Highly sensitive chemiluminescent sensing of intracellular Al3+ based on the phosphatase mimetic activity of cerium oxide nanoparticles

https://doi.org/10.1016/j.bios.2020.112027Get rights and content

Highlights

  • The catalytic dephosphorylation of chemiluminescent substrate CDP-star by nanoceria was demonstrated.

  • The nanoceria possessed considerable phosphatase mimetic activity at neutral pH.

  • The chemiluminescent sensing of Al3+ was achieved based on the inhibition of the phosphatase mimetic activity of nanoceria.

  • The chemiluminescent sensing of Al3+ in living cells was demonstrated.

Abstract

Nanomaterials with enzyme-like characteristics (also called nanozymes) have attracted increasing attention in the area of analytical chemistry. Nevertheless, most of the nanozymes used for analytical applications are oxidoreductase mimics, and their enzyme-like activities are usually demonstrated by using chromogenic and/or fluorogenic substrates. Herein, the phosphatase mimetic activity of cerium oxide nanoparticles (nanoceria) was investigated by using CDP-star as the chemiluminescent (CL) substrate. Interestingly, we found that the phosphatase mimetic activity of nanoceria can be remarkably inhibited by the addition of Al3+. Based on this finding, a highly sensitive and selective CL method for Al3+ detection is proposed. The CL intensity of the nanoceria/CDP-star system decreased with the increasing Al3+ concentrations in the range from 30 nM to 3.5 μM. A detection limit as low as 10 nM was obtained. Finally, the CL detection of intracellular Al3+ was achieved, demonstrating the utility of the CL method in complex biological samples.

Introduction

Aluminium (Al) is one of the most abundant metallic element in the earth and is widespreadly existed in daily life and industries (Singh et al., 2017). The Al-based containers and packaging materials are frequently used, which may result in the increase of Al3+ levels in food and drinking water. Nevertheless, the high concentration of Al3+ in organism can cause severe diseases such as Alzheimer's disease, Parkinson's disease, and osteoporosis (Hornung et al., 2008; Verstraeten et al., 2008). The maximum Al3+ concentration allowed in drinking water is limited to 200 μg/L (7.41 μM) by the World Health Organization. Conventional methods for Al3+ determination include atomic absorption spectroscopy and inductively-coupled plasma mass spectrometry (Frankowski et al., 2010; Sanz-Medel et al., 2002). These methods usually need complicated sample preparation process and relatively expensive instruments. In recent years, fluorescent method has attracted increasing attention for Al3+ detection by using organic dyes as fluorescent probes (Boonkitpatarakul et al., 2016; Gui et al., 2015; Han et al., 2012; Samanta et al., 2014; Wang et al., 2016; Xu et al., 2016). The fluorescent methods showed good sensitivity and selectivity, but most of them only worked well in organic solvents or organic-water mixed solvents. Thus, the development of new method for the sensitive detection of Al3+ in aqueous solutions is still highly desired.

Chemiluminecence (CL) is the light-emission phenomenon initiated by chemical reactions (Siraj et al., 2016; Saqib et al., 2018). It is known as a powerful analytical technology with inherent features such as high sensitivity, wide linear range, and does not require an additional light source (Lan et al., 2019; Gao et al., 2017). Commonly used CL luminophores include luminol, lucigenin, peroxyoxalate esters, and acridinium esters derivatives, etc (Kong et al., 2017; Zangheri et al., 2019). These CL compounds produce light through oxidation-dependent mechanisms, and their CL emissions are usually activated by the addition of strong oxidizing agents such as H2O2 (Saqib et al., 2017; Gao et al., 2015). This may result in poor reproducibility and selectivity in bio-analysis as H2O2 is unstable and can react with many metal ions and biological redox active molecules. Phenoxy 1,2-dioxetane compounds are one kind of the promising CL luminophores that do not need the assistance of oxidizing agents (Gnaim et al., 2018, 2019). In commercial enzyme-linked immunoassays, the light emission of phenoxy 1,2-dioxetane luminophores is usually activated by the enzyme catalyzed deprotection of phenol-protecting group. Nevertheless, natural enzymes are expensive and susceptible to environmental conditions (eg. solution pH), which limits the wide applications of enzyme catalyzed phenoxy 1,2-dioxetane CL system.

