A novel smartphone-based colorimetric biosensor for reliable quantification of hydrogen peroxide by enzyme-inorganic hybrid nanoflowers
Graphical abstract
A novel smartphone-based colorimetric biosensor based on enzyme-inorganic hybrid nanoflowers was applied for ultrasensitive determination of hydrogen peroxide.
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
References (33)
- et al.
Dual-emission ratiometric probe combining carbon dots and CdTe quantum dots for fluorometric and visual determination of H2O2
Sens. Actuators B Chem.
(2019) - et al.
Enzyme-like catalysis of polyoxometalates for chemiluminescence: application in ultrasensitive detection of H2O2 and blood glucose
Talanta
(2019) - et al.
Tuning polyelectrolyte-graphene interaction for enhanced electrochemical nonenzymatic hydrogen peroxide sensing
Anal. Chim. Acta
(2019) - et al.
TMB-assembly as nanosubstrate construction colorimetric kit for highly sensitive and selective detection of H2O2 and monoamine oxidase-a based on Fenton reaction
Microchem. J.
(2019) - et al.
Real-time in vitro detection of cellular H2O2 under camptothecin stress using horseradish peroxidase, ionic liquid, and carbon nanotube-modified carbon fiber ultramicroelectrode
Sens. Actuators B Chem.
(2017) - et al.
Covalent immobilization of cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive amperometric detection of cholesterol in real samples
Biosens. Bioelectron.
(2011) - et al.
Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties
Enzyme Microb. Technol.
(2016) - et al.
Synthesis of catalase-inorganic hybrid nanoflowers via sonication for colorimetric detection of hydrogen peroxide
Enzyme Microb. Technol.
(2019) - et al.
Self-assembled organic-inorganic hybrid glucoamylase nanoflowers with enhanced activity and stability
Int. J. Biol. Macromol.
(2016) - et al.
A hierarchical assembly of flower-like hybrid Turkish black radish peroxidase-Cu2+ nanobiocatalyst and its effective use in dye decolorization
Chemosphere
(2017)