Elsevier

Talanta

Volume 195, 1 April 2019, Pages 40-45
Talanta

Facile approach to prepare HSA-templated MnO2 nanosheets as oxidase mimic for colorimetric detection of glutathione

https://doi.org/10.1016/j.talanta.2018.11.024Get rights and content

Highlights

  • We developed a simple, rapid, and highly sensitive colorimetric assay for the determination of glutathione (GSH).

  • The colorimetric assay is based on human serum albumin (HSA)-templated MnO2 nanosheets as an artificial oxidase.

  • The practicality of the proposed method was demonstrated by the detection of GSH in complex matrix.

Abstract

In this work, a simple, rapid, and highly sensitive colorimetric assay for the determination of glutathione (GSH) was developed. It employs human serum albumin (HSA)-templated MnO2 nanosheets as an artificial oxidase. HSA-templated MnO2 nanosheets can oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue oxTMB product with a significant increase in absorbance at 652 nm in the absence of H2O2. When GSH is introduced, the MnO2 nanosheets are reduced to Mn2+ ions, thereby inhibiting the formation of oxTMB. Based on these findings, a simple colorimetric assay was developed for the detection GSH in the range of 10 nM to 5 μM with a low detection limit of 5.6 nM. Importantly, the proposed method was successfully used for quantitative determination of GSH in biological fluids, such as human serum samples.

Graphical abstract

A colorimetric method for facile sensing of GSH based on HSA-templated MnO2 nanosheets with oxidase-like activity.

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Introduction

Biothiols play a crucial role in pathological processes and biochemical pathways [1], [2], [3]. Glutathione (GSH), the most abundant intracellular biothiol, consists of a tripeptide of glutamic acid, cysteine, and glycine [4], [5]. GSH plays a crucial role in biological systems and is involved in many cellular functions, such as intracellular signal transduction, maintenance of intracellular redox activities, gene regulation, and xenobiotic metabolism [6], [7], [8]. Cellular levels of GSH are associated with many diseases, such as liver damage, cancer, aging, Parkinson's disease, autism in children, and HIV infection [9], [10], [11], [12], [13]. Therefore, based on its clinical and biological importance, the rapid and sensitive detection of GSH has attracted continuing interest for monitoring the levels of GSH in real samples. To date, a variety of methods have been proposed for the detection of GSH, including liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) [14], electrochemistry [15], flow injection analysis [16], high-performance liquid chromatography (HPLC) [17], and spectrofluorimetry [18], [19]. However, these methods are time-consuming and involve tedious sample preparation and utilization of sophisticated instruments, making them inconvenient for practical use in certain circumstances. Thus, it is still highly desirable to develop simpler, more rapid and sensitive methods for GSH detection.

Colorimetric analysis is popular because of the advantages of simplicity, without sophisticated instrumentation, rapidity and low cost [20], [21]. One of the most common types of colorimetric methods focuses on enzymatic chromogenic substrates. However, owing to susceptibility to environmental conditions and high cost in preparation and purification, natural enzymes have restricted applications. Over the past few decades, nanomaterial-based artificial enzymes have gradually become important research tools because of their excellent performance and size effect. Such nanomaterials are based on carbon [22], [23], metal oxides [24], or noble metals [25]. However, the catalytic activity of these nanomaterials is sometimes unsatisfactory, and their preparation is complicated [26]. In addition, these methods usually require additional destructive and unstable H2O2 as an oxidant.

Manganese dioxide (MnO2) is a kind of functional transition metal oxide. Because of its unique physicochemical properties and biological compatibility, MnO2 has attracted wide research interest, and it has found a variety of potential applications in cell batteries [27], catalysts [28], sensors [29], [30], magnetic resonance imaging, and drug delivery [31]. Several techniques for synthesizing MnO2 nanosheets have been reported, such as the chemical reduction of MnO4 and ultrasound-assisted methods. The sizes and morphologies of MnO2 nanosheets synthesized from the above methods are quite variable, depending on the conditions and methods applied. In order to find an eco-friendly technique for production of well-characterized MnO2 nanosheets, we have developed a simple and green route for the synthesis the MnO2 nanosheets with better sizes and morphologies using human serum albumin (HSA) as a biotemplate.

In this work, we describe a novel and rapid colorimetric assay for the detection of GSH based on HSA-templated MnO2 nanosheets as an oxidase-like mimic. As shown in Scheme 1, we have developed a simple and green route for the synthesis the MnO2 nanosheets by using human serum albumin (HSA) as biotemplate. The HSA-templated MnO2 nanosheets can directly catalyze oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue oxTMB product with a characteristic absorption peak located at 652 nm, implying oxidase-like activity. In the presence of GSH, MnO2 nanosheets are reduced to produce Mn2+ ions, owing to the decomposition of the MnO2 nanosheets by GSH, thereby inhibiting the oxidization of TMB. Thus, the GSH assay could be realized by a bioassay in which absorbance changes occur in a concentration-dependent manner. In addition, MnO2 nanosheets could oxidize substrates using oxygen as a green oxidant without the need for H2O2. Therefore, we herein describe a facile and fast colorimetric method based on MnO2 nanosheets for the quantitative determination of GSH. Importantly, the assay is selective for GSH and unaffected by common amino acids, proteins and or coexisting compounds in human serum.

Section snippets

Materials and apparatus

Glutathione (GSH), glucose, ascorbic acid (AA), 30% hydrogen peroxide (H2O2), human serum albumin (HSA), bovine serum albumin (BSA), manganese (II) chloride, 3,3′,5,5′-tetramethylbenzidine (TMB), sodium hydroxide, and other amino acids were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) and had an electric resistance > 18.2 MΩ.

Characterization of HSA-templated MnO2 nanosheets

In this work, the as-prepared oxidase-like activity of MnO2 nanosheets was synthesized using HSA as template. The HSA-templated MnO2 nanosheets were characterized using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and UV–vis spectroscopy. As shown in Fig. 1A, the as-prepared HSA-templated MnO2 nanosheets showed a large two-dimensional structure with average lateral dimension of nearly 50 nm. The large surface area of MnO2 nanosheets provides a correspondingly

Conclusions

In summary, we developed a rapid, selective and quantitative colorimetric method for facile sensing of GSH based on HSA-templated MnO2 nanosheets with oxidase-like activity, which were, for the first time, synthesized for direct catalysis of TMB to a blue oxTMB product. GSH was able to trigger the decomposition of MnO2 nanosheets, inhibiting the generation of oxTMB. The as-proposed colorimetric method provides a wide linear range from 0.01 to 5 μM and a low detection limit of 5.6 nM for GSH

Acknowledgements

The authors are grateful to Dr. Kathryn Williams for her critical comments during the preparation of this manuscript. This work is supported by grants awarded by the National Natural Science Foundation of China (21505122), the National Institutes of Health (GM079359 and CA133086), the NSFC grants (NSFC 21221003 and NSFC 21327009), the China Scholarship Council, the Education Department of Henan Province, and Zhengzhou University.

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