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Non-enzymatic colorimetric biosensor for hydrogen peroxide using lignin-based silver nanoparticles tuned with ionic liquid as a peroxidase mimic
⁎Corresponding authors at: Department of Chemistry, Kohat University of Science and Technology (KUST), Kohat, KPK, Pakistan. umarnishan85@gmail.com (Umar Nishan), rahimkhan533@gmail.com (Abdur Rahim)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Hydrogen peroxide (H2O2) is a byproduct of oxidative metabolism, acts as a signaling molecule, and can induce protein and DNA damage by oxidative stress. Ionic liquid-coated lignin stabilized silver nanoparticles (LAgNPs) have been used for the colorimetric detection of H2O2. Both ionic liquid and lignin moieties have a synergistic effect on the conductivity of silver nanoparticles to display intrinsic peroxidase-like activity with the enhanced potential to catalyze the oxidation of substrate 3,3′,5,5′-tetramethylbenzidine (TMB). LAgNPs have been synthesized by the green synthesis method and the prepared nanoparticles have been characterized by different techniques such as FTIR, EDX, TGA, SEM, and XRD. 1-H-3-methylimidazolium acetate (ionic liquid) was prepared and used for tuning of AgNPs. By using ionic liquid capped LAgNPs the colorimetric detection of H2O2 was done with the assistance of TMB solution and results were analyzed by UV–Vis spectrophotometer. Different parameters such as (amount of capping agent and TMB, pH, H2O2 concentration, and time) were optimized to get the best results of the proposed sensor. The H2O2 biosensor exhibited a linear response in the range of 1 × 10−9–3.6 × 10−7 M, with a detection limit of 1.379 × 10−8 M and a quantification limit of 4.59 × 10−8 M, and R2 of 0.999. The sensor gave a short response time of 5 min for colorimetric detection of H2O2 at pH 7.5 and room temperature. For the detection of hydrogen peroxide, the proposed sensor showed good sensitivity and selectivity and was successfully employed for the detection of H2O2 in the blood serum samples of hypertension patients.
Keywords
Lignin mediated synthesis
Ionic liquid
Silver NPs
Colorimetric sensor
Hydrogen peroxide
Peroxidase mimic
1 Introduction
Hydrogen peroxide (H2O2) is formed as a byproduct of oxidative metabolism. In the biological system, peroxidases play important role in the catalytic degradation of H2O2 (Wang et al., 2015). The concentration of hydrogen peroxide is regulated by peroxidases, which are generally generated from human red blood cells and kidneys (Kirkman and Gaetani, 2007). The concentration of hydrogen peroxide is regulated through peroxidases but for their functioning, they still need ambient conditions of temperature and pH, etc. (Favre et al., 2011). The monitoring of hydrogen peroxide is also critical in many other fields such as biological processes, paper bleaching, chemical, sterilization processes, pulp, clinical and food production, etc·H2O2 also plays an important role in food security, environmental protection as well as in bioanalysis (Nossol and Zarbin, 2009) therefore, it is also important to monitor the concentration of hydrogen peroxide (Salimi et al., 2007). To monitor, the concentration of hydrogen peroxide is also possible through multiple routes. The determination of hydrogen peroxide can be done through enzymatic methods (Tian et al., 2013). Due to certain limitations of the enzymatic methods such as low shelf life, high cost, less stability, difficult transportation, and delicate handling enzymatic sensing of the analyte become undesirable. On the other hand, nanomaterials-based detection is gaining more interest owing to its cost-effectiveness, ease in preparations, stability, and easy transportation (Tian et al., 2013) (see Scheme 1).
Proposed reaction mechanism for the detection of H2O2.
Various sophisticated techniques have been utilized for the determination of hydrogen peroxide such as chromatographic (Nakashima et al., 1994), spectrophotometric (Lu et al., 2016), chemiluminescence (Haddad Irani-nezhad et al., 2019), and electrochemical methods (Chen et al., 2014; Theyagarajan et al., 2020). Compared with other techniques colorimetric method is low cost, highly sensitive, and selective for hydrogen peroxide determination (Wang et al., 2016; Chen et al., 2016). Colorimetric analysis is commonly used for field analysis and point-of-care diagnosis owing to its efficiency, simple operation, and sensitivity (Liu et al., 2012).
