Enzyme mimics are developed as an alternative to natural enzymes to overcome the inherent limitations of natural enzymes. Among different types of enzyme mimics, nanozymes gained importance due to their tuneable catalytic properties. In this article, we discuss the peroxidase behaviour of different shape V2O5 nanoparticles (NPs). A simple spectrophotometric method is presented for the quantification of glucose and H2O2 using novel chromogenic reagents. The NPS are characterized with SEM, DLS, EDS, FTIR and XRD. From SEM images, based on the morphology, the NPs were named as vanadium nanosheets (VNShs), nanoflowers (VNFws) and nanospheres (VNSps). The average crystalline size of the nanoparticles is calculated using XRD data from Scherrer’s equation and Williamson-Hall plot and was found to be 45.42, 45.7nm for VNShs, 29.14, 32.5nm for VNFws, and 39.83, 38.7nm for VNSps respectively. The linearity of glucose was ranged from 0.0289 to 0.925mM for HRP, VNShs VNFws, and 0.925 to 0.0528mM for VNSps. The H2O2 was in good linear range between 0.003 to 1.9383mM in both rate and fixed time method for all nanozymes and HRP. For recovery study 10µL serum sample was directly used without dilution. The Km values were found to be 1.6239 mM for HRP, 0.7843 mM for VNShs, 0.6514 mM for VNFws, ands 0.6398 mM for VNSps concluding that NZs have better affinity towards substrate molecule. The detection limit and quantification limits were found to be 0.0548mM and 0.1662mM for HRP, 0.066mM and 0.2002mM for VNShs, 0.0425mM and 0.1287mM for VNFws and 0.1474mM and 0.4465mM for VNSps.
Research Article
Peroxidase Mimicking V2O5 Nanozymes as the Spectrophotometric Sensor for the Determination of Glucose in Human Serum Sample Employing New Chromogenic Co-Substrates
https://doi.org/10.21203/rs.3.rs-5318695/v1
This work is licensed under a CC BY 4.0 License
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vanadium oxide nanoparticle
nanozyme
peroxidase mimics
Horseradish peroxidase
glucose
glucose oxidase
Reactive oxygen species (ROS), are a collective form of various reactive molecules and free radicals (species with unpaired electrons) which are intermediates of molecular oxygen such as hydrogen peroxide (H2O2), hydroxyl radicals (.OH), and super oxides (−O2)[1, 2]. ROS are produced by physiological processes in biological systems during which molecular oxygen is converted into oxygen radicals which can cause hepatitis [3], glomerulonephritis [4], and inflammatory bowel disease[5]. Several literatures have exposed those diseases like cancer, and neurodegenerative disorders due to the unevenness between the production and removal of hydrogen peroxide (H2O2) and other reactive species[6, 7]. H2O2 is an ROS, that is produced from the combination of hydroperoxyl radicals (HO2·) and their hydrated form in the atmosphere[8]. In humans and animals, it is produced in small amounts as a byproduct of normal cellular metabolism through certain enzyme activities like oxidase which can transfer electrons to oxygen molecules[9]. H2O2 is present in the human body in the kidney, urinary tract, and bladder[10]. The concentration of H2O2 is a significant parameter giving an edge between harmless and harmful impacts on the body. In small amounts, it plays a prominent role in the immune system of animals. However, the high level of concentration of H2O2 is cytotoxic and can also cause damage to cells[11], biomolecules like DNA [12], tissues[13], proteins & lipids which can lead to inflammation and cell death[14]. Because of that, it is important to quantify the H2O2 in real samples. Natural enzymes are used for the quantification of these ROS. Enzymes act as biocatalysts that catalyse biochemical reactions and enable the cells to function properly[15]. Enzymes like peroxidase[16] and catalase[17] catalyse the reduction of hydrogen peroxide to water and organic hydroperoxides to corresponding alcohols[18]. Horseradish peroxidase (HRP) is a porphyrin enzyme that contains iron as a central metal ion that catalyses the oxidation of a variety of electron donors by H2O2.
Extraction of enzymes is a tedious process making them costly and these enzymes are sensitive towards limited reaction conditions like pH and temperature makes them bit selective[19]. In this regard enzyme mimics, which are synthetic compounds that can mimic the enzyme's catalytic properties and offer more cost-effective, greater stability, low cost, and the ability to function in wide range of conditions for catalysing specific chemical reactions. The Nanoscience and nanotechnology provide us with a new range of features that are often not formed in bulk materials, since metal oxides are the most fascinating functional materials, they have drawn a lot of attention from many researchers in synthesizing and characterizing the metal oxide nanostructures with different morphologies to expose them to a wide variety of applications in medicine, biosensors, nanozymes (NZs), and environmental science, among many others. Among different types of enzyme mimics, NZs (NPs with enzyme activity) are recently emerged enzyme mimics. Recent exploration of enzymatic behaviour of NPs has provided a greater insight towards the mimic enzyme behaviour. Ferromagnetic iron oxide nanoparticles (NPs) are the first reported NZs for peroxidase mimics[20], after that many NPs are reported as NZs for different enzyme mimics, Layered vanadium(IV) disulfide nanosheets[21], Fe-MIM/ZIF-8 [22], Pt/cube-CeO2 nanocomposite [23], Bi2Fe4O9 NPs [24], FA@Ag NPs [25], Cu2+-modified hollow carbon nanosphere[26], Ru–N–C NZs [27] for peroxidase activity, Fe3+/AMP NPs [28], Cu-MOFs [29], Nanostructured binuclear Fe(III) and Mn(III) porphyrin materials [30], Co3O4 nanocrystals [31], Fe–N4 NZs [32] for catalase activity, polydopamine-decorated CuO [33], Au NRs-Pd@HA [34], Au-integrated Fe single-atom nanozyme [35], MMSN/Au NPs [36], GOD-GO/MnO2 [37] for GOD activity, are recently reported NZs. In most of the NPs reported for the peroxidase mimics, the reaction is optimum at certain reaction conditions like pH, temperature, or the reagents are being costly.
In contrast of the above demerits, in this article we report the peroxidase mimicking behaviour of vanadium oxide NPs which has been synthesised with three different shapes, its kinetic parameters have been evaluated and compared each other and also with HRP to analyse its efficiency, and the same is applied for the glucose quantification using glucose oxidase (GOD) in human serum sample without dilution. Novel chromogenic co-substrates, P-aminophenol sulphate (PAP) & N-(1-Naphthyl) ethylenediamine dihydrochloride (NEDA) are used for the quantification of glucose and H2O2 using simple UV-Vis spectrophotometric method.
Reagents and their preparation
All chemicals used in the assay were of analytical grade. Reagents were freshly prepared using double distilled water. PAP was purchased from Himedia. Ltd, India, and the stock solution (72.39mM) was prepared by dissolving 150mg PAP in 10m of distilled water. NEDA was purchased from Sisco Research Laboratories Pvt. Ltd. (SRL) – India, and the stock solution (7.717mM) was prepared by dissolving 20 mg NEDA in 10mL of distilled water. Horseradish peroxidase (139 U mg–1) was purchased from SRL chemicals (Mumbai, India), and the stock solution was prepared by dissolving 1mg of peroxidase in 100mL of KH2PO4/NaOH buffer of pH-6 and the solution was stored in the fridge at -4oC until use. GOD with 175 U/mg activity was purchased from Sigma–Aldrich and the stock solution was prepared by dissolving 5mg in 5mL of distilled water. Glucose was purchased from SRL chemicals and the stock solution (55.5mM) was prepared by dissolving 99.9mg glucose in 10 mL of the solution. H2O2 was purchased from Molychem (Mumbai, India), the stock solution (116.3mM) was prepared by diluting 1mL of H2O2 to 100mL with double distilled water and the concentration was verified by titrating it with standardized potassium permanganate solution. The stock solutions were diluted to required concentrations with double distilled water.
Instrumentation
LABMAN (LMSP-UV1200) spectrophotometer, 3cm3 quartz cells was used to record all absorbance measurements. The prepared materials were subjected to characterization using scanning electron microscope (SEM, Hitachi S 3400 N) for Particle size and morphology, elemental composition and purity, Dynamic Light Scattering (DLS, MicrotracNanotrawave) for Size, size distribution, and stability of NPs, FT-IR spectroscopy (PerkinElmer Spectrum), X-ray diffraction spectrophotometry (XRD, RegakuSmartLab) for the crystallinity and crystal size of NPs. From the XRD spectra, the crystalline diameter (D) was obtained from the XRD spectra using Scherrer’s equation, i.e., d=\(\:\frac{K\lambda\:}{{\beta\:}cos\theta\:}\) and Williamson-Hall plot.
Collection and processing of human serum sample for recovery study
A pre-quantified human blood sample was collected from a clinical diagnostic centre from a healthy volunteer who was in a good condition and not suffered from any disease with their approval. After that the acquired blood sample was centrifuged in a Remi R-24 Centrifuge above 10000 rpm speed at room temperature. The supernatant solution of serum was collected in a heparin container and kept at -40C until use. The remaining was discorded after adding Trichloroacetic acid as per the clinical laboratory guidelines.
Synthesis of different morphological Vanadium oxide nanostructures.
The synthesis of vanadium oxide NPs followed the method [38] with slight modifications.
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Vanadium Nanosheets (VNShs); Simple wet synthesis approach was incorporated to synthesis vanadium oxide VNShs. 300mg of vanadium pentoxide (V2O5) was sonicated in 30mL of double distilled water and later stirred for 15 minutes at 1200rpm. 15mL of 30% H2O2 was added dropwise at the rate of 1.5mL/min. The change in color of the reaction mixture from yellow to orange and finally to dark brown confirms the formation of hydrogen diperoxodioxovanadate(III)[39]. The stirring was continued for three hours after the formation of dark brown colour. The reaction mixture was diluted by adding 10 mL of double distilled water. The temperature was raised to 600C to accelerate the gelation process of hydrated V2O5 to get viscous brownish gel. The obtained gel was dried at 1000C for 12 hours and calcinated at 4000C for 6hrs to obtain the vanadium oxide VNShs.
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Vanadium Nanoflowers (VNFws); Hydrothermal method was adopted to synthesis the vanadium oxide VNFws. 180mg of V2O5 precursor in 50mL of double distilled water was initially sonicated for 10 min. To the suspended mixture 8mL of 30% H2O2 was added drop wise at the rate of 0.5mL/min with continuous stirring. Formation of 4[39]was confirmed by the change in color of the reaction mixture to dark brown. To the above reaction mixture 170mg of sodium dihydrogen orthophosphate was added and continued stirring for 30 min, the mixture was then transferred to the Teflon lined autoclave and placed in oven at 1800C for six hours and finally allowed to cool to room temperature. The formed precipitate was separated by centrifuging at 5000 rpm and washing with double-distilled water later was dried at 700C and calcinated at 4000C for four hours.
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Vanadium Nanospheres (VNSps): Precipitation process was used to synthesis the vanadium oxide nanospheres. 350mg of ammonium metavanadate was dissolved in 100mL of double distilled water and stirred for 15min. The reaction mixture turns yellow transparent solution by the addition of 1.5mL of 1M HCl dropwise confirming the formation of Vanadium oxytrichloride (VOCl3). The VOCl3 is reduced to vanadyl trichloride by adding 4.5mL hydrazine hydrate added dropwise at the rate of 1mL / 5min. The grey-colored precipitate formed confirms the formation of Vanadyl trichloride which is separated by centrifugation and repeatedly cleaning with double distilled water. The obtained precipitate was dried for 12 hours at 700C and roasted at 4000C for 5 hours. During roasting process, the vanadyl trichloride undergoes air oxidation to V2O5 With the liberation of Cl2 gas.
Optimization of reaction parameters
Absorption spectra; The maximum absorbance of the resultant product obtained by HRP and vanadium oxide NZs of the reaction mixture was identified by using the proposed assay method with a scanning rate of 2 nm/S in the wavelength range of 400-700nm and was found to be 470nm for HRP and all NZs as shown in the Fig. 1. This confirms the produced product is the same in all cases.
Buffer & pH; Different buffers of various concentrations from 0.01mM to 50 mM included A) CH3COOH/CH3COONa buffer, B) citric acid/potassium citrate buffer, C) KH2PO4/K2HPO4 buffer, D) Tris buffer E) KH2PO4/NaOH buffer were used with reaction mixture and incubated for 10minutes. The absorbance of KH2PO4/NaOH and tris buffer (buffers with higher pH range) with VNShs and VNFws is less due to development of blank reagent colour, with HRP the absorbance is very less because of lower activity of HRP at higher pH. V2O5 NZs shows good activity with wide range of buffers but maximum was with CH3COOH/CH3COONa buffer. Among different buffers, 3.33mM CH3COOH/CH3COONa buffer showed better absorbance for all NZs and HRP, as shown in inset of Figure (2). Different pH solutions of CH3COOH/CH3COONa buffer were prepared and mixed with reaction mixture. The result shows a good absorbance at pH 3.8 for all NZs and HRP, VNFws shows good absorbance from pH 3.8 to 4.5 with less deviation, as shown in Fig. 2. Hence, 3.33mM CH3COOH/CH3COONa buffer of pH 3.8 in the final 3mL of the reaction mixture was chosen as optimum buffer for future work with all NZs and HRP.
Temperature; The effect of temperature was studied for HRP and all NZs from 10 to 50oC. The result shows a maximum absorbance at 20oC for HRP, whereas NZs show a considerable absorbance from 10 to 25oC as shown in Fig. 3. Hence the reaction with HRP is optimum at 20oC whereas the reaction with NZs can be performed with variable temperature range from 10 to 25oC with less deviation.
Linearity of glucose
A calibration graph for glucose was constructed for HRP and all V2O5 NZs, using fixed-time assay method. Absorbance of the reaction mixture contains optimum concentration of all the reagents [as mentioned in table (1)] and 17.5 units of GOD with varying concentrations of glucose (0.0144 to 3.7mM) was recorded at 470nm with respect to blank reagent mixture after the incubation period of 10 minutes. The result shows good linearity between 0.0289 to 0.925mM for HRP, VNShs VNFws, and 0.0528 to 0.925mM for VNSps. The linearity of glucose with respect to HRP and all NZs is showing in the Fig. 4.
Linearity of HO
A calibration graph for H2O2 was constructed for HRP and three V2O5 NZs using both rate and fixed-time assay methods. In the rate method, the absorbance was recorded with respect to the control blank for 5 minutes with a time interval of 1 minute at 470nm. In fixed time assay method, the reaction mixture was incubated for 10 minutes and the absorbance was recorded with respect to the control blank at 470nm. The linearity for the assay of H2O2 was investigated in 3 mL of the reaction mixture and the concentration of reagents used are tabulated in Table (1) for HRP and all NZs.
With respect to rate method, HRP and VNFws showed a limited linearity range between 0.0242-0.3877mM. VNSps and VNShs showed a very good response towards H2O2 with a large linearity range between 0.0606-0.9692mM and 0.0969-1.5507mM respectively. This avoids the dilution of real samples for its application studies. Due to the different linearity range of H2O2 for enzyme and NZs, depending on the sample, selectivity of NZs can be done for the quantification of H2O2.
In fixed time method, HRP showed linearity between 0.0030-0.3877mM, linearity in very lesser concentration of H2O2, whereas in NZs, VNSps showed a linearity in lower concentration of H2O2 between 0.0076-0.9692mM. VNShs and VNFws showed a linearity upto same lower concentration i.e. between 0.0121-0.7753mM and 0.0121–0.3877 mM respectively. The linearity of H2O2 with respect to HRP and all NZs bye rate and fixed time method are showing in the Fig. 5a & Fig. 5b respectively.
| Reagents | HRP | VNShs | VNFws | VNSps | |
|---|---|---|---|---|---|
| PAP (mM) | 2.413 | 1.206 | 1.206 | 1.609 | |
| NEDA (mM) | 0.193 | 0.257 | 0.129 | 0.129 | |
| Buffer (mM) | 0.33 | 0.33 | 0.33 | 0.33 | |
| HRP or NZs | 0.0185 units | 0.3299mM | 0.1099mM | 0.2199mM | |
| Linearity of H2O2 (mM) | Rate method | 0.0242–0.3877 | 0.0969–1.5507 | 0.0242–0.3877 | 0.0606–0.9692 |
| Fixed time method | 0.0030–0.3877 | 0.0121–0.7753 | 0.0121–0.3877 | 0.0076–0.9692 | |
| Regression equation (Y=) | Rate method | 0.2504x + 0.0063 | 0.0823x − 0.0004 | 0.3027x − 0.0013 | 0.072x − 0.0011 |
| Fixed time method | 1.8423x + 0.0374 | 1.1963x + 0.0113 | 1.5118x − 0.0077 | 0.389x + 0.0272 | |
| Regression coefficient (R2) | Rate method | 0.9954 | 0.9957 | 0.9978 | 0.9993 |
| Fixed time method | 0.9973 | 0.9992 | 0.9991 | 0.9991 | |
Table (1): linearity range of H2O2 and concentrations of reagents used.
Effect of concentration of analytical reagents on the rate of reaction
The effect of varying concentrations of analytical reagents (PAP, NEDA, and H2O2) on the rate of the reaction was investigated under experimental settings with 3mL solution by varying the concentration of one reagent at a time. The results show an increase in the absorbance with an increase in the concentration of analytical reagents up to optimized concentration afterward, there is no significant change in the rate of the reaction or the rate of the reaction decreases slightly. As a result, the final optimal concentration was fixed at the same level for all further experiments. Optimized concentrations of all analytical reagents are tabulated below (Table 2). The graphs of rate versus concentration of PAP, NEDA, and H2O2 are shown in Fig. S1, S2, S3. & S4.
Therefore, the concentrations of reagents needed by NZs are far lower than those needed by HRP enzyme. The PAP concentration needed by VNShs and VNFws are same and lower than that of VNSps, which is lower than that of HRP. NEDA required by the VNFws and VNSps are same, and greater than the HRP, which is greater than VNShs. H2O2 required by the HRP and VNFws is same and lesser than the VNSps and which is lesser then the VNShs.
| Reagents | HRP | VNShs | VNFws | VNSps |
|---|---|---|---|---|
| PAP (mM) | 2.413 | 1.206 | 1.206 | 1.609 |
| NEDA (mM) | 0.193 | 0.257 | 0.129 | 0.129 |
| H2O2(mM) | 0.3877 | 1.551 | 0.3877 | 0.9692 |
| NZs | 0.0185 units | 0.3299mM | 0.1099mM | 0.2199mM |
Table (2): Optimized values of analytical reagents.
Evaluation of analytical characteristics of the proposed assay
The Michaelis–Menten constant (Km) for PAP, NEDA & H2O2 was determined by Lineweaver–Burk plot, keeping all the reagents at optimized condition & by varying one reagent concentration (PAP, NEDA & H2O2) at a time. The Km values for PAP, and NEDA are 1.0867 and 0.3142mM for HRP, 0.3179 and 0.0327mM for VNShs, 0.2108 and 0.07499mM for VNFws, 0.4625 and 0.3085mM for VNSps and the Lineweaver–Burk plots are showed in Fig. S5, S6, S7, & S8. Km values with respect to H2O2, Vmax, Kcat, and Keff are tabulated (Table 3). Lineweaver–Burk plots for the determination of Km values with respect to H2O2 were showed in the Fig. 6.
Km values for all NZs is lesser than that of the HRP enzyme in the increasing order of VNSps, VNFws, VNShs and HRP which indicates the large affinity of NZs towards substrate (H2O2) than HRP enzyme. Vmax is maximum for VNShs is greater than the HRP followed by VNFws and VNSps. Kcat and Keff of HRP is much greater than all NZs which is in the increasing order of VNShs, VNSps and VNFws. Comparison of catalytic parameters are showen in Table (4) indicates that the synthesised NZs have higher affinity towards substrates than some reported NZs.
| Reagents | Km for H2O2 (mM) | Vmax (mM/sec) | Kcat (sec− 1) | Keff (mM− 1sec− 1) |
|---|---|---|---|---|
| HRP | 0.8051 | 0.3042 | 16.4166 | 20.3908 |
| VNShs | 0.7843 | 0.3369 | 1.0212 | 1.3021 |
| VNFws | 0.6514 | 0.2508 | 2.2817 | 3.5034 |
| VNSps | 0.6398 | 0.2133 | 1.9408 | 3.0334 |
Table (3): Catalytic parameters.
| Sl. No. | Nanozyme | H2O2 linearity, LOD | Km | Reference |
|---|---|---|---|---|
| 1 | Nickel metal-organic framework 2D NShs | 0.04–160µM LOD-8 nM | TMB-0.365mM H2O2-2.49mM | [40] |
| 2 | porous PtCu dendrites | 0.3–325µM LOD-0.1 µM | TMB- 0.08 mM H2O2- 0.26 mM | [41] |
| 3 | Pt NPs | 1–50µM LOD-1µM | TMB-0.091 mM H2O2-80.25 mM | [42] |
| 4 | rhodium nanoparticles | 1-100µM LOD- 0.20 µM | TMB-0.198 mM H2O2- 0.38 mM | [43] |
| 5 | VNShs | 0.0969-1.5507mM LOD-0.066mM | H2O2- 0.7843mM PAP- 0.3179 and NEDA-0.0327mM | Present work |
| VNFws | 0.0242-0.3877mM LOD-0.0425mM | H2O2- 0.6514mM PAP- 0.2108 and NEDA- 0.0749mM | ||
| VNSps | 0.0606-0.9692mM LOD-0.1474mM | H2O2- 0.6398mM PAP- 0.4625 and NEDA- 0.3085mM |
Table (4): comparison of catalytic parameters.
Characterization of vanadium oxide Nano crystals.
SEM and EDS analysis
FE-SEM images of vanadium oxide NPs synthesized by different methods were showed in Fig. 7 & S9, S10, S11. Based on the type of the morphology, the NPs were named as VNShs, VNFws and VNSps. During heat treatment of hydrogen diperoxodioxovanadate (III) complex to form brownish gel (V2O5.nH2O), sheet like NPs is formed. When brown solution of hydrogen diperoxodioxovanadate (III) complex was treated with sodium dihydrogen orthophosphate followed by hydrothermal process, flower like NPs were formed. When the yellow transparent solution of ammonium metavanadate and HCl was treated with reducing agent like hydrazine hydrate forms spheres like NPs. Elemental composition of the NPs was showing the presence of 41.97% of vanadium and 58.03% of oxygen in VNShs, 51.56% vanadium and 48.44% of oxygen in VNFws, 36.2% of vanadium and 63.8% of oxygen in VNSps, which indicates the formation of pure vanadium oxide NPs. EDS spectres with percentage compositions were showed in Fig. 8.
XRD
The XRD pattern of the NPs were shown in the Fig. 9. The XRD peaks of VNFws and VNSps, the different peaks were exactly matched with standard card JCPDS no. 41-1426, representing vanadium pentoxide NPs in orthorhombic phase. whereas VNShs were matched with [44], which correspondence to rhombohedral structure of the V2O5. The average crystalline size of the synthesised NPs was calculated using Scherrer’s and W-H equations and was found to be 45.42, 45.7nm for VNShs, 29.14, 32.5nm for VNFws, and 39.83, 38.7nm for VNSps. The degree of crystallinity index was calculated and was found to be 0.7978 for VNShs, 0.7856 For VNFws and 0.8570for VNSps. Therefore, in VNShs 79.78% is in crystalline and 20.22% is in amorphous form, in VNFws 78.57% is in crystalline and 21.43% is in amorphous form, and in VNSps, 85.70% is in crystalline and 14.30% is in amorphous form. Raw XRD patterns were showed in Fig. S15, S16 & S17.
DLS
The variations in the intensity of scattered light resulting from Brownian motion in synthesized NPs, size, and size distribution can be predicted with the help of the distribution curve in DLS spectra. The resultant DLS spectrums and size distribution of different morphological vanadium oxide NPs are shown below in Table (5). It showed that the size distribution by the analysis of DLS are in good agreement with histogram plot data which is showed in Table S1, S2 & S3. Zeta potential is a stability indicator of NPs, which is influenced by the surface charge and is measured by analysing particles' electrophilic mobility in an electric field. The Zeta potential of VNShs is 4.0mv, VNFws is -4.5mv and VNSps is 4.6mv which are almost neutral[45]. The Polydispersity index (PDI) is a parameter used to measure the size distribution, specifying the uniformity of NPs. International standards organizations have established that PDI values of 0.1 to 0.25 indicate a small size distribution (monodisperse samples) and values greater than 0.5 are common to broad distribution (polydisperse samples)[46, 47]. PDI values of VNShs is 1.144, VNFws is 1.834 and VNSps is 1.896 which indicates polydispersity with multiple-sized particles, which might be due to the rapid agglomeration of the NPs due to low zeta potentials[48]. Size distribution spectres as obtained by DLS analysis is showed in the Fig. 10. DLS result on Surface charge measurements are showed in Fig. S12, S13, & S14.
| NZs | Volume % | Diameter(nm) |
|---|---|---|
| VNShs | 100 | 349 |
| VNFws | 44.7 | 390 |
| 55.3 | 180.8 | |
| VNSps | 100 | 254.1 |
Table (5): size distribution of different shaped vanadium oxide NZs.
FT-IR Spectra analysis
To study the nature of binding between vanadium and oxygen in V2O5 NPs, FT-IR was recorded in the range of 400 to 4000cm− 1 wave number, which identifies the chemical bonds as well as functional groups in the compound. FTIR spectra of V2O5 NPs exhibited three characteristic vibration modes. The peaks near 470 cm− 1 are due to the V-O-V bond symmetric stretch, 800cm− 1 is due to the V-O-V asymmetric stretch and the peak around 1000cm− 1 can be assigned to the V = O stretching, the peaks around 600 to 700cm− 1 is potentially originate from the V − O bond, and the peaks from 1950 to 2350cm− 1 are due to δ(HOH) and ט γ(OH) in water. FRIR spectres of V2O5 NPs were showed in Fig. 11.
Influence of interfering species
To study the effect of interfering species, optimized experimental conditions were used by taking several cations, anions and amino acids using hydrogen peroxide concentration of 0.0969mM for HRP, 0.1938mM for both VNShs and VNFws and 0.4845mM for VNSps. Influence of interfering species is tabulated in Table (6) as tolerant ratio. It is the ratio of limit of interfering species concentration to that of the concentration of hydrogen peroxide. The result shows high interference for Fe2+, Fe3+, and FAS (iron-containing salts) with HRP and all NZs. Zr4+, Na2CO3, Glycine, Glucose, and Alanine show average interference, whereas NH4Cl, Na+, Mg2+, Zn2+, Zr4+, KNO3, and Urea show less interference to a large extent.
|
| Tolerant ratio | |||||
|---|---|---|---|---|---|---|
| HRP | VNShs | VNSps | VNFws | |||
| Cl− 1 | 360 | 715 | 1429 | 360 | ||
| Na+, Mg2+ | 280 | 550 | 1100 | 270 | ||
| Zn2+ | 55 | 210 | 395 | 90 | ||
| Se4+ | 20 | 163 | 325 | 90 | ||
| Ni2+ | 20 | 210 | 430 | 50 | ||
| Zr4+ | 40 | 70 | 250 | 20 | ||
| Co2+ | 55 | 15 | 20 | 5 | ||
| Cu2+ | 10 | 10 | 0.05 | 0.05 | ||
| Na2CO3 | 60 | 10 | 250 | 35 | ||
| KNO3 | 165 | 330 | 660 | 165 | ||
| Urea | 430 | 210 | 430 | 105 | ||
| Glycine | 80 | 85 | 170 | 50 | ||
| Glucose | 40 | 70 | 140 | 35 | ||
| EDTA | 20 | 10 | 85 | 5 | ||
| Alanine | 80 | 70 | 85 | 20 | ||
| Leucine, Isoleucine | 55 | 50 | 50 | 10 | ||
| L-Histidine | 45 | 40 | 85 | 10 | ||
| cysteine | 10 | 25 | 110 | 10 | ||
| Methionine | 45 | 50 | 85 | 50 | ||
| Cetric acid | 40 | 15 | 140 | 5 | ||
| D-Valine | 55 | 55 | 110 | 25 | ||
| glutamic acid | 10 | 50 | 50 | 25 | ||
| L-ornithine HCl | 40 | 40 | 20 | 20 | ||
| Fe3+ | 0.0282 | 0.0029 | 0.0058 | 0.0283 | ||
| Fe2+ | 0.2861 | 0.2887 | 0.0231 | 0.0014 | ||
| FAS | 0.2861 | 0.0029 | 0.0058 | 0.0282 | ||
Table (6): Interference study of different metal ions, organic and organic compounds.
Precision study
Precision studies were performed at optimized conditions of all the reagents along with four different concentrations of H2O2 within the linearity range. The study included 10 runs in a day with a time interval of 1 h for within-day precision and 10 days run for day-to-day precision with one day interval. All solutions were prepared freshly every day(n = 10). The standard deviation (SD) and percentage standard deviation (%SD) were showed in Table (7). The results show Intraday %SD is greater than the Inter-day %SD in all cases and VNSps has highest %SD when compared to other NZs and HRP. The Limit of detection (LOD), and Limit of quantification (LOQ) were determined by taking the readings of blank regent versus blank reagent. LOD and LOQs were found to be 0.0548 mM and 0.1662 mM for HRP, 0.066 mM and 0.2002 mM for VNShs, 0.0425 mM and 0.1287 mM for VNFws and 0.1474 mM and 0.4465 mM for VNSps.
| HRP | VNShs | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| [H2O2] in mM | SD | %SD | [H2O2] in mM | SD | %SD | ||||
| Intraday | Inter-day | Intraday | Inter-day | Intraday | Inter-day | Intraday | Inter-day | ||
| 0.3877 | 0.022 | 0.0252 | 1.7933 | 2.0629 | 0.1938 | 0.0066 | 0.0106 | 0.8221 | 1.3616 |
| 0.1938 | 0.0043 | 0.0078 | 0.7192 | 1.3288 | 0.0485 | 0.0072 | 0.0081 | 1.0815 | 1.1883 |
| 0.0969 | 0.0037 | 0.0038 | 1.584 | 1.6163 | 0.0242 | 0.004 | 0.0055 | 0.8156 | 1.0317 |
| 0.0484 | 0.0016 | 0.0021 | 1.4266 | 1.8851 | 0.0121 | 0.0048 | 0.0060 | 1.1507 | 1.4792 |
| VNFws | VNSps | ||||||||
| [H2O2] in mM | SD | %SD | [H2O2] in mM | SD | %SD | ||||
| Intraday | Inter-day | Intraday | Inter-day | Intraday | Inter-day | Intraday | Inter-day | ||
| 0.3877 | 0.005 | 0.0054 | 0.865 | 0.9475 | 0.2423 | 0.0169 | 0.0253 | 1.7465 | 2.652 |
| 0.1938 | 0.0043 | 0.0052 | 1.301 | 1.5629 | 0.1211 | 0.0187 | 0.0207 | 2.088 | 2.3316 |
| 0.0969 | 0.0014 | 0.0016 | 1.048 | 1.1694 | 0.0606 | 0.0182 | 0.0195 | 2.264 | 2.4247 |
| 0.0485 | 0.0005 | 0.0012 | 0.908 | 1.8965 | 0.0151 | 0.0131 | 0.0134 | 2.614 | 2.6942 |
| Table (7): Inter & Intraday precision study of the proposed method. (n = 10) | |||||||||
Recovery and applicability
With glucose
Recovery studies were conducted by inoculating the standard glucose to serum sample in the proposed method. The serum sample has been analysed by spiking 10µL directly to the reaction mixture. The result shows recovery rate ranging between 71.51 and 96.8% for HRP, 74.58 and 97.65% for VNShs, 75.29 and 97.28% for VNFws, 76.89 and 97.79% for VNSps. The recovery percentage is high with higher concentration of glucose, decreases with decrease in concentration and the percentage recovery is almost similar for HRP and NZs. The results are showen in the Table (8).
| Blood sample in µL | HRP | VNShs | ||||||
| Glucose (mM) | Added (mM) | Found (mM) | Recovered*(%) | Glucose (mM) | Added (mM) | Found (mM) | Recovered*(%) | |
| 10 | 0.4625 | 0.4742 | 0.4591 | 96.8 | 0.4625 | 0.462 | 0.4512 | 97.65 |
| 0.2312 | 0.2079 | 0.1927 | 92.71 | 0.2312 | 0.236 | 0.2252 | 95.39 | |
| 0.0578 | 0.0532 | 0.038 | 71.51 | 0.0578 | 0.0427 | 0.0319 | 74.58 | |
| VNFws | VNSps | |||||||
| Glucose (mM) | Added (mM) | Found (mM) | Recovered*(%) | Glucose (mM) | Added (mM) | Found (mM) | Recovered*(%) | |
| 0.4625 | 0.47 | 0.4572 | 97.28 | 0.4625 | 0.4353 | 0.4257 | 97.79 | |
| 0.2312 | 0.2188 | 0.2059 | 94.16 | 0.2312 | 0.3242 | 0.3146 | 97.03 | |
| 0.0578 | 0.0516 | 0.0389 | 75.29 | 0.1156 | 0.0417 | 0.03205 | 76.89 | |
Table (8): recovery study of glucose in blood sample.
With H 2 O 2
Recovery studies were conducted by inoculating the standard H2O2 to serum sample in the proposed method. The experiment demonstrated repeatability and minimal interference from inhibitory species, resulting in a recovery rate ranging from 99.72–99.93% for HRP, 94.99–98.92% for VNShs, 95.70–99.16% for VNFws and 98.13–99.55% for VNSps as shown in the Table (9). The results show the recovery percentage is maximum when the added H2O2 concentration was maximum, minimum when added H2O2 concentration was minimum and highest recovery percentage with HRP and least with VNShs.
| Blood sample in µL | HRP | VNShs | ||||||
| H2O2 (mM) | Added (mM) | Found (mM) | Recovered*(%) | H2O2 (mM) | Added (mM) | Found (mM) | Recovered*(%) | |
| 10 | 0.1938 | 0.1952 | 0.1951 | 99.93 | 0.7753 | 0.7702 | 0.7618 | 98.92 |
| 0.0969 | 0.0929 | 0.0928 | 99.85 | 0.3877 | 0.4118 | 0.4034 | 97.98 | |
| 0.0484 | 0.0509 | 0.0508 | 99.72 | 0.1938 | 0.1666 | 0.1583 | 94.99 | |
| VNFws | VNSps | |||||||
| H2O2 (mM) | Added (mM) | Found (mM) | Recovered*(%) | H2O2 (mM) | Added (mM) | Found (mM) | Recovered*(%) | |
| 0.1938 | 0.1898 | 0.18824 | 99.16 | 0.4845 | 0.4841 | 0.4819 | 99.55 | |
| 0.0969 | 0.1095 | 0.1079 | 98.54 | 0.2422 | 0.2495 | 0.2438 | 99.11 | |
| 0.0484 | 0.0371 | 0.0355 | 95.70 | 0.121 | 0.1167 | 0.1145 | 98.13 | |
Table (9): recovery study of H2O2 in blood sample.
Probable reaction mechanism
V2O5 [V(V)] NZs oxidises P-amino phenol to 4-iminocyclohexa-2, 5-dien-1-one (oxidized P-amino phenol) and converted into V(IV). V(IV) donates electron to H2O2 and convert them to H2O. Oxidized P-amino phenol coupled with NEDA to form 4-((4-((2-aminoethyl) amino) naphthalen-1-yl)imino)cyclohexa-2,5-dien-1-one which is an orange-red coloured product with maximum absorbance at 470nm.
The proposed method which involves coupling of oxidized PAP and NEDA using H2O2, is very easy to use, quick and sensitive to H2O2 assay. The reagents utilized are affordable, widely accessible, and soluble in water. Since the coupled product's maximum absorbance occurs at 470nm, this method can also be used for the quantification of H2O2 in biological samples. The linearity of H2O2 in the range of 0.0242 to 0.3877mM for HRP, 0.0969 to 1.5507mM for VNShs, 0.0242 to 0.3877mM for VNFws and 0.0606 to 0.9692mM for VNSps which indicates the sensitivity of the proposed method.
Ethical Approval
The institutional Human Ethical Committee (IHEC-UOMNo.22/Ph.D/2008-09) of the university of mysore has granted us permission to use of human blood serum sample
Funding
Not applicable
Author Contribution
NYG prepared the manuscript, RHS helped to prepare the manuscript and all authors are reviewed the manuscript
Acknowledgement
One of the authors, Nikhil Y Gangadhara would like to thank ATME college of engineering, mysore, Karnataka, for providing the research laboratory facilities.
Availability of data and materials
The authors affirm that the data supporting the discoveries of the study are accessible within the paper and its supplementary information file.
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