Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2021 Nov 22;6(48):32754–32762. doi: 10.1021/acsomega.1c04548

Ionic-Liquid-Stabilized TiO2 Nanostructures: A Platform for Detection of Hydrogen Peroxide

Umar Nishan †,*, Shams Ul Haq †, Abdur Rahim ‡, Muhammad Asad †, Amir Badshah †, Azhar-ul-Haq Ali Shah †, Anwar Iqbal §, Nawshad Muhammad ∥,*
PMCID: PMC8655897  PMID: 34901624

Abstract

graphic file with name ao1c04548_0017.jpg

Hydrogen peroxide (H2O2) acts as a signaling molecule to direct different biological processes. However, its excess amount results in oxidative stress, which causes the onset of different types of cancers. TiO2 nanostructure was synthesized by a facile hydrothermal method. The prepared material was characterized by FTIR spectroscopy, XRD, SEM, EDX, TGA, and Raman spectroscopy, which confirmed the formation of nanostructured material. Subsequently, the prepared nanoparticles (NPs) were capped with 1-H-3-methylimidazolium acetate ionic liquid (IL) to achieve its deagglomeration and functionalization. A new colorimetric sensing probe was prepared for the detection of H2O2 based on ionic liquid-capped TiO2 nanoparticles (TiO2/IL) and 3,3′,5,5′-tetramethylbenzidine (TMB) dye, which acts as an oxidative chromogenic substrate. H2O2 reacts with TMB, in the presence of ionic liquid-coated TiO2 NPs, to form a blue-green product. The color was visualized with the naked eye, and the colorimetric change was confirmed by a UV–vis spectrophotometer. To obtain the best response of the synthesized sensor, different parameters (time, pH, concentrations, loading of nanomaterials) were optimized. It showed a low limit of detection 8.61 × 10–8 M, a high sensitivity of 2.86 × 10–7 M, and a wide linear range of 1 × 10–9–3.6 × 10–7 M, with a regression coefficient (R2) value of 0.999. The proposed sensor showed a short incubation time of 4 min. The sensing probe did not show any interference from the coexisting species. The TiO2/IL sensor was effectively used for finding H2O2 in the urine samples of cancer patients.

1. Introduction

Hydrogen peroxide (H2O2) is one of the important analytes in the medical community due to its high reactivity.1 H2O2 is the byproduct of almost all oxidative metabolic reactions in living organisms. Naturally, existing peroxidases assume a key role in the catalytic degradation of H2O2 in biological systems.2 They regulate the concentration of H2O2 and require ambient conditions (temperature, pH) for their working.2 H2O2 can act as a signaling compound to direct different types of biological processes, such as activation of immune cells, vascular remodeling, etc.3 However, the higher concentration of H2O2 in the body creates oxidative stress-inducing protein and DNA damage. Ultimately, this DNA damage also increases our susceptibility toward the onset of different types of cancers. The human body can be affected by more than 100 types of cancers.4 Therefore, it is essentially imperative to monitor the concentration of H2O2 in the body.5

Over the past decades, various techniques have been utilized for H2O2 detection, such as chromatographic,6 spectrophotometric,7 chemiluminescence,8 and electrochemical methods.9 Nevertheless, some of these techniques are hazardous to living cells, making them unsuitable for in situ sensing of H2O2 in biological samples. Some of these methods are time-consuming, expensive, and complex, thus limiting their use in resource-limited laboratories.10 The detection of H2O2 via the colorimetric biosensor method is a very rapid, simple, low-cost, highly selective, and sensitive technique compared to the aforementioned techniques.

NPs play an important role in the development of different types of biosensors.11 Nanostructures show exceptional features because of their nanoeffects like the mini size effect, larger surface-to-volume ratio, quantum effect, macro-quantum tunneling effect, and surface plasmon resonance (SPR). Various materials including metal oxides, sulfide, and selenide nanomaterials, such as polymer-coated CeO2 NPs,12 CuO NPs,13 Co3O4 NPs,14 V2O5 nanowires,15 BiFeO3 NPs,16 CoFe2O NPs,17 and sheet-like FeS NPs,18 have been used as peroxidase mimics. However, novel ionic liquid-capped TiO2 NPs are recyclable, highly stable, and highly efficient, which give excellent sensing properties and catalytic activity that shows strong potential to replace expensive noble-metal NPs for biosensing.

In the class of metal oxides, TiO2 nanostructures are most commonly used in industrial products, such as cosmetics, sunscreens, food products, paints, and drugs as well as in medical diagnosis.19,20 TiO2 nanostructures are well-known semiconducting materials as well as photocatalysts.21 TiO2 photocatalysts exhibit bactericidal activity22−25 and degradation of chemical pollutants such as superoxide, H2O2, etc.26 The nanomaterials based on TiO2 are used commonly on priority bases in the field of energy due to their high band gap (2.8). Ionic liquids (ILs) are considered magical chemicals because of their wide temperature stability, negligible vapor pressure, and tunable properties through appropriate modification of cation and anion.27,28 The capping of NPs with ionic liquid enhances their catalytic activity manyfold. Among ILs, 1-H-3-methylimidazolium acetate has a lot of uses, reported, in diagnosis.29

In the present study, we synthesized TiO2 NPs by the hydrothermal method and characterized by various analytical techniques such as FTIR, XRD, SEM, TGA, and Raman spectroscopy. The prepared NPs were dispersed in ionic liquid to enhance their sensing activities. The association of the ionic liquid with NPs improves both the sensing and catalytic properties of the system.30,31 For H2O2 detection, a new simple, rapid, highly sensitive, and selective method is being developed, which is based on the oxidation of chromogenic substrate, i.e., 3,3′,5,5′-tetramethylbenzidine, by H2O2 in the presence of TiO2 NPs coated with ionic liquid. To obtain the best performance of the proposed sensor, different reaction conditions such as (a) amount of capped NPs; (b) pH; (c) TMB concentration; and (d) time of incubation were optimized. Using the above optimum conditions, sensitivity and selectivity of the proposed sensor were also analyzed. The urine samples of cancer patients were analyzed for H2O2 detection.