Over the last few years, researchers have been focused on the synthesis of metal nanoparticles and their application because of their novel properties such as optical, electronic, magnetic, thermal, sensor devices, and catalysis, etc (Daniel and Astruc, 2004; Schmid et al., 2004). According to literature the monitoring of hydrogen peroxide has become more important due to its wide applications such as food industries, textile paper, and cleaning products. Hydrogen peroxide is a powerful oxidizing agent as well as its derivatives, which can be employed to treat environmental pollutants such as aldehydes, chlorine, phenols, and other aromatic compounds and also used in the synthesis of many organic compounds (Usui et al., 2003; Hasebe and Eguchi, 2003). As several enzymatic reactions produce H2O2 as an end product, its concentration can be used as a measure of the progress of a reaction (Jiang and Miles, 1993).
For the preparation and stabilization of nanoparticles, ionic liquids (ILs) have been explored as excellent media for their applications (Wang et al., 2015). Although ILs are solvents for different nanomaterials due to their low vapor pressure and a wide range of stable temperatures (Rogers and Seddon, 2003; Wang and Hao, 2016). Capping of nanomaterials with ionic liquid is used to get catalytic reactions that are not possible with the help of other solvents because IL prevents the nanomaterials from aggregations and enhances the electrical conductivity as recently reported by our group (Nishan et al., 2019; Nishan et al., 2021).
Due to their large surface-to-volume ratio, high catalytic performance, structure-dependent properties, and tunable optical properties, nanoparticles have received significant attention over the last few years. (Wen et al., 2008). According to the literature, different materials have been used as peroxidase mimics for H2O2 detection, including metal oxide and magnetic nanoparticles, conjugated polymers, bimetallic alloy nanoparticles, carbon nanotubes, and noble metal nanoparticles (He et al., 2017; Xie et al., 2018; Shin et al., 2017).
In the present work, silver nanoparticles were prepared by using lignin as stabilizing and reducing agent, furthermore a conductive layer of lignin was obtained on the surface of the AgNPs (Chen et al., 2017). The LAgNPs were characterized by diverse techniques like FTIR, SEM, TGA, EDX, and XRD. After characterization LAgNPs were capped/tuned with ionic liquid (1-H-3-methylimidazolium acetate) to further enhance the catalytic activity, stability, and conductivity, and dispersibility in comparison to other peroxidase mimics for the colorimetric detection of hydrogen peroxide. The proposed material LAgNPs was used as promising peroxidase mimics for the colorimetric determination of H2O2. To achieve the optimum performance of the IL capped lignin-based silver nanoparticles, different parameters have been optimized, such as (a) pH (b) amount of LAg/IL (c) incubation period (d) TMB and (e) H2O2 concentration. The proposed sensor was applied for the determination of hydrogen peroxide in the real samples (in hypertension patient's blood).
2 Materials and methods
2.1 Reagents and materials
All the chemicals used were of analytical grade: silver nitrate, 3,3′,5,5′-tetramethylbenzidine (TMB), acetic acid CH3COOH (99.9%), and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich. Hydrogen peroxide (H2O2, 35%) was obtained from Merch KGaA. 1-methylimidazole C4H6N2 (99.2%) was purchased from ACROS. The saline phosphate buffer (PBS, pH-7.4) was acquired from Bio World. Cuvettes (Disposable) with 2 mL of volume capacity were purchased from Kartell. Without further purification, all chemicals were used and they were of analytical grade. Deionized water was utilized for the preparation of the different solutions.
2.2 Instrumentation
The characteristic peaks were recorded on UV–Vis double beam spectrophotometer (Shimadzu, UV-1800, Germany) using quartz cuvettes. Functional groups of the prepared NPs were investigated by using FTIR MX-300 (USA). The size and morphology of the synthesized nanoparticles were evaluated by using SEM (JSM-5910, Japan) and elemental analysis was performed on EDX equipped with SEM. The crystal structure of the prepared nanoparticles was studied by using XRD Bruker Smart Apex CCD. On a Perkin-Elmer STA 6000 thermal analyzer with a heating rate of 10 °C/min in a temperature range from 30 to 800 °C under the nitrogen atmosphere, thermogravimetric analysis (TGA) was carried out. The Raman spectrum of the prepared nanoparticles were recorded at room temperature using a portable Raman instrument (i-Raman, Bwtek Inc., U.S.A.) attached with a microscope (20X objectives).
