Gold functionalised attapulgite for discrimination of hydrogen peroxide and oxidising ions

Abstract

Aiming to monitor hydrogen peroxide and oxidizing ions in aqueous circumstance, functionalized attapulgite [i.e. gold‐modified attapulgite nanocomposites (Au/ATP NCs)] as peroxidase mimics were prepared by loading β‐cysteamine‐capped gold nanoparticles onto attapulgite with the use of electrostatic interactions. As‐prepared Au/ATP NCs were used for the detection of H₂ O₂. The linear range and detection limit for H₂ O₂ were determined by quantitative experiments emerging excellent stability and recyclability. The peroxidase‐like activity of Au/ATP NCs could be expanded for the detection of some oxidative ions (Fe³⁺ and Ag⁺) was also been discovered. Based on the absorption spectrum and steady‐state kinetics, the mechanism for peroxidase mimic reaction is investigated. This work represents the first example of multitarget detection for molecule and cations (H₂ O₂, Fe³⁺ and Ag⁺) using Au/ATP NCs as peroxidase enzyme mimics and holds a promise for future biomedical and analytical applications.

1 Introduction

Looking back through the 20th century, there emerged two ground breaking technologies which have enabled the vast growth of human activity in terms of population and economy. Nanotechnology is one of the most important keywords in current science and technology. The novel technologies concept of nanotechnology has been established over the past few decades with a view to exploit nanometre‐sized phenomena and to fabricate functional materials by precisely controlling the materials’ structures at the nanoscale. Furthermore, machine fabrication based on other concepts such as supra‐molecular self‐assembly or biomimetic approaches have been developed [1, 2]. Artificial enzyme mimics have been well suited in chemistry, medicine and biochemistry [3, 4, 5], owing to their high catalytic performance, outstanding chemical stability and strong environmental insensitivity compared with enzymes occurred naturally [6, 7, 8]. Especially, inorganic materials possessing intrinsic peroxidase‐like activity, such as magnetic nanoparticles [9, 10], noble metal nanoparticles [11, 12], carbon materials [13, 14, 15] and polymer‐coated inorganic nanoparticles [16] have exhibited considerable potentials in various fields.

Metal nanoparticles have attracted much attention in the fields of catalysis, separation, magnetism, optoelectronics and microelectronics owing to their unique physical and chemical properties [17, 18]. Herein, gold nanoparticles (Au NPs) are easily fabricated by controlling shape and size, unique optical and electronic properties and excellent biocompatibility. With the aid of their exceptional capacity towards nucleophilic attacks, Au NPs have aroused tremendous interests in the applications related to organic reactions, like CO oxidation, diboration, selective hydrogenation and reduction and oxidation processes [19, 20, 21, 22]. For instance, Cao et al. reported on that positively charged Au NPs exhibited promising potential in the detection of H₂ O₂ and glucose with peroxidase‐like activity [11]. However, most artificial substratum supporting Au NPs demand for sophisticated synthesis procedure. Thus, the intricate preparation process would limit their applications. Satisfactorily, attapulgite (ATP), a natural phyllosilicate with special spatial structure and surface state, has proved to be a candidate as substrate. This particular colloidal material possesses unique chain structure, high temperature endurance, salt and alkali resistance and high adsorption and penetrability [23, 24, 25]. Furthermore, the inverted structure endows ATP with pre‐eminent absorption capacity for positively charged ions [26, 27].

Herein, we reported the preparation of gold‐modified ATP nanocomposites (Au/ATP NCs) via the reduction of chloroauric acid by sodium borohydride. To investigate whether the as‐prepared functionalised nanomaterials preserve the intrinsic peroxidase‐like activity of Au NCs, the catalytic oxidation of 3,3,5,5‐tetramethylbenzidine (TMB) by H₂ O₂ was monitored using colorimetric method. Furthermore, the detection of Fe³⁺ and Ag⁺ ions was also evaluated. The recyclability and catalytic ability of nanoparticles still maintained after eight‐batch repetitions. The possible mechanism of peroxidase activity of Au/ATP NCs was presumed via the absorption spectra.

2 Materials and methods

2.1 Materials

Cysteamine (CA, 95%), chloroauric acid (HAuCl₄ ·4H₂ O), sodium borohydride (NaBH₄, 96%), hydrogen peroxide (H₂ O₂, 30%), ferric chloride (FeCl₃ ·6H₂ O), silver nitrate (AgNO₃), acetate (HAc) and sodium acetate anhydrous (NaAc) were purchased from Sinopharm Chemical Reagent Co., Ltd. TMB was obtained from Aladdin. ATP was obtained from Changzhou University. All the reagents were used without purification. The deionised water was directly obtained from Millipore‐Q water system.

2.2 Preparation of Au/ATP NCs

HAuCl₄ solution and CA were then dissolved in 25 ml water (AuCl₄ ⁻ : CA=1:1) using 1 M HCl to adjust the pH to 5.6. The mixture was kept stirring for 20 min in ice bath to allow the coordinate reaction sufficiently proceed between –SH and AuCl₄ ⁻, followed by the addition of ATP suspension (25 ml in 20 min) and AuCl₄ ⁻ was thus absorbed onto the surface of ATP through electrostatic interaction. Finally, AuCl₄ ⁻ was reduced by NaBH₄ to produce Au NPs on the surface of ATP. The resulted nanomaterials were collected by centrifugation and washed with water for three times. The composites were then re‐dispersed in water for further use. For controlled experiment, CA‐capped Au nanoparticles (CA‐Au NPs) were prepared without ATP as substrate.

2.3 Characterisations

The morphology and structure test of catalysts were carried on transmission electron microscope (TEM, JEOL‐JEM2100), X‐ray powder diffractometer (XRD, Bruker D8 ADVANCE) with Cu‐Kα radiation (40 kV, λ  = 1.54060 Å). Ultraviolet–visible absorption spectra were performed on 1201 spectrophotometer (Shimadzu, Japan) with a quartz cell of 1 cm light path.

2.4 Activity evaluation

Peroxidase‐like activity tests were performed as follows: 70 μl Au/ATP NCs suspensions (10 μgml⁻¹), 200 μl of H₂ O₂ (or Fe³⁺, Ag⁺) with different concentrations and 330 μl of TMB solution (dissolve in 0.5 M acetate buffer, pH 4.0). N₂ was bubbled into the reaction solutions to exclude the oxygen prior to use. The mixture was placed in a water bath for a few minutes and the resulting solution was used for adsorption spectroscopy measurement.

3 Analysis and results

3.1 Characteristics of Au/ATP NCs

Compared with Fig. 1 a, numerous small spots can be found in Fig. 1 b, indicating that Au NPs were mono‐dispersed nanospheres adsorbed to the surface of ATP. The corresponding Au NPs size distribution of Au/ATP NCs was shown in Fig. 1 c with a size distribution of 4.41 ± 0.74 nm. The high‐resolution TEM (HRTEM) micrograph was also presented in Fig. 1 d, which shows a perfectly crystallised nanoparticle with a lattice spacing of ca. 0.23 nm, corresponding to the (111) face of metallic gold [28]. The results evidenced the successful preparation of Au/ATP NCs with uniform size and good crystallinity.

Fig. 1.

TEM images and corresponding Au NPs size distribution

(a) TEM images of raw ATP, (b) Au/ATP NCs, (c) Au NPs size distribution of Au/ATP NCs, (d) HRTEM image of Au NPs absorbed on ATP

CA was a key impact in the preparation process. If AuCl₄ ⁻ solution was directly mixed with ATP suspension, the surface of ATP surface would not only be lack of Au NPs, but also adsorbed Au NPs could easily peel off from the surface of ATP when washed by water. Since natural ATP has an isoelectric point (IEP) at pH 3.1 and the surface charge became increasingly negative, the pH values of the suspension increased [29]. Neaman et al. suggested that the IEP of ATP is 4–4.5 at pH 7.4 [30]. ATP nanostructures thus have a net negative surface charge in water [31]. CA‐stabilised gold ions at pH 5.6 were electropositive. Therefore, the addition of CA enhanced the distribution of gold ions to the surface of ATP through electrostatic adsorption.

The loading amount and dispersion of Au on ATP surface can be tuned by adding different volume of AuCl₄ ⁻ stock solution to ATP suspension. The evolution of the composites with different reactant ratio was characterised by XRD patterns (see Fig. 2). When the amount of AuCl₄ ⁻ solution increased from 0.2 to 2 ml, the corresponding Au loading amount for NCs increased from 1 to 10 wt% expressed as the enhanced Au peaks. As the amount of AuCl₄ ⁻ solution further increased to 5 ml, the supernatant upon the reaction completed turned deep wine red, demonstrating Au NPs were not completely adsorbed on the surface of ATP. Furthermore, 20 wt% of AuCl₄ ⁻ solution would lead to Au NPs aggregated and produced larger particles as sharp strong peaks shown in XRD pattern. As a result, the optimal loading amount for Au NPs was 10 wt% for further study. As shown in Fig. 2, the peaks at 38°, 44°, 64.5° and 77.5° were assigned to (111), (200), (220) and (311) planes of bulk gold, respectively. (111) planes for cubic Au NPs were also consistent with the lattice planes in the HRTEM images.

Fig. 2.

XRD patterns of ATP/Au NCs with different reactant ratio

3.2 Catalytic activity of Au/ATP NCs

The mixture of TMB and H₂ O₂ presented as a blue colour liquid with maximum absorbance at 652 nm in the presence of as‐prepared Au/ATP NCs. Similar to natural enzymes, the catalytic efficiency of the Au/ATP NCs was strongly dependent on environmental conditions such as pH and temperature. The effect of these factors on the catalytic activity of Au/ATP NCs was investigated by measuring the absorbance intensity of the resulting solution while varying the pH from 1 to 12 and the temperature from 20 to 60°C. Upon the optimisation of experimental conditions, the absorbance reached the highest when pH increased to 4.0 and subsequently dropped at higher pH due to the proper solubility of TMB at pH 4.0. Meanwhile, Au/ATP NCs exhibited the best catalytic activity at 45°C. Therefore, we employed pH = 4.0 and 45°C as our following reaction condition.

Resembling natural enzymes, the peroxidase‐like activity of Au/ATP NCs was also inhibited by high H₂ O₂ concentrations (see Fig. 3 a). The normalised absorbance increased in the H₂ O₂ concentration range from 0 to 0.5 molml⁻¹, while increased concentration turned to restrain the catalytic activity of the Au/ATP NCs. The detection limit of H₂ O₂ reached as low as 2 × 10⁻⁶ moll⁻¹ with a linear range from 5 × 10⁻⁶ to 1 × 10⁻⁴ moll⁻¹. Moreover, the inhibited concentration was about two orders of magnitude higher than natural peroxidise [9, 10, 14], demonstrating that Au/ATP NCs can suffer from a higher H₂ O₂ concentration. In addition, Fig. 3 b showed the time‐dependent absorbance change with different concentrations of Au/ATP NCs used, while single ATP cannot efficiently oxidise TMB, revealing the peroxidase activity not originate from ATP. Meanwhile, the catalytic reaction was accelerated at higher Au/ATP NCs concentration.

Fig. 3.

Time‐dependent absorbance change with different concentrations of Au/ATP NCs

(a) Peroxidase‐like activity of Au/ATP NCs dependent on H₂ O₂ concentration, (b) Au/ATP amount, (c) Absorbance evolution for CA‐Au NPs and Au/ATP NCs with different storage time, (d) Catalytic activity of Au/ATP NCs over number of cycles

To evaluate the stability of catalyst, CA‐Au NPs and Au/ATP NCs were used as enzyme mimic after different store time (see Fig. 3 c), respectively. Significant activity decrease was observed for CA‐Au NPs after 10 h store, while the activity of Au/ATP NCs only slightly receded. The activity of enzyme mimics declined significantly, which is attributed to Au NPs of CA‐Au NPs in solution tended to aggregate into large particle. ATP provided a stable matrix for Au NPs that prevented the aggregation of Au NPs. Au/ATP NCs were centrifuged out when the reaction completed and washed three times before applied in the repeated reaction. Fig. 3 d showed that the catalytic activity of Au/ATP NCs remained unchanged over eight cycles (more than 95%). This slightly reduced activity for cycle test was due to the inevitable Au/ATP NCs loss in the process and also evidenced the stability of catalysts.

Besides, two oxidative ions (Fe³⁺, Ag⁺) could also be oxidised at the presence of Au/ATP NCs. The detection limit of concentration for Fe³⁺ and Ag⁺ was 1 × 10⁻⁶ moll⁻¹ and 5 × 10⁻⁷ moll⁻¹ and the linear range was 2 × 10⁻⁶ –1 × 10⁻⁴ moll⁻¹ and 1 × 10⁻⁶ –1 × 10⁻⁴ moll⁻¹, respectively. This interesting discovery paved a way for the detection of oxidising ions in solution using inorganic enzymes mimics.

3.3 Mechanism

Yan et al. have already excluded the possibility that enzyme‐like activity is caused by iron ions in the solution [10]. Huang and his coworkers proved that the catalytic mechanism of carbon dots involves electron transfer from lone‐pair electrons in TMB amino groups to the catalyst [14]. Ito et al. have presented that electron transfer took place between C60 and TMB upon laser photolysis [32]. Based on these findings, we proposed a possible mechanism for Au NPs mediated TMB oxidation. When Au NP is irradiated by light, the oscillating electric field causes the electrons of Au NPs to oscillate coherently, generating surface plasma. Surface plasma mediated charge transfer from hole sacrificial agent to TMB, resulting in blue product. TMB oxidation took place through nucleophilic attack on the surface of Au NPs.

Herein, steady‐state kinetic assay was conducted for H₂ O₂, Fe³⁺ and Ag⁺ to investigate the possible catalytic mechanism. The velocity of the reaction was measured using 10 μgml⁻¹ Au/ATP NCs in 0.4 ml of 0.2 M HAc‐NaAc buffer (pH 4.0) at room temperature. The concentration of H₂ O₂ (Fe³⁺ and Ag⁺) was 250 mM while varying TMB concentration. The concentration of TMB was 0.8 mM while changed H₂ O₂ (Fe³⁺ and Ag⁺) concentration. The results indicated that typical Michaelis–Menten curves can be obtained in a certain range of concentrations. Maximum initial velocity (V _max) and Michaelis–Menten constant (K _m) were fitted to the Michaelis–Menten model to obtain the kinetic parameters (shown in Table 1). The apparent K _m value of Au/ATP NCs for H₂ O₂ was slightly lower than that for Fe³⁺ and Ag⁺, indicating that higher Fe³⁺ and Ag⁺ concentrations were required to achieve maximal activity for Au/ATP NCs [12], which was consistent with the catalytic results in Figs. 3 and 4. The corresponding K _m values with TMB were almost the same, showing that Au/ATP NCs has almost the same activity for TMB [10, 12]. For natural enzymes, the lower K _m value is related to a higher affinity for the substrate. Compared with reported inorganic enzyme mimics, such as Fe₃ O₄ (K _m  = 0.098) [10], graphene oxide (K _m  = 0.024) [13], carbon dots (K _m  = 0.039) [14], Au/ATP NCs structure has a relatively lower K _m value.

Table 1.

Kinetic parameters of H₂ O₂, Fe³⁺ and Ag⁺

Items	H2 O2 /Fe3+ /Ag+		TMB
	K m, mM	V max (10−8 Ms−1)	K m, mM	V max (10−8 Ms−1)
H2 O2	10.09 ± 0.81	10.48 ± 0.79	0.019 ± 0.0012	3.68 ± 0.21
Fe3+	12.54 ± 1.31	12.54 ± 1.03	0.017 ± 0.0021	4.36 ± 0.32
Ag+	13.87 ± 0.94	13.87 ± 0.92	0.016 ± 0.0014	4.56 ± 0.43

K _m is the Michaelis constant, V _max is the maximal reaction velocity.

Fig. 4.

Dose‐response curves for

(a) Fe³⁺ and (b) Ag⁺ detection using Au/ATP NCs as enzyme mimic

To further explore the mechanism of catalysis of Au/ATP NCs, we measured the reaction kinetics over a range of TMB and H₂ O₂ (Fe³⁺, Ag⁺) concentrations. Figs. 5 a –f showed the double reciprocal plots of initial velocity versus one substrate concentration, which was obtained for a range of concentrations of the second substrate. It was evident that the slopes of the lines are parallel, which is characteristic of a ping‐pong mechanism, as was observed for HRP [9, 10, 33]. This indicated that, similar to horseradish peroxidise (HRP), Au/ATP NCs binded and reacted with one substrate, and released the first product before binding and reacting with the second substrate.

Fig. 5.

Double reciprocal plots of activity of Au/ATP with the concentration of one substrate (H₂ O₂ /Fe³⁺ /Ag⁺ or TMB) fixed and the other varied

4 Conclusion

In summary, Au/ATP NCs possessing peroxidase‐like activity was prepared and used for the effective and sensitive detection of H₂ O₂, Fe³⁺ and Ag⁺. Our method provides a colorimetric assay for target (H₂ O₂, Fe³⁺ and Ag⁺). Importantly, the catalytic activity of as‐prepared catalyst was almost identical even after eight cycles. Based on the results, catalytic reaction by Au/ATP NCs was consistent with typical ping‐pong mechanism with more effective than the reported inorganic enzyme mimics. These results proved that Au/ATP NCs can act as a robust and effective peroxidase mimetic as well as a versatile capture and detection tool for some ions. Our method showed great potential applications in biosensors, environmental chemistry, biotechnology and medicine in the future.