Mineralizing gold-silver bimetals into hemin-melamine matrix: A nanocomposite nanozyme for visual colorimetric analysis of H₂O₂ and glucose

Highlights

• A rod-like nanocomposite nanozyme was fabricated by mineralizing Au–Ag bimetals into Hemin-melamine matrix.

• The mineralized Au–Ag bimetals could act as “nanowires” to promote the electron transferring within nanocomposites.

• The nanozyme with enhanced peroxidase-like catalysis was coupled with GOD towards a complex enzyme.

• High adsorption capacity could be expected for the substrates and targeting analytes.

• The catalysis-based colorimetric method can detect glucose with the level down to 1.8 μM.

Abstract

A nanocomposite nanozyme has been fabricated through mineralizing gold-silver bimetals into Hemin (Hem)-coupled melamine (MA) polymer matrix for visual colorimetric analysis of H₂O₂ and glucose. Catalytic Hem was cross-linked onto MA scaffold for the mineralization of Au–Ag bimetals yielding the rod-like nanocomposite of MA-Hem/Au–Ag. It was discovered that the resulting nanocomposite could present high aqueous stability and especially improved catalysis, which was more than four-fold higher than that of native Hem. Catalytic kinetics studies indicate that the prepared nanocomposite nanozyme could present much higher affinities to the substrates than those of native Hem or even horseradish peroxidase. Herein, the so mineralized Au–Ag bimetals with the “silver effect” would act as “nanowires” for promoting the electron transferring of nanocomposite nanozyme. Moreover, the Hem-coupled MA polymer matrix with high specific surface area could ensure the high adsorption capacity for the reactant substrates and targeting analytes. The application feasibility of the developed nanocomposite nanozyme was demonstrated subsequently by the colorimetric assays for H₂O₂ and glucose separately in milk and blood samples, with the linear ranges of 0.010–2.50 mM and 0.0050–2.0 mM, respectively. Such a bimetal mineralization-based fabrication route may open a new door toward the design of diverse nanocomposites nanozymes with improved catalysis and adsorption performances.

Introduction

Due to the high catalysis efficiencies and specificities, natural enzymes such as horseradish peroxidase (HRP) and glucose oxidase (GOD), have been widely applied in the catalysis, food, biomedicine, and environment fields [1]. Nevertheless, they may suffer from some inherent drawbacks, such as cost-ineffectiveness, storage instability and environmentally-affected catalysis [2]. Alternatively, many researchers have been focused on the fabrications of various artificial enzymes as more stable and low-cost alternatives to the natural ones by using porphyrins, supramolecules, biomolecules, and metal complexes to mimic natural enzymes [[3], [4], [5], [6]]. As a representative, hemin (Hem), the heme-redox active sites of catalytic proteins such as HRP and hemoglobin, has been widely applied in catalysis field, but showing some disadvantages like low catalysis and poor aqueous solubility [[7], [8], [9], [10], [11]]. Also, increasing efforts have been devoted to the development of catalytic nanomaterials, known as nanozymes, such as Fe₃O₄ nanoparticles (NPs), carbon nanotubes, graphenes and ultra-small noble metals (Pt, Au and Ag) [6,[12], [13], [14], [15], [16], [17], [18], [19], [20]]. In particular, Au NPs have been utilized to label or anchor some enzymes (i.e., HRP) or catalytic derivatives to accelerate the electron transferring toward the improved catalysis [9,[21], [22], [23]]. Moreover, recent decades have witnessed the rapid development of nanocomposites-based nanozymes that are formed by noble metals or transition metal oxides showing enhanced peroxidase-like activities [14,[24], [25], [26], [27], [28], [29], [30]]. For example, Saeed Y and coworkers designed a nanocomposite nanozyme of C-dots/Fe₃O₄ for the determination of H₂O₂ in nanomolar levels [26]. Huang et al. have fabricated a graphene oxide-Se nanocomposite with glutathione peroxidase-like catalysis for cytoprotection [27]. Liu’ group reported a nanocomposite nanozyme consisting of cobalt oxide and carbon for the colorimetric detection of glucose [28]. However, most of the nanocomposites-based nanozymes may suffer from a formidable limitation regarding the poor integration of different components or low environmental stability, which may greatly prevent them from being used on a large scale.

It is well recognized that with the synergetic effects, the integration of Au and Ag metals towards bimetallic Au/Ag NPs can achieve better electronic, optical and catalytic performances over the monometallic ones [17,[31], [32], [33], [34]]. For example, Shi et al. discovered that a “silver effect” could be obtained for the bimetallic Au/Ag NPs presenting much higher catalysis than Au NPs alone [32]. Our group also established that bimetallic Au–Ag nanoclusters could display a “silver effect”-enhanced red fluorescence [17]. In addition, noble metals like silver could conduct the strong interaction with melamine (MA), a nitrogen-rich polymer molecule for forming nanocomposites [35], to yield the diverse functional nanocomposites [[36], [37], [38]]. For instance, Li's group synthesized the hierarchical silver nanochains by the Ag-MA self-assembly [36].

Inspired by the pioneering works above, in the present work, catalytic Hem was first covalently attached onto MA by the cross-linking chemistry to obtain the Hem-coupled MA polymer matrix. The in-site encapsulation of bimetallic Au–Ag was then conducted by the minerization route to yield the nanocomposite nanozyme. It was discovered that the resulting MA-Hem/Au–Ag nanocomposite could present the robust environmental stability and especially strong catalytic activities, which was more than four-fold higher than that of native Hem. Herein, the mineralized Au–Ag bimetals were thought to act as the “nanowires” for promoting the electron transferring of Hem-containing nanocomposites with the improved catalysis, in which the “silver effect” could be expected for Au NPs at a vital Au-to-Ag molar ratio (i.e., 5/2). Moreover, the catalysis performances of the MA-Hem/Au–Ag nanozyme were studied by catalyzing chromogenic reactions of 3, 3′, 5, 5′-tetramethyl benzidine (TMB) and H₂O₂. Also, steady-state kinetic studies were carried out to explore the catalysis and substrate affinities of MA-Hem/Au–Ag, of which the calculated parameters were compared with those of Hem and HRP. Subsequently, the feasibility of the developed catalysis-based colorimetric strategy for probing H₂O₂ and glucose was demonstrated with high sensitivity. To the best of our knowledge, this is the first report on the fabrication of nanocomposite nanozyme by the bimetallic mineralization into catalytic Hem-binding polymer matrix, showing greatly improved catalysis and adsorption capacity for the colorimetric assays of H₂O₂ and glucose.