2.3 Synthesis of lignin doped silver nanoparticles
For the synthesis of Lignin Doped Ag NPs, 50 mL of Longan fruit (Dimocarpuslongan Lour) was prepared in 100 mL double-distilled water. The obtained extract of longan juice was added dropwise to 50 mM solution of lignin and 50 mM solution of AgNO3. The reaction mixture was magnetically stirred for 3 h at room temperature and the solution was stored at 5 °C for 24 h. Lignin doped Ag nanoparticles were then separated by centrifugation at 800 rpm for 10 min at room temperature and were collected in the pellet. After that, the prepared nanoparticles were dried in an oven at 40 °C as shown in Fig. 1 (A). The nanoparticles were stored at room temperature in an Eppendorf tube for further use. The reproducibility of the process was confirmed by repeating the synthesis of the nanoparticles to get a good amount for different experiments.
(A) Synthesis of Lignin doped silver nanoparticles (B) Synthesis of ionic liquid (C) Capping of Lignin doped silver nanoparticles with ionic liquid.
2.4 Capping of lignin doped silver nanoparticles with ionic liquid
The ionic liquid synthesis was carried out according to the protocol earlier reported by our group (Nishan et al., 2020). Lignin-doped silver nanoparticles dispersion was done in ionic liquid. To achieve the desired dispersion, 3 mL of ionic liquid (1-H-3-methylimidazolium acetate) was taken via a micro pipette in a china dish and 18 mg of silver nanoparticles doped with lignin were taken and then applied to the ionic liquid. Maceration of the nanoparticles and the ionic liquid was done for around 20 min with the aid of the pestle and mortar as shown in Fig. 1(C). A greyish mixture of lignin-doped Ag NPs with ionic liquid was collected and the mixture was placed for further use in an Eppendorf tube.
2.5 Colorimetric detection of hydrogen peroxide
Hydrogen peroxide and TMB solutions were prepared in deionized water. 30 µL ionic liquid capped lignin doped silver nanoparticles were taken and then 100 µL of hydrogen peroxide solution (3.6 × 10−7 M) was added into the ionic liquid coated lignin doped silver nanoparticles followed by the addition of PBS solution. The reaction was done at room temperature. By increasing concentrations of H2O2, the colorimetric change was observed and the color change from transparent to a greenish color, and the spectrum of absorption was captured by UV–Vis spectrophotometer. The detection performance of the proposed sensor was evaluated with respect to ionic liquid coated lignin doped silver nanoparticles, H2O2 concentration, TMB, pH, and buffer solution concentration.
3 Results and discussions
3.1 Characterization of the lignin doped silver nanostructures
3.1.1 FTIR analysis of the prepared nanoparticles
The FTIR spectrum of the prepared nanoparticles is shown in Fig. 2. The possible biomolecules take part in the stabilization and capping of the lignin doped silver NPs synthesized by Longan fruit juice. The FTIR spectrum displays absorption bands at 3365, 2911, 1633, 1521, and 1035 cm−1 as shown in Fig. 2. The 3370 cm−1 and 2911 cm−1 absorption bands reflect stretching of hydrogen-bonded-OH and vibration of sp2 C—H (alkane) respectively. N—H bend vibration can be attributed to the absorption at 1521 cm−1 and the peak at 1633 cm−1 can be attributed to the C—C symmetric stretching vibration of aromatic rings. The band might be contributed by the C—N bonds at 1035 cm−1. It is evident from the FTIR spectrum that the OH group is present in Lignin juice, which is responsible for reducing silver ions to silver nanoparticles (Badiya and Ramamurthy, 2018).
FTIR spectrum of L/AgNPs.
