• Intercalating hemoglobin in mixed hemi/ad-micelles (MHAM) of CTAB coated Mag-NPs.

• Zeta potential isotherm study was performed to gain the MHAM configuration of CTAB.

• The Hb/MHAM@Mag-NPs biocomposite was captured at SPCE by a super magnet.

• The biosensor exhibited high sensitivity and great affinity towards H₂O₂ reduction.

Abstract

An efficient procedure for the physical entrapment of proteins within a biocompatible matrix and their immobilization on electrode surfaces is of utmost importance in the fabrication of biosensors. In this work, the magnetic entrapment of hemoglobin (Hb) at the surface of a screen-printed carbon electrode (SPCE), through mixed hemi/ad-micelles (MHAM) array of positively charged surfactant supported iron oxide magnetic nanoparticles (Mag-NPs), is reported. The Hb/MHAM@Mag-NPs biocomposite is captured at SPCE by a super magnet (Hb/MHAM@Mag-NPs/SPCE). To gain insight in the configuration of the mixed hemi/ad-micelles of CTAB at Mag-NPs, zeta-potential measurements were performed. The entrapment of Hb at MHAM@Mag-NPs was confirmed by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Fourier transform infrared spectroscopy (FT-IR). Direct electron transfer of the Hb intercalated into the composite film showed a pair of well-defined quasi-reversible redox peak at formal potential of −0.255 V vs. Ag/AgCl corresponding to heme Fe(III)/Fe(II) redox couple. It shows that the MHAM@Mag-NPs composite could increase the adsorption ability for Hb, thus provides a facile direct electron transfer between the Hb and the substrate. The proposed biosensor showed excellent electrocatalytic activity to the H₂O₂ reduction in the wide concentration range from 5.0 to 300.0 µM obtained by amperometric measurement. The Michaelis–Menten constant (K_m) value of Hb at the modified electrode is 55.4 µM, showing its high affinity. Magnetic entrapment offers a promising design for fast, convenient and effective immobilization of protein within a few minutes for determination of the target molecule in low sample volume at disposable cost-effective SPCE.

Introduction

Direct electron transfer (DET) of proteins has been a research focus for many years because of its ability to provide a good model for mechanistic studies of electron transfer in biological systems, and its significant role in the field of developing mediator-free bioelectrochemical devices such as third generation biosensors (Gorton et al., 1999, Rusling, 1998), biofuel cells (Ikeda and Kano, 2003, Ramanavicius et al., 2005), heterogeneous catalysts (Vincent et al., 2007) and biomedical devices (Katz and Privman, 2010). Hemoglobin (Hb) is probably the protein whose DET is the most extensively studied. However, redox proteins show a slow rate of electron transfer on a conventional electrode due to the deep bury of the electroactive prosthetic group, the adsorptive denaturation and the unfavorable orientations when directly adsorbed on the electrode surface (Heller, 1990). Therefore, optimization of the electron transfer between the heme center in the large three-dimensional structure of Hb and the electrode surface is challenging as well as finding ideal electrode materials and suitable protein immobilization methods. For this purpose, entrapment or encapsulation of a protein within a biocompatible material using simple procedures so without the need of complicated covalently attachment or time consuming intermediate step, is certainly desirable.

Biocompatible materials such as natural biomolecules (Paolucci-Jeanjean et al., 2005), biopolymers (Peniche et al., 2003), hydrogels (De Wael et al., 2012), surfactants (Cai and Chen, 2004, Li et al., 2002, Wang et al., 2009a, Wang et al., 2009) and nanomaterials (Baghayeri et al., 2013, Kafi and Crossley, 2013) would present a favorable microenvironment to keep the activity of the entrapped proteins, and consequently to fabricate eligible biosensors. Considerable attention has been paid in magnetic nanoparticle (Mag-NPs) based electrochemical biosensors in the recent years (Geng et al., 2011, Wang et al., 2013a, Wang et al., 2013b). As an ideal bimolecular immobilizing carrier, Mag-NPs enable quick and efficient isolation or extraction of a target molecule or substance by an external magnetic field (Palecek and Fojta, 2007). Additionally, Mag-NPs can provide a low detection limit due to their large surface area to immobilize biomolecules.

Surfactants can be used as a physical promoter for modifying the nanoparticles. On the other side, the biocompatible characteristics of surfactants would provide a favorable microenvironment to retain the activities of the immobilized proteins (Cai and Chen, 2004). For instance, electrocatalysis of hemoglobin at C₇₀/didodecyldimethylammonium bromide (C₇₀/DDAB) films casted on glassy carbon electrode in an aqueous solution was reported by Li et al. (2002) and Wang et al., 2009a, Wang et al., 2009) studied direct electrochemistry and electrocatalysis of heme proteins with single walled carbon nanotubes–cetyltrimethylammonium bromide (SWCNT/CTAB) nanocomposite film modified glassy carbon electrodes. However, surfactants can make three different coating shells around the Mag-NPs core including hemi-micelle, ad-micelle and mixed hemi-micelle/ad-micelle, which are formed by the adsorption of ionic surfactants on oppositely charged surface of the metal oxides (Merino et al., 2003). In the hemi-micelle state, the adsorbed surfactant molecules spread themselves on the oxides surface to form a monolayer coverage, probably through columbic attraction between the charged Mag-NPs surface and the oppositely charged surfactant head group. With increasing the adsorbed surfactants onto the mineral oxide surface, the hydrophobic interactions between tails of surfactant hydrocarbon chains resulted in the formation of bilayer of surfactant called ad-micelles. Mixed hemi/ad-micelles (MHAM) are an intermediate state between the two previously described terms which is providing a two-fold mechanism for adsorption of amphiphilic analytes (Merino et al., 2003). Major advantage of MHAM respect to the two other states is ability of MHAM in establishing of both electrostatic and hydrophobic interactions with target analytes.

To the best of our knowledge, mixed hemi/ad-micelles supported magnetic nanoparticles (MHAM@Mag-NPs) have not yet been reported, as a protein immobilizing carrier and for direct electron transfer of Hb. For this purpose positively charged surfactant, cetyltrimethylammonium bromide (CTAB), was used for preparing a MHAM coating shell and its formation was controlled by using zeta potential (ZP) measurements. The immobilized Hb at MHAM@Mag-NPs (Hb/MHAM@Mag-NPs) is magnetically entrapped onto disposable screen-printed carbon electrodes (SPCE) via an extremely fast and simple procedure. This magnetic immobilization strategy is employed for the first time as a simple, efficient and disposable cost-effective method for the preparation of a hemoglobin based biosensor. Finally, integrating the unique properties of MHAM@Mag-NPs with Hb magnetic entrapment allowed an accurate and sensitive determination of hydrogen peroxide in a low sample volume at Hb/MHAM@Mag-NPs/SPCE. The suggested magnetic entrapment offers a promising design for the fast, convenient and effective immobilization of proteins within a few minutes for determination of the target molecule in low sample volume at disposable cost-effective SPCE.