2. Experimental Section

2.1. Chemicals and Reagent

Oxalic acid (98%), hydrogen fluoride (99.9%), HClO4 (70%), NaOH (≥97.0%), sodium sulfate (99.0%), 3,3′,5,5′-tetramethylbenzidine (TMB), 1-methylimidazole (C4H6N2) (99%), and acetic acid (99.8%) were obtained from Sigma-Aldrich. H2O2 (35%) was obtained from Merck KGaA (https://www.merckgroup.com/en). PBS of different pHs was obtained from BioWorld. All of these chemicals were found in pure form and were used without further purification. Solutions were prepared in deionized water obtained from an Elga Purelab Ultra water deionizer.

2.2. Instrumentation

The formation of the synthesized TiO2 NPs was confirmed by the FT-IR spectral data. The FT-IR spectrometer used was from Agilent Technologies Danbury, Conn. The range chosen for getting FTIR spectra of the samples was 4000–600 cm–1. The morphology and the size of the NPs were studied through a coupled scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS) on a TESCAN VEGA (LMU) SEM with INCAx-act (Oxford Instruments) EDS attachment operating at 20 kV. The analysis and phase identification of the synthesized NPs was carried out by X-ray powder diffraction (XRD; PAN analytical, X’pert Powder). The Raman spectra of the prepared NPs were recorded by employing a convenient Raman instrument (i-Raman, Bwtek, Inc.) connected with a microscope (20×). The thermal stability of the synthesized NPs was assessed via Pyris-1, V-3.81 PerkinElmer Thermal gravimetric analysis (TGA), under nitrogen atmosphere having a temperature range of 40–800 °C with 10 °C min–1 heating rate. UV–vis spectra of both the synthesized TiO2 NPs and experimental samples were recorded on a UV–vis spectrophotometer (Shimadzu, UV, 1800, Japan).

2.3. Synthesis of TiO2

To prepare TiO2 NPs, the titanium plate is subjected to hydrothermal treatment and pretreated with an oxalic acid solution to remove the oxides on the surface. The hydrothermal reaction medium is prepared to utilize 110 mL of Milli-Q water, eosin dye, and 50 mL of isopropanol. The pH of the medium was balanced at 2.62 by adding 25 mL of NaOH solution having 0.1 M concentration and 60 mL of HF solution having 0.1 M solutions. The synthesized solution was hydrothermally treated at a temperature of 180 °C for 3 h. After that, TiO2 NPs were calcined at a temperature of 600 °C for 2 h to obtain a fluorine-free surface.32

2.4. Synthesis of Ionic Liquid

Preparation of 1-H-3-methylimidazole acetate ionic liquid was done using the modified protocol previously reported by our group.29

2.5. Capping of TiO2 NPs with Ionic Liquid

TiO2 NPs were modified with ionic liquid as follows. First of all, TiO2 NPs (6 mg) was added to 1 mL of ionic liquid. The mixture was macerated thoroughly in a mortar for around 30 min. As a result, a reddish dark mixture was obtained and stored in an Eppendorf tube for further use.

2.6. Colorimetric Sensing of H2O2

The detection of H2O2 was evaluated by colorimetric changes, in which 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized by H2O2 to form a blue-green product. Capped NPs (25 μL) were taken in an Eppendorf tube, and 190 μL of TMB (14 mM) was added to the solution, and finally 550 μL of pH 7 phosphate buffer was added. Subsequently, 90 μL of H2O2 (3.6 × 10–7 M) was added to the reaction solution and incubated for 4 min under the optimal temperature condition to detect optical changes. The resulting solution was subjected to a UV–vis spectrophotometer to record the absorption spectrum. To obtain the ideal performance, some experimental parameters have been optimized, such as response time, pH, amount of capped NPs, and concentration of TMB solution.

3. Results and Discussion

The synthesized material TiO2 NPs were characterized by FTIR spectroscopy as shown in Figure 1. A peak appears in the range of 1800–1600 cm–1, corresponding to stretching vibrations of adsorbed carbonyl (background peak of adsorbed carbon dioxide). The stretching vibrations of Ti–O–Ti and Ti–O were confirmed by the peak at 780 cm–1, which is almost the same as reported.33

Figure 1.

Figure 1

Open in a new tab

FTIR spectrum of the TiO2 NPs confirming the presence of the Ti–O bond.

Figure 2 shows the XRD pattern of the TiO2 NPs calcined at 600 °C. From XRD studies, it is to confirm that the materials synthesized are in the rutile TiO2 phase. The crystal structures are in complete agreement with the corresponding reported JCPDS database Card-No. 21-1272. The diffraction peak of the NPs was recorded at a 2θ value of 25.8 confirms its rutile phase. The diffraction angles (2θ) of 25.35, 37.75, 48.11, 62.71, and 75.008 correspond to the (110), (121), (111), (210), and (211) crystal faces of rutile.34

Figure 2.

Figure 2

Open in a new tab

XRD pattern of the prepared TiO2 NPs.

Nanoparticles size: The average crystalline size of TiO2 was estimated using the Scherrer equation.

3.

where D is the crystal size of the catalyst, λ is the X-ray wavelength, β is the full width at half-maximum (FWHM) of the diffraction peak (radian), k is the coefficient (0.89), and θ is the diffraction angle at the peak maximum.

The average crystalline size of rutile phase NPs is 43.88 nm.