3.1.2 The synthesized nanostructures' XRD pattern
The XRD spectrum of the nanoparticles prepared is shown in Fig. 3. X-ray diffraction analysis at 20-80° confirmed the crystalline structure of lignin-doped silver nanoparticles. Different numbers of lattice planes represented at (1 1 1), (2 0 0), (2 2 0) and (3 1 1) and different numbers of Braggs' reflection with 2 theta values at 38.31°, 44.44°, 64.7°, and 77° show that the prepared nanoparticles have faced centered cubic structure (Zhang et al., 2019). All the peaks are in good agreement with JCPDS file number 00-004-0783. XRD spectrum show strong diffraction peaks at 38.31°, 44.44°, and 64.7°. The peak that corresponds to (1 1 0) is more prominent than the other planes, implying that the dominant orientation is (1 1 0).
XRD pattern of L/AgNPs.
The average particle size of the prepared lignin-doped silver nanoparticles was calculated through the Scherrer equation on the face center cubic phase. D = Kλ/βCosθ
K = coefficient (0.89) and θ represent the angle of diffraction at the maximum peak.
D = crystal size of the catalyst,
λ = X-ray wavelength,
β = full width at half maximum (FWHM) of the diffraction peak (radian),
The average crystal size of face center cubic phase lignin doped silver NPs were calculated to be ∼ 30 nm.
3.1.3 SEM analysis of the nanostructures prepared
Fig. 4 demonstrates the morphology of lignin doped silver nanoparticles was characterized by Scanning Electron Microscopy (SEM). SEM is used to characterize the surface morphology, particle shape, and size (Asnis, 2018). The SEM image shows that the synthesized lignin doped nanoparticles are granular agglomerated cluster structures owing to the strong tendency of agglomeration.
SEM image of the prepared L/AgNPs, magnification of 15 kx.
3.1.4 EDX analysis of lignin doped silver NPs
Fig. 5 demonstrates the chemical composition of lignin-doped silver NPs that have been examined by EDX analysis. This confirmed the existence of silver and oxygen in the sample of prepared nanoparticles (Hoyo et al., 2020). The silver and oxygen content by EDX analysis were found to be 87.84 and 12.16% by weight, respectively as shown in Table 1.
EDX spectrum of the synthesized L/AgNPs.
Element
Line Type
Weight %
Weight % Sigma
Atomic %
Ag
L series
87.84
1.89
51.72
O
K series
12.16
1.89
48.28
Total
100.00
100.00
3.1.5 Thermal gravimetric assessment of the nanoparticles prepared
Fig. 6 demonstrates that the prepared lignin-doped silver NPs were analyzed by thermogravimetric analysis (TGA). The thermal stability of the lignin-doped silver NPs is shown in Fig. 5. To investigate the combustion properties of the prepared nanoparticles TGA-Q5000 apparatus (TA Co., USA) was used from 50 to 800 °C at a heating rate of 10 °C min−1. From the TGA curve, it is observed that the weight loss of the nanoparticles occurred in the 50 to 800 °C temperature range. The weight of all samples was maintained within 2–4 mg in an open platinum pan (Shankar and Rhim, 2017).
TGA curves of the synthesized L/AgNPs.
3.2 Colorimetric detection of hydrogen peroxide
For the colorimetric detection of hydrogen peroxide, a simple assay was designed as shown in Fig. 7. For the sensing of hydrogen peroxide, ionic liquid capped lignin doped silver nanoparticles (30 µL) were used. TMB solution 180 µL (10 mM) and PBS solution 550 µL (6 mM) was mixed with 100 µL hydrogen peroxide solution (3.6 × 10−7 M). After the addition of hydrogen peroxide solution to the capped lignin doped silver nanoparticles, the color changes from transparent to greenish. The UV–Vis spectrum and the colorimetric change can be seen in Fig. 7.
Shows the UV–Vis spectra and the colorimetric change. The colorimetric change was observed with a solution containing 30 µL ionic liquid capped nanoparticles, 180 µL TMB solution (10 mM), 550 µL buffer solution (6 mM), and 100 µL hydrogen peroxide solution (3.6 × 10−7 M). (A) In the absence of hydrogen peroxide (B) in the presence of 100 µL hydrogen peroxide solution (3.6 × 10−7 M), the color changes from transparent to greenish.
