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Colorimetric method for clenbuterol detection based on the enzyme-like activity of magnetic Fe3O4@mSiO2@MMT-SDS nanoparticles
⁎Corresponding author. zs@zqu.edu.cn (Shuai Zhang)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Abstract
Fe3O4 has enzyme-like activities, can be used as an alternative for natural enzymes. Fe3O4@mSiO2@MMT-SDS nanoparticles were prepared by modifying Fe3O4@mSiO2 with MMT and SDS to impart negative charges to the particle surfaces. Fe3O4@mSiO2@MMT-SDS nanoparticles exhibited excellent catalytic activity for the catalytic oxidation of clenbuterol (CLB) by H2O2, turning the solution blue in the presence of the 3,3ʹ, 5,5ʹ-tetramethylbenzidine chromogen.. A novel colorimetric method for clenbuterol detection were demonstrated based on the peroxidase-like activity of Fe3O4@mSiO2@MMT-SDS.
Abstract
This study demonstrates a novel colorimetric method for clenbuterol (CLB) detection based on the peroxidase-like activity of SiO2-coated Fe3O4 nanoparticles (Fe3O4@mSiO2) modified with montmorillonite (MMT) and sodium dodecyl sulfate (SDS), namely, Fe3O4@mSiO2@MMT-SDS. Fe3O4@mSiO2@MMT-SDS nanoparticles were prepared by modifying Fe3O4@mSiO2 with MMT and SDS to impart negative charges to the particle surfaces. The structural properties, steady-state kinetics, catalytic performance, and reaction conditions of the as-prepared nanoparticles were analyzed. Fe3O4@mSiO2@MMT-SDS nanoparticles exhibited excellent dispersion and enzyme-like activity, which improved the affinity with substrate (TMB and H2O2) for the catalytic oxidation of CLB by H2O2, turning the solution blue in the presence of the 3,3ʹ, 5,5ʹ-tetramethylbenzidine chromogen. Under optimal reaction conditions, a strong linear correlation between the absorbance of solution and CLB concentration (R2 = 0.9983) was observed. Here, the limit of detection, spike recovery, and relative standard deviation were 9.6 × 10-9 mol·L-1, 94.2 %–102.2 %, and 2.2 %–4.4 %, respectively. The proposed method is a simple, fast, cost-efficient, and sensitive assay, which can be used to rapidly and accurately screen CLB in pork and animal feeds.
Keywords
Magnetic nanoparticle
Enzyme-like
Colorimetry
Clenbuterol
- CLB
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clenbuterol
- mSiO2
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mesoporous silica
- MMT
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montmorillonite
- SDS
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sodium dodecyl sulfate
- CTAB
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cetrimonium bromide
- TMB
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3,3′,5,5′-tetramethylbenzidine
- FHB
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fenoterol hydrobromide
- RAC
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ractopamine
Abbreviations
1 Introduction
Clenbuterol (CLB) is a sympathomimetic (beta agonist) bronchodilator that can be used to treat asthma and animal respiratory diseases (Zhao et al., 2023; Duan et al., 2020). It is a commonly used type of synthetic adrenaline, which can significantly increase the rate of muscle growth and promote the conversion of fat into muscle (Wang et al., 2023; Cai et al., 2023). When used as an animal feed additive, CLB increases the lean meat percentage of pork. However, the long-term consumption of CLB-contaminated pork has deleterious effects on human health, as it can cause heart palpitations, tachycardia, oversensitive nerves, and muscle tremors (Brambilla et al., 2000; Barbosa et al., 2005; Salpeter et al., 2006). Many countries, including the European Union and China, have prohibited the use of CLB as a feed additive or veterinary drug for livestock (Xu et al., 2020; Li et al., 2023; Shenashen et al., 2022). Therefore, the development of a rapid, simple, and sensitive method to detect and monitor CLB in animal feed and other products is considerably important.
As the concerns related to food safety are increasing, stringent standards have been enforced for the CLB concentration, and various methodologies have been developed for CLB detection. Among these methods, high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), and liquid chromatography–mass spectrometry (LC–MS) are widely used owing to their high sensitivity and low detection limits (Velasco-Bejarano et al., 2023; Medellín-Martínez et al., 2018; Ye et al., 2016). However, they require complex instruments and skilled technicians, in addition to expensive and time-consuming procedures. Furthermore, these methods are not suitable for on-site detection, limiting their scalability. Diverse novel methods, such as the enzyme-linked immunosorbent assay (ELISA), surface-enhanced Raman spectroscopy (SERS), molecular imprinting and electrochemical surface plasmon resonance (SPR), have been developed for CLB detection and monitoring in food, feed, and environmental wastewater (Bui et al., 2016; Liu et al., 2022; Cheng et al., 2019; Lin and Li, 2023; Kabiraz et al., 2018). Owing to the novelty, speed, and simplicity of these methods, these methods have garnered significant research and industrial attention. In particular, ELISA has high selectivity and sensitivity and is often used to detect CLB residues in animal feed, food, and urine. However, the enzymes used for ELISA are expensive, susceptible to deactivation, and difficult to store, which limit its usefulness.