The surface morphology, i.e., particle size and shape of the prepared TiO2 NPs calcined at 600 °C was characterized using the cross-sectional SEM image as shown in Figure 3. SEM image confirmed that the prepared TiO2 NPs are in a crystalline state and they have a strong tendency to agglomerate. These trends are very consistent with the reported literature.35

Figure 3.

Figure 3

Open in a new tab

Cross-sectional SEM image of the synthesized TiO2 NPs calcined at 600 °C and 15 000× magnification.

Using EDX analysis, the chemical composition of TiO2 NPs has been examined as shown in Table 1 and Figure 4. This confirmed the existence of Ti and O in the NPs samples. The Ti and O contents by EDX analysis were found to be 29.84 and 70.16 by weight.36

Table 1. EDX Analysis of TiO2 NPs.

element line type weight % weight % sigma atomic %
Ti K series 56.01 3.81 29.84
O K series 43.99 3.81 70.16
total   100.00   100.00
Open in a new tab

Figure 4.

Figure 4

Open in a new tab

EDX analysis of the prepared TiO2 NPs.

The thermal property of the prepared NPs was determined using TGA. The TGA curves of the synthesized TiO2 NPs are shown in Figure 5. The weight loss observed at 125 °C corresponds to the loss of moisture from the surface. The weight loss observed at 170 °C corresponds to the loss of the alkyl part in the synthesized TiO2 and crystallized water. The weight loss occurred at 615 °C corresponds to the thermal decomposition of residual organic groups in the as-synthesized TiO2.37

Figure 5.

Figure 5

Open in a new tab

TGA analysis of the prepared TiO2 NPs.

Using Raman spectroscopy, the crystalline phases of TiO2 NPs were identified. Figure 6 shows the Raman spectrum of TiO2 NPs prepared at different ratios calcined at 400 °C. The main characteristic Raman bands of the rutile crystal phase were observed at 167, 399, 515, 519, and 638 cm–1 in all samples calcined at 400 °C, which is completely consistent with earlier work.38 For the photocatalyst, a sharp peak was observed near 638 cm–1, indicating that the NPs have higher crystallinity. This can minimize charge recombination during the photoreaction process.39 The samples also show a fraction of the rutile phase. The sample also showed part of the rutile phase. After the two samples were calcined at 400 °C, a faint rutile peak was observed at 399 cm–1. The higher calcination temperature results in the anatase phase of TiO2 NPs.40 The presence of glycerol has no obvious effect on the crystal phase formation of TiO2 NPs. However, with increasing the concentration of water in the system, the crystallinity also increases.

Figure 6.

Figure 6

Open in a new tab

Raman spectrum of TiO2 nanostructures.

3.1. Colorimetric Detection of H2O2

A simple and selective colorimetric method based on the ionic liquid-coated TiO2 NPs was used for the detection of H2O2. The optical sensing and UV–vis absorption spectra are shown in Figure 7I. The sensor system effectively detects H2O2 by achieving blue-green products of colorless TMB (oxidative substrate) upon the addition of H2O2. Also, the adsorption of H2O2 on the surface of NPs generates OH radicals, which are subsequently involved in the oxidation of TMB to give blue-green products. In Figure 7II, it can be seen that when only ionic liquid (A) and only TiO2 (B) were used, the colorimetric change was very low. However, when ionic-liquid-coated TiO2 (C) was used, a clearly visible colorimetric change can be observed. The UV–vis spectra confirm the change.

Figure 7.

Figure 7

Open in a new tab

(I) UV–vis spectra obtained with a solution containing 25 μL of capped TiO2 NPs, 190 μL of TMB (14 mM), 550 μL of PBS of pH 7, and 90 μL of H2O2 (3.6 × 10–7 M). Curve (A) was obtained in the absence, and curve (B) was obtained in the presence of H2O2. The inset shows the colorimetric change of H2O2. (II) Absorption spectra of (A) only ionic liquid, (B) only TiO2 solution, and (C) ionic-liquid-coated TiO2 NPs.

3.2. Proposed Mechanism of H2O2 Sensing

The peroxidase-like activity of ionic liquid-coated TiO2 NPs was confirmed from the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to form a blue-green product. The reaction was also monitored using a spectrophotometer, which shows a broad peak at 653 nm for oxidized TMB. The proposed mechanism is followed by the reaction: IL-coated TiO2 NPs absorb photon (light) whose energy is equal to its band gap energy. Due to this absorption of photon, excitation of electron takes place from the valence band to the conduction band to yield electrons and h+ pair. H2O2 having a strong oxidation ability scavenges the excited electron to prevent the recombination of the electron and h+ and generates OH radical and OH– ions. Moreover, H2O2 adsorbed on the surface of TiO2 NPs to form a peroxo complex between Ti and H2O2 that enhances the absorption capability of TiO2 NPs under visible light. As a result, more OH radicals are formed, which leads to increasing TMB oxidation to form blue-green products (Scheme 1).41

Scheme 1. Proposed Mechanism for the Colorimetric Detection of H2O2.