3.3 Proposed mechanism of hydrogen peroxide detection
The peroxidase-like activity of ionic liquid capped lignin doped silver nanoparticles was confirmed from the catalytic oxidation of a 3,3′,5,5′-Tetramethylbenzidine (TMB) by H2O2 to form a bluish-green product. Their absorbance was also recorded by UV–Vis spectrophotometer, which demonstrates a broad peak at 653 nm for the oxidized TMB. The mechanism of the proposed reaction is as follows; Ionic liquid capped lignin doped silver nanoparticles absorb photon (light) whose energy is equal to its bandgap energy. Due to this absorption of photon excitation of electron takes place from the valence band to conduction band to yield electrons and H+ pair. After this H2O2 having a strong oxidation ability to scavenge the excited electron to prevent the recombination of the electron and H+ and generate OH radicals and OH– ions. Moreover, hydrogen peroxide adsorbs on the surface of ionic liquid capped lignin doped silver nanoparticles to form peroxo complex between Ag and H2O2 that enhance the absorption capability of ionic liquid capped lignin doped silver nanoparticles under visible light. As a result, more OH radicals are formed that lead to increasing TMB oxidation to form blue-green products as shown in equation 3 (Velegraki et al., 2006).
3.4 Optimization-of the method proposed
3.4.1 Effect of capping amount
The optimization of ionic liquid-coated nanostructures is shown in Fig. 8(A). The colorimetric detection of H2O2, the peroxidase-like activity was evaluated using TMB as a substrate. The reaction was carried out as follow; 30 μL of IL coated NPs, 180 μL of TMB solution (10 mM), 550 μL of phosphate-buffer solution (6 mM), and 100 μL H2O2 solution having concentration (3.6 × 10−7 M). The reaction was incubated for 5 min to detect the colorimetric change. 30 µL ionic liquid capped lignin doped silver nanoparticles are compatible with 100 µL hydrogen peroxide (3.6 × 10−7 M). In short 30 µL, the capped amount is optimum at which the color completely changes from transparent to greenish. In 2018, Zarif et al. reported that 35 μL IL coated iron NPs were the optimum amount for H2O2 detection (Zarif et al., 2018). The quantitative relationship between IL-coated NPs, TMB, and varying concentrations of hydrogen peroxide was also recorded by UV–Vis spectroscopy as revealed in Fig. 8(A).
(A) The effect of capping amount (μL). The best colorimetric change was observed at 30 μL capped nanoparticles, 180 μL TMB solution (10 mM), 550 μL buffer solution (6 mM), and 100 μL H2O2 solution (3.6 × 10−7 M). (B) Optimization of pH in the range of pH (3–11), under optimized conditions: 30 μL capped nanoparticles, 180 μL TMB solution (10 mM), 550 μL buffer solution (6 mM) and 100 μL H2O2 solution (3.6 × 10−7 M). (C) Optimization of the TMB solution under optimized conditions: 30 μL capped nanoparticles, 180 μL TMB solution (6–14 mM), 550 μL buffer solution (6 mM), and 100 μL H2O2 solution (3.6 × 10−7 M). (D) Time optimization (1–10 min), under optimized conditions: 30 μL capped nanoparticles, 180 μL TMB solution (10 mM), 550 μL buffer solution (6 mM), and 100 μL H2O2 solution (3.6 × 10−7 M).
3.4.2 Effect of pH on the response of the sensor
Fig. 8 (B) shows the optimization of pH for the proposed reaction. With different pH values of pH 3–11, the response of the proposed sensor was optimized. The pH optimization was performed by using sodium hydroxide and hydrochloric acid solutions. The best colorimetric change of the proposed sensor occurred at the following optimum conditions; 30 μL IL-based lignin doped silver nanoparticles, 180 μL TMB solution (10 mM), pH 7.5, incubation time 5 min, and 100 μL hydrogen peroxide (3.6 × 10−7 M). By increasing the concentration of H2O2 the best colorimetric change of our proposed sensor occurred at pH 7.5. So the pH 7.5 was selected as optimum for the proposed sensor. According to the literature, the optimal pH for the proposed sensor was reported to be 7.4 (Zarif et al., 2020).
