A novel smartphone-based colorimetric biosensor for reliable quantification of hydrogen peroxide by enzyme-inorganic hybrid nanoflowers

https://doi.org/10.1016/j.bej.2021.107925Get rights and content

Highlights

  • Smartphone-based colorimetric detection of H2O2 by HRP-Cu3(PO4)2⋅3H2O HNFs.

  • HRP-Cu3(PO4)2⋅3H2O HNFs were facilely fabricated by self-assembly.

  • The proposed colorimetric assay showed wide H2O2 detection range (5–500 μM).

  • HRP-Cu3(PO4)2⋅3H2O HNFs had desirable storage stability and reusability.

  • The colorimetric system can realize H2O2 detection in tap water sample.

Abstract

A novel smartphone-based colorimetric biosensor based on enzyme-inorganic hybrid nanoflowers was applied for ultrasensitive determination of of hydrogen peroxide (H2O2). Horseradish peroxidase-inorganic hybrid nanoflowers (HRP-Cu3(PO4)2⋅3H2O HNFs) were facilely synthesized by a one-pot incubation using HRP and Cu3(PO4)2⋅3H2O as organic and inorganic components, respectively. Characterization results revealed HRP molecules were well immobilized in HRP-Cu3(PO4)2⋅3H2O HNFs via self-assembly. The results of smartphone-based colorimetric assay successfully demonstrated the good sensing performance of HRP-Cu3(PO4)2⋅3H2O HNFs. The linear detection range of H2O2 was from 5 to 500 μM, and the detection limit was 0.5 μM. Further, the proposed assay showed good anti-interference ability and high sensitivity for H2O2 detection owing to the high specificity of HRP and excellent accuracy of color parameters obtained by Color Detector software, respectively. In addition, HRP-Cu3(PO4)2⋅3H2O HNFs had desirable storage stability and reusability, and this colorimetric system can realize the H2O2 detection in tap water, which indicated the good applicability of HRP-Cu3(PO4)2⋅3H2O HNFs in real sample.

Graphical abstract

A novel smartphone-based colorimetric biosensor based on enzyme-inorganic hybrid nanoflowers was applied for ultrasensitive determination of hydrogen peroxide.

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Introduction

The facile, accurate and rapid determination of hydrogen peroxide (H2O2) has practical significance in various fields (e.g., food, textile, environment, diagnostics and minerals) [1]. In addition, H2O2 particitates with several biological activities and is the by-product of oxidases. Therefore, several analytical techniques including fluorescence, chemiluminescence, electrochemiluminescence, chromatography, electrochemistry and spectrophotometry have been employed to quantify H2O2 [[2], [3], [4], [5], [6]]. Among above detection strategies, colorimetric detection possesses merits of simple analysis and usage, cost effective, easy operation and monitoring [[7], [8], [9], [10]]. However, despite above advantages, its application to H2O2 detection has been limited by interferences from other matrix components. Therefore, the synthesis of new materials for the selective detection of H2O2 is a critical priority. Meanwhile, traditional colorimetric detection based on spectrophotometer can not realize on-site instant detection, so the development of portable and compact testing device is important for H2O2 detection on some special situations.

Although non-enzyme catalysts show good activity for H2O2 detection, the selectivity for H2O2 detection is relatively poor. Compared with non-enzyme catalysts, the naturally evolved enzymes as green biocatalysts have unparalled advantages of high catalytic activity at mild pH and ambient temperature, good substrate specificity and enantioselectivity [11]. However, the practical applications of free enzymes are still challenging due to their inherent disadvantages including difficult separation and recovery, poor operational stability and product contamination [12]. To compensate above shortcomings of free enzymes, enzyme immobilization techniques including physisorption, cross-linking, covalent binding and entrapment are developed [[13], [14], [15], [16]]. Nevertheless, the activity and stability of immobilized enzymes can not be simultaneously improved via above traditional methods. Therefore, seeking for an optimal enzyme immobilization method has always been the goal pursued by researchers.

Since the first report of enzyme-inorganic hybrid nanoflowers by Ge et al., this new kind of material has gained increasing attention due to their enhanced enzymatic activity, better stability, desirable reusability and durability compared to free enzymes [[17], [18], [19], [20]]. Moreover, our previous study has demonstrated enzyme-inorganic hybrid nanoflowers can be used to detect H2O2 [21]. Here in this work, horseradish peroxidase-inorganic hybrid nanoflowers (HRP-Cu3(PO4)2⋅3H2O HNFs) with a smartphone as a colorimetric biosensor were applied for fast, accurate and real-time detection of H2O2. HRP-Cu3(PO4)2⋅3H2O HNFs can be facilely fabricated via room temperature incubation without any harsh conditions. Further, smartphone as analytical device can not only decrease detection cost but also achieve in-field and on-demand analysis, which is in accordance with the future development direction of analytical science. A series of detection results demonstrated this easy-to-operate colorimetric platform showed high sensitivity and accuracy, good selectivity, desirable storage stability and reusability for H2O2 detection.

Section snippets

Chemicals

Copper sulfate (CuSO4), ferric sulfate (Fe2(SO4)3), sodium dihydrogen phosphate dihydrate (NaH2PO4⋅2H2O), disodium hydrogen phosphate dodecahydrate (Na2HPO4⋅12H2O), sodium acetate trihydrate (NaAc⋅3H2O), acetic acid (HAc), sodium chloride (NaCl), 3,3’,5,5’-tetramethylbenzidine (TMB), ethanol (EA), acetone (CP), hypochloric acid (HClO), glucose (Glu), ascorbic acid (AA) and H2O2 were purchased from Sinopharm Chemical Reagent Co., Ltd. HRP (>300 units mg−1) was purchased from Shanghai Aladdin

Materials characterization

Previous studies have investigated the formation of enzyme-inorganic hybrid nanoflowers [[22], [23], [24]]. Similarly, here the formation of HRP-Cu3(PO4)2⋅3H2O HNFs can be divided into three stages, as shown in Fig. 1. At early nucleation stage, copper ions coordinate with amide groups of HRP, which provide locations for the nucleation of primary Cu3(PO4)2⋅3H2O crystals. At the second growth stage, primary HRP-Cu3(PO4)2⋅3H2O crystals combine into large agglomerates, and the kinetically

Conclusions

HRP-Cu3(PO4)2⋅3H2O HNFs were fabricated via room temperature incubation using HRP and Cu3(PO4)2⋅3H2O as organic and inorganic components, respectively. EDS mapping and FT-IR demonstrated HRP can direct the growth of Cu3(PO4)2⋅3H2O crystals to form the nanoflowers via self-assembly rather than covalent bonding. Smartphone-based colorimetric assay based on HRP-Cu3(PO4)2⋅3H2O HNFs showed good selectivity and high accuracy for H2O2 detection due to the specificity of HRP and accuracy of color

CRediT authorship contribution statement

Miaorong Zhang: Conceptualization, Methodology, Funding acquisition, Writing - original draft, Writing - review & editing. Yan Zhang: Writing - original draft, Writing - review & editing. Chuankai Yang: Investigation, Methodology. Chunyun Ma: Investigation, Methodology. Jianguo Tang: Supervision.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This work was supported by (1) Scientific Research Starting Foundation of Qingdao University, Qingdao Postdoctoral Applied Research Project and China Postdoctoral Science Foundation (2019M662298; 2019M652325); (2) National Scientific Foundation of China (51473082); (3) State Key Project of International Cooperation Research (2017YFE0108300, 2016YFE0110800); (4) Shandong Double-Hundred Project (2018); (5) National Plan for Introducing Talents of Discipline to Universities (“111” plan); (6) The 1

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