Scheme 1

Open in a new tab

3.3. Optimization of Parameters

3.3.1. Optimization of Capped NPs

Protic ionic liquids have a very versatile role in enhancing the catalytic activity as well as stabilization of metal oxide NPs because it prevents NPs from undesirable agglomeration by providing steric and electrostatic stabilization. Previously, TiO2 NPs were doped with nitrogen and used as a colorimetric sensing platform for H2O2 detection.42 Here, we have successfully synthesized TiO2 through an alternate method giving a different nanopore size and without any doping. Moreover, in the reported work, optimization results provided were not comprehensive and lacked necessary details. The reported work42 also did not provide any real sample analysis, which is a very vital step for testing the applications of a fabricated biosensor. In the current work, we have provided comprehensive optimizations and application for the proposed simple TiO2-based biosensor. To prepare a more efficient catalyst to the characteristics of different reaction systems, ionic liquid design ability has become the latest research hotspot.43,44 A significant role of ionic liquids in enhancing catalytic power, i.e., stabilization of NPs, is possibly connected with their good dispersion and solvation power, electrostatic attraction, π–π stretching, weak interaction with substrate and product, and involvement of cationic part of ILs containing acidic proton, thereby facilitating the decomposition of H2O2 to generate OH radicals in the oxidation of chromogenic substrate TMB.31,43,45,46 In our research work, the ionic liquid is chosen as a stabilizing agent. The amount of capped TiO2 NPs was optimized in microliters by changing its amount in the range of 10–40 μL. The best colorimetric change and the highest peak were obtained when 25 μL of capped NPs was used as shown in Figure 8. Using a small amount of capping agent, there are not enough OH radicals to oxidize the whole TMB. It was seen that increasing the amount of capped NPs helps in accelerating TMB oxidation and color change gradually. However, as the concentration of the capping agent is increased further, the change in color starts to disappear. This indicates that when the concentration of the capped amount is very high, the excess amount will be dispersed in the reaction medium and agglomeration will not occur, which indicates that the reaction is incomplete, which is consistent with the reported literature.46

Figure 8.

Figure 8

Open in a new tab

Optimization of the amount of capped TiO2 NPs (in μL). The best response was observed at 25 μL of capped TiO2. COND: TMB = [14 mM] (190 μL); PBS pH = 7 (550 μL); H2O2 = [3.6 × 10–7 M] (90 μL).

3.3.2. Optimization of TMB Concentration

Figure 9 shows the effect of TMB concentration on biosensor activity. By increasing the concentration of the TMB solution, the absorbance increased quickly till point D as shown in Figure 9. At a concentration of 14 mM (190 μL), the maximum colorimetric change took place. When the concentration of TMB solution was increased further, the growth rate slows down and the reaction mixture appeared in the precipitate because the available TiO2 molecule gets utilized in the oxidation of TMB. Therefore, 14 mM was selected as the optimum TMB concentration to produce a noticeable colorimetric change.

Figure 9.

Figure 9

Open in a new tab

Optimization of TMB concentration. Conditions: [capped TiO2 NPs loading] = 25 μL, [PBS pH] = 7 (550 μL); [H2O2] = 3.6 × 10–7 M (90 μL).

3.3.3. Optimization of pH

In the biosensor system, the pH of the solution is a key factor. It increases or decreases the efficiency of the biosensor. To find the optimal pH of the sensor we proposed, the response of the sensor was analyzed in the pH range of 3–11. HCl and NaOH were used for variation in the pH, where the best response was obtained at pH 7.2. At pH 7.2, the sensing time was reduced to 4 min and the color of the mixture completely changed to blue-green, as shown in Figure 10.

Figure 10.

Figure 10

Open in a new tab

Optimization of pH. Conditions: [capped TiO2 NPs loading] = 25 μL; [TMB] = 14 mM (190 μL); [H2O2 conc.] = 3.6 × 10–7 M (90 μL).

Therefore, 7.2 pH was chosen as the optimum pH for further experimental work. It is concluded that our sensor works favorably around physiological pH, which is in complete agreement with the available literature.46

3.3.4. Optimization of Time

The impact of time on sensor response was examined at various time intervals 1–8 min. Figure 11 shows that the excellent response was observed at 4 min because all of the available TiO2 is utilized during this time and reached the maximum response. After 4 min, no further change was observed. Hence, we selected 4 min as the optimum time for our proposed sensor and all other experiments were conducted at this time.

Figure 11.

Figure 11

Open in a new tab

Optimization of time with respect to absorption obtained from UV–vis spectra. Conditions: [capped TiO2NPs loading] = 25 μL; [TMB] = 14 mM (190 μL); [PBS pH] = 7 (550 μL); [H2O2] = 3.6 × 10–7 M (90 μL); inset: colorimetric changes observed with time.

3.4. Colorimetric Determination of H2O2

Under the optimum experimental conditions, a rapid and simple colorimetric method based on TiO2 NPs coated with ionic liquid was used for H2O2 detection. Keeping in mind the color change for the quantitative assay of H2O2, a sensitive and selective colorimetric method has been used based on the relationship between H2O2 concentration and absorbance intensity at 652 nm. For the detection of H2O2, the sensitivity of the developed sensor was investigated with different concentrations of H2O2. Figure 12 shows the response of colorimetric biosensors toward various H2O2 concentrations. The sensor response and peak intensity were low at a lower concentration of H2O2, and they increased linearly by increasing its concentration. This technique enabled H2O2 detection with a linear range of 1 × 10–9–3.60 × 10–7 M with an R2 value of 0.999. The limit of quantification (LOQ) and limit of detection (LOD) were calculated as 2.86 × 10–7 and 8.61 × 10–8 M, respectively. An effective NP-based sensor shows a linear response to minor changes in analyte concentration.47Table 2 presents a comparison of the proposed work with the previous studies reported in the literature,48 which gives a proof of concept that our designed sensor can work at higher as well as lower concentrations. These observations were also compared with the previous study for H2O2 detection based on double molecular recognition, and the results were comparable to our proposed sensor.31

Figure 12.

Figure 12

Open in a new tab

(A) UV–vis spectra and the corresponding change concerning change in the concentration of H2O2. (B) Calibration plot of H2O2 concentration versus absorbance.

