MoOx quantum dots with peroxidase-like activity on microfluidic paperbased analytical device for rapid colorimetric detection of H2O2 released from PC12 cells
Meng-Meng Liua, Shan-Hong Lia, Dan-Dan Huanga, Zhi-Wei Xua, Ya-Wei Wub, Yun Leia,*,
Ai-Lin Liua,*
a Department of Pharmaceutical Analysis, Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Faculty of Pharmacy, Fujian Medical University, Fuzhou, 350122, China
b Key Laboratory of Optoelectronic Materials Chemistry and Physics and Key Laboratory of Design and Assembly of Functional Nanostructures, Fujian Institute of Research on the Structure of Matter Chinese Academy of Sciences, Fuzhou, 350002 China
A R T I C L E I N F O A B S T R A C T
Keywords:
MoOx quantum dots
Peroxidase-like activity
Microfluidic paper-based analytical device
H2O2
PC12 cells Diagnostic assays made of microfluidic paper-based analytical devices have become the focus of considerable attention in developing countries because of simplicity, low cost, user friendliness, and no requirement for complex equipment, which is suitable to point-of-care testing. Considering the intrinsic shortcomings of nature enzyme, synthesis of artificial peroxidase mimics possessing improved catalytic activities relative to native enzymes is of considerable interest for the development of highly sensitive and stable sensor. In this paper, water-soluble molybdenum oxide quantum dots (MoOx QDs) were developed as highly effective biomimetic catalysts which were prepared by a facile ultrasonic-assisted hydrothermal method. The resulted MoOx QDs displayed peroxidase-like activity with high sensitivity and specificity, and were constructed for efficient colorimetric quantitative detection of H2O2 based on microfluidic paper-based device. As low as 0.175 μmol/L H2O2 could be determined at a linear range from 1 to 20 μmol/L. Moreover, this biosensing device was successfully applied for visual detection of H2O2 released from PC12 cells with the advantages of low cost, rapid response and portability, indicating that the designed sensor of H2O2 has great potential in biotechnology and clinical diagnosis.
1. Introduction
Reactive oxygen species (ROS) are the major cellular intermediates produced as by-products of numerous enzymatic reactions in various cellar compartments, and play a well-established role in mediating both physiological and pathophysiological signal transduction [1,2]. Moreover, these versatile functions of ROS are greatly dependent on its amount, duration and localization [3]. It has been proved that excessive accumulation of ROS probably contributes to the progression of cancer and neurodegenerative diseases for oxidative damage to proteins, DNA and lipids, despite having beneficial effects by regulating intracellular signaling and homeostasis at lower levels [4,5]. This abnormal biochemical alteration in the disease sites has inspired researchers to understand the role of ROS signaling in the regulation of metabolic activity. Hydrogen peroxide (H2O2), as the most stable and representative ROS, is gaining increasing recognition as a rapid neuromodulatory signaling molecule. Once diffused out of the cell membrane, it will influence cell migration, immunity generation and cellular communications [6,7]. Therefore, the selective and quantitative detection of H2O2 content in neuron-like cells is of great significance in our pursuit of novel therapies to treat some central nervous system diseases.
⁎ Corresponding authors.
E-mail addresses: lypiglet@163.com (Y. Lei), ailinliu@fjmu.edu.cn (A.-L. Liu).
https://doi.org/10.1016/j.snb.2019.127512
Received 16 August 2019; Received in revised form 18 November 2019; Accepted 30 November 2019 Available online 30 November 2019
0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Various methods and strategies have been reported for the measurement of H2O2, including liquid chromatography [8], fluorescence [9], chemiluminescence [10], electrochemical methods [11]. However, these methods suffer from some drawbacks such as cumbersome operating procedures, long-term analysis and expensive instruments involved. In response to the need of rapid, simple and sensitive detection techniques, colorimetric method is highly developed and favored because of its simplicity, fast response for analysis as well as low cost. In addition, visual detection could be directly realized by the color change instead of using expensive readout instruments [12]. More importantly, when combined with the advantages of microfluidic paper-based analytical devices (μPADs) which has been a burgeoning research field due to the portability, easy operation, low sample/regent consumption [13–15], it would make it possible to perform high-throughput sampleto-answer tests in a cost-effective way. The most significant contribution of such a proper integration lies in their ability to complete multiplexed analyses at the point-of-care testing (POCT) [16,17].