Nanomaterials with enzyme-like characteristics (also called nanozymes) have received increasing attention due to their possibility as alternatives to natural enzymes (Wei and Wang, 2013). Compared with natural enzymes, nanozymes possess several advantages such as chemical stability, low cost, and easy to large-scale preparation (Wu et al., 2019). Nanozymes have found wide applications in the areas of biomedical analysis, imaging, therapeutics, and environmental protection (Lin et al., 2014; Zhou et al., 2017). Most of these applications are limited to redox-type enzyme mimics, although natural enzymes are known to catalyze various types of reactions including redox reaction, hydrolytic reaction, ligation reaction, decomposition reaction, etc (Gao et al., 2007; Qin et al., 2018; Zhang et al., 2019). On the other hand, most developed nanozyme systems are demonstrated by their enzyme-like activities toward chromogenic and/or fluorogenic substrates. For example, the peroxidase-mimicking nanozymes are usually studied by using 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), or Amplex Red as the chromogenic or fluorogenic substrates (Hu et al., 2018; Li et al., 2018; Wang et al., 2019). There are few works focusing on the CL substrates of natural enzymes (Zhong et al., 2018).

Cerium oxide nanoparticles (nanoceria) have attracted considerable attention in the field of nanozymes due to their high catalytic activity (Asati et al., 2009; Cheng et al., 2016; Liu et al., 2016). Recently, the phosphatase mimetic activity of nanoceria has been demonstrated by using p-nitrophenyl phosphate as the chromogenic substrate or 4-methylumbelliferyl phosphate as the fluorogenic substrate (Dhall et al., 2017; Kuchma et al., 2010; Yao et al., 2019). Herein, the phosphatase mimetic activity of nanoceria toward the dephosphorylation of chemiluminescent substrate CDP-star was demonstrated and used for the detection of Al3+. CDP-star is a commercially used CL substrate of alkaline phosphatase (ALP). It is one of the promising phenoxy 1,2-dioxetane CL luminophores. As shown in Scheme 1, nanoceria possessed considerable catalytic activity toward the dephosphorylation of CDP-star, and strong CL emission was observed. In comparison, the catalytic activity of nanoceria was remarkably inhibited upon the addition of Al3+, which resulting in relatively weak CL emission. Based on this phenomenon, the highly sensitive CL detection of intracellular Al3+ was achieved, suggesting the great potential of the phosphatase nanozyme in analytical applications.

Section snippets

Reagents and instruments

CDP-star, alkaline phosphatase, 4-methylumbelliferyl phosphate disodium salt (4-MUP), and the malachite green phosphate assay kit were purchased from Sigma. Cerium(III) nitrate hexahydrate, polyethyleneimine, dextran, polyacrylic acid, ammonium hydroxide and the metal salts were obtained from Sinopharm Chemical Reagent Co., Ltd. Mito-Tracker Green and DiIC18(3) (DiI) were supplied by Beyotime Biotechnology Co., Ltd. The solutions were prepared with water purified by a Milli-Q system.

CL data

Catalytic dephosphorylation of CDP-star by nanoceria

The CL emission mechanism of CDP-star is illustrated in Fig. S1. The ALP triggered dephosphorylation of CDP-star leads to the formation of the meta-stable dioxetane phenolate anion, which decomposes and emits light at 466 nm. Previous studies show that surface modification plays an important role on the catalytic property of nanoceria. Different polymer-coated nanoceria have been used as peroxidase mimics and oxidase mimics for various applications (Zhang et al., 2016). Therefore, the

Conclusion

In conclusion, the catalytic dephosphorylation of CL substrate CDP-star by nanoceria was demonstrated and applied for the CL sensing of Al3+. The phosphatase nanozyme based CL system has several advantages. Firstly, nanoceria exhibits considerable phosphatase mimetic activity at pH 7, while ALP only works at alkaline solution which is not suitable for CL detection in physiological pH condition. Secondly, the proposed nanoceria/CDP-star CL system does not need the use of unstable H2O2 as the

CRediT authorship contribution statement

Xue Tian: Methodology, Validation, Investigation. Hong Liao: Data curation, Investigation. Min Wang: Writing - original draft, Resources. Lingyan Feng: Writing - original draft. Wensheng Fu: Writing - original draft, Project administration. Lianzhe Hu: Conceptualization, Project administration, Funding acquisition, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (NSFC, 21605012 and 21705012), the Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN201900522), and the Venture & Innovation Support Program for Chongqing Overseas Returnees.

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