Table 2. Comparison of Colorimetric Biosensors for the Detection of H2O2 with Some Recently Reported Studies.

s. no. materials used method applied limit of detection (μM) linear range (μM) references
1 RhNPs colorimetric 0.75 5–125 (49)
2 AgNPs/GQDs colorimetric 0.162 0.5–50 (41)
3 Fe3S4-MNPs colorimetric 0.16 2–100 (50)
4 Fe2(MoO4)3-F colorimetric 0.7 1–30 (51)
5 NiFe LDH colorimetric 4.4 10–500 (52)
6 β-CD/Cu-NCsa colorimetric 0.2 0.02–10 (53)
7 NiO NPs colorimetric 8 20–100 (32)
8 CeO2 NPs colorimetric 0.5 0.6–1.5 (54)
9 [Pyr]Ac-NiO colorimetric 120 400–4000 (46)
10 TiO2 NPs colorimetric 0.086 0.001–0.36 present work
Open in a new tab

3.5. Selectivity Study of the Sensor

The selectivity of the proposed sensor was analyzed with potential interfering species including, ascorbic acid, folic acid, urea, potassium ions, calcium ions, dopamine, and methanol as depicted in Figure 13. Interference studies of the developed sensor play a key role in its productivity having diverse biomedical applications toward clinical diagnoses. Urine has a wide range of potentially interfering species; hence, urine presents huge challenges to various analytical approaches for H2O2 detection. These challenges are not only restricted to the detection limit and sensitivity of biosensors but more essentially to the selectivity of the sensor. Selective detection of H2O2 by the proposed strategy was performed by taking into consideration the coexisting biomolecules and ions in urine. Compared to H2O2, the absorbance values for interfering species such as ascorbic acid, folic acid, urea, uric acid, potassium, calcium ions, dopamine, and methanol were very small. The absorption value observed at 652 nm increases manyfold by adding H2O2. This analytical platform shows high selectivity toward H2O2 in the presence of other coexisting species and does not affect the response even in the presence of twofold of these interfering species. All of the experiments were conducted in the presence of 3.6 × 10–7 M H2O2 and the double concentration of interfering species. The results of selectivity are in good agreement with the reported literature.31 Furthermore, the stability of the developed sensor was assessed by measuring the response with H2O2 after 5 months; there was no remarkable difference observed in sensitivity and selectivity of the sensor. This experiment confirms that our sensor is highly stable and reproducible.

Figure 13.

Figure 13

Open in a new tab

Interfering study of H2O2 with other analytes (ascorbic acid, folic acid, urea, uric acid, potassium, calcium ions, dopamine, and methanol).

3.6. Real Sample Analysis

The proposed colorimetric method was applied to monitor H2O2 in urine samples of cancer patients. In the previous work reported for H2O2 sensing, no real sample analysis was performed.42 By adding different amounts of H2O2 to the samples, the recoveries and quantitative results of the proposed method are shown in Table 2. The standard solution of H2O2 was spiked with different ratios such as 59, 168, 247, and 329 nm to the urine samples of cancer patients obtained from IRNUM hospital Peshawar, KP, Pakistan (Table 3). The present amount of H2O2 in urine samples of cancer patients is determined from an already established calibration plot, using different concentrations of H2O2 under the same optimized conditions generated at 652 nm. The obtained results are summarized in Table 2 using the percentage recovery formula as shown in Figure 14.

Table 3. Recovery Tests for H2O2 Analysis in Cancer Patients’ Urine Samples Using the Proposed Assay (n = 3).

samples detected (nm) H2O2 added (nm) H2O2 found (nm) recovery (%) RSD (%)
1 1 59 60 101.69 0.838
2 2 168 170 101.19 1.009
3 3 247 250 101.21 0.459
4 1 329 330 100.30 0.923
Open in a new tab

Figure 14.

Figure 14

Open in a new tab

UV–vis spectra of the real samples.

4. Conclusions

Ionic-liquid-capped TiO2 NPs were synthesized and characterized by various analytical techniques such as FTIR, XRD, SEM, TGA, and Raman spectroscopy. The characteristic peaks related to TiO2 have been identified by FTIR, Raman, and XRD analyses. A strong tendency to achieve agglomeration and round morphology of the materials was observed by SEM analysis. The weight loss that occurred at 615 °C corresponds to the thermal decomposition of residual organic groups in the as-synthesized TiO2. The ionic liquid utilized not only provides stabilization to NPs but also enhances the conductivity and enzyme mimic properties of the NPs. The proposed colorimetric sensor TiO2/IL exhibits a low limit of detection of 8.61 × 10–8 M, a high sensitivity of 2.86 × 10–7 M, and a wide linear range of 1 × 10–9–3.6 × 10–7 M. The sensor showed a short incubation time of 4 min. The proposed sensor did not show any interference in results from the coexisting species present in the urine sample. The sensing probe was effectively applied for the determination of H2O2 in cancer patients’ urine samples.

Acknowledgments

The authors are grateful to the Department of Chemistry, Kohat University of Science and Technology, Kohat, for providing necessary funding and infrastructure for the project.

The authors declare no competing financial interest.

