Core–shell nanocluster films of hemoglobin and clay nanoparticle: Direct electrochemistry and electrocatalysis

Abstract

A novel core–shell protein nanocluster film, designated as clay–(Hb/PSS)_n, was fabricated on pyrolytic graphite (PG) electrodes. Positively charged hemoglobin (Hb) at pH 5.5 and negatively charged poly(styrenesulfonate) (PSS) were first assembled layer by layer on surface of clay nanoparticles from their solutions mainly by electrostatic attraction, forming a core–shell nanocluster structure in which clay nanoparticles were the “cores” and (Hb/PSS)_n multilayers were the “shells”. The aqueous dispersion of clay–(Hb/PSS)_n nanoclusters was then cast on surface of PG electrodes, forming clay–(Hb/PSS)_n nanocluster films after evaporation of solvent. Hb in clay–(Hb/PSS)_n films exhibited a pair of well-defined and reversible cyclic voltammetric (CV) peaks at about − 0.36 V vs. SCE in pH 7.0 buffers, characteristic of Hb heme Fe(III)/Fe(II) redox couples. Compared with other Hb-containing clay films, clay–(Hb/PSS)_n films displayed smaller CV peak separation (ΔE_p), indicating the better electrochemical reversibility of Hb in these nanocluster films. The partially ordered structure of the films was characterized by X-ray diffraction (XRD) experiments. UV–VIS and reflection absorption infrared (RAIR) spectroscopy suggests that Hb retains its near-native structure in clay–(Hb/PSS)_n films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at clay–(Hb/PSS)_n film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on protein direct electrochemistry. The electrochemical and electrocatalytic activity of the films could be tailored by controlling the number of bilayers of the (Hb/PSS)_n shells on the surface of clay nanoparticle cores.

Introduction

Since 1990s, the direct electrochemistry of redox proteins in thin films modified on electrode surface has aroused increasing interest among researchers for their theoretical significance and perspective application in electrochemical biosensors and bioreactors [1], [2], [3], [4]. The thin films may provide a suitable microenvironment for the proteins and enhance the direct electron transfer between the proteins and underlying electrodes [5], [6]. Recently, layer-by-layer assembly based on alternate adsorption of oppositely charged polyions from their solutions has also been used to build up ultrathin protein films [7], [8]. One of the advantages of layer-by-layer assembly over the cast method is the precise control of film composition and thickness at molecular level or in nanometer size. The direct electrochemistry of proteins in layer-by-layer films has also been studied. For example, Lvov et al. [9] constructed layer-by-layer films of myoglobin (Mb) or cytochrome P450_cam (Cyt P450) with oppositely charged DNA or poly(styrenesulfonate) (PSS) on gold electrodes. Reversible cyclic voltammograms of Mb and Cyt P450 in these films were achieved. We reported the assembly of layer-by-layer films of heme proteins with various polyions or nanoparticles on pyrolytic graphite (PG) electrodes [10], [11], [12], [13], [14], [15], [16]. Direct and reversible voltammograms of the proteins in these films were observed and used to electrocatalyze various substrates. The protein layer-by-layer films assembled on planar solid supports have also been extended to curved surfaces of submicrometer-sized particles or nanometer-sized colloids [17], [18], [19], forming “core–shell” clusters where the small particles are “cores” and the multibilayer {protein/polyion}_n films assembled on the core surface are “shells”. In our laboratory, the core–shell clusters with heme protein shells assembled on polystyrene latex or silica nanoparticle cores were further cast or assembled layer by layer on PG electrodes [20], [21], [22], and the direct electrochemistry of the proteins in these films was realized. The electrochemical and electrocatalytic activity of the proteins in these core–shell cluster films could be tailored by controlling the number of bilayers of the shells.

Clays are stable aluminosilicates with high cation-exchange capacity, and exfoliated clay particles have a platelet shape with nanoscopic size [23]. Compared with organic polyelectrolytes, clay has the advantages of high chemical stability, good adsorption property due to its appreciable surface area, special structural feature, and unusual intercalation property. The interaction of proteins with clay has been studied extensively, and the direct electrochemistry of redox proteins such as hemoglobin (Hb), cytochrome c (Cyt c), horseradish peroxidase (HRP), and Mb in clay-related films has also been reported [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. For example, our group has studied the electrochemical and electrocatalytic properties of heme proteins in cast protein–clay films and layer-by-layer {clay/proteins}_n films modified on PG electrodes [32], [33], [34].

In the present work, the strategy of construction of the protein core–shell nanocluster films was extended to clay nanoparticles, and this new kind of clay films was used to immobilize Hb and realize its direct electrochemistry. Oppositely charged hemoglobin (Hb) and PSS were first assembled layer by layer on the surface of clay nanoparticles, forming core–shell clay–(Hb/PSS)_n nanoclusters. The nanoclusters were then cast on PG electrodes, forming clay–(Hb/PSS)_n films. The construction of the clay–(Hb/PSS)_n nanoclusters and the clay–(Hb/PSS)_n films were characterized by UV–VIS spectroscopy, cyclic and square wave voltammetry, X-ray diffraction (XRD), scanning electron microscopy (SEM), and reflection absorption infrared spectroscopy (RAIR). The feasibility of the protein films in electrochemical catalysis toward various substrates of biological or environmental significance was also studied. To the best of our knowledge, while the direct electrochemistry of Hb in different types of clay films was reported previously, the direct electrochemistry of Hb in this new type of core–shell nanocluster films concerning clay nanoparticles has not been studied until now. We expected that Hb in clay–(Hb/PSS)_n films would demonstrate some unique and better properties in voltammetry and electrocatalysis than in other Hb-containing clay films.