3.4.3 Effect of TMB on the sensor
Fig. 8(C) shows the optimization of the TMB solution. By using TMB as a substrate the peroxidase-like properties of the synthesized lignin doped silver nanoparticles were evaluated. Fig. 8(C) demonstrate that when 100 μL H2O2 solution having concentration (3.6 × 10−7 M) is added to 30 μL capped nanoparticles, 180 μL TMB (10 mM) and 550 μL of phosphate-buffer solution (6 mM) together, the color change from colorless to bluish-green, and the absorbance was also recorded through UV–Vis spectrum at 652 nm. Thus the TMB solution having a concentration of 10 mM (180 μL) was selected optimum for the proposed sensor. While in literature 0.5 mM concentration of TMB solution was reported (Zhang et al., 2018). In the absence of TMB, the reaction of H2O2, and lignin doped silver NPs does not occur. This phenomenon shows that the intrinsic peroxidase-like activity of the synthesized lignin-doped silver nanoparticles, similar to other noble metal-based peroxidase mimics.
3.4.4 Time impact on sensitivity
The time optimization study was conducted in the range of (1–10 min) as shown in Fig. 8(D). The proposed sensor LAgNPs gave the best response at a time of 5 min because maximum conversion (oxidation) occurred at this time. Thus 5 min was chosen as the optimal time for the colorimetric detection of H2O2. In the case of the reported sensor (Zhang et al., 2019), however, the optimum time for the reported sensor was 30 min. Our proposed sensor showed a rapid detection of H2O2 compared to the already published literature.
3.4.5 Analytical specifications of the proposed sensor
The analytical features of the proposed sensor for hydrogen peroxide concentration were evaluated by applying a calibration graph. As shown in Fig. 9(A), the optimization was achieved with various concentrations of hydrogen peroxide. The peroxidase-like the activity of IL coated lignin doped silver nanoparticles under the optimum experimental condition was conducted for colorimetric detection of hydrogen peroxide. It can be observed that by rising the concentration of hydrogen peroxide the UV–Vis spectrum shows a peak at 652 nm that increases linearly with the rise in hydrogen peroxide concentration as shown in Fig. 9(A). The proposed sensor showed a linear response to hydrogen peroxide in the range of 1 × 10−9–3.6 × 10−7 M, with a LOD of 1.37 × 10−8 M and LOQ 4.59 × 10−8 M, with an R2 value of 0.999. By using the formulas (10σ/slope) for LOQ and (3σ/slope) for LOD the values were calculated. The σ represents the standard deviation of the blank and the slope value is derived from the linear curve. By comparing the proposed sensor results with the reported literature (Lin et al., 2015), the limit of detection (5.3 μM) is low and the R2 value is 0.994. Fig. 9 (B) demonstrates the linear curve obtained with different concentrations (1 × 10−9–3.6 × 10−7 M) of H2O2 as a function of absorbance (nm), calculated at 652 nm to detect the strong optical changes as shown in Fig. 9 (A). Compared with the literature already published, Table 2 shows the analytical efficiency of the proposed sensor (Wang et al., 2016; Nguyen et al., 2018; Choleva et al., 2018; Ding et al., 2016; Zhong et al., 2016; Xiang et al., 2016; Liu et al., 2015).
(A). Colorimetric change concerning different concentrations of hydrogen peroxide and UV–Vis spectra; (B) Calibration plot of hydrogen peroxide concentration (1 × 10−9–3.6 × 10−7 M) versus absorbance.