Although there are many H2O2 chromogenic agents, for the rapid and precise detection of H2O2, natural enzymes are well-known to be used for colorimetric detection by catalyzing the oxidation of chromogenic substrate to produce the colored product with specific high activity under eco-friendly and mild conditions [18]. Nevertheless, the inherent instability will lead to inactivation, not to mention the expensive preparation and complex purification, which greatly hampers their practical applications [19]. Nanostructured materials have drawn considerable attention in biomedical and biosensing field after long being misunderstood to be biologically and chemically inert [12]. Since Fe3O4 nanoparticles (NPs) were firstly found to harbor the peroxidase activity and were used for detection of H2O2 [20], inorganic sensors have opened a new avenue for colorimetric detection owing to their remarkable superiorities over traditional natural enzymes for high stability and reproducibility, higher binding affinity for the substrate and even better catalytic activities under harsh conditions [21]. Up to date, increasing effort has been concentrated to the synthesis of peroxidase mimics and numerous nanomaterials including metallic nanomaterials (e.g. Au [22]), Au-Ag bimetallic alloy nanostructure [23], and FePt-Au ternary metallic nanoparticles [18], metal oxide (e.g. Co3O4 nanoparticles [24], CuO nanoclusters [25] and CeO2 nanocomposites [26], carbon nanotubes and graphene quantum dots [27] have been explored as peroxidase mimics.
Molybdenum oxide (MoOx) nanomaterial, as an n-type semiconductor, has demonstrated various applications including catalysis [28], gas sensors [29], excellent field emitters [30], electrochemical capacitor [31], reversible lithium-ion batteries [32]. Despite that molybdenum is regarded as key component of enzymes in biological systems [33], biocatalytic behavior of MoOx is still in its infancy to be explored as a biological detection platform [34]. Encouragingly, Ruben Ragg and coworkers have reported that surface functionalized MoO3 nanoparticles exhibited an intrinsic biomimetic sulfite oxidase (SuOx) activity [35]. The fascinating electronic properties of MoO3 originating from the changeable oxidation states (Mo6+↔Mo5+) allow MoOx to be a good promoter of free radical reactions [36], which may facilitate electron transfer between the substrate and H2O2. In addition to the good biocompatibility [37] and low cytotoxicity of MoOx [38], which are the prerequisites of natural peroxidases, it can be safely concluded that MoOx nanomaterial holds great promise to be good candidate for robust peroxidase mimics.
Herein, an ultrasonic-assisted hydrothermal method was developed for synthesis of water-soluable MoOx quantum dots (MoOx QDs) using commercial molybdenum disulfide (MoS2) as precursor. The obtained MoOx QDs showed efficient peroxidase-like activity with high sensitivity and specificity, which was proved by the ability to catalyze the oxidation of Diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) to give a color change. As shown in Scheme 1, this work provided a truly low-cost, easy-to-operate μPAD for visual detection of cellular H2O2, which held potential utility to cellular biology and pathophysiology.
2. Experimental
2.1. Materials
MoS2 powder, ascorbic acid (AA), dopamine (DA), uric acid (UA) and Whatman paper were purchased from Sigma-Aldrich (Shanghai, China). Sodium hydroxide (NaOH), sodium dihydrogen phosphate (NaH2PO4·2H2O), dibasic sodium phosphate (Na2HPO4·12H2O), barium chloride (BaCl2), 30 wt % hydrogen peroxide (H2O2), phorbol ester (PMA) were supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Glucose was purchased from Sangon Biotech Co. Ltd. (Shanghai, China). ABTS was purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). The rat adrenal pheochromocytoma (PC12) cell line was obtained from China Center for Type Culture Collection (Shanghai, China), and the green fluorescent protein labelled PC12 cells (GFP-PC12) was obtained from Hanheng Biotechnology (Shanghai) Co., Ltd (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin (10,000 units/mL penicillin and 10,000 μg/mL streptomycin) and 0.25 % trypsin containing 0.53 mmol/L sodium ethylenediaminetetraacetate (Na2EDTA) were purchased from Gibco (New York, USA). Phosphate buffer solution was purchased from Hyclone (Utah, USA).
2.2. Apparatus
Transmission electron microscope (TEM, Tecnai G2 F20) equipped with an energy dispersive X-ray spectroscope (EDS) was employed to investigate the morphology of the prepared sample and the statistic analysis of the size distribution was carried out on Nano Measure software. X-ray photoelectron spectroscopy (XPS) measurements were performed on ESCALAB 250XI (Thermo, USA) and standard C 1s peak was used as a reference for correcting the shifts. UV–vis absorption (UV/Vis) spectra of the MoOx QDs solution were obtained by UV-2450 spectrophotometer (Shimadzu, Japan). Photoluminescence (PL) spectra were recorded on Cary Eclipse spectrophotometer (Agilent, France). Fourier transform infrared (FTIR) spectra were carried out on a Vertex 70 spectrophotometer (Bruker, Germany).
2.3. Synthesis of MoOx QDs
The MoOx QDs were synthesized using commercial MoS2 powder as the precursor. In brief, 30.0 mg of black MoS2 powder was added into 200 mL of deionized water at 65 °C. After ultrasonication for 17 h, the obtained solution was subjected to suction filtration with micropore filter membrane of 0.22 μm in diameter. The resultant was collected and resolved in 30 mL of deionized water, and then the solution was transferred to a 50 mL Teflon autoclave that was placed in an oven and kept at 200 °C for 12 h. After cooling to room temperature, the water soluble MoOx QDs were obtained by collecting the filtrate.
2.4. Patterned paper-based device
The patterns were designed with Adobe Illustrator CS6 software and printed on a filter paper with the Xerox ColorQube 8870 wax printer. Once printed, the paper substrate was placed in an oven at a temperature about 90 °C for 30 min. The molten wax spread through the entire thickness of the paper by capillary action to form the hydrophobic wall, allowing the reaction reagents to flow along the boundaries. In our design, the microfluidic paper-based device was the same size as A4 paper, containing 6 mm circular detection zones which formed a 21 × 29 mm rectangular array on the surface of filter paper at regular interval of 4 mm. The patterned paper-based device was stored at room temperature and cut to the size you want before experiment.
2.5. Paper-based device for H2O2 detection with MoOx QDs
Scheme 1. Schematic illustration of colorimetric technology on paper-based microfluidic chips for detecting H2O2 released from PC12 cells.
In this work, MoOx QDs was used as peroxidase mimic and ABTS as chromogenic substrate. Typically, 10 μL of PBS (20 mmol/L, pH 6.0), 4 μL of ABTS (1 mmol/L), 4 μL of H2O2 with different concentrations and 2 μL of MoOx QDs solution were subsequently added into each detection zone. Then, the paper-based device was incubated for 10 min at 37 °C and the color of the detection zone turned from colorless to blue. To obtain the quantitative results, the images were collected by scanner. The gray intensity of the colored spot was measured using ImageJ software. The relative gray intensity (ΔG) was calculated as the difference between the gray value of the sample group (G) and the control group (G0). Before the experiment, nitrogen was used to remove the interference of oxygen.
2.6. Detection of H2O2 released from stimulated PC12 cells
PC12 cells were maintained in a humidity incubator (5 % CO2) at 37 °C with a complete medium DMEM consisting of 10 % FBS and 1 % penicillin-streptomycin. For H2O2 colorimetric assay, the PC12 cells in logarithmic phase were chosen as object of study. PBS (pH 7.4) containing PMA (200 ng/mL) was dropped into the PC12 cells and then incubated for 5 min at 37 °C to stimulate the H2O2 released from PC12 cells in situ. The solution with H2O2 was collected for further colorimetric assay.
3. Results and discussion
3.1. Characterization of the obtained MoOx QDs
The morphology of the as-prepared MoOx QDs was presented in Fig. 1A. As depicted in TEM images, the obtained MoOx QDs are highly uniform and monodisperse nanocrystals with the average size about 3.42 nm. The size was statistically calculated from more than 200 MoOx QDs in TEM images. In addition, the high-resolution transmission electron microscope (HRTEM) image (Fig. 1B) displayed a lattice spacing of 0.185 nm, well coincident with the (002) plane of MoOx QDs (JCPDF no.35-0609). EDS was further employed to confirm the elemental composition of MoOx QDs, and Mo and O elements were detected in Fig. S1, while S element vanished, implying the oxidation of MoS2 precursor and the formation of sulfate ions. The MoOx QDs were synthesized via split-step hydrothermal method involving the oxidation of molybdenum disulfide, along with the production of sulfate ions, which could be proved by adding BaCl2 solution into the as-synthesized product to obtain white acid-insoluble precipitate (Eqs. (2) and (3) and (4)) (Fig. S2). Thus, the oxidation reaction of MoS2 and the provement of sulfate ions formed in synthesis process of MoOx QDs might be described as the following equations [39]:
2MoS2 + 9O2 + 4H2O = 2MoO3 + H2SO4 (1)
H2SO4 + BaCl2 = BaSO4 ↓ + 2HCl (2)
Na2CO3 + BaCl2 = BaCO3 ↓ + 2NaCl (3)
BaCO3 + 2HCl = BaCl2 + CO2 ↑ + H2O (4)
Fig. 1. (A) TEM and (B) HRTEM images of MoOx QDs. Inset of A: The corresponding particle size distribution histogram.
In order to demonstrate the oxidation state of MoOx QDs, XPS were employed. The XPS spectrum of MoOx QDs (Fig. 2A) reveals the Mo 3d, Mo 3p and O 1s peaks at 232, 399 and 532 ev, respectively. Meanwhile, the deconvolution of Mo 3d peak (Fig. 2B) shows that there are two different molybdenum element valences in MoOx QDs. As shown in Fig. 2B, the Mo 3d5/2 and Mo 3d3/2 doublets at 231.48 and 234.38 eV can be ascribed to Mo5+, while those at 232.88 and 235.98 eV are the characteristic peaks of Mo6+ [37]. The proportion of Mo6+ and Mo5+ was determined to be 91.7 % and 8.3 % according to the XPS peak area of Mo 3d, respectively. The formation of Mo−O bonds was further confirmed by high-resolution spectra of O 1s (Fig. 2C) and FTIR spectrum (Fig. S3). In the typical FTIR spectrum, the three characteristic peaks of 995, 829 and 536 cm−1 are attributed to the stretching vibration of Mo-O, the doubly coordinated oxygen (Mo2-O) stretching
Fig. 2. (A) XPS spectrum of the MoOx QDs. High-resolution spectrum of Mo3d (B) peak and O1 s (C) peak of the MoOx QDs.
mode and the triply coordinated oxygen (Mo3-O) stretching mode, respectively [40]. Besides, the absorption peaks at 3443 cm-1 and 1636 cm-1 are the characteristic of the stretching vibration and bending vibration of −OH group [41], which accounts for the water solubility of MoOx QDs.
The optical properties of MoOx QD were investigated by UV/Vis absorption and PL spectra. There is an absorption band between 200–400 nm in the UV range (Fig. S4A), which originates from the charge transfer of the Mo-O band in the MoO66− octahedron [42]. It was worth mentioning that when excited with a 355 nm beam, the MoOx QDs exhibited a strong photoluminescence peak at 459 nm with a Stokes shift of 104 nm (Fig. S4B).
Fig. 3. The physical photographs (A) and gray value curves (B) of three different reaction systems. a: MoOx QDs-ABTS system; b: ABTS-H2O2 system; c: MoOx QDs-ABTS-H2O2 system.
3.2. Peroxidase-like activity of MoOx QDs on paper-based microfluidic chip
To verify the peroxidase-like activity of MoOx QDs on paper-based microfluidic chip, ABTS was used as the peroxidase substrate in the chromogenic reaction. Different chromogenic reaction systems including MoOx QDs-ABTS (a), H2O2- ABTS (b) and MoOx QDs-ABTSH2O2 (c) were evaluated. As displayed in the physical pictures (Fig. 3A), the experimental systems without H2O2 or MoOx QDs (system a and b) exhibit a negligible color change, suggesting that the MoOx QDs or H2O2 alone is not able to oxidize the ABTS substrate. In contrast, an obvious color change can be observed in system c, where both MoOx QDs and H2O2 are present at the same time, and the absolute value of gray decreased with the reaction time (Fig. 3B). A very small slope value was observed in system b indicating a slow decomposition of H2O2 in the absence of MoOx QDs. Conversely, a large slope value was obtained in system c, in the presence of MoOx QDs, which is 7.45-fold of system b, certifying the high peroxidase-like catalytic activity of MoOx QDs. It is thought that such an intrinsic peroxidase-like activity originates from their ability of facilitating electron transfer between reducing substrates and H2O2, thus accelerating the oxidization of ABTS into a colored product (ABTS·+), similar to other NPs-based sensors reported in previous literatures [18,27].
3.3. Optimization of experimental parameter
In general, the peroxidase-like catalytic activity of nanomaterialbased materials depends on pH, the concentration of oxide, peroxidase substrate and reaction time, similar to that of the natural enzyme.
Fig. 4. Effect of pH (A), ABTS concentration (B), H2O2 concentration (C) and reaction time (D) on the MoOx QDs-ABTS-H2O2 colored system. Inset of B: Effect of
ABTS concentration on the MoOx QDs-ABTS system.
Therefore, the peroxidase-like activity of the MoOx QDs is first investigated by varying the pH from 3 to 9 under otherwise identical conditions (Fig. 4A). Within a pH value of 3–6, the color intensity gradually increases with the increase of pH, and reaches strongest when the pH value is 6.0, while rapidly decreases with the further increase of pH value. Therefore, the optimal pH was 6, indicating the oxidation of ABTS occurs easily under acidic conditions which might be conductive to MoOx QDs-catalyzed decomposition of H2O2 into superoxide radicals. Additionally, we also evaluated the effect of ABTS concentration in the range of 0.2–4.0 mmol/L in the MoOx QDs-ABTS-H2O2 system and MoOx QDs-ABTS system. As shown in Fig. 4B, the relative intensity of MoOx QDs-ABTS-H2O2 system increases with the enhancement of ABTS concentration, but it levels off when ABTS concentration is over 1 mmol/L. Considering that the relative intensity in blank group (MoOx QDs-ABTS) also increases with the concentration of ABTS, the ABTS concentration was selected as 1 mmol/L (inset of Fig. 4B). Similarly, the higher the concentration of H2O2, the more obvious the color intensity is. However, the rate of growth became slow under high H2O2 concentration (shown in Fig. 4C). In order to enhance the efficiency of the catalysts as well as avoid the self-decomposition of H2O2 at high concentration, the H2O2 concentration of 1 mmol/L was adopted for the following assays. The effect of reaction time on the colored system was also investigated under the above-mentioned optimal conditions. The color intensity enhances with the increase of reaction time within 10 min, and gradually became stable after 10 min (Fig. 4D). Thus, the reaction time of the colored system in this experiment was chosen as 10 min.
3.4. Analytical performance of paper-based analytical device
To evaluate the sensitivity and the potential application of MoOx QDs-based μPAD, various concentrations of H2O2 were assayed. Based on the optimal conditions, the color intensity of H2O2 concentrations was measured in the range of 1 - 104 μmol/L. It was demonstrated that there was a logarithmic response curve between the color intensity and H2O2 concentration (ΔG = |G-G0|, G is the gray value of colored system containing different concentrations of H2O2, G0 is the gray value without H2O2), as illustrated in Fig. S5. The linear regression equation was ΔG = 14.70 lg[H2O2] + 74.10, with a correlation coefficient of 0.9909. The limit of detection (LOD) as low as 0.175 μmol/L (> 3σ) was achieved. To validating the applicability of the biosystem for H2O2 detection in complex biological samples, PBS containing PMA incubated with PC12 cells was collected to prepare a series of H2O2 concentrations. PC12 cells cultured in petri dish was imaged by inverted fluorescence microscope, displaying good state for cell adhesion and proliferation (shown in Fig. 5A and Fig. S5B). The results demonstrated that there was a good linear relationship between color intensity and H2O2 concentration range from 1 to 20 μmol/L (R2 = 0.9940) with the LOD of 0.3896 μmol/L (> 3σ) (shown in Fig. 5A). From the above analysis, the high sensitivity of MoOx QDs is superior to that of other nanomaterials with peroxidase-like activity [18]. Another advantage is that the visual variation could be observed by naked eyes, as shown in the inserted photographs (Fig. 5A and Fig. S5A) of colored products corresponding to the different concentrations of H2O2.
To examine the specificity of the colored system in this work, the influence of other analytes including ascorbic acid, urea, dopamine and glucose, which may co-exist with H2O2, was investigated. Noticeably, the color intensity in control group (PMA) was negligibly weak, and only H2O2 could lead to intense color intensity (Fig. 5B). No such remarkable changes were seen upon addition of the interfering substances, indicating that this biosensing system is highly selective for H2O2.
3.5. Colorimetric detection of H2O2 released from PC12 cells
The approach was applied to the measurement of H2O2 released from PC12 cells stimulated by PMA. According to the linear relationship between color intensity and H2O2 concentration, it was calculated that each PC12 cell can release 259.16 ± 8.6 amol H2O2, which is close to the value of 105.3 ± 3.3 amol reported in literature [43]. Meanwhile,
Fig. 5. (A) Linear relationship between color intensity and different concentrations of H2O2 in PBS incubated with PC12 cells. Concentrations: 1, 4, 8, 12, 16 and 20 μmol/L. Inset of A: GFP-PC12 cells photographed by inverted fluorescence microscope with magnification 100 times. (B) The selectivity of the MoOx QDs-ABTS-H2O2 colored system on paper substrate. Analysts: 0.5 mmol/L H2O2, 1 mmol/L AA, 1 mg/mL PMA, 1 mmol/L DA, 1 mmol/L UA and 1 mmol/L Glucose.
the analytical accuracy and reliability of the biosensing system were evaluated by standard addition recoveries. Different concentrations of H2O2 standard solution were added into PBS incubated with PC12 cells. The recoveries were obtained in the range of 91.5–107.04 % (Table S1), which is within the acceptance criteria (recoveries in the range of 90–110 %) set for bioanalytical method validation. Taken together, the findings indicated that the developed colorimetric sensing platform established in this work can be employed to monitor the activity of PC12 cells.
4. Conclusions
In summary, water-soluable MoOx QDs were fabricated by a facile and eco-friendly process, exhibiting intrinsic peroxidase-like activity to catalyze a chromogenic substrate ABTS for the detection of H2O2 released from PC12 cells. Moreover, the peroxidase-like activity of MoOx QDs is dependent on pH, concentration of ABTS and H2O2, and reaction time. The new technology established in this work is based on microfluidic paper-based analytical device, and thus has many remarkable advantages of low cost, rapid response, high sensitivity and good selectivity, which manifests great prospects in monitoring cell activity. In addition, the successful attempt of MoOx QDs as mimic enzyme might also boost its renewed utilization in biotechnology and clinical diagnosis in the future.
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.
Acknowledgments
The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grant No. 21775023), the Natural Science Foundation of Fujian Province of China (2014J07009 and 2019J01301), Social Development Guiding Programs of Fujian Province of China (2019Y0012), and the Fujian Provincial University-Industry Cooperation Science & Technology Major Program (2019Y4006).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.127512.
References
[1] S. Prasad, S.C. Gupta, A.K. Tyagi, Reactive oxygen species (ROS) and cancer: role of antioxidative nutraceuticals, Cancer Lett. 387 (2017) 95–105.
[2] Z.H. Liao, D.M. Chua, N.S. Tan, Reactive oxygen species: a volatile driver of field cancerization and metastasis, Mol. Cancer 18 (2019) 65, https://doi.org/10.1186/ s12943-019-0961-y.
[3] R. Shen, P.P. Liu, Y.Q. Zhang, Z. Yu, X.Y. Chen, L. Zhou, B.Q. Nie, A. Zaczek, J. Chen, J. Liu, Sensitive detection of single-cell secreted H2O2 by integrating a microfluidic droplet sensor and Au nanoclusters, Anal. Chem. 90 (2018) 4478–4484.
[4] A. Acharya, I. Das, D. Chandhok, T. Saha, Redox regulation in cancer: a doubleedged sword with therapeutic potential, Oxid. Med. Cell. Longev. 3 (2010) 23–34.
[5] G. Saravanakumar, J. Kim, W.J. Kim, Reactive‐oxygen‐species‐responsive drug delivery systems: promises and challenges, Adv. Sci. 4 (2016) 1600124, , https:// doi.org/10.1002/advs.201600124.
[6] F. Antunes, E. Cadenas, Estimation of H2O2 gradients across biomembranes, FEBS Lett. 475 (2000) 121–126.
[7] G.P. Bienert, J.K. Schjoerring, T.P. Jahn, Membrane transport of hydrogen peroxide, BBA-Biomembranes 1758 (2006) 994–1003.
[8] H.F. Yue, X. Bu, M.H. Huang, J. Young, T. Raglione, Quantitative determination of trace levels of hydrogen peroxide in crospovidone and a pharmaceutical product using high performance liquid chromatography with coulometric detection, Int. J. Pharmaceut. 375 (2009) 33–40.
[9] B.C. Dickinson, C. Huynh, C.J. Chang, A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells, J. Am.
Chem. Soc. 132 (2010) 5906–5915.
[10] J. Wang, L. Li, W. Huang, J. Cheng, Development and evaluation of a rotary cell for capillary electrophoresis-chemiluminescence detection, Anal. Chem. 82 (2010) 5380–5383.
[11] Z.H. Dai, S.H. Liu, J.C. Bao, H.X. Ju, Nanostructured FeS as a mimic peroxidase for biocatalysis and biosensing, Chem. Eur. J. 15 (2009) 4321–4326.
[12] D.Q. Ma, J. Yu, W.Y. Yin, X. Zhang, L.Q. Mei, Q. Gao, L. Yan, Z.J. Gu, X.Y. Ma, Y.L. Zhao, Peroxidase-like activity of MoS2 nanoflakes with different modifications and their application for H2O2 and glucose detection, J. Mater. Chem. B 6 (2018) 487–498.
[13] X. Chen, J. Chen, F. Wang, X. Xiang, M. Luo, X. Ji, Z. He, Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices, Biosens. Bioelectron. 35 (2012) 363–368.
[14] R.L. Cromartie, A. Wardlow, G. Duncan, B.R. Mccord, Development of a microfluidic device (μPADs) for forensic serological analysis, Anal. Methods 11 (2019) 587–595.
[15] P. Lisowski, P.K. Zarzycki, Microfluidic paper-based analytical devices (μPADs) and micro total analysis systems (μTAS): development, applications and future trends, Chromatographia 76 (2013) 1201–1214.
[16] J. Zhou, C.M. Yu, Z. Li, P.P. Peng, D.T. Zhang, X. Han, H.Z. Tang, Q. Wu, L. Li, W. Huang, A rapid and highly selective paper-based device for high-throughput detection of cysteine with red fluorescence emission and a large Stokes shift, Anal. Methods 11 (2019) 1312–1316.
[17] T.Y. Dong, G.A. Wang, F. Li, Shaping up field-deployable nucleic acid testing using microfluidic paper-based analytical devices, Anal. Bioanal. Chem. 411 (2019) 4401–4414.
[18] Y.N. Ding, B.H. Yang, H. Liu, Z.X. Liu, X. Zhang, X.W. Zheng, Q.Y. Liu, FePt-Au ternary metallic nanoparticles with the enhanced peroxidase-like activity for ultrafast colorimetric detection of H2O2, Sensor. Actuat. B-Chem. 259 (2018) 775–783.
[19] R. Breslow, Biomimetic chemistry and artificial enzymes: catalysis by design, Accounts Chem. Res. 28 (1995) 146–153.
[20] L.Z. Gao, J. Zhuang, L. Nie, J.B. Zhang, Y. Zhang, N. Gu, T.H. Wang, J. Feng, D.L. Yang, S. Perrett, X.Y. Yan, Intrinsic peroxidase-like activity of ferromagnetic nanoparticles, Nat. Nanotechnol. 2 (2007) 577–583.
[21] S.L. Dean, Encoded anisotropic particles for multiplexed bioanalysis, Wires.
Nanomed. Nanobiol. 2 (2010) 578–600.
[22] Y. Jv, B. Li, R. Cao, Positively-charged gold nanoparticles as peroxidiase mimic and their application in hydrogen peroxide and glucose detection, Chem. Commun. 46 (2010) 8017–8019.
[23] J. Chen, J.M. Mclellan, A. Siekkinen, Y. Xiong, Z.Y. Li, Y. Xia, Facile synthesis of gold-silver nanocages with controllable pores on the surface, J. Am. Chem. Soc. 128 (2006) 14776–14777.
[24] J. Mu, Y. Wang, M. Zhao, L. Zhang, Intrinsic peroxidase-like activity and catalaselike activity of Co3O4 nanoparticles, Chem. Commun. 48 (2012) 2540–2542.
[25] L. Hu, Y. Yuan, L. Zhang, J. Zhao, S. Majeed, G. Xu, Copper nanoclusters as peroxidase mimetics and their applications to H2O2 and glucose detection, Anal. Chim.
Acta 762 (2013) 83–86.
[26] L.H. Sun, Y.Y. Ding, Y.L. Jiang, Q.Y. Liu, Montmorillonite-loaded ceria nanocomposites with superior peroxidase-like activity for rapid colorimetric detection of H2O2, Sens. Actuator B-Chem. 239 (2017) 848–856.
[27] F. Honarasa, F.H. Kamshoori, S. Fathi, Z. Motamedifar, Carbon dots on V2O5 nanowires are a viable peroxidase mimic for colorimetric determination of hydrogen peroxide and glucose, Microchim. Acta 186 (2019) 234.
[28] J. Haber, E. Lalik, Catalytic properties of MoO3 revisited, Catal. Today 33 (1997) 119–137.
[29] Q.Y. Ouyang, L. Li, Q.S. Wang, Y. Zhang, T.S. Wang, F.N. Meng, Y.J. Chen, P. Gao, Sonochemical synthesis and ppb H2S sensing performances of CuO nanobelts, Sens. actuat B-Chem. 169 (2012) 17–25.
[30] J. Zhou, S.Z. Deng, N.S. Xu, J. Chen, J.C. She, Synthesis and field-emission properties of aligned MoO3 nanowires, Appl. Phys. Lett. 83 (2003) 2653–2655.
[31] B. Yao, L. Huang, J. Zhang, X. Gao, J.B. Wu, Y.L. Cheng, X. Xiao, B. Wang, Y. Li, J. Zhou, Flexible transparent molybdenum trioxide nanopaper for energy storage, Adv. Mater. 28 (2016) 6353–6358.
[32] M.A. Ibrahem, F.Y. Wu, D.A. Mengistie, C.S. Chang, L.J. Li, C.W. Chu, Direct conversion of multilayer molybdenum trioxide to nanorods as multifunctional electrodes in lithium-ion batteries, Nanoscale 6 (2014) 5484–5490.
[33] R. Hille, The molybdenum oxotransferases and related enzymes, Dalton Trans. 42 (2013) 3029–3042.
[34] G. Song, J. Shen, F. Jiang, R. Hu, W. Li, L. An, R. Zou, Z. Chen, Z. Qin, J. Hu, Hydrophilic molybdenum oxide nanomaterials with controlled morphology and strong plasmonic absorption for photothermal ablation of cancer cells, ACS Appl.
Mater. Interfaces 6 (2014) 3915–3922.
[35] R. Ragg, F. Natalio, M.N. Tahir, H. Janssen, A. Kashyap, D. Strand, S. Strand, W. Tremel, Molybdenum trioxide nanoparticles with intrinsic sulfite oxidase activity, ACS Nano 8 (2014) 5182–5189.
[36] B. Halliwell, Occupational exposure to chemicals and oxidative toxic stress, Am. J.
Med. 91 (1991) S14–S22.
[37] S.J. Xiao, X.J. Zhao, P.P. Hu, Z.J. Chu, C.Z. Huang, L. Zhang, Highly photoluminescent molybdenum oxide quantum dots: one-pot synthesis and application in TNT determination, ACS Appl. Mater. Inter. 8 (2016) 8184–8191.
[38] Y. Anh Tran, K. Krishnamoorthy, Y.W. Song, S.K. Cho, S.J. Kim, Toxicity of nano molybdenum trioxide toward invasive breast cancer cells, ACS Appl. Mater.
Interfaces 6 (2014) 2980–2986.
[39] H.P. Peng, P. Liu, D.W. Lin, Y.N. Deng, Y. Lei, W. Chen, Y.Z. Chen, X.H. Lin, X.H. Xia, A.L. Liu, Fabrication and multifunctional properties of ultrasmall watersoluble tungsten oxide quantum dots, Chem. Commun. 52 (2016) 9534–9537.
[40] B.M. Reddy, P. Saikia, P. Bharali, Physicochemical characteristics and catalytic activity of alumina-supported nanosized ceria-terbia solid solutions, J. Phys. Chem.
C 113 (2009) 2452–2462.
[41] A. Klinbumrung, T. Thongtem, S. Thongtem, Characterization and gas sensing properties of CuO synthesized by DC directly applying voltage, Appl. Surf. Sci. 313 (2012) 640–646.
[42] T. Xia, Q. Li, X.D. Liu, J. Meng, X.Q. Cao, Morphology-controllable synthesis and characterization of single-crystal molybdenum trioxide, J. Phys. Chem. B 110 (2006) 2006–2012.
[43] V.P. Shah, K.K. Midha, J.W.A. Findlay, H.M. Hill, J.D. Hulse, I.J. Mcgilveray, G. Mckay, K.J. Miller, R.N. Patnaik, M.L. Powell, Bioanalytical method validation-a revisit with a decade of progress, Pharm. Res. 17 (2000) 1551–1557.
Meng-Meng Liu is now studying for a PhD degree of pharmacology in Fujian Medical University. Her research focuses on the development for hybridization of paper and high polymer on μ-TAS.
Shan-Hong Li is a research associate in Fujian Medical University. She received her MS degree in Pharmaceutical Analysis from Fujian Medical University in 2018, China. Her research focuses on electrochemical biosensor and μ-TAS.
Dan-Dan Huang is studying for a MS degree of Pharmaceutical Analysis in Fujian Medical University. Her research focuses on electrochemical biosensor and μ-TAS.
Zhi-Wei Xu is studying for a MS degree of Pharmaceutical Analysis in Fujian Medical University. His research focuses on electrochemical biosensor and μ-TAS.
Ya-Wei Wu is a research associate in Fujian Institute of Research on the Structure of Matter Chinese Academy of Sciences. She received her MS degree in Analytical Chemistry from Fuzhou University in 2018, China. Her current research focuses on functional nanomaterials, electrochemical sensor and biosensor.
Yun Lei is associate professor of Pharmaceutical Analysis in Fujian Medical University. She received her PhD degree in Analytical Chemistry from Wuhan University in 2009, China. Her current research focuses on functional nanomaterials, electrochemical sensor and biosensor.
Ai-Lin Liu is a professor of Pharmaceutical analysis in Fujian Medical University. He received his PhD degree in Analytical Chemistry from NanjingUniversity in 2006, China. His current interests include the development of biosensors, chemically modified electrodes and μ-TAS.