Magnetic nanoparticles are widely used in fields such as material, biomedicine, and environmental engineering due to their superparamagnetism, fast mass transfer, high efficiency, and selective adsorption (Fayazi & Ghanbarian, 2020; Fayazi et al., 2022; Sadeghi et al., 2023). Fe3O4 is a typical magnetic nanoparticle with enzymatic-like activity, which can be used as an alternative for natural enzymes (Tu et al., 2023; Wu et al., 2023; Xing et al., 2023). For example, 3,3′,5,5′-tetramethylbenzidine (TMB) can be catalyzed by Fe3O4 nanoparticles and show distinct blue color in the presence of H2O2. The concurrence of Fe2+ and Fe3+ in Fe3O4 accelerates the catalytic process where Fe3+ is partially reduced to Fe2+, activating H2O2 to generate.OH firstly, and then Fe2+ is oxidized to Fe3+ by H2O2. The enzyme-like activity of Fe3O4 can be further improved by structural modifications with noble metals (Liu et al., 2023; Tarhan et al., 2022; Guan et al., 2021). However, bare Fe3O4 tends to aggregate, which rapidly reduces its specific surface area and the number of catalytically active centers, rendering it significantly less active than natural enzymes (Radoń et al., 2017). Therefore, identifying a suitable coating for Fe3O4 that can improve its dispersion and enzyme-like activity is essential. SiO2 is a promising coating material. In current study, mesoporous silica (mSiO2) was used to modify and coat Fe3O4 nanoparticles to improve dispersion and prevent aggregation (Li et al., 2020; Sadeghi et al., 2023; Wang et al., 2022; Wan et al., 2022), but its enzyme activity still needs to be improved.
The objective of this study is to develop a fast and sensitive colorimetric method for CLB detection in animal feeds and pork. For this purpose, magnetic SiO2-coated Fe3O4 nanoparticles modified with montmorillonite (MMT) and sodium dodecyl sulfate (SDS), labeled as, Fe3O4@mSiO2@MMT-SDS nanoparticles, with excellent dispersion and enzyme-like activity were prepared using the sol–gel method. MMT was used considering its excellent absorptivity and numerous mesopores that freely allow H2O2 ingress and egress. The nanoparticles were further modified with SDS to alter their charge and reduce the surface energy of their solid–liquid interface, which improved affinity to TMB and H2O2 and enhanced the enzyme-like activity. The resulting Fe3O4@mSiO2@MMT-SDS particles were then used to catalyze the oxidation of CLB by H2O2. The excess H2O2 then oxidizes the TMB chromogen and forms a blue solution. Within a certain range of concentration, a linear relationship is observed between CLB and H2O2 concentrations.
2 Materials and methods
2.1 Materials
The materials used in this study were: 99 % 3,3′,5,5′-TMB purchased from J&K Scientific (Gangzhou, China); tetraethoxysilane (TEOS) and disodium hydrogen phosphate dodecahydrate obtained from Tianjin Guangcheng Chemical Reagent Co., Ltd. (Tianjin, China); sodium dihydrogen phosphate dihydrate procured from Tianjin Fuchen Chemical Reagent Co., Ltd. (Tianjin, China); cetrimonium bromide (CTAB), ammonium ferrous sulfate hexahydrate, ferric chloride hexahydrate, SDS, 30 % peroxide, ammonia, citric acid, hydrochloric acid, sodium hydroxide, diethyl ether, methanol, anhydrous ethanol, acetone, and anhydrous sodium sulfate purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China); MMT obtained from Zhejiang Feng Hong (Huzhou, China); CLB standard solution purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.(Shanghai, China); nitrogen gas procured from Zhaoqing Gaonengda Chemical Industry Co. Ltd.(Zhaoqing, China); solid phase extraction (SPE)-1 eluent (5 % aqueous methanol with 2 % ammonia); and SPE-2 eluent (30 % aqueous methanol with 2 % ammonia). All reagents used are analytical pure, and double-distilled water was used in this study.
2.2 Preparation of magnetic and mesoporous Fe3O4@mSiO2@MMT-SDS nanoparticles
2.2.1 Synthesis of magnetic mesoporous Fe3O4 nanoparticles
Fe3O4 was prepared via the co-precipitation method. Briefly, 6.0 mL of 0.5 mol·L-1 Fe2+ and 10.0 mL of 0.5 mol·L-1 Fe3+ aqueous solutions were added to a 250 mL three-neck flask under N2 protection, and the mixture was mechanically stirred at 600 r·min−1. To adjust pH, 25 % NH3·H2O was added dropwise until the pH reached 11–12, and the mixture was heated to 80 °C and maintained at that temperature for 30 min. The reaction was then allowed to mature with N2 purging for 1 h. A magnet was used to collect the reaction product (i.e., the magnetic Fe3O4 nanoparticles), which were subsequently washed using double-distilled water and anhydrous ethanol until their pH reached 7. Finally, they were vacuum dried at 60 °C for 12 h.
2.2.2 Synthesis of magnetic mesoporous Fe3O4@mSiO2 nanoparticles
The sol–gel method was used to prepare the Fe3O4@mSiO2 nanoparticles. In a beaker, 50 mg of dry Fe3O4 and 500 mg of CTAB were dissolved in 50.0 mL of water. The mixture was ultrasonicated for 30 min (using an SK8200H ultrasonic cleaning machine from Shanghai Kudos Ultrasonic Instrument Co., Ltd., China). Subsequently, 400 mL of water was added along with 50.0 mL of 0.01 mol·L-1 NaOH solution, followed by ultrasonic dispersion for 5 min. The mixture was then reacted isothermally at 60 °C in a water bath for 30 min. Next, 2.5 mL of TEOS/ethanol mixture (v/v = 1:4) was added dropwise to the beaker, and the mixture was homogenized with mechanical stirring and then placed in a 60 °C water bath for 12 h. The product was recovered using a strong magnet and dried for 6 h at 60 °C, followed by re-dispersion in acetone, and it was extracted at 80 °C in a water bath (using a GM-0.33II from Tianjin Tengda Filtration Instrument Co., Ltd., China) to remove all surfactants. Next, the product was extracted five times (20 mL and 5 min in each instance) using acetone and double-distilled water. Finally, the product was vacuum dried at 50 °C for 12 h to obtain the magnetic mesoporous Fe3O4@mSiO2 nanoparticles.
2.2.3 Synthesis of magnetic and mesoporous Fe3O4@mSiO2@MMT-SDS nanoparticles
The sol–gel method was used to prepare Fe3O4@mSiO2@MMT-SDS nanoparticles. To prepare Solution A, 2.0 g of Fe3O4@mSiO2 was dispersed in 20 mL of anhydrous ethanol. Solution B was prepared by adding 0.6 g of MMT in 40 mL of double-distilled water and ultrasonically dispersing the mixture for 30 min. Solution C was prepared by uniformly mixing Solutions A and B and subsequently stirring the solution in a thermostatic water bath at 50 °C for 1 h. Next, 0.2 g of SDS was dispersed in 10 mL of double-distilled water and added dropwise to Solution C, followed by 12 h of stirring at 60 °C. Finally, the mixture was centrifugally filtered, and the filtrate was dried in a vacuum at 60 °C for 6 h. The dried filtrate was ground into a powder.
2.3 Characterizations
2.3.1 Fourier transform infrared (FT-IR) analysis
FT-IR analysis (Nexus 670 from Thermo Nicolet, USA) was performed on Fe3O4, Fe3O4@mSiO2, and Fe3O4@mSiO2@MMT-SDS nanoparticle samples at a scan range of 400–4000 cm−1.
2.3.2 Scanning electron microscopy (SEM)
SEM was performed on Fe3O4, Fe3O4@mSiO2, and Fe3O4@mSiO2@MMT-SDS samples using a Philips XL-30 scanning electron microscope (Phillips, the Netherlands) to observe their morphologies.
2.4 Performance and chromogenic reaction conditions
2.4.1 Catalytic performance
Four tubes with 3.0 mL of citrate–phosphate buffer (pH 4) were prepared. For Tube a, 30 mg of Fe3O4@mSiO2@MMT-SDS, 4 μL of 0.8 mol·L-1 H2O2, and 20 μL of TMB were added. Tube b received 4 μL of 0.8 mol·L-1 H2O2 and 20 μL of TMB (without any catalyst). Tube c received 30 mg of Fe3O4@mSiO2@MMT-SDS and 20 μL of TMB (without H2O2). To compare the enzyme-like activities of Fe3O4@mSiO2 and Fe3O4@mSiO2@MMT-SDS, 30 mg of Fe3O4@mSiO2, 4 μL of 0.8 mol·L-1 H2O2, and 20 μL TMB were added into Tube d. All four tubes were reacted in a 40 °C thermostatic water bath for 60 min and then placed in an ice bath for 20 min to stop the reaction. Thereafter, each solution was analyzed using ultraviolet–visible (UV–vis) spectroscopy (UV–VIS 916, GBC Scientific Equipment, Australia).
2.4.2 Steady-state kinetics
We combined 30 mg of Fe3O4@mSiO2@MMT-SDS and 3.0 mL of citrate–phosphate buffer (pH = 4) inside a 30 °C water bath. Experiments were then performed on the TMB–H2O2 system. In the first experiment, the concentration of H2O2 was varied (0, 0.1, 0.2, 0.3, and 0.4 mmol·L-1) whereas the concentration of the TMB substrate was fixed at 0.2 mmol·L-1. The maximum absorptions of these reaction systems were measured at 1 min interval for 5 min. In the second experiment, the concentration of H2O2 was fixed (0.2 mmol·L-1), whereas the concentration of TMB was varied (0, 0.1, 0.2, 0.3, and 0.4 mmol·L-1), and the maximum absorption of these reaction systems were measured at 1 min interval for 5 min. Thereafter, Michaelis constant Km was computed using the Lineweaver–Burk formula: 1/v = Km/vm(1/[s] + 1/Km).
2.4.3 Optimization of the chromogenic reaction conditions
To the citrate–phosphate buffer (pH = 4) placed in a 30 °C water bath, 30 mg of Fe3O4@mSiO2@MMT-SDS and 200 μL of 0.8 mol·L-1 H2O2 were added to obtain a 3.0 mL reaction system. This mixture was homogenized and then left to react for 30 min at 30 °C. Afterward, the mixture was placed in an ice bath to terminate the reaction. TMB was then added at a concentration of 2.0 mg·mL−1, and the mixture was reacted at 30 °C for 10 min. At the end of the reaction, the nanoparticles were magnetically separated from the solution, and 1.0 mL of the supernatant was pipetted out and diluted with double-distilled water. The maximum absorption at 652 nm was then measured to characterize the enzyme-like activity of the Fe3O4@mSiO2@MMT-SDS nanoparticles.
Next, the effects of different temperatures (10, 20, 30, 40, 50, and 60 °C), TMB concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg.mL−1), pH values (2, 3, 4, 5, 6, 7, and 8), reaction times (10, 20, 30, 40, 50, 60, and 70 min), H2O2 concentrations (0.05, 0.1, 0.8, 1.0, 1.5, 2.0, and 2.5 mol.mL−1) and nanoparticle masses (10, 15, 20, 25, 30, 35, and 40 mg) on the absorption at 652 nm were evaluated, while maintaining all other parameter values constant.
2.5 Determination of the CLB content
2.5.1 Calibration curve
Standard solutions containing 0.3, 1.2, 3.6, 7.2, 9.6, 13.2, and 14.4 × 10-7 mol·L-1 of CLB were prepared via the serial dilution method using methanol. For each standard solution, a 3.0 mL reaction system was prepared by adding the standard solution to the pH 4 citrate–phosphate buffer, along with 30 mg of Fe3O4@mSiO2@MMT-SDS and 200 μL of 0.8 mol·L-1 H2O2. The mixture was homogenized and allowed for a reaction at 30 °C for 30 min. Subsequently, ice cubes were added to the water bath to terminate the reaction. Thereafter, 20 μL of TMB was added, and the mixture was reacted at 30 °C for another 10 min. The nanoparticles were magnetically separated from the solution, and 1.0 mL of the supernatant was aliquoted. The absorption values at 652 nm were used to plot the calibration curve.
2.5.2 Sample processing
2.5.2.1 Extraction of CLB from pork
In a 50.0 mL stoppered glass tube, 10.0 g of minced pork was mixed with 20.0 mL of 0.1 mol·L-1 HCl. After the mixture was well shaken and stirred, 20.0 mL of anhydrous ethanol was added, and ultrasonic extraction was performed for 30 min. The mixture was centrifugated at 4000 r·min−1 for 10 min, and the supernatant was transferred to a separatory funnel. Residue was extracted using anhydrous ethanol, and the extracts from two extractions were combined. Next, 2 mol·L-1 NaOH was added dropwise to the supernatant in the separatory funnel to adjust the pH to 12, followed by the addition of ether. Ether extraction was performed three times, and the extracts were combined. Subsequently, 1–2 g of anhydrous sodium sulfate was added to dehydrate the extract, which was then placed in an evaporating dish in a water bath. Finally, anhydrous ethanol was used to dissolve the resulting substance, and the as-obtained ethanol-dissolved extract was stored for future use.
2.5.2.2 Extraction of CLB from animal feed
Animal feed (5.0 g) was placed in a 100 mL conical flask, to which 50.0 mL of a 0.1 mol mol·L-1 HCl/methanol (80:20 v/v) extraction solvent was added. The mixture was shaken to wet the animal feed. Subsequently, extraction was performed in an ultrasonic bath for 15 min. The mixture was hand-shaken once every 5 min. After ultrasonic extraction, the mixture was centrifugated at 4000 r·min−1 for 10 min. Afterward, 10.0 mL of the supernatant was placed in a 150 mL separatory funnel, and the pH was adjusted using NaOH to pH 11–12. Two cycles of extraction were sequentially performed using 30 and 25 mL of ether, and the ether layer was passed through anhydrous sodium sulfate in each instance. Finally, the volume of the extract was diluted to 50 mL with ether. The final extract (25.0 mL) was added to a 50 mL beaker, and it was evaporated until dry in a < 50 °C water bath. Finally, the resulting substrate was dissolved and stored in anhydrous ethanol.
2.5.2.3 Purification
An SPE-C18 column was activated and equilibrated using 3.0 mL of methanol and 3.0 mL of double-distilled water. The sample was then loaded onto the column and eluted with 3.0 mL of SPE-1 eluent and 3.0 mL of SPE-2 eluent. After the column was dried with a vacuum pump, it was eluted with 5.0 mL of methanol. Finally, the eluent was evaporated to 1.0 mL with nitrogen gas (KL512, Beijing Kanglin Science and Technology Co., Ltd.).
2.5.3 Determination of the CLB content in a sample
The chromogenic reaction described in Section 2.5.1 was performed on 1.5 mL of the pretreated sample solution. The absorption maxima at 652 nm were measured to calculate the CLB content of the samples.
2.5.4 Interference testing
Interference testing was performed with a fixed CLB concentration of 7.2 × 10-7 mol·L-1. The interfering components (additives) include structural analogues of CLB, such as fenoterol hydrobromide (FHB) and ractopamine (RAC), and pork/animal feed matrix components, such as potassium chloride (KCl), potassium nitrate (KNO3), calcium chloride (CaCl2), potassium sulfate (K2SO4), sodium nitrate (NaNO3), glucose, vitamins, and tyrosine. The additives used in this test are listed in Table 1.
Coexisting component concentration (μmol·L-1)
Founded (10-7mol·L-1)
Relative deviation (%, n = 3)
7.2 μmol·L-1CLB
7.14 ± 0.567
1.4
0.26 μmol·L-1 FHB + 0.72 μmol·L-1 CLB
7.84 ± 0.442
8.3
5.2 μmol·L-1 FHB + 0.72 μmol·L-1CLB
10.02 ± 0.991
39.1
7.8 μmol·L-1 FHB + 0.72 μmol·L-1 CLB
11.56 ± 0.846
60.6
2.6 μmol·L-1 RAC + 7.2 μmol·L-1 CLB
8.40 ± 0.583
16.7
5.2 μmol·L-1 RAC + 0.72 μmol·L-1 CLB
10.30 ± 1.054
43.1
7.8 μmol·L-1 RAC + 0.72 μmol·L-1 CLB
11.55 ± 1.022
60.4
30.0 μmol·L-1 KCl + 0.72 μmol·L-1 CLB
6.95 ± 0.918
1.3
30.0 μmol·L-1KNO3 + 0.72 μmol·L-1 CLB
7.00 ± 0.842
1.2
30.0 μmol.L-1 CaCl2 + 0.72 μmol·L-1 CLB
6.68 ± 0.479
2.5
30.0 μmol·L-1 K2SO4 + 0.72 μmol•L-1 CLB
6.72 ± 0.655
2.3
30.0 μmol·L-1 NaNO3 + 0.72 μmol·L-1 CLB
6.82 ± 0.826
1.9
0.1 μmol·L-1 glucose + 0.72 μmol·L-1 CLB
6.95 ± 0.393
1.2
8.0 μmol·L-1 vitamins + 0.72 μmol·L-1 CLB
6.95 ± 0.262
1.2
8.0 μmol·L-1 tyrosine + 0.72 μmol·L-1 CLB
6.95 ± 0.589
1.8
2.5.5 Reproducibility and precision tests
Extracts were prepared from pork and animal feeds, which were randomly selected and purchased from the market, according to the procedures stated in Section 2.5.2. CLB standards with concentrations of 0.1, 1.0, and 2.0 × 10-7 mol·L-1 were added to these extracts, and the chromogenic reaction was performed according to the experimental procedures described in Section 2.5.1. The maximum absorption at 652 nm was measured in each sample, and the CLB content was determined using the calibration curve.
2.5.6 Stability and lifespan of magnetic Fe3O4@mSiO2@MMT-SDS nanoparticles
To test the stability of Fe3O4@mSiO2@MMT-SDS nanoparticles, the chromogenic reaction was performed by adding nanoparticles stored for 15, 30 and 60 d to a 0.4 × 10-7 mol·L-1 CLB solution.
To examine the lifespan of Fe3O4@mSiO2@MMT-SDS nanoparticles, 30 mg of Fe3O4@mSiO2@MMT-SDS nanoparticles were reused 30 times, employing the same concentration of TMB and H2O2 in each cycle. The maximum absorption at 652 nm was measured once every 5 cycles to quantify the service life of the material to observe the oxidative color changes.
2.6 Statistical analysis
All statistical analyses were performed using SPSS 21.0 software. Mean ± standard deviation was used to indicate the results, and Duncan’s multiple range test and one-way analysis of variance were used to examine means differences. Statistical significance was set at p < 0.05.
3 Results and discussion
3.1 Characterizations
The FT-IR spectra of Fe3O4@mSiO2 and Fe3O4 are shown in Fig. 1A(a,b), respectively. The peaks at 788.31, 964.14, 464.70, and 1090.26 cm−1 correspond to the C–H absorption of TEOS, Si–O–H vibration, Si–O–Si stretching, and Si–O–Si vibration, respectively. The peak at 570.18 cm−1 is the characteristic absorption peak of Fe–O (Li et al., 2010), and it is substantially weaker in the Fe3O4@mSiO2 spectrum (a) than in the Fe3O4 spectrum (b). This observation implies that the Fe3O4 nanoparticles in Fe3O4@mSiO2 are coated with a layer of SiO2. The FT-IR spectra also indicate that the sample contains small amounts of impurities, as the peak at 1392.96 cm−1 represents the O–H bending mode of FeO(OH), and several absorption peaks are observed at approximately 1000 cm−1. The absorptions at 3425.76 and 1617.75 cm−1 correspond to the stretching and bending modes of surface hydroxyl groups (Long et al., 2013).
(A) FT-IR spectra. Fe3O4@mSiO2 (a) and Fe3O4 (b); (B) FT-IR spectra. Fe3O4@mSiO2 (a) and Fe3O4@mSiO2@MMT-SDS (b); (C) SEM images. Fe3O4@mSiO2; (D)SEM images. Fe3O4@mSiO2@MMT-SDS.
In Fig. 1B(b), the absorptions at 460.71 and 1032.71 cm−1 are the bending and stretching absorptions of Si–O–Si, whereas the peak at 588.31 cm−1 is the characteristic absorption peak of Fe3O4 (Mu et al., 2018). The sharp peaks at 2919.12 and 2851.10 cm−1 correspond to the asymmetric and symmetric stretching of C–H groups in SDS, respectively (Qiao et al., 2016). The presence of the 1221.42 cm−1 peak, which corresponds to the asymmetric stretching of sulfonate, indicates that SDS is successfully intercalated into MMT, and the peak at 1457.68 cm−1 represents the characteristic absorption mode of Na–MMT. The absorption peaks at 3633.01 and 1726.12 cm−1 correspond to the stretching and bending modes of MMT surface hydroxyls (Li et al., 2010), indicating that MMT is successfully coated onto the Fe3O4@mSiO2 nanoparticles.
Fig. 1C and D shows that the Fe3O4@mSiO2 nanoparticles have a neat and regular arrangement. They are well-dispersed and have coarse surfaces. Therefore, the SiO2 coating could successfully prevent the agglomeration of Fe3O4 particles. The MMT-modified Fe3O4@mSiO2 nanoparticles have a well-defined spherical shape and are well-dispersed, which indicate that MMT modification further improves the dispersion of these magnetic nanoparticles.
3.2 Catalytic performance
After 60 min of reaction in a 40 °C thermostatic water bath, the solution in Tube c was colorless. The colors of the solutions of Tubes b and a were light blue and deep blue, respectively. The UV–vis spectra of the samples are shown in Fig. 2 (A). The UV–vis spectrum of the solution in Tube c shows a plateau from 300 to 800 nm, whereas the spectra of the solutions in Tubes a and b show significant absorption peaks at 380 and 652 nm, with Tube a solution spectrum exhibiting stronger peaks than Tube b solution spectrum. Therefore, Fe3O4@SiO2@MMT-SDS nanoparticles do not react directly with TMB and can catalyze the oxidation of TMB by H2O2.
(A) UV–vis spectra. (a)TMB + H2O2 + Fe3O4@mSiO2@MMT-SDS, (b) TMB + H2O2, and (c) Fe3O4@mSiO2@MMT-SDS + TMB; (B) Enzyme activity. Catalytic color reaction of Fe3O4@mSiO2 and Fe3O4@mSiO2@MMT-SDS.
The enzyme-like activities of Fe3O4@mSiO2 and Fe3O4@mSiO2@MMT-SDS (Tubes a and d solutions) are shown in Fig. 2(B), where absorbance is used as a measure of enzyme-like activity. Fe3O4@mSiO2@MMT-SDS has a higher activity than Fe3O4@mSiO2, demonstrating that MMT modification significantly enhances the enzyme-like activity of Fe3O4@mSiO2.
3.3 Michaelis constant (Km)
Here, Km was used as an indicator of enzyme-like catalytic kinetics; the smaller the value of Km, the better the nanoparticle affinity for the substrate, and the stronger the enzyme-like activity of the nanoparticles. The results of the experiments performed under the conditions of a fixed TMB concentration in the substrate buffer and varying H2O2 concentrations are shown in Fig. 3A, and the results of experiments performed under the conditions of a fixed H2O2 concentration and varying TMB concentrations are shown in Fig. 3B. Fig. 3C and 3D were plotted by considering the double reciprocal of Fig. 3A and 3B, and the Lineweaver–Burk formula was used to calculate Km. When the substrate was H2O2, Km was 0.05175, and the maximum velocity (Vmax) was 6.99 × 10-8. When TMB was used as the substrate, Km was 0.4302, and Vmax was 13.3 × 10-8. The lower Km values indicate the excellent enzyme-like activity of Fe3O4@SiO2@MMT-SDS nanoparticles (Wang et al., 2023).
Reaction kinetics. Catalytic kinetics of Fe3O4@SiO2@MMT-SDS nanoparticles.
3.4 Detection procedure
The underlying principles of CLB colorimetry using the proposed method are illustrated in Fig. 4. (1) The Fe3O4@SiO2@MMT-SDS nanoparticles catalyzes the oxidation of CLB by H2O2 (Ji et al., 2018). (2) The excess H2O2 then oxidizes the TMB chromogen and forms a blue solution. Within a certain range of concentration, a linear relationship is observed between CLB and H2O2 concentrations. This relationship can be used to create a calibration curve, which is then used to measure CLB concentration (Liu et al., 2022; Wang et al., 2023).
CLB colorimetry. Schematic of the Fe3O4@mSiO2@MMT-SDS catalytic process.
3.5 Optimizing the conditions for the H2O2–TMB–CLB reaction system
3.5.1 Effects of temperature on the H2O2–TMB–CLB reaction system
From Fig. 5A, the absorption of the reaction system at 652 nm increases with increasing temperature. Therefore, the enzyme-like activity of Fe3O4@mSiO2@MMT-SDS increases with temperature. This could be attributed to the increase in the rate of hydroxyl radical generation from H2O2 with increasing temperatures. However, H2O2 rapidly decomposes at temperatures higher than 30 °C, which reduces hydroxyl radical generation. As the absorbance of the reaction system is the highest at 30 °C, it may be concluded that its enzyme-like activity is the highest at this temperature and that the optimal temperature for the H2O2–TMB–CLB reaction is 30 °C.
Influence of reaction conditions on the H2O2–TMB–CLB reaction system. Panel (A) temperature, (B) TMB concentration, (C) pH, (D) reaction time, (E) H2O2 concentration, and (F) amount of nanoparticles.
3.5.2 Effects of the TMB concentration on the H2O2–TMB–CLB reaction system
From Fig. 5B, the absorbance of the reaction system increases with the TMB concentration and plateaus after 2.0 mg·mL−1. This indicates that the rate at which TMB is oxidized by hydroxyl radicals increases with the TMB concentration (Jin et al., 2018). Although absorbance should decrease with a decrease in the number of hydroxyl radicals, the presence of Fe3O4@mSiO2@MMT-SDS nanoparticles in the reaction system prevents the hydroxyl radicals from disappearing, thereby stabilizing the system. Hence, the enzyme-like activity of Fe3O4@mSiO2@MMT-SDS is maximal when TMB concentration is 2.0 mg·mL−1.
3.5.3 Effects of pH on the H2O2–TMB–CLB reaction system
The absorbance of the reaction system increased with pH until pH = 4 and then decreased with further increases in pH (Fig. 5C). Hence, the enzyme-like activity of Fe3O4@mSiO2@MMT-SDS is affected by pH. This is because H2O2 is weakly acidic and readily produces hydroxyl radicals under weakly acidic conditions. Therefore, the optimal pH for the reaction system is pH = 4.
3.5.4 Effects of the reaction time on the H2O2–TMB–CLB reaction system
In Fig. 5D, absorbance increases with the reaction time, until 30 min. As the reaction time increases, the degree of contact between the Fe3O4@mSiO2@MMT-SDS nanoparticles and H2O2, increases. The increase in the reaction time will promote the production of hydroxyl radicals and, therefore, the oxidation of TMB, which renders the reaction system a deep-blue color (Zhang et al., 2016). However, the reaction system will reach equilibrium after the substrate has been exhausted. Hence, the optimal reaction time for this reaction system is 30 min.
3.5.5 Effects of the H2O2 concentration on the H2O2–TMB–CLB reaction system
In Fig. 5E, the absorbance of the reaction system increases with increasing H2O2 concentration up to 0.8 mol·L-1 and then decreases with further increases in the H2O2 content until it reaches an equilibrium. This is because an increase in the H2O2 concentration increases hydroxyl radical generation. However, H2O2 rapidly decomposes at concentrations above 0.8 mol·L-1, which reduces hydroxyl radical generation. As the absorbance of the reaction system is the highest when the H2O2 concentration is 0.8 mol·L-1, the enzyme-like activity of Fe3O4@mSiO2@MMT-SDS is maximal at this concentration of H2O2.
3.5.6 Effects of the Fe3O4@SiO2@MMT-SDS dosage on the H2O2–TMB–CLB reaction system
In Fig. 5F, the absorbance of the reaction system increases with increasing catalyst dosage and plateaus after 30 mg. Hence, a catalyst dosage of 30 mg affords an optimal catalytic performance and enzyme-like activity.
3.6 Detection performance of the Fe3O4@SiO2@MMT-SDS
3.6.1 Linear equation and limit of detection
The calibration curves of CLB showed adequate linearity (Fig. 6) over the studied concentration range (0.3–14.4 × 10-7 mol·L-1). The current assay offered a limit of detection (LOD) of 9.6 × 10-9 mol·L-1, slightly below the LOD obtained by Medellín-Martínez et al. (0.108 ng/g) (Medellín-Martínez et al., 2018), but this method does not require large instruments and is convenient and fast. Hence, our method can be used for rapid CLB screening.
Correlation between the CLB concentration and the absorbance at 652 nm.
3.6.2 Interference test
From Table 1, structural analogues of CLB, such as FHB and RAC, have comparably strong impacts on CLB determination. However, pork or animal feed matrix components do not have a significant impact on CLB determination using our method (p < 0.05).
3.6.3 Reproducibility and precision
From Table 2, the spike recovery and relative standard deviation obtained using the developed method are ≥ 92.4 % and < 4.0 %, respectively. Hence, the proposed method is sufficiently accurate and precise for CLB detection in real-world applications.
Sample matrix
Added (10-7mol·L-1)
Founded (10-7mol·L-1)
Recovery (%)
RSD (%, n = 6)
Pork
0.1
0.09 ± 0.037
92
4.0
1.0
1.10 ± 0.157
112
1.4
2.0
2.10 ± 0.432
108
2.0
Feed
0.1
0.095 ± 0.004
95.5
0.4
1.0
0.92 ± 0.185
92.4
2.0
2.0
1.95 ± 0.352
97.7
1.8
3.7 Stability and lifespan
The maximum absorbance of the Fe3O4@SiO2@MMT-SDS nanoparticles decreases slightly over 60 d (p > 0.05). Therefore, we assume that the Fe3O4@SiO2@MMT-SDS nanoparticles are stable and have a long shelf-life of up to 60 d. After 30 cycles of re-use, the maximum absorbance of the Fe3O4@SiO2@MMT-SDS nanoparticles decreased slightly (p > 0.05). Hence, the Fe3O4@SiO2@MMT-SDS nanoparticles have highly repeatable enzyme-like activity and can be reused.
4 Conclusions
Magnetic, mesoporous, and well-dispersed Fe3O4@mSiO2 nanoparticles were prepared using the sol–gel method, and they were subsequently modified with MMT for catalytic applications. The nanoparticles were further modified using SDS to impart negative charges to their surfaces. The resulting Fe3O4@mSiO2@MMT-SDS nanoparticles exhibit excellent dispersion and enzyme-like activity as well as improved substrate (TMB and H2O2) affinity, compared with Fe3O4@mSiO2. Utilizing the catalytic properties of these nanoparticles, we constructed a H2O2–TMB–CLB colorimetric system, where H2O2 oxidized CLB, and the excess H2O2 oxidized the chromogen TMB to form a blue-colored solution. Within a certain range of the CLB concentration, a strong linear correlation was observed between the peak absorbance of this solution and CLB concentration. Hence, in this study, we established a novel colorimetric method for CLB detection and pioneered a new approach for the construction of cost-efficient, rapid, and accurate colorimetric devices.
Acknowledgments
This work was supported by the funds provided by the Science and Technology Program of Guangdong Province of China (Grant No. 2020B121202014), and the Open fund for scientific research platform of Chongqing City (Grant No. KFJJ2018052).
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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