References

  1. Rivero P. J.; Ibañez E.; Goicoechea J.; Urrutia A.; Matias I. R.; Arregui F. J. A self-referenced optical colorimetric sensor based on silver and gold nanoparticles for quantitative determination of hydrogen peroxide. Sens. Actuators, B 2017, 251, 624–631. 10.1016/j.snb.2017.05.110. [DOI] [Google Scholar]
  2. Wang N.; Sun J.; Chen L.; Fan H.; Ai S. A Cu2(OH)3Cl-CeO2 nanocomposite with peroxidase-like activity, and its application to the determination of hydrogen peroxide, glucose and cholesterol. Microchim. Acta 2015, 182, 1733–1738. 10.1007/s00604-015-1506-8. [DOI] [Google Scholar]
  3. Veal E. A.; Day A. M.; Morgan B. A. Hydrogen peroxide sensing and signaling. Mol. Cell 2007, 26, 1–14. 10.1016/j.molcel.2007.03.016. [DOI] [PubMed] [Google Scholar]
  4. Lisanti M. P.; Martinez-Outschoorn U. E.; Lin Z.; Pavlides S.; Whitaker-Menezes D.; Pestell R. G.; Howell A.; Sotgia F. Hydrogen peroxide fuels aging, inflammation, cancer metabolism and metastasis: the seed and soil also needs “fertilizer”. Cell Cycle 2011, 10, 2440–2449. 10.4161/cc.10.15.16870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Scherf U.; Ross D. T.; Waltham M.; Smith L. H.; Lee J. K.; Tanabe L.; Kohn K. W.; Reinhold W. C.; Myers T. G.; Andrews D. T.; et al. A gene expression database for the molecular pharmacology of cancer. Nat. Genet. 2000, 24, 236–244. 10.1038/73439. [DOI] [PubMed] [Google Scholar]
  6. Nakashima K.; Wada M.; Kuroda N.; Akiyama S.; Imai K. High-performance liquid chromatographic determination of hydrogen peroxide with peroxyoxalate chemiluminescence detection. J. Liq. Chromatogr. 1994, 17, 2111–2126. 10.1080/10826079408013535. [DOI] [Google Scholar]
  7. Lu Y.; Yu J.; Ye W.; Yao X.; Zhou P.; Zhang H.; Zhao S.; Jia L. Spectrophotometric determination of mercury (II) ions based on their stimulation effect on the peroxidase-like activity of molybdenum disulfide nanosheets. Microchim. Acta 2016, 183, 2481–2489. 10.1007/s00604-016-1886-4. [DOI] [Google Scholar]
  8. Haddad Irani-nezhad M.; Khataee A.; Hassanzadeh J.; Orooji Y. A chemiluminescent method for the detection of H2O2 and glucose based on intrinsic peroxidase-like activity of WS2 quantum dots. Molecules 2019, 24, 689 10.3390/molecules24040689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen X.; Wu G.; Cai Z.; Oyama M.; Chen X. Advances in enzyme-free electrochemical sensors for hydrogen peroxide, glucose, and uric acid. Microchim. Acta 2014, 181, 689–705. 10.1007/s00604-013-1098-0. [DOI] [Google Scholar]
  10. Gul U.; Kanwal S.; Tabassum S.; Gilani M. A.; Rahim A. Microwave-assisted synthesis of carbon dots as reductant and stabilizer for silver nanoparticles with enhanced-peroxidase like activity for colorimetric determination of hydrogen peroxide and glucose. Microchim. Acta 2020, 187, 135 10.1007/s00604-019-4098-x. [DOI] [PubMed] [Google Scholar]
  11. Luo X.; Morrin A.; Killard A. J.; Smyth M. R. Application of nanoparticles in electrochemical sensors and biosensors. Electroanalysis 2006, 18, 319–326. 10.1002/elan.200503415. [DOI] [Google Scholar]
  12. Asati A.; Santra S.; Kaittanis C.; Nath S.; Perez J. M. Oxidase-like activity of polymer-coated cerium oxide nanoparticles. Angew. Chem. 2009, 121, 2344–2348. 10.1002/ange.200805279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen W.; Chen J.; Feng Y.-B.; Hong L.; Chen Q.-Y.; Wu L.-F.; Lin X.-H.; Xia X.-H. Peroxidase-like activity of water-soluble cupric oxide nanoparticles and its analytical application for detection of hydrogen peroxide and glucose. Analyst 2012, 137, 1706–1712. 10.1039/c2an35072f. [DOI] [PubMed] [Google Scholar]
  14. Mu J.; Wang Y.; Zhao M.; Zhang L. Intrinsic peroxidase-like activity and catalase-like activity of Co3O4 nanoparticles. Chem. Commun. 2012, 48, 2540–2542. 10.1039/c2cc17013b. [DOI] [PubMed] [Google Scholar]
  15. Liu Q.; Jiang Y.; Zhang L.; Zhou X.; Lv X.; Ding Y.; Sun L.; Chen P.; Yin H. The catalytic activity of Ag2S-montmorillonites as peroxidase mimetic toward colorimetric detection of H2O2. Mater. Sci. Eng.: C 2016, 65, 109–115. 10.1016/j.msec.2016.04.007. [DOI] [PubMed] [Google Scholar]
  16. Lin X.; Liu Y.; Tao Z.; Gao J.; Deng J.; Yin J.; Wang S. Nanozyme-based bio-barcode assay for high sensitive and logic-controlled specific detection of multiple DNAs. Biosens. Bioelectron. 2017, 94, 471–477. 10.1016/j.bios.2017.01.008. [DOI] [PubMed] [Google Scholar]
  17. Shi W.; Zhang X.; He S.; Huang Y. CoFe2O4 magnetic nanoparticles as a peroxidase mimic mediated chemiluminescence for hydrogen peroxide and glucose. Chem. Commun. 2011, 47, 10785–10787. 10.1039/c1cc14300j. [DOI] [PubMed] [Google Scholar]
