Synthesis of Au–Cu Alloy Nanoparticles as Peroxidase Mimetics for H2O2 and Glucose Colorimetric Detection

The detection of hydrogen peroxide (H2O2) is essential in many research fields, including medical diagnosis, food safety, and environmental monitoring. In this context, Au-based bimetallic alloy nanomaterials have attracted increasing attention as an alternative to enzymes due to their superior catalytic activity. In this study, we report a coreduction synthesis of gold–copper (Au–Cu) alloy nanoparticles in aqueous phase. By controlling the amount of Au and Cu precursors, the Au/Cu molar ratio of the nanoparticles can be tuned from 1/0.1 to 1/2. The synthesized Au–Cu alloy nanoparticles show good peroxidase-like catalytic activity and high selectivity for the H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB, colorless) to TMB oxide (blue). The Au–Cu nanoparticles with an Au/Cu molar ratio of 1/2 exhibit high catalytic activity in the H2O2 colorimetric detection, with a limit of detection of 0.141 μM in the linear range of 1–10 μM and a correlation coefficient R2 = 0.991. Furthermore, the Au–Cu alloy nanoparticles can also efficiently detect glucose in the presence of glucose oxidase (GOx), and the detection limit is as low as 0.26 μM.


Introduction
Hydrogen peroxide (H 2 O 2 ), an essential representative of reactive oxygen species, plays a critical role in various biological processes, including cell proliferation, differentiation, and migration [1][2][3]. Moreover, H 2 O 2 is a vital component in modern industries applications such as chemical synthesis, water treatment, and textile bleaching, in which H 2 O 2 is used as a versatile and environmentally benign oxidant [4,5]. Meanwhile, H 2 O 2 is also a byproduct of many enzymatic reactions in living beings. The generation and accumulation of H 2 O 2 may cause several diseases, e.g., cancer, Alzheimer's disease, and Parkinson's disease, endangering the health and life of human beings [6]. Therefore, the detection of H 2 O 2 is essential in various areas including medical diagnosis, food safety, and environmental monitoring [7][8][9].
In terms of H 2 O 2 detection, numerous analytical strategies have been developed, such as optical sensing, electrochemical analysis, and colorimetric methods [10][11][12][13]. Among these, colorimetric methods show great potential in practical applications due to advantages including simplicity, low cost, and unsophisticated instrumentation [14][15][16][17]. In colorimetric assays, a natural enzyme is typically needed to develop an optical signal. However, practical applications of natural enzymes are restricted by their high cost and low stability against denaturation and protease [18,19]. Consequently, many studies have been devoted to the development of alternative approaches based on nanozymes, whose intrinsic properties such as large specific surface area, high stability, good durability, low cost, and tunable catalytic activity render them suitable for various practical applications [20][21][22]. Since the first Fe 3 O 4 -based nanozyme was reported [23], several kinds of nanomaterials have been developed, e.g., metallic oxide nanoparticles [24][25][26][27], carbon-based nanomaterials [28,29], and noble metal nanoparticles (Au, Ag, and Pt) [30][31][32][33].
Among these nanomaterials, Au nanoparticles are often used in fundamental research due to their good enzyme-like activities as both oxidase and peroxidase mimics [34]. However, naked Au nanoparticles generally show high chemical inertness in many catalytic reactions of enzyme mimics [35]. Moreover, aggregation of Au nanoparticles often occurs, reducing their catalytic efficiency and hampering their practical application. To circumvent these problems, Au-based bimetallic alloy nanomaterials have attracted significant attention because of their superior catalytic activity to that of their single-phase counterparts [36,37] [40]. As can be extracted from these reports, Au-based bimetallic alloy nanomaterials with good dispersion degree and excellent enzyme-like performance are relevant for H 2 O 2 detection in various research fields.
In this paper, we report a coreduction method for the synthesis of well-mixed Au-Cu alloy nanoparticles in aqueous phase, in which the Au/Cu ratio can be tuned from 1/0.1 to 1/2 by controlling the amount of Au and Cu precursors. In a typical colorimetric reaction, that is, the H 2 O 2 -mediated oxidation of the peroxidase substrate 3,3 ,5,5tetramethylbenzidine (TMB, colorless) to produce TMB oxide (TMBox, blue), the prepared Au-Cu