S. No
Materials used
Method applied
Limit of detection (μM)
Linear range (μΜ)
Ref
1
AgNPs/GQDs
Colorimetric
0.162
0.5–50
(Nguyen et al., 2018)
2
RhNPs
Colorimetric
0.75
5–125
(Choleva et al., 2018)
3
Fe2(MoO4)3-F
Colorimetric
0.7
1–30
(Wang et al., 2016)
4
Fe3S4-MNPs
Colorimetric
0.16
2–100
(Ding et al., 2016)
5
β-CD/Cu-NCsa
Colorimetric
0.2
0.02–10
(Zhong et al., 2016)
6
AgVO3 nanobelts
Colorimetric
5
75–500
(Xiang et al., 2016)
7
NiO NPs
Colorimetric
8
20–100
(Liu et al., 2015)
8
NiFe LDH
Colorimetric
4.4
10–500
(Zhan et al., 2018)
9
NiSnanocubes
Colorimetric
1.72
4–40
(Liu et al., 2019)
10
Lignin Doped AgNPs
Colorimetric
0.013
0.001–0.36
Present work
3.5 Interference studies
The selectivity of the proposed sensor (IL coated lignin doped silver nanoparticles) was explored for the selective detection of hydrogen peroxide in the presence of the co-existing species. In the presence of chemicals that usually co-exist, such as ascorbic acid, urea, Ca+, K+, and folic acid, the absorbance responses of the proposed sensor were investigated as shown in Fig. 10. It demonstrates that the absorbance value of hydrogen peroxide is very high as compared to the absorbance value of ascorbic acid, urea, Ca+, K+, and folic acid. By increasing the H2O2 concentration the absorbance spectra increase at 652 nm even in the presence of 3-fold of co-existing species, and no obvious change in absorbance was observed. The experiments were carried out in the same experimental circumstances by retaining the 3-fold concentration (1.0 × 10−6 M) of co-existing species as compared to hydrogen peroxide (3.6 × 10−7 M). It is confirmed from these findings that, in the presence of commonly co-existing species, the sensor proposed is highly selective and precise for the detection of hydrogen peroxide. While in the case of reported literature the selectivity of the stated method for hydrogen peroxide detection, the effects of various interfering species, such as galactose, ascorbic acid, uric acid, and citric acid (Zn2+, Ni2+, Co2+, Na+, Fe3+, Hg2+) have been examined. It was noted that the species described had no detectable effect on the detection of hydrogen peroxide up to 1 mM concentration (Swaidan et al., 2020).
Shows the interference study of hydrogen peroxide (3.6 × 10−7 M) with other analytes such as ascorbic acid, urea, Ca+, K+, and folic acid having concentration 1.0 × 10−6 M.
3.6 Real samples analysis
The proposed sensor is applied to confirm the detection of H2O2 in real samples. The detection of H2O2 was conducted in hypertension patient’s blood serum samples under the optimum experimental conditions. By using the standard addition method different concentrations of H2O2 such as 69, 137, 208, 241, 274, and 310 nM were spiked into the blood serum samples of hypertension patients as shown in Fig. 11. The present amount of H2O2 in blood serum samples solution is determined from an already established calibration plot, by using different concentrations of H2O2 under the same optimized condition generated at 652 nm (Swaidan et al., 2020). The percentage recovery formula is applied and the obtained results are summarized in Table 3.
Recovery % = H2O2 found/H2O2 added × 100
shows the UV–Vis spectra of the real samples of blood serum at optimized conditions by the addition of different concentrations of hydrogen peroxide (7 × 10−8–3.15 × 10−7 M).
Samples
Detected (nm)
H2O2 added (nm)
H2O2 found (nm)
Recovery (%)
RSD (%)
1
1
69
70
101.44
0.838
2
3
137
140
102.18
1.009
3
2
208
210
100.96
0.459
4
4
241
245
101.65
0.923
5
1
274
275
100.36
1.203
6
5
310
315
101.61
1.101
4 Conclusions
We report a simple method for the colorimetric detection of hydrogen peroxide based on IL capped lignin stabilized silver nanoparticles. The synthesized material lignin stabilized silver nanoparticles were confirmed through different characterization techniques. The designed protocol having ionic liquid coated nanoparticles (IL-NPs) along with 3,3′,5,5′-tetramethylbenzidine (TMB) solution and buffer solution to introduce colorimetric hydrogen peroxide sensor. Under optimized conditions, the proposed sensor showed good applicability with a wide linear range (1 × 10−9–3.6 × 10−7 M), low limit of detection 1.37 × 10−8 M and limit of quantification 4.59 × 10−8 M with an R2 value of 0.999. The proposed sensing probe is a simple, rapid, highly sensitive, selective stable biomimetic catalyst method for the colorimetric sensing of H2O2 that can be applied in medical diagnostics. The proposed sensor was employed for the selective detection of hydrogen peroxide in the presence of co-existing species. Furthermore, it was successfully applied for the detection of H2O2 in the real samples (blood serum of hypertension patients).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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