  18. Dai Z.; Liu S.; Bao J.; Ju H. Nanostructured FeS as a mimic peroxidase for biocatalysis and biosensing. Chem. - Eur. J. 2009, 15, 4321–4326. 10.1002/chem.200802158. [DOI] [PubMed] [Google Scholar]
  19. Rajakumar G.; Rahuman A. A.; Jayaseelan C.; Santhoshkumar T.; Marimuthu S.; Kamaraj C.; Bagavan A.; Zahir A. A.; Kirthi A. V.; Elango G.; et al. Solanum trilobatum extract-mediated synthesis of titanium dioxide nanoparticles to control Pediculus humanus capitis, Hyalomma anatolicum anatolicum and Anopheles subpictus. Parasitol. Res. 2014, 113, 469–479. 10.1007/s00436-013-3676-9. [DOI] [PubMed] [Google Scholar]
  20. Fei Yin Z.; Wu L.; Yang H. G.; Su Y. H. Recent progress in biomedical applications of titanium dioxide. Phys. Chem. Chem. Phys. 2013, 15, 4844–4858. 10.1039/c3cp43938k. [DOI] [PubMed] [Google Scholar]
  21. Wei X.; Zhu G.; Fang J.; Chen J. Synthesis, characterization, and photocatalysis of well-dispersible phase-pure anatase TiO2 nanoparticles. Int. J. Photoenergy 2013, 2013, 726872 10.1155/2013/726872. [DOI] [Google Scholar]
  22. Yan G.; Chen J.; Hua Z. Roles of H2O2 and OH radical in bactericidal action of immobilized TiO2 thin-film reactor: An ESR study. J. Photochem. Photobiol., A 2009, 207, 153–159. 10.1016/j.jphotochem.2009.03.003. [DOI] [Google Scholar]
  23. Fujishima A.; Rao T. N.; Tryk D. A. Titanium dioxide photocatalysis. J. Photochem. Photobiol., C 2000, 1, 1–21. 10.1016/S1389-5567(00)00002-2. [DOI] [Google Scholar]
  24. Liu K.; Cao M.; Fujishima A.; Jiang L. Bio-inspired titanium dioxide materials with special wettability and their applications. Chem. Rev. 2014, 114, 10044–10094. 10.1021/cr4006796. [DOI] [PubMed] [Google Scholar]
  25. Kikuchi Y.; Sunada K.; Iyoda T.; Hashimoto K.; Fujishima A. Photocatalytic bactericidal effect of TiO2 thin films: dynamic view of the active oxygen species responsible for the effect. J. Photochem. Photobiol., A 1997, 106, 51–56. 10.1016/S1010-6030(97)00038-5. [DOI] [Google Scholar]
  26. Coral J. A.; Kitchens C. L. Quantitative measurements of hydroxyl radicals generated by irradiated titanium dioxide nanoparticle suspensions. bioRxiv 2019, 10.1101/2019.12.27.889618. [DOI] [Google Scholar]
  27. Fei Z.; Geldbach T. J.; Zhao D.; Dyson P. J. From dysfunction to bis-function: on the design and applications of functionalised ionic liquids. Chem. - Eur. J. 2006, 12, 2122–2130. 10.1002/chem.200500581. [DOI] [PubMed] [Google Scholar]
  28. Ahrens S.; Peritz A.; Strassner T. Tunable aryl alkyl ionic liquids (TAAILs): The next generation of ionic liquids. Angew. Chem., Int. Ed. 2009, 48, 7908–7910. 10.1002/anie.200903399. [DOI] [PubMed] [Google Scholar]
  29. Nishan U.; Gul R.; Muhammad N.; Asad M.; Rahim A.; Shah M.; Iqbal J.; Uddin J.; Shujah S.; et al. Colorimetric based sensing of dopamine using ionic liquid functionalized drug mediated silver nanostructures. Microchem. J. 2020, 159, 105382 10.1016/j.microc.2020.105382. [DOI] [Google Scholar]
  30. He Z.; Alexandridis P. Nanoparticles in ionic liquids: interactions and organization. Phys. Chem. Chem. Phys. 2015, 17, 18238–18261. 10.1039/C5CP01620G. [DOI] [PubMed] [Google Scholar]
  31. Zarif F.; Rauf S.; Qureshi M. Z.; Shah N. S.; Hayat A.; Muhammad N.; Rahim A.; Nawaz M. H.; Nasir M. Ionic liquid coated iron nanoparticles are promising peroxidase mimics for optical determination of H2O2. Microchim. Acta 2018, 185, 302 10.1007/s00604-018-2841-3. [DOI] [PubMed] [Google Scholar]
  32. Liu Q.; Yang Y.; Li H.; Zhu R.; Shao Q.; Yang S.; Xu J. NiO nanoparticles modified with 5, 10, 15, 20-tetrakis (4-carboxyl pheyl)-porphyrin: promising peroxidase mimetics for H2O2 and glucose detection. Biosens. Bioelectron. 2015, 64, 147–153. 10.1016/j.bios.2014.08.062. [DOI] [PubMed] [Google Scholar]
  33. Nishan U.; Bashir F.; Muhammad N.; Khan N.; Rahim A.; Shah M.; Nazir R.; Sayed M. Ionic liquid as a moderator for improved sensing properties of TiO2 nanostructures for the detection of acetone biomarker in diabetes mellitus. J. Mol. Liq. 2019, 294, 111681 10.1016/j.molliq.2019.111681. [DOI] [Google Scholar]
  34. Pratheepa M. I.; Lawrence M. X-Ray Diffraction Analyses of Titanium Dioxide Nanoparticles. Int. J. Sci. Res. Sci. Technol. 2017, 3, 83–88. [Google Scholar]
  35. Haider A. J.; Jameel Z. N.; Taha S. Y. Synthesis and characterization of TiO2 nanoparticles via sol-gel method by pulse laser ablation. Eng. Technol. J. 2015, 33, 761–771. [Google Scholar]
  36. Askari M. B.; Banizi Z. T.; Seifi M.; Dehaghi S. B.; Veisi P. Synthesis of TiO2 nanoparticles and decorated multi-wall carbon nanotube (MWCNT) with anatase TiO2 nanoparticles and study of optical properties and structural characterization of TiO2/MWCNT nanocomposite. Optik 2017, 149, 447–454. 10.1016/j.ijleo.2017.09.078. [DOI] [Google Scholar]
