V₂O₅ nanowires as a robust and efficient peroxidase mimic at high temperature in aqueous media

Abstract

For the first time, this article demonstrated that V₂O₅ nanowires served as a robust and efficient peroxidase mimic catalyst in luminol-based chemiluminescence reaction in aqueous media at high temperature. Over 90% of the maximum catalytic activity remained at 70 °C. Singlet oxygen was involved in the CL reaction catalysed by V₂O₅ nanowires, which was quite different from the traditional catalysts.

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Recently, nanomaterials as enzyme mimetics have attracted increasing interest owing to their stability under harsh conditions, low cost and resistance to denaturation compared with natural enzymes.¹ Various nanomaterials have been found to exhibit unexpected enzyme-like activity (so-called nanozymes) and widely applied in medical diagnosis, biosensing and environmental analysis.² However, except the study by Qu et al.,³ recently reporting that Au NPs encapsulated in porous support serve as a robust and efficient peroxidase mimic at high temperature by utilizing ionic liquid as an efficient modulator, almost all of the reported nanozymes cannot realize high-temperature catalytic reactions in aqueous media. This drawback will obviously limit the applications of nanozymes as catalysts. Hence, searching for new and efficient nanozyme mimics for realizing high-temperature catalytic reactions in aqueous media is highly valuable.

Meanwhile, the increasing applications of nanozymes lead to a great demand for developing an efficient method to screen the enzyme-like activities of various nanomaterials.^1c,4 A well-established method for screening the nanomaterial-based enzyme mimetics is mainly based on colorimetric method, in particular, the color reaction of 3,3,5,5-tetramethylbenzidine (TMB) oxidation catalyzed by various nanozymes.^2a–2l,5 However, it suffers from the drawbacks of low sensitivity, the use of toxic chromogenic substrate and long reaction time. Chemiluminescence (CL) approach is one of the most promising screening methods due to its high sensitivity, simplicity, rapidity and low cost.⁶ However, only rare investigations have documented the screening of the nanomaterials-based enzyme mimetics by the CL method.^2m

Herein, for the first time, by using the luminol-based CL reaction as a screening tool, we demonstrated that V₂O₅ nanowires possess intrinsic enzyme-like activity and could catalyze luminol oxidation to produce intensified CL. It was found that V₂O₅ nanowires could serve as a robust and efficient peroxidase mimic in aqueous media at high temperature without need of any modulator such as ionic liquid. This unique feature indicated that the V₂O₅ nanowires had great potential applications in catalyzing high-temperature reactions in aqueous solution. V₂O₅ is selected due to its unique layered structure and distinctive redox behavior. It is a multifunctional material with a broad range of applications,⁷ such as in lithium batteries,^7a gas sensors,^7b electrochromic devices,^7c and supercapacitors.^7d

The V₂O₅ nanowires were synthesized by hydrothermal method.^7c The SEM and TEM images confirmed the nanowire structures of V₂O₅ samples (Fig. S1A and S1B ESI†). The XRD peak at 2θ = 9.1° (Fig. S1C ESI†) was signed to the (001) reflection, corresponding to a layered structure of the hydrated V₂O₅.^7c The appearance of the (001) reflection with higher intensity indicated a higher crystallinity with a preferential orientation and highly ordered structure.⁸ The FT-IR spectra in the band range of 1008 to 900 cm⁻¹ was associated with the unshared V–O stretching vibration, and the bands at 847 and 533 cm⁻¹ corresponded to the V–O–V bending vibration and edge-sharing V–O stretching vibration, respectively (Fig. S1D ESI†).⁹

The oxidase-like activity of V₂O₅ nanowires was evaluated by traditional luminol CL reaction. Luminol could be oxidized by dissolved O₂ to produce a weak emission (Fig. S2 ESI†), which could be enhanced by V₂O₅ nanowires with one peak situating at around 430 nm (the maximum emission spectra of 3-aminophthalate). This fact suggested that the V₂O₅ nanowires worked as a catalytic reagent due to absence of any new emitter produced in the reaction. The result also confirmed the oxidase-like activity of V₂O₅ nanowires to catalyse the reaction between luminol and dissolved O₂. Interesting, upon the addition of H₂O₂, the CL intensity of the V₂O₅ nanowires–luminol–H₂O₂ system was ca. 18 times larger than that of the V₂O₅ nanowires–luminol system after deducting blank data (Fig. S2 ESI†), showing that V₂O₅ nanowires had peroxidase-like catalytic activity.

The oxidase/peroxidase-like activity of V₂O₅ nanowires was evaluated in the catalytic reaction systems with typical substrates TMB, OPD and ABTS in the absence and presence of H₂O₂. Clearly, V₂O₅ nanowires could catalyze the oxidation of TMB, OPD and ABTS by dissolved O₂ in NaAc buffer, and produce the typical color reaction (Fig. S3 ESI†). Similar to other enzyme mimic reaction, the typical absorbance peak of oxidation products of TMB was located at 652 nm.^2a–2l The absorbance of the oxidized product of TMB at 652 nm significantly increased in the presence of V₂O₅ nanowires. These results confirmed the oxidase-like activity of V₂O₅ nanowires toward TMB, OPD and ABTS. Upon the addition of H₂O₂, intensified color reactions were observed (Fig. S3 ESI†), which evidenced the peroxidase-like catalytic activity of V₂O₅ nanowires.

Luminol CL reaction was proceeded quite rapidly, and thus valuable for fast screening of the enzyme-like activity of nanomaterials. The kinetic curve of the present system was obtained from the static injection experiments. The result suggested that a strong CL peak appeared immediately after H₂O₂ solution or mixture of H₂O₂ and V₂O₅ nanowires was injected. It took less than 5 s for the signal to reach baseline (Fig. S4 ESI†). However, it took about 30 min when using a colorimetric method (Fig. S3 ESI†).

In further investigation of the characteristics of peroxidase-like of V₂O₅ nanowires, we found that the catalytic behavior of V₂O₅ nanowires depended on temperature and solution pH. As shown in Fig. 1A, the catalytic activity of V₂O₅ nanowires increased with increasing temperature from 5 to 60 °C, followed by a plateau from 60 to 65 °C, then decreased above 65 °C. If the maximum catalytic activity of V₂O₅ nanowires was set to 100% at 65 °C, then it could be concluded that over 90% of the maximum catalytic activity was remained even at 70 °C. In contrast to previously reported nanozymes which can not realize high-temperature reactions, to our knowledge, this paper shows for the first time that the V₂O₅ nanowires could serve as a robust and efficient peroxidase mimic in aqueous media without the need of a modulator such as ionic liquid.³ This shows great potential of V₂O₅ nanowires as nanozyme in high-temperature applications in catalysis. The catalytic activities of V₂O₅ nanowires were determined by adjusting the solution pH from 3.08 to 9.27 by 0.1 M HCl or 0.1 M NaOH. The maximal catalytic activity was obtained at pH 4.40 (Fig. S5 ESI†). Besides the temperature and pH, it was found that the peroxidase activity of V₂O₅ nanowires depended on H₂O₂ concentration. The maximal level of peroxidase activity was found at 300 μM H₂O₂ (Fig. 1B and S6 ESI†). Further increase in the H₂O₂ concentration inhibited its activity, which was similar to that of HRP. This further confirmed that the peroxidase-like activity of V₂O₅ nanowires. In addition, the Michaelis–Menten behavior of the V₂O₅ nanowires was investigated to further understand the peroxidase-like activity of the V₂O₅ nanowires. The kinetic data obtained with 18.2 mg L⁻¹ V₂O₅ nanowires was fitted to the Michaelis–Menten kinetic model using a nonlinear least squares fitting routine. The K_m value with H₂O₂ was 58.9 ± 2.9 μM (n = 3) for the V₂O₅ nanowires, which was significantly lower than that for CoFe₂O₄ NPs,^2m showing that V₂O₅ nanowires had higher affinity to H₂O₂ than CoFe₂O₄ NPs (Table S1 ESI†).

Fig. 1 (A) Effect of temperature on catalytic activity of V₂O₅ nanowires. (B) Steady-state kinetic assay of V₂O₅ nanowires as catalysts. Conditions: 8 μM luminol in 0.2 M Na₂CO₃ buffer (pH 11.3), 1.0 μM H₂O₂, 18.2 mg L⁻¹ V₂O₅ (pH 4.40). Error bars represent one standard deviation for three measurements.

The effect of different structures of V₂O₅ products, such as V₂O₅ nanowires, V₂O₅ nanorods, V₂O₅ nanoparticles, V₂O₅ nanosheets and V₂O₅ nanobelts (Fig. S7 ESI†), on their catalytic activities were studied. The catalytic activity follows the order of V₂O₅ nanowires > V₂O₅ nanorods > V₂O₅ nanoparticles > V₂O₅ nanosheets > V₂O₅ nanobelts (Fig. S8 ESI†). It is noted that V₂O₅ nanowires had the highest catalytic activity as compared with other nanostructures. Puvvada et al. observed that the peroxidase mimetic activity of the truncated octahedral Fe₃O₄ nanocrystals was superior to that of spherical-shaped nanoparticles, which was due to high surface energy facets.¹⁰ Hence, we speculate that the high catalytic activity of V₂O₅ nanowires is probably related to their surface facets. In addition, the higher catalytic performance of the V₂O₅ nanorods than that of V₂O₅ nanoparticles is probably due to their larger specific surface area per unit volume compared to spherical shaped nanostructure.¹¹

The reaction conditions were optimized in the present CL system (Fig. S9 ESI†) and the optimal reaction conditions were obtained as follows: 8 μM luminol in 0.2 M Na₂CO₃ buffer (pH 11.3), 18.2 mg L⁻¹ V₂O₅. Under optimal conditions, the calibration data for 0.001–3 μM H₂O₂ were well fitted by the equation: ΔI = 364.6 + 2709.5 [H₂O₂] (μM) (n = 12, r² = 0.990) (Fig. S10A ESI†), where ΔI is the net CL intensity. The limit of detection for H₂O₂ was 0.5 nM. The selectivity of the present sensing system for H₂O₂ detection was examined by the addition of other ions into the system. Table S2† shows that the additions of commonly existing ions had minor effect on the quantitation of H₂O₂ (ESI†).

The glucose detection was performed in two separated steps (ESI†). Because H₂O₂ was the main product of glucose oxidase (GOx)-catalyzed reaction, thus, when the CL reaction of luminol–H₂O₂ catalyzed by V₂O₅ nanowires was coupled with the glucose catalytic reaction by GOx, CL detection of glucose could be realized. The linear range was from 0.05 to 10 μM (Fig. S10B ESI†), and the regression equation was ΔI = 251.7 + 443 [glucose] (μM) (n = 9, r² = 0.991). Glucose was detected as low as 1 nM, which was more sensitive than most of the previous works using the luminol–H₂O₂ CL system for glucose detection.^2m,12 For glucose detection, the selectivity experiments were carried out using 10 mM lactose and 10 mM fructose in place of glucose (1.0 μM). No detectable signals were observed compared with that of glucose, showing high selectivity for glucose detection.

The present method was also used for glucose detection in blood serum samples to evaluate its applicability and reliability. Validation of the method was performed by comparison study, which was carried out by an OneTouch Ultra glucose meter. The results obtained by the proposed CL method agreed well with those obtained by the conventional electrochemical method (Table S3 ESI†). This implied the potential applicability of the V₂O₅ nanowires as nanozymes mediated CL method for fast, sensitive and accurate sensing of glucose in biological samples. Further, the method was employed to rainwater and lake water samples for H₂O₂ determination. The recovery for spiked water samples range from 90 to 108%, (Table S4 ESI†). Thus the proposed method proved to be satisfactory for the routine estimation of H₂O₂ in water samples.

The most probable mechanism for the CL enhancement of luminol by V₂O₅ was mainly through the intermediate of ¹O₂, which was suggested in the carbon nanodots-catalyzed luminol–H₂O₂ CL reaction and graphene oxide-catalyzed luminol–H₂O₂ CL reaction.¹³ In order to confirm this, the different active oxygen species as intermediates of the present CL reaction were studied. As seen, 5.0 mM NaN₃, a well-known scavenger of singlet O₂, quenched about 76% of the CL intensity (Table S5 ESI†). This indicated that singlet ¹O₂ contributed to the observed CL.

The ¹O₂ was probably from the recombination reaction of ˙OH and O₂˙⁻.^13a With regard to ˙OH generation, as shown in Table S5,† the CL intensity was almost totally quenched by the addition of 1.0 μM ascorbic acid (scavengers of ˙OH radical and O₂˙⁻ radical) or 5 mM thiourea (scavengers of ˙OH radical), indicating that abundant ˙OH radicals were generated in the reaction.^2m This was confirmed by the result of ESR (Fig. S11 ESI†). As a specific target molecule of ˙OH radical, DMPO was used to identify the formation of ˙OH radical during the CL reaction. The ESR spectra in the presence or absence of V₂O₅ nanowires displayed a 4-fold characteristic peak of the typical DMPO-˙OH adduct with an intensity ratio of 1 : 2 : 2 : 1. However, the DMPO-˙OH adduct signal intensity in the presence of V₂O₅ was much higher than that in the absence of V₂O₅ (Fig. S11 ESI†). This served to evidence that the V₂O₅ nanowires exhibited high catalyse-mimic activity for the decomposition of H₂O₂ to produce ˙OH radical. With regard to O₂˙⁻ generation, more than 90% of the CL emission was inhibited by the addition of 0.07 mg mL⁻¹ SOD (Table S5 ESI†), suggesting that O₂˙⁻ was produced and played a role in the CL emission process. In addition, O₂˙⁻ might come from oxygen dissolved in the solution or H₂O₂ decomposition.^13b

After production, the recombination reaction of ˙OH and O₂˙⁻ would generate ¹O₂.^13a The reaction of the produced ¹O₂ with luminol might be responsible for the final CL emission at ∼430 nm. In the presence of V₂O₅ nanowires, much more active radical species such as O₂˙⁻ or ˙OH were produced, which was reflected by the much higher signal intensity of the DMPO-˙OH adduct (Fig. S11 ESI†). This would lead to yield more ¹O₂ on the surface of V₂O₅ nanowires, which generated strong CL emission centered at ∼430 nm (Scheme 1).

Scheme 1 Possible CL mechanism from the reaction between V₂O₅ nanowires and luminol.

In summary, for the first time, we demonstrated that V₂O₅ nanowires could serve as robust and efficient peroxidase mimic catalyst in aqueous media at high temperature without the aid of any modulator such as ionic liquid, showing its great potential applications as a nanozyme in high-temperature catalysis. Also, we developed a luminol-based CL method for fast screening of the enzyme-like activity of nanozyme. Compared with the colorimetric method based screening method with low sensitivity, the use of toxic chromogenic substrate and long reaction time, the proposed CL-based screening method not only provided the advantages of simplicity and rapidity, but also embraced high sensitivity and sample throughput. Hence, it could be used for screening of the nanoparticles-based mimetic enzymes in a large scale.

The financial support of the research by the Natural Science Foundation of China (no. 21075099) is acknowledged.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c4ra03118k

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