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Two dimensional α-MoO3-x nanoflakes as bare eye probe for hydrogen peroxide in biological fluids
Accepted Manuscript Two dimensional α-MoO3-x nanoflakes as bare eye probe for hydrogen peroxide in biological fluids Amit Kumar Sharma, Sunil Pandey, Krishna Hari Sharma, Yowan Nerthigan, M. Shahnawaz Khan, Da-Ren Hang, Hui-Fen Wu PII:
S0003-2670(18)30162-4
DOI:
10.1016/j.aca.2018.01.057
Reference:
ACA 235704
To appear in:
Analytica Chimica Acta
Received Date: 10 May 2017 Revised Date:
12 January 2018
Accepted Date: 22 January 2018
Please cite this article as: A.K. Sharma, S. Pandey, K.H. Sharma, Y. Nerthigan, M.S. Khan, D.-R. Hang, H.-F. Wu, Two dimensional α-MoO3-x nanoflakes as bare eye probe for hydrogen peroxide in biological fluids, Analytica Chimica Acta (2018), doi: 10.1016/j.aca.2018.01.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Amit Kumar Sharma†1, Sunil Pandey†1, Krishna Hari Sharma5, Yowan Nerthigan1, M. Shahnawaz Khan1,3, Da-Ren Hang5, Hui-Fen Wu*1, 2, 3, 4
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Two dimensional α-MoO3-x nanoflakes as bare eye probe for hydrogen peroxide in biological fluids
Department of Chemistry, National Sun Yat-sen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan
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School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, 80424, Taiwan
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Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan Department of Materials and Optoelectronic Science, College of Engineering, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan
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*Corresponding author: [email protected] (HFWu)
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Phone: +886-7-5252000-3955; Fax: +886-7-5253909
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† Authors share equal contributions for the first authors.
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Abstract
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We report a rapid and facile method for detection of hydrogen peroxide (H2O2) in biological
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fluids using sub-stoichiometric two-dimensional (2D) molybdenum trioxide (α-MoO3-x)
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nanoflakes. The two-dimensional nanoflakes, initially blue in color, is oxidized after interaction
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with hydrogen peroxide thereby changing its oxidation state to form α-MoO3. The change in
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oxidation state of nanoflakes transforms from blue to a visually distinct hazy blue color with
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change in absorption spectrum. The phenomenal property is explored here in sensing up to 34
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nM as limit of detection. The efficacy of the detection system was analyzed by “zone of
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inhibition” based agar diffusion assay with different concentrations of H2O2. The current
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approach is highly accurate, effective and reproducible for quantification of physiological
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concentration of H2O2 in biological fluid such as human urine.
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Keywords: two-dimensional nanomaterial; bare eye; rapid detection; H2O2; colorimetric assay;
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urine bioassay.
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Introduction
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Hydrogen peroxide (H2O2) is one of the most widely used chemical constituent in various
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industrial processes [1]. Apart from this, hydrogen peroxide plays quintessential role in variety
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of biomolecular processes such as ageing [2,3], molecular signaling [4,5], membrane damage
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[6], genetic mutation [7], carcinogenesis [8,9], neurological ailments [10,11] etc. This intricate
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involvement makes H2O2 a molecular alarm for serious physiological disorders. There are
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various techniques for detecting H2O2 using fluorescence [12,13], electrochemistry [14], bare eye
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sensor using gold nanoparticles [15]. A recent review reported by Chen et al have highlighted
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different approach for detection of hydrogen peroxide through metal oxides by electrochemical
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methods [16]. These methods require typically tedious procedures and critical observations to
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detect low concentration of H2O2. Hence facile and rapid detection of H2O2 with high sensitivity
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is in great demand for screening its presence in biological samples. Despite substantial amount
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of research reported on hydrogen peroxide sensing, rapid detection with high sensitivity still has
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a room for improvement.
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After the discovery of Graphene [17–19], other layered materials such as Transition-Metal
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Dichalcogenides (TMDs): Tungsten disulfide (WS2) [20,21], Molybdenum disulfide (MoS2)
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[22,23] and Transition-Metal Oxides (TMOs): Molybdenum trioxide (MoO3) [24,25]; there is an
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essential leaf towards development of sensors for detection of biological analytes [26]. Amongst
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the 2D materials, TMDs have been widely studied as fluorescent probes for sensing of biological
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analytes [27] and reported to be highly biocompatible [28]. The necessary transformation from
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+4 to +6 oxidation states to obtain a color of the nanomaterial is a tedious process thereby
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making it unfit for use in rapid visual detection sensing strategies [29,30]. TMOs, on the other
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hand, exhibit properties such as photochromism, electrochromism and thermochromism which
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attribute to chromogenic coloration of nanomaterial. The photochromic [31,32], electrochromic
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[33] and thermochromic [34] properties of Molybdenum oxide have been extensively studied.
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Moreover, the transition between the oxidation states in MoO3 is comparatively easy [35] thus
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making is a choice of material in this detection system .
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Exploring over molybdenum oxides, Wagner et al highlighted increased catalytic activity of
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orthorhombic MoO3 under appropriate surface defect. This property of MoO3 is still being
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explored for several applications in environmental studies [36–38] and in electrochemical
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storage devices [39]. The defects on surface can be controlled by subjecting MoO3 to mixtures of
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varying ratios of aqueous and organic solvents [40], exposure to visible light [41], xenon lamp
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[42,43], solar simulator [40] to obtain catalytically active reduced form of α-MoO3-x [44]. The
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oxygen vacancies created on the surface of α-MoO3-x nanoflakes have been shown to have
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enhanced charge storage [45] and await its applications in biological field. α-MoO3-x is the least
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explored material in biological field due to its low stability in aqueous environment [36] and
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delicate interaction chemistry with other molecules in solution. α-MoO3-x, like other 2D
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materials, is a planar structure with high aspect ratio [40]. The optical property such as surface
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plasmon resonance in α-MoO3-x, are sensitive to number of planes, which are held together by
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weak van der Waals forces and to the duration of exposure of light during the synthesis of α-
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MoO3-x [46]. This interlayer spacing is highly sensitive to interaction of protons and/or removal
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of oxygen from α-MoO3-x (Fig. 1, Panel A). The holistic impact of these processes is alteration in
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electronic band gap of the α-MoO3-x. These unique attributes enables α-MoO3-x nanoflakes as a
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promising platform for developing novel sensitive probe for biological analytes [47].
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We report the use of α-MoO3-x as a sensitive probe for H2O2 in biological fluids. To date, to the
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best of our knowledge, detection of H2O2 in biological fluid has not been reported. The detection
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system has been able to reach extremely low concentration of H2O2 with remarkable sensitivity
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ever reported before for a bare eye based colorimetric probe and for the first time in biological
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fluid. The mechanism of detection is outlined in Fig. 1. As discussed earlier, the electron
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concentration in 2D materials like MoO3 can be enhanced by encouraging oxygen vacancies,
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which forms α-MoO3-x. In the present context, this reaction is reversed as per our speculation in
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presence of H2O2. This leads to re-conversion of α-MoO3-x, a blue colored solution to oxidized
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form α-MoO3. The transformation is distinctly observed in the ambient light photograph of
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samples. The sensitivity of α-MoO3-x towards availability of oxygen makes it a favorable
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sensitive probe for H2O2. Parallel to the colorimetric assay, we have also developed an agarose
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gel diffusion method for detection of H2O2 based on its “zone of inhibition” (Fig. 1). We have
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found that our method is competitively more sensitive than most of the earlier reports as
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represented graphically in Fig. 2.
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Materials and Methods
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1. Materials
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Molybdenum trioxide (MoO3) powder, Hydrogen peroxide (H2O2), Agarose was purchased from
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Sigma, UK. All the chemicals used in experiments were of analytical grade and used without
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further purification.
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2. Instrumentation
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The morphological details of nanoparticles were elucidated using transmission electron
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microscope (TEM) (Philips, The Netherlands). 2 µl of the samples were drop coated on formvar
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coated copper grids to form uniform layer and dried under ambient temperature to ensure the
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integrity of the film. X-ray Photoelectron Spectroscopy (XPS) was performed (Photoelectron
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spectrometer ESCA, JEOL Ltd., Japan). The sample was drop casted on silicon substrate
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followed by observing the corresponding spectrum. UV-Vis spectroscopy (Thermo,
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EVOLUTION 201, USA) was carried out using standard quartz cuvette having path length 1 cm.
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Particle size analysis (dynamic light scattering DLS) was done on Zeta potential & Particle Size
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Analyzer ELSZ-2000 series, Otsuka Electronics Co. Ltd., Japan.
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3. Preparation of α-MoO3-x
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α-MoO3-x nanoflakes was synthesized using modified protocol [36]. Briefly, 0.5 g MoO3 was
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dispersed in 100 ml equal volume of water and ethanol. The mixture was sonicated for 2 h at 5
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secs ON and 2 secs OFF pulse at 125 W (Ultrasonic Processor, Qsonica probe Sonicator, USA).
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The obtained solution was then centrifuged for 30 min at 3000 rpm to collect the supernatant.
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The solution was subjected exposed to xenon lamp (Newport, USA) [44] at a power of 600 W till
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the solution turned deep blue. This solution was washed three times and dialyzed against
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ethanolic water for 48 h to obtain purified solution contained α-MoO3-x nanoflakes. To remove
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traces of ethanol and to avoid its interference in the reaction mixtures, the purified solution of
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nanoflakes was dried in rotary vacuum evaporator. The dried sample was dispersed in DI water
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followed by sonication for 15 min before further experimental use.
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4. Concentration dependent studies
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The initial test for visual detection was done in presence of 300 µl of as prepared α-MoO3-x and
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100 mM H2O2 with total reaction volume of 1000 µl. To study the sensitivity of the
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nanomaterial, H2O2 was serially diluted as 100 mM – 100 nM using DI water. This was followed
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by adding 300 µl of as prepared α-MoO3-x in a total reaction volume of 1000 µl. The mixture was
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vortexed and incubated for 10 min at room temperature after which the corresponding
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absorbance was recorded in UV-Vis spectrophotometer. The change in the particle size of α-
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MoO3-x in presence of H2O2 was analyzed by particle size analyzer. A day-light photograph of
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the reaction mixtures was captured.
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5. Understanding the sensitivity in urine samples
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A pilot study for test of H2O2 in biological fluid was done by spiking 100 mM H2O2 in urine
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sample of a lab volunteer and mixed with 300 µl as prepared α-MoO3-x in reaction volume of
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1000 µl. To further identify the sensitivity of α-MoO3-x for H2O2 in urine samples, we spiked
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H2O2 in range of 10 mM – 100 nM into the urine sample. To this, 300 µl of the as prepared α-
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MoO3-x was introduced in a 1000 µl reaction volume as mentioned above. The samples were
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vortexed and incubated for 10 mins at room temperature. The absorbance was recorded in UV-
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Vis spectrophotometer and I/I0 was calculated based on peak maxima.
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6. “Zone of inhibition” study and precedence over conventional methods for H2O2
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To exhibit the visual detection of H2O2, α-MoO3-x was introduced to 0.8% agarose solution. The
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as formed solution was then plated on petri dish and allowed to solidify. Wells of 100 µl
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volumes were bored using a borer. This was followed by adding H2O2 as test species from 100
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mM – 100 nM following ten-fold dilution in each well to finally measure colorless “zone of
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inhibition” formed on the α-MoO3-x gel.
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To assess the sensitivity of the assay system against other reactive oxygen species such as
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hydroxyl radicals, we used Fenton reaction mediated generation of OH• radicals. FeCl2 (5µM)
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and H2O2 (100mM) were mixed in a reaction volume of 1ml followed by addition of α-MoO3-x
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after 5 mins of incubation. The corresponding visual changes and spectroscopic readings were
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recorded. To comprehend the precedence of the current assay system over conventional methods
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for H2O2 detection, we used earlier reported FOX (ferrous ion oxidation in xylenol orange)
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reagent assay method [48]. Initially, urine samples from a healthy subject was collected followed
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by three concentrations of 100µM, 10µM and 1µM spiked into the urine sample to record percent
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recovery. The sensitivity of the current method was also tested on urine samples collected in
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different intervals of day such as morning (DM), afternoon (DA), evening (DE) and night (DN)
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from a donor under consideration. The test also compares with healthy sleep and sleep deprived
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urine samples collected in the following morning.
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Results & Discussions
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The optical and physical properties of 2D nanomaterials particularly transition metal oxides and
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dichalcogenides have attracted its widespread use in development of different biological sensors.
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The flexibility and easy tunability of metal oxide nanoflakes have an advantage over other
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nanomaterials that demand various molecular markers on its surface for increasing affinity
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[49,50]. Also, the use of complex nanostructures (as compared in Fig. 2) involves tedious
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synthesis processes. Apart from other metal oxides and nanocomposites [51,52], α-MoO3-x
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nanoflakes is a novel material synthesized with surface defects and enhanced oxygen vacancy
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thereby making it sensitive and selective probe for the presence of H2O2. The sensitivity of this
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material is attributable to the oxidation potential and its tendency to achieve oxidized state of α-
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MoO3. The use of α-MoO3-x also has other advantages of facile synthesis and “rapid” visual
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detection of H2O2 hence overcoming use of other metal oxides with electrochemical approach.
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Figure 1: Schematic representation of the detection system at a glance. Panel A shows the reactivity of H2O2 with α-MoO3-x nanoflakes. The oxygen a vacancy on the surface of α-MoO3-x is occupied rapidly by H2O2 thus transforming the blue colored nanoflakes to pale blue/colorless α-MoO3. Panel B shows diffusion based quantification of H2O2 in an agarose gel embedded αMoO3-x forming a “zone of inhibition”.
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Figure 2: Graphical representation: Comparing the sensitivity of colorimetric methods earlier reported for detection of H2O2 [53–63]
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Characterization of α-MoO3-x
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The TEM image (Fig. 3A) shows the formation of 2D α-MoO3-x nano-flakes. The lattice fringe
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width of 0.37 nm supports the formation of 2D nano-flakes (Fig. 3B). The associated SAED
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display diffraction spots from [2 0 0] and [0 0 2] planes confirms the formation of α-MoO3-x
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nano-flakes [36]. XPS was performed to understand their sub-stoichiometry of α-MoO3-x nano9
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flakes (Fig. 3C). The XPS analysis shows absence of impurities during the process of xenon
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lamp assisted synthesis. The presence of doublet peaks 231 eV and 234.38 eV are assigned to
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Mo+5 3d5/2 and Mo+5 3d3/2 of sub-stoichiometric α-MoO3-x nanoflakes. Fig. 3D shows the XPS
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spectra of nanoflakes after addition of H2O2. The presence of doublets at 232.58 and 235.48 eV
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are attributed to the presence of oxidized form α-MoO3. The peaks are assigned as the oxidation
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states of Mo+6 3d5/2 and Mo+6 3d3/2 [64]. DLS studies (Table 1), shows superficial morphological
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changes in α-MoO3-x nanoflakes in presence of various concentrations of H2O2.
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Figure 3: (A) TEM representative of α-MoO3-x nanoflakes, Inset: reduced image of nanoflakes; (B) SEAD pattern of α-MoO3-x nanoflakes; (C) XPS spectrum of α-MoO3-x; (D) XPS spectra after addition of H2O2
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The optical properties of α-MoO3-x, H2O2 and their mixture are displayed in Fig. 4A. The optical
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spectra of α-MoO3-x shows broad hump peak between 600-900 nm. The characteristic absorption 10
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of α-MoO3-x in visible range (600-1000 nm) is due to change in its optical properties and hence
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the deep blue color of the solution [40,45]. Interestingly, in the presence of H2O2, the peaks in
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visible domain were decreased and its intensity in UV regime was found to be enhanced. This is
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the peculiar property of oxidized form α-MoO3 [65]. Fig. 4B shows the ambient light photograph
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of color changes in presence of H2O2. This confirms the rapid bare eye detection of H2O2 in an
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effective way.
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Table 1: DLS studies of the α-MoO3-x nanoflakes in presence of various concentration of H2O2.
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Concentration of H2O2
100mM
10mM
1mM
100µM
10µM
1µM
100nM
Mean Diameter
708.9
451.3
568.3
1100.8
859.0
427.9
1257.1
Polydispersity index
0.427
0.295
0.280
0.641
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*All samples were recorded at 25°C in water
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Sensitivity of α-MoO3-x against varying concentration of H2O2
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The optical-chemistry behind the appearance of plasmon peak in visible range (600-900 nm) is
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due to the proton intercalation in the interlayer spaces of α-MoO3-x [64]. During the synthesis of
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α-MoO3-x from MoO3 under the influence of xenon lamp (600 W; 200-1500 nm), the water splits
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to H+ and OH-. The proton from this reaction sits into the interlayer spacing and initiate the
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removal of oxygen from MoO3 [66]. The overall effect of the reaction is alteration in the
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thickness of α-MoO3 nanoflakes and shift of the plasmon resonance in visible range [67]. In the
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presence of H2O2, however, this reaction gets reversed. H2O2 is an exceptionally potent oxidizer
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in acidic medium. Its presence in minute concentration can replenish the oxygen vacancies and
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transform α-MoO3-x to α-MoO3 nanoflakes.
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To comprehend the sensing capacity of α-MoO3-x, the optical response of α-MoO3-x in presence
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of different concentration of H2O2 (Fig. 4C) is proposed. As evident from the figure, there was
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proportional decrease in absorption of α-MoO3-x. The plot of I/I0 vs. concentration of H2O2 shows
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linear relationship with R2 = 0.9937 (Fig. 4D). The limit of detection was calculated to be 34 nM
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(S/N=3).
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Figure 4: (A) UV-Vis spectra of α-MoO3-x (black), α-MoO3 (red); Inset: Zoom with reduced intensity of the graph (B) Day-light photograph of H2O2 concentration dependent color change of α-MoO3-x; (C) UV-Vis spectral change of α-MoO3-x in presence of varying concentrations of H2O2; (D) Relative intensity curve (I/I0) and Regression analysis.
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Sensitivity in biological fluids
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The physiological stress from sleeplessness instigates the production of H2O2 [3,68] and is
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abundantly present in urine under physiological stress. Hence, to detect the presence of
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physiological concentration of H2O2 in human urine (biological fluid); we collected samples
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from four volunteers in lab working sleeplessly overnight. The urine samples were spiked with
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varying concentration of H2O2 and the UV-Vis absorbance was recorded (Fig. S1). The change in 12
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concentration of H2O2 was found to be proportional with the spectra in presence of biological
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fluid. Fig. 5A represents the relative intensity I/I0 plot against concentration with R2 = 0.9333.
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Fig. 5B demonstrates the transformation of solution from blue to colorless after addition of
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biological fluid collected from stressed volunteers to α-MoO3-x. A set of test sample was also
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compared with 100 mM H2O2 to observe the visible change as shown in Fig. 4B.
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Figure 5: (A) Relative intensity I/I0 vs. Concentration of H2O2 spiked in biological fluid. (B) Day-light photograph of color change in H2O2 spiked biological sample and its comparison with controls (colorless – 100 mM H2O2; blue – α-MoO3-x).
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“Zone of inhibition” study and precedence over conventional methods for H2O2
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A simple and effective method was designed for determination of the concentration of H2O2 in
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unknown samples like biological fluids. α-MoO3-x is often found to be highly unstable in solution
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due to rapid exchange of protons from the surrounding. To avoid this, we incorporated α-MoO3-x
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in agarose and poured in petri dish (Fig. S2). After punching holes in the gel, different
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concentration of H2O2 was filled in the wells and zone of inhibition was recorded (Table 2). A
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clear zone was apparent on the agarose gel due to diffusion of H2O2 and the size of the zone
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varied corresponding to different concentration of H2O2 (Fig, 6A).
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The sensitivity of the assay system against other reactive oxygen species such as OH• radical was
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assessed using Fenton reaction. Fe2+ in presence of H2O2 generates OH• radicals and Fe3+. After
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addition of α-MoO3-x, the relative absorbance obtained suggest clear transformation of the
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nanoflakes to α-MoO3 corresponding to stabilization of oxygen vacancies (Fig. S2A). This is
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indicative that the current assay system is sensitive to reactive oxygen species. In comparison to
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conventional methods, current assay method shows comparable response to the FOX reagent
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based assay of H2O2 spiked urine samples (Fig. S2B). The percent recovery is attributable the
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inherent H2O2 concentration in urine sample. To comprehend the impact of physiological stress
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on analytical performance of proposed method, the urine samples were collected at different time
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intervals which indicate the varying levels of stress (each stress are explained in Fig. 6B). Based
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on the interpretation of results, the ability of the proposed probe was excellent to discriminate
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differential level of hydrogen peroxide in the urine (indicative of different physiological stress).
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Fig. 6: (A) Representative of “zone of inhibition” recorded in petri dish containing α-MoO3-x
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infused 0.8% agarose. Wells contain various concentration of H2O2 from 100 mM – 0.0001 mM;
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(B) Relative absorbance showing selective analysis of urine samples for H2O2 using α-MoO3-x in
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Control (healthy donor), samples collected in morning (DM), afternoon (DA), evening (DE), night
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(DN) and with proper sleep (DS), sleep deprived (DSD).
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Table 2: “Zone of inhibition” recorded in petri dish containing α-MoO3-x infused 0.8% agarose. Wells contain various concentration of H2O2.
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Concentration
Zone of Inhibition (cm)* 14
RSD
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100 mM
1.4
0.2
10 mM
1.34
0.0529
1 mM
1.21
0.041
0.1 mM
0.97
0.02
*Mean of zone of inhibition (n=3) Zone of inhibition = -0.1417 Concentration + 1.585 R2 = 0.9217
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Conclusion
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α-MoO3-x has been reported widely for its use in development of electrochemical storage devices
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and environmental applications. The visually distinct blue color in the solution corresponds to
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optical characteristics observed. The oxidation property of α-MoO3-x has been explored in a
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facile assay method for colorimetric detection of H2O2. The method is based on “rapid” bare eye
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detection and hence more informative in case of physiological ailments. This study establishes a
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new platform in detecting biological samples such as urine or blood by bare eyes using sub-
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stoichiometric α-MoO3-x nanoflakes. We believe that this is a significant contribution to be first
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of its kind report in biological application of α-MoO3-x at such high sensitivity.
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Acknowledgement
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We thank the Ministry of Science and Technology of Taiwan for financial support with the grant
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number of MOST 104-2113-M-110-003-MY3.
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Figure 1: Schematic representation of the detection system at a glance. Panel A shows the reactivity of H2O2 with α-MoO3-x nanoflakes. The oxygen a vacancy on the surface of α-MoO3-x is occupied rapidly by H2O2 thus transforming the blue colored nanoflakes to pale blue/colorless α-MoO3. Panel B shows diffusion based quantification of H2O2 in an agarose gel embedded αMoO3-x forming a “zone of inhibition”.
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Figure 2: Graphical representation: Comparing the sensitivity of colorimetric methods earlier reported for detection of H2O2 [53–63]
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Figure 3: (A) TEM representative of α-MoO3-x nanoflakes, Inset: reduced image of nanoflakes; (B) SEAD pattern of α-MoO3-x nanoflakes; (C) XPS spectrum of α-MoO3-x; (D) XPS spectra after addition of H2O2
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Figure 4: (A) UV-Vis spectra of α-MoO3-x (black), α-MoO3 (red); Inset: Zoom with reduced intensity of the graph (B) Day-light photograph of H2O2 concentration dependent color change of α-MoO3-x; (C) UV-Vis spectral change of α-MoO3-x in presence of varying concentrations of H2O2; (D) Relative intensity curve (I/I0) and Regression analysis.
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Figure 5: (A) Relative intensity I/I0 vs. Concentration of H2O2 spiked in biological fluid. (B) Day-light photograph of color change in H2O2 spiked biological sample and its comparison with controls (colorless – 100 mM H2O2; blue – α-MoO3-x).
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Fig. 6: (A) Representative of “zone of inhibition” recorded in petri dish containing α-MoO3-x
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infused 0.8% agarose. Wells contain various concentration of H2O2 from 100 mM – 0.0001 mM;
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(B) Relative absorbance showing selective analysis of urine samples for H2O2 using α-MoO3-x in
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Control (healthy donor), samples collected in morning (DM), afternoon (DA), evening (DE), night
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(DN) and with proper sleep (DS), sleep deprived (DSD). 5
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Fig. S1: UV-Vis absorbance spectra of α-MoO3-x in presence of varying concentration of urine
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spiked H2O2.
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g. S2: (A) Selective assay of OH• radicals generated using Fenton Reaction (Control: α-MoO3-x).
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Inset: (a) α-MoO3-x, (b) FeCl2, (c) Test sample α-MoO3-x+FeCl2+H2O2; (B) Comparing percent
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recovery of three concentrations of H2O2 in urine samples using current method and
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conventional method using FOX reagent. 6
ACCEPTED MANUSCRIPT Highlights 1. We have developed a bare eye sensor for Hydrogen peroxide 2. α-MoO3-x is used as Rapid detection probe for Hydrogen peroxide 3. The method has also been tested for the first time on biological fluids to affirm the
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4. It has also been tested on agar well diffusion assay detection based on its “zone of
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inhibition”
S0003-2670(18)30162-4
DOI:
10.1016/j.aca.2018.01.057
Reference:
ACA 235704
To appear in:
Analytica Chimica Acta
Received Date: 10 May 2017 Revised Date:
12 January 2018
Accepted Date: 22 January 2018
Please cite this article as: A.K. Sharma, S. Pandey, K.H. Sharma, Y. Nerthigan, M.S. Khan, D.-R. Hang, H.-F. Wu, Two dimensional α-MoO3-x nanoflakes as bare eye probe for hydrogen peroxide in biological fluids, Analytica Chimica Acta (2018), doi: 10.1016/j.aca.2018.01.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Amit Kumar Sharma†1, Sunil Pandey†1, Krishna Hari Sharma5, Yowan Nerthigan1, M. Shahnawaz Khan1,3, Da-Ren Hang5, Hui-Fen Wu*1, 2, 3, 4
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Two dimensional α-MoO3-x nanoflakes as bare eye probe for hydrogen peroxide in biological fluids
Department of Chemistry, National Sun Yat-sen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan
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School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, 80424, Taiwan
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Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan Department of Materials and Optoelectronic Science, College of Engineering, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan
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*Corresponding author: [email protected] (HFWu)
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Phone: +886-7-5252000-3955; Fax: +886-7-5253909
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† Authors share equal contributions for the first authors.
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Abstract
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We report a rapid and facile method for detection of hydrogen peroxide (H2O2) in biological
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fluids using sub-stoichiometric two-dimensional (2D) molybdenum trioxide (α-MoO3-x)
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nanoflakes. The two-dimensional nanoflakes, initially blue in color, is oxidized after interaction
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with hydrogen peroxide thereby changing its oxidation state to form α-MoO3. The change in
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oxidation state of nanoflakes transforms from blue to a visually distinct hazy blue color with
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change in absorption spectrum. The phenomenal property is explored here in sensing up to 34
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nM as limit of detection. The efficacy of the detection system was analyzed by “zone of
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inhibition” based agar diffusion assay with different concentrations of H2O2. The current
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approach is highly accurate, effective and reproducible for quantification of physiological
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concentration of H2O2 in biological fluid such as human urine.
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Keywords: two-dimensional nanomaterial; bare eye; rapid detection; H2O2; colorimetric assay;
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urine bioassay.
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Introduction
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Hydrogen peroxide (H2O2) is one of the most widely used chemical constituent in various
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industrial processes [1]. Apart from this, hydrogen peroxide plays quintessential role in variety
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of biomolecular processes such as ageing [2,3], molecular signaling [4,5], membrane damage
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[6], genetic mutation [7], carcinogenesis [8,9], neurological ailments [10,11] etc. This intricate
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involvement makes H2O2 a molecular alarm for serious physiological disorders. There are
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various techniques for detecting H2O2 using fluorescence [12,13], electrochemistry [14], bare eye
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sensor using gold nanoparticles [15]. A recent review reported by Chen et al have highlighted
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different approach for detection of hydrogen peroxide through metal oxides by electrochemical
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methods [16]. These methods require typically tedious procedures and critical observations to
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detect low concentration of H2O2. Hence facile and rapid detection of H2O2 with high sensitivity
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is in great demand for screening its presence in biological samples. Despite substantial amount
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of research reported on hydrogen peroxide sensing, rapid detection with high sensitivity still has
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a room for improvement.
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After the discovery of Graphene [17–19], other layered materials such as Transition-Metal
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Dichalcogenides (TMDs): Tungsten disulfide (WS2) [20,21], Molybdenum disulfide (MoS2)
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[22,23] and Transition-Metal Oxides (TMOs): Molybdenum trioxide (MoO3) [24,25]; there is an
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essential leaf towards development of sensors for detection of biological analytes [26]. Amongst
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the 2D materials, TMDs have been widely studied as fluorescent probes for sensing of biological
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analytes [27] and reported to be highly biocompatible [28]. The necessary transformation from
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+4 to +6 oxidation states to obtain a color of the nanomaterial is a tedious process thereby
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making it unfit for use in rapid visual detection sensing strategies [29,30]. TMOs, on the other
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hand, exhibit properties such as photochromism, electrochromism and thermochromism which
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attribute to chromogenic coloration of nanomaterial. The photochromic [31,32], electrochromic
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[33] and thermochromic [34] properties of Molybdenum oxide have been extensively studied.
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Moreover, the transition between the oxidation states in MoO3 is comparatively easy [35] thus
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making is a choice of material in this detection system .
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Exploring over molybdenum oxides, Wagner et al highlighted increased catalytic activity of
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orthorhombic MoO3 under appropriate surface defect. This property of MoO3 is still being
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explored for several applications in environmental studies [36–38] and in electrochemical
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storage devices [39]. The defects on surface can be controlled by subjecting MoO3 to mixtures of
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varying ratios of aqueous and organic solvents [40], exposure to visible light [41], xenon lamp
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[42,43], solar simulator [40] to obtain catalytically active reduced form of α-MoO3-x [44]. The
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oxygen vacancies created on the surface of α-MoO3-x nanoflakes have been shown to have
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enhanced charge storage [45] and await its applications in biological field. α-MoO3-x is the least
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explored material in biological field due to its low stability in aqueous environment [36] and
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delicate interaction chemistry with other molecules in solution. α-MoO3-x, like other 2D
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materials, is a planar structure with high aspect ratio [40]. The optical property such as surface
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plasmon resonance in α-MoO3-x, are sensitive to number of planes, which are held together by
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weak van der Waals forces and to the duration of exposure of light during the synthesis of α-
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MoO3-x [46]. This interlayer spacing is highly sensitive to interaction of protons and/or removal
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of oxygen from α-MoO3-x (Fig. 1, Panel A). The holistic impact of these processes is alteration in
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electronic band gap of the α-MoO3-x. These unique attributes enables α-MoO3-x nanoflakes as a
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promising platform for developing novel sensitive probe for biological analytes [47].
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We report the use of α-MoO3-x as a sensitive probe for H2O2 in biological fluids. To date, to the
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best of our knowledge, detection of H2O2 in biological fluid has not been reported. The detection
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system has been able to reach extremely low concentration of H2O2 with remarkable sensitivity
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ever reported before for a bare eye based colorimetric probe and for the first time in biological
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fluid. The mechanism of detection is outlined in Fig. 1. As discussed earlier, the electron
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concentration in 2D materials like MoO3 can be enhanced by encouraging oxygen vacancies,
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which forms α-MoO3-x. In the present context, this reaction is reversed as per our speculation in
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presence of H2O2. This leads to re-conversion of α-MoO3-x, a blue colored solution to oxidized
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form α-MoO3. The transformation is distinctly observed in the ambient light photograph of
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samples. The sensitivity of α-MoO3-x towards availability of oxygen makes it a favorable
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sensitive probe for H2O2. Parallel to the colorimetric assay, we have also developed an agarose
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gel diffusion method for detection of H2O2 based on its “zone of inhibition” (Fig. 1). We have
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found that our method is competitively more sensitive than most of the earlier reports as
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represented graphically in Fig. 2.
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Materials and Methods
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1. Materials
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Molybdenum trioxide (MoO3) powder, Hydrogen peroxide (H2O2), Agarose was purchased from
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Sigma, UK. All the chemicals used in experiments were of analytical grade and used without
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further purification.
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2. Instrumentation
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The morphological details of nanoparticles were elucidated using transmission electron
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microscope (TEM) (Philips, The Netherlands). 2 µl of the samples were drop coated on formvar
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coated copper grids to form uniform layer and dried under ambient temperature to ensure the
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integrity of the film. X-ray Photoelectron Spectroscopy (XPS) was performed (Photoelectron
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spectrometer ESCA, JEOL Ltd., Japan). The sample was drop casted on silicon substrate
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followed by observing the corresponding spectrum. UV-Vis spectroscopy (Thermo,
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EVOLUTION 201, USA) was carried out using standard quartz cuvette having path length 1 cm.
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Particle size analysis (dynamic light scattering DLS) was done on Zeta potential & Particle Size
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Analyzer ELSZ-2000 series, Otsuka Electronics Co. Ltd., Japan.
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3. Preparation of α-MoO3-x
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α-MoO3-x nanoflakes was synthesized using modified protocol [36]. Briefly, 0.5 g MoO3 was
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dispersed in 100 ml equal volume of water and ethanol. The mixture was sonicated for 2 h at 5
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secs ON and 2 secs OFF pulse at 125 W (Ultrasonic Processor, Qsonica probe Sonicator, USA).
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The obtained solution was then centrifuged for 30 min at 3000 rpm to collect the supernatant.
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The solution was subjected exposed to xenon lamp (Newport, USA) [44] at a power of 600 W till
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the solution turned deep blue. This solution was washed three times and dialyzed against
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ethanolic water for 48 h to obtain purified solution contained α-MoO3-x nanoflakes. To remove
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traces of ethanol and to avoid its interference in the reaction mixtures, the purified solution of
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nanoflakes was dried in rotary vacuum evaporator. The dried sample was dispersed in DI water
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followed by sonication for 15 min before further experimental use.
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4. Concentration dependent studies
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The initial test for visual detection was done in presence of 300 µl of as prepared α-MoO3-x and
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100 mM H2O2 with total reaction volume of 1000 µl. To study the sensitivity of the
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nanomaterial, H2O2 was serially diluted as 100 mM – 100 nM using DI water. This was followed
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by adding 300 µl of as prepared α-MoO3-x in a total reaction volume of 1000 µl. The mixture was
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vortexed and incubated for 10 min at room temperature after which the corresponding
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absorbance was recorded in UV-Vis spectrophotometer. The change in the particle size of α-
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MoO3-x in presence of H2O2 was analyzed by particle size analyzer. A day-light photograph of
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the reaction mixtures was captured.
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5. Understanding the sensitivity in urine samples
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A pilot study for test of H2O2 in biological fluid was done by spiking 100 mM H2O2 in urine
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sample of a lab volunteer and mixed with 300 µl as prepared α-MoO3-x in reaction volume of
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1000 µl. To further identify the sensitivity of α-MoO3-x for H2O2 in urine samples, we spiked
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H2O2 in range of 10 mM – 100 nM into the urine sample. To this, 300 µl of the as prepared α-
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MoO3-x was introduced in a 1000 µl reaction volume as mentioned above. The samples were
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vortexed and incubated for 10 mins at room temperature. The absorbance was recorded in UV-
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Vis spectrophotometer and I/I0 was calculated based on peak maxima.
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6. “Zone of inhibition” study and precedence over conventional methods for H2O2
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To exhibit the visual detection of H2O2, α-MoO3-x was introduced to 0.8% agarose solution. The
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as formed solution was then plated on petri dish and allowed to solidify. Wells of 100 µl
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volumes were bored using a borer. This was followed by adding H2O2 as test species from 100
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mM – 100 nM following ten-fold dilution in each well to finally measure colorless “zone of
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inhibition” formed on the α-MoO3-x gel.
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To assess the sensitivity of the assay system against other reactive oxygen species such as
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hydroxyl radicals, we used Fenton reaction mediated generation of OH• radicals. FeCl2 (5µM)
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and H2O2 (100mM) were mixed in a reaction volume of 1ml followed by addition of α-MoO3-x
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after 5 mins of incubation. The corresponding visual changes and spectroscopic readings were
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recorded. To comprehend the precedence of the current assay system over conventional methods
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for H2O2 detection, we used earlier reported FOX (ferrous ion oxidation in xylenol orange)
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reagent assay method [48]. Initially, urine samples from a healthy subject was collected followed
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by three concentrations of 100µM, 10µM and 1µM spiked into the urine sample to record percent
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recovery. The sensitivity of the current method was also tested on urine samples collected in
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different intervals of day such as morning (DM), afternoon (DA), evening (DE) and night (DN)
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from a donor under consideration. The test also compares with healthy sleep and sleep deprived
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urine samples collected in the following morning.
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Results & Discussions
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The optical and physical properties of 2D nanomaterials particularly transition metal oxides and
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dichalcogenides have attracted its widespread use in development of different biological sensors.
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The flexibility and easy tunability of metal oxide nanoflakes have an advantage over other
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nanomaterials that demand various molecular markers on its surface for increasing affinity
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[49,50]. Also, the use of complex nanostructures (as compared in Fig. 2) involves tedious
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synthesis processes. Apart from other metal oxides and nanocomposites [51,52], α-MoO3-x
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nanoflakes is a novel material synthesized with surface defects and enhanced oxygen vacancy
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thereby making it sensitive and selective probe for the presence of H2O2. The sensitivity of this
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material is attributable to the oxidation potential and its tendency to achieve oxidized state of α-
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MoO3. The use of α-MoO3-x also has other advantages of facile synthesis and “rapid” visual
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detection of H2O2 hence overcoming use of other metal oxides with electrochemical approach.
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Figure 1: Schematic representation of the detection system at a glance. Panel A shows the reactivity of H2O2 with α-MoO3-x nanoflakes. The oxygen a vacancy on the surface of α-MoO3-x is occupied rapidly by H2O2 thus transforming the blue colored nanoflakes to pale blue/colorless α-MoO3. Panel B shows diffusion based quantification of H2O2 in an agarose gel embedded αMoO3-x forming a “zone of inhibition”.
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Figure 2: Graphical representation: Comparing the sensitivity of colorimetric methods earlier reported for detection of H2O2 [53–63]
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Characterization of α-MoO3-x
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The TEM image (Fig. 3A) shows the formation of 2D α-MoO3-x nano-flakes. The lattice fringe
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width of 0.37 nm supports the formation of 2D nano-flakes (Fig. 3B). The associated SAED
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display diffraction spots from [2 0 0] and [0 0 2] planes confirms the formation of α-MoO3-x
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nano-flakes [36]. XPS was performed to understand their sub-stoichiometry of α-MoO3-x nano9
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flakes (Fig. 3C). The XPS analysis shows absence of impurities during the process of xenon
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lamp assisted synthesis. The presence of doublet peaks 231 eV and 234.38 eV are assigned to
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Mo+5 3d5/2 and Mo+5 3d3/2 of sub-stoichiometric α-MoO3-x nanoflakes. Fig. 3D shows the XPS
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spectra of nanoflakes after addition of H2O2. The presence of doublets at 232.58 and 235.48 eV
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are attributed to the presence of oxidized form α-MoO3. The peaks are assigned as the oxidation
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states of Mo+6 3d5/2 and Mo+6 3d3/2 [64]. DLS studies (Table 1), shows superficial morphological
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changes in α-MoO3-x nanoflakes in presence of various concentrations of H2O2.
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Figure 3: (A) TEM representative of α-MoO3-x nanoflakes, Inset: reduced image of nanoflakes; (B) SEAD pattern of α-MoO3-x nanoflakes; (C) XPS spectrum of α-MoO3-x; (D) XPS spectra after addition of H2O2
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The optical properties of α-MoO3-x, H2O2 and their mixture are displayed in Fig. 4A. The optical
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spectra of α-MoO3-x shows broad hump peak between 600-900 nm. The characteristic absorption 10
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of α-MoO3-x in visible range (600-1000 nm) is due to change in its optical properties and hence
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the deep blue color of the solution [40,45]. Interestingly, in the presence of H2O2, the peaks in
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visible domain were decreased and its intensity in UV regime was found to be enhanced. This is
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the peculiar property of oxidized form α-MoO3 [65]. Fig. 4B shows the ambient light photograph
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of color changes in presence of H2O2. This confirms the rapid bare eye detection of H2O2 in an
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effective way.
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Table 1: DLS studies of the α-MoO3-x nanoflakes in presence of various concentration of H2O2.
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Concentration of H2O2
100mM
10mM
1mM
100µM
10µM
1µM
100nM
Mean Diameter
708.9
451.3
568.3
1100.8
859.0
427.9
1257.1
Polydispersity index
0.427
0.295
0.280
0.641
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*All samples were recorded at 25°C in water
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Sensitivity of α-MoO3-x against varying concentration of H2O2
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The optical-chemistry behind the appearance of plasmon peak in visible range (600-900 nm) is
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due to the proton intercalation in the interlayer spaces of α-MoO3-x [64]. During the synthesis of
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α-MoO3-x from MoO3 under the influence of xenon lamp (600 W; 200-1500 nm), the water splits
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to H+ and OH-. The proton from this reaction sits into the interlayer spacing and initiate the
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removal of oxygen from MoO3 [66]. The overall effect of the reaction is alteration in the
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thickness of α-MoO3 nanoflakes and shift of the plasmon resonance in visible range [67]. In the
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presence of H2O2, however, this reaction gets reversed. H2O2 is an exceptionally potent oxidizer
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in acidic medium. Its presence in minute concentration can replenish the oxygen vacancies and
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transform α-MoO3-x to α-MoO3 nanoflakes.
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To comprehend the sensing capacity of α-MoO3-x, the optical response of α-MoO3-x in presence
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of different concentration of H2O2 (Fig. 4C) is proposed. As evident from the figure, there was
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proportional decrease in absorption of α-MoO3-x. The plot of I/I0 vs. concentration of H2O2 shows
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linear relationship with R2 = 0.9937 (Fig. 4D). The limit of detection was calculated to be 34 nM
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(S/N=3).
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Figure 4: (A) UV-Vis spectra of α-MoO3-x (black), α-MoO3 (red); Inset: Zoom with reduced intensity of the graph (B) Day-light photograph of H2O2 concentration dependent color change of α-MoO3-x; (C) UV-Vis spectral change of α-MoO3-x in presence of varying concentrations of H2O2; (D) Relative intensity curve (I/I0) and Regression analysis.
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Sensitivity in biological fluids
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The physiological stress from sleeplessness instigates the production of H2O2 [3,68] and is
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abundantly present in urine under physiological stress. Hence, to detect the presence of
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physiological concentration of H2O2 in human urine (biological fluid); we collected samples
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from four volunteers in lab working sleeplessly overnight. The urine samples were spiked with
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varying concentration of H2O2 and the UV-Vis absorbance was recorded (Fig. S1). The change in 12
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concentration of H2O2 was found to be proportional with the spectra in presence of biological
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fluid. Fig. 5A represents the relative intensity I/I0 plot against concentration with R2 = 0.9333.
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Fig. 5B demonstrates the transformation of solution from blue to colorless after addition of
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biological fluid collected from stressed volunteers to α-MoO3-x. A set of test sample was also
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compared with 100 mM H2O2 to observe the visible change as shown in Fig. 4B.
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Figure 5: (A) Relative intensity I/I0 vs. Concentration of H2O2 spiked in biological fluid. (B) Day-light photograph of color change in H2O2 spiked biological sample and its comparison with controls (colorless – 100 mM H2O2; blue – α-MoO3-x).
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“Zone of inhibition” study and precedence over conventional methods for H2O2
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A simple and effective method was designed for determination of the concentration of H2O2 in
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unknown samples like biological fluids. α-MoO3-x is often found to be highly unstable in solution
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due to rapid exchange of protons from the surrounding. To avoid this, we incorporated α-MoO3-x
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in agarose and poured in petri dish (Fig. S2). After punching holes in the gel, different
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concentration of H2O2 was filled in the wells and zone of inhibition was recorded (Table 2). A
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clear zone was apparent on the agarose gel due to diffusion of H2O2 and the size of the zone
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varied corresponding to different concentration of H2O2 (Fig, 6A).
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The sensitivity of the assay system against other reactive oxygen species such as OH• radical was
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assessed using Fenton reaction. Fe2+ in presence of H2O2 generates OH• radicals and Fe3+. After
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addition of α-MoO3-x, the relative absorbance obtained suggest clear transformation of the
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nanoflakes to α-MoO3 corresponding to stabilization of oxygen vacancies (Fig. S2A). This is
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indicative that the current assay system is sensitive to reactive oxygen species. In comparison to
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conventional methods, current assay method shows comparable response to the FOX reagent
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based assay of H2O2 spiked urine samples (Fig. S2B). The percent recovery is attributable the
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inherent H2O2 concentration in urine sample. To comprehend the impact of physiological stress
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on analytical performance of proposed method, the urine samples were collected at different time
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intervals which indicate the varying levels of stress (each stress are explained in Fig. 6B). Based
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on the interpretation of results, the ability of the proposed probe was excellent to discriminate
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differential level of hydrogen peroxide in the urine (indicative of different physiological stress).
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Fig. 6: (A) Representative of “zone of inhibition” recorded in petri dish containing α-MoO3-x
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infused 0.8% agarose. Wells contain various concentration of H2O2 from 100 mM – 0.0001 mM;
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(B) Relative absorbance showing selective analysis of urine samples for H2O2 using α-MoO3-x in
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Control (healthy donor), samples collected in morning (DM), afternoon (DA), evening (DE), night
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(DN) and with proper sleep (DS), sleep deprived (DSD).
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Table 2: “Zone of inhibition” recorded in petri dish containing α-MoO3-x infused 0.8% agarose. Wells contain various concentration of H2O2.
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Concentration
Zone of Inhibition (cm)* 14
RSD
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100 mM
1.4
0.2
10 mM
1.34
0.0529
1 mM
1.21
0.041
0.1 mM
0.97
0.02
*Mean of zone of inhibition (n=3) Zone of inhibition = -0.1417 Concentration + 1.585 R2 = 0.9217
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Conclusion
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α-MoO3-x has been reported widely for its use in development of electrochemical storage devices
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and environmental applications. The visually distinct blue color in the solution corresponds to
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optical characteristics observed. The oxidation property of α-MoO3-x has been explored in a
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facile assay method for colorimetric detection of H2O2. The method is based on “rapid” bare eye
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detection and hence more informative in case of physiological ailments. This study establishes a
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new platform in detecting biological samples such as urine or blood by bare eyes using sub-
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stoichiometric α-MoO3-x nanoflakes. We believe that this is a significant contribution to be first
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of its kind report in biological application of α-MoO3-x at such high sensitivity.
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Acknowledgement
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We thank the Ministry of Science and Technology of Taiwan for financial support with the grant
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number of MOST 104-2113-M-110-003-MY3.
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Figure 1: Schematic representation of the detection system at a glance. Panel A shows the reactivity of H2O2 with α-MoO3-x nanoflakes. The oxygen a vacancy on the surface of α-MoO3-x is occupied rapidly by H2O2 thus transforming the blue colored nanoflakes to pale blue/colorless α-MoO3. Panel B shows diffusion based quantification of H2O2 in an agarose gel embedded αMoO3-x forming a “zone of inhibition”.
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Figure 2: Graphical representation: Comparing the sensitivity of colorimetric methods earlier reported for detection of H2O2 [53–63]
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Figure 3: (A) TEM representative of α-MoO3-x nanoflakes, Inset: reduced image of nanoflakes; (B) SEAD pattern of α-MoO3-x nanoflakes; (C) XPS spectrum of α-MoO3-x; (D) XPS spectra after addition of H2O2
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Figure 4: (A) UV-Vis spectra of α-MoO3-x (black), α-MoO3 (red); Inset: Zoom with reduced intensity of the graph (B) Day-light photograph of H2O2 concentration dependent color change of α-MoO3-x; (C) UV-Vis spectral change of α-MoO3-x in presence of varying concentrations of H2O2; (D) Relative intensity curve (I/I0) and Regression analysis.
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Figure 5: (A) Relative intensity I/I0 vs. Concentration of H2O2 spiked in biological fluid. (B) Day-light photograph of color change in H2O2 spiked biological sample and its comparison with controls (colorless – 100 mM H2O2; blue – α-MoO3-x).
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Fig. 6: (A) Representative of “zone of inhibition” recorded in petri dish containing α-MoO3-x
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infused 0.8% agarose. Wells contain various concentration of H2O2 from 100 mM – 0.0001 mM;
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(B) Relative absorbance showing selective analysis of urine samples for H2O2 using α-MoO3-x in
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Control (healthy donor), samples collected in morning (DM), afternoon (DA), evening (DE), night
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(DN) and with proper sleep (DS), sleep deprived (DSD). 5
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Fig. S1: UV-Vis absorbance spectra of α-MoO3-x in presence of varying concentration of urine
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g. S2: (A) Selective assay of OH• radicals generated using Fenton Reaction (Control: α-MoO3-x).
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Inset: (a) α-MoO3-x, (b) FeCl2, (c) Test sample α-MoO3-x+FeCl2+H2O2; (B) Comparing percent
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recovery of three concentrations of H2O2 in urine samples using current method and
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conventional method using FOX reagent. 6
ACCEPTED MANUSCRIPT Highlights 1. We have developed a bare eye sensor for Hydrogen peroxide 2. α-MoO3-x is used as Rapid detection probe for Hydrogen peroxide 3. The method has also been tested for the first time on biological fluids to affirm the
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4. It has also been tested on agar well diffusion assay detection based on its “zone of
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inhibition”