  37. Muniz E.; Góes M.; Silva J.; Varela J. A.; Joanni E.; Parra R.; Bueno P. R. Synthesis and characterization of mesoporous TiO2 nanostructured films prepared by a modified sol–gel method for application in dye solar cells. Ceram. Int. 2011, 37, 1017–1024. 10.1016/j.ceramint.2010.11.014. [DOI] [Google Scholar]
  38. Li W.; Zeng T. Preparation of TiO2 anatase nanocrystals by TiCl4 hydrolysis with additive H2SO4. PLoS One 2011, 6, e21082 10.1371/journal.pone.0021082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kudo A.; Miseki Y. Heterogeneous Photocatalyst Materials for Water Splitting. Chem. Soc. Rev. 2009, 38, 253–278. 10.1039/B800489G. [DOI] [PubMed] [Google Scholar]
  40. Sugapriya S.; Sriram R.; Lakshmi S. Effect of annealing on TiO2 nanoparticles. Optik 2013, 124, 4971–4975. 10.1016/j.ijleo.2013.03.040. [DOI] [Google Scholar]
  41. Achilleos A.; Hapeshi E.; Xekoukoulotakis N. P.; Mantzavinos D.; Fatta-Kassinos D. Factors affecting diclofenac decomposition in water by UV-A/TiO2 photocatalysis. Chem. Eng. J. 2010, 161, 53–59. 10.1016/j.cej.2010.04.020. [DOI] [Google Scholar]
  42. Nasir M.; Rauf S.; Muhammad N.; Nawaz M. H.; Chaudhry A. A.; Malik M. H.; Shahid S. A.; Hayat A. Biomimetic nitrogen doped titania nanoparticles as a colorimetric platform for hydrogen peroxide detection. J. Colloid Interface Sci. 2017, 505, 1147–1157. 10.1016/j.jcis.2017.07.014. [DOI] [PubMed] [Google Scholar]
  43. Van Dao D.; Nguyen T. T.; Song H.-Y.; Yang J.-K.; Kim T.-W.; Yu Y.-T.; Lee I.-H. Ionic liquid-assisted preparation of Ag-CeO2 nanocomposites and their improved photocatalytic activity. Mater. Des. 2018, 159, 186–194. 10.1016/j.matdes.2018.08.042. [DOI] [Google Scholar]
  44. Yang B. In Synthesis and Characterization of Brønsted Acidic Ionic Liquids, Proceedings of the AASRI International Conference on Industrial Electronics and Applications (2015); Atlantis Press, 2015.
  45. Welton T. Room-temperature ionic liquids. Solvents for synthesis and catalysis. Chem. Rev. 1999, 99, 2071–2083. 10.1021/cr980032t. [DOI] [PubMed] [Google Scholar]
  46. Zarif F.; Rauf S.; Khurshid S.; Muhammad N.; Hayat A.; Rahim A.; Shah N. S.; Yang C. P. Effect of pyridinium based ionic liquid on the sensing property of Ni0 nanoparticle for the colorimetric detection of hydrogen peroxide. J. Mol. Struct. 2020, 1219, 128620 10.1016/j.molstruc.2020.128620. [DOI] [Google Scholar]
  47. Peng G.; Tisch U.; Adams O.; Hakim M.; Shehada N.; Broza Y. Y.; Billan S.; Abdah-Bortnyak R.; Kuten A.; Haick H. Diagnosing lung cancer in exhaled breath using gold nanoparticles. Nat. Nanotechnol. 2009, 4, 669–673. 10.1038/nnano.2009.235. [DOI] [PubMed] [Google Scholar]
  48. Zarif F.; Khurshid S.; Muhammad N.; Zahid Qureshi M.; Shah N. S. Colorimetric Sensing of Hydrogen Peroxide Using Ionic-Liquid-Sensitized Zero-Valent Copper Nanoparticle (nZVCu). ChemistrySelect 2020, 5, 6066–6074. 10.1002/slct.202001470. [DOI] [Google Scholar]
  49. Choleva T. G.; Gatselou V. A.; Tsogas G. Z.; Giokas D. L. Intrinsic peroxidase-like activity of rhodium nanoparticles, and their application to the colorimetric determination of hydrogen peroxide and glucose. Microchim. Acta 2018, 185, 22 10.1007/s00604-017-2582-8. [DOI] [PubMed] [Google Scholar]
  50. Ding C.; Yan Y.; Xiang D.; Zhang C.; Xian Y. Magnetic Fe3S4 nanoparticles with peroxidase-like activity, and their use in a photometric enzymatic glucose assay. Microchim. Acta 2016, 183, 625–631. 10.1007/s00604-015-1690-6. [DOI] [Google Scholar]
  51. Wang B.; Ju P.; Zhang D.; Han X.; Zheng L.; Yin X.; Sun C. Colorimetric detection of H2O2 using flower-like Fe2(MoO 4)3 microparticles as a peroxidase mimic. Microchim. Acta 2016, 183, 3025–3033. 10.1007/s00604-016-1955-8. [DOI] [Google Scholar]
  52. Zhan T.; Kang J.; Li X.; Pan L.; Li G.; Hou W. NiFe layered double hydroxide nanosheets as an efficiently mimic enzyme for colorimetric determination of glucose and H2O2. Sens. Actuators, B 2018, 255, 2635–2642. 10.1016/j.snb.2017.09.074. [DOI] [Google Scholar]
  53. Zhong Y.; Deng C.; He Y.; Ge Y.; Song G. Exploring a monothiolated β-cyclodextrin as the template to synthesize copper nanoclusters with exceptionally increased peroxidase-like activity. Microchim. Acta 2016, 183, 2823–2830. 10.1007/s00604-016-1915-3. [DOI] [Google Scholar]
  54. Jiao X.; Song H.; Zhao H.; Bai W.; Zhang L.; Lv Y. Well-redispersed ceria nanoparticles: promising peroxidase mimetics for H2O2 and glucose detection. Anal. Methods 2012, 4, 3261–3267. 10.1039/c2ay25511a. [DOI] [Google Scholar]

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES