Clay/au nanoparticle composites as acetylcholinesterase carriers and modified-electrode materials: A comparative study

Highlights

• The first comparative study of four types of clay/gold nanoparticle composites

• Novel acetylcholinesterase biosensors for chlorpyrifos detection were constructed.

• Surface area, charges and hydrophobicity of composites strongly affect performances.

• Montmorillonite/gold nanoparticles was the most suitable composite for this study.

• Clay/gold nanoparticles benefit enzyme immobilization and electrode modification.

Abstract

This work presents the first comparative study on syntheses, characterization, and applications of different clay/gold nanoparticle composites (clay/AuNPs) as acetylcholinesterase (AChE) carriers and modified-electrode materials to detect chlorpyrifos, an organophosphate pesticide. The selected based-clays were plate-like kaolinite (Kaol; 1:1 aluminum phyllosilicate), globular montmorillonite (Mt; 2:1 aluminum phyllosilicate), globular bentonite (Bent; 2:1 aluminum phyllosilicate), and fibrous sepiolite (Sep; 2:1 inverted ribbons of magnesium phyllosilicate). The clay/AuNPs were synthesized using in situ chemical reduction of the gold precursor with anchoring 3-aminopropyl triethoxysilane (APTES) on the clay surfaces. The studied materials were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), nitrogen adsorption/desorption, Fourier transformed infrared (FTIR) spectroscopy, thermogravimetric analyzer (TGA), zeta-potential analysis, and electrochemical impedance spectroscopy (EIS). Surface area, surface charges, and hydrophilic/hydrophobic nature of based-clays and the clay/AuNPs played major roles on AChE loading, residual activity, and sensor storage stability. The Mt/AuNPs was determined, among the studied clays, as the best supports for AChE loading and residual activities due to its high specific surface area and weak electrostatic interaction with the immobilized enzyme. Since apparent catalytic efficiencies k_cat^app/K_M^appof the immobilized AChE in the four clay/AuNPs were found statistically similar, Mt/AuNPs which contained the highest enzyme loading was the favorite modified-electrode material. For chlorpyrifos detection, Kaol/AuNPs-based sensors resulted in the lowest detection limit, presumably due to the strong hydrophobic surface that helps to concentrate the nonpolar pesticide to the vicinity of the immobilized enzyme. Unfortunately, the highest electrostatic repulsion between the Kaol/AuNPs and AChE, and the dehydrated Kaol surface resulted in the lowest storage stability of the AChE pesticide sensors. Overall, Mt/AuNPs showed the highest potential as the AChE carrier and the modified electrode material for chlorpyrifos determination.

Introduction

Clay minerals, classified as hydrous aluminum or magnesium phyllosilicates, are resourceful materials for enzyme immobilization. Clay minerals and their modified forms have been widely proved to be excellent supports for enzyme catalyses which result in tremendous enhancement of enzyme activity and stability (An et al., 2015; Kadam et al., 2017; Li et al., 2015b; Öztürk et al., 2016; Pandey et al., 2017; Tzialla et al., 2010). This is because they offer high specific surface area, excellent biocompatibility, ion-exchangeability, high adsorption capacity as well as mechanical and chemical stability (Zhou and Keeling, 2013; Zhou et al., 2017). Furthermore, their internal and external surfaces bear different functional groups such as SiOSi, SiOOH, and Al (or Mg)OH) (An et al., 2015), thus tailored surface modification can be accomplished. Clay minerals are also abundant in nature, hence, are low-cost and attractive for commercial applications in biocatalysis and biosensors.

Nonetheless, modification of clay minerals prior to enzyme immobilization has been considered essential for improved catalytic efficiency. Organo-modified clays have been the most extensively utilized as enzyme carriers. With incorporated organic compounds, clay minerals become more hydrophobic leading to different attachments and amounts of adsorbed enzymes as well as enhancements of catalytic activity and stability (Chao et al., 2017; Öztürk et al., 2016). Moreover, stronger adsorption and holding capability for non-ionic molecules can be achieved (Abolhasani et al., 2017).

Apart from general catalysis systems, modified clay minerals have also been applied as platforms for enzyme biosensors, but to a limited extent. Again, organo-modified clays have been the most popularly utilized. However, electroconductive materials may also be combined to improve sensor sensitivity. Calixalene modified (Sonmez et al., 2014), amino acid intercalated (Demir et al., 2014), and histidine modified (Songurtekin et al., 2013) montmorillonite were used as modified electrode materials for enzyme immobilization. Examples of incorporation of electroconductive materials with the modified clay minerals include carbon nanotubes/aminopropyltriethoxysilane and carbon nanotubes/ octadecylamine modified montmorillonite for peroxidase biosensors (Oliveira et al., 2012), and gold nanoparticles/chitosan-montmorillonite/horseradish peroxidase biosensor (Zhao et al., 2008). These reports have demonstrated that clay minerals are suitable biosensing materials with enhanced enzyme immobilization capacity, and display good biosensor performances in terms of linearity and low detection limits.

Hybridization of clay minerals and gold nanoparticles (AuNPs) provides remarkable composite materials with high electrochemical conductivity, the excessive surface to volume ratio, high chemical reactivity, and chemisorption capacity of various types of molecules (Jlassi et al., 2018). Hence, clay/gold nanoparticle composites (clay/AuNPs) have shown numerous applications in various fields such as drug delivery systems (Rao et al., 2018; Zhao et al., 2019), therapeutic systems (Banerjee et al., 2018), renewable source of energy (Jlassi et al., 2018), chemical catalyses (Rocha et al., 2018), sensors (Yadav et al., 2017), etc. Unfortunately, applications of the clay/AuNPs for enzyme immobilization are very scarce. This is surprising since AuNPs are known to provide a biocompatible microenvironment to the immobilized enzymes, give freedom to the enzyme orientation and enhance catalytic activity and stabililty (Hong et al., 2019; Liu et al., 2007; Mukhopadhyay et al., 2003; Qiao et al., 2013).

Therefore, this article presents the first utilization of the clay/AuNPs for acetylcholinesterase (AChE) immobilization, and further assessment of their capability as enzyme carriers and modified-electrode materials for detection of an organophosphate pesticide chlorpyrifos. A comparative study among four different types of clay-based minerals is the focus of this work. We carefully investigated effects of physical characteristics (using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Brunauer–Emmett–Teller (BET)), chemical (Fourier transformed infrared (FTIR) spectroscopy, thermogravimetric analyzer (TGA) and zeta potential), and electrochemical (electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and amperometry) of four different types of clays and their modified forms on the clay/AuNPs syntheses, and applications in enzyme immobilization and biosensors. The selected clay minerals were plate-like kaolinite (Kaol; 1:1 aluminum phyllosilicate), globular montmorillonite (Mt; 2:1 aluminum phyllosilicate), globular bentonite (Bent; 2:1 aluminum phyllosilicate), and fibrous sepiolite (Sep; 2:1 inverted ribbons of magnesium phyllosilicate). The synthesized clay/AuNPs were then tested as carriers for AChE, a model enzyme. Loadings, activities, and kinetics of the immobilized AChE were assessed by mass balance and hydrolysis of acetylthiocholine (ATCh). The immobilized AChE was implemented for the determination of chlorpyrifos. To the best of our knowledge, there have been no reports on a comparative study of different clay/AuNPs for enzyme immobilization. Therefore, this work will be fruitful for the development of low-cost and easy-to-synthesize clay/AuNPs as platforms for enzyme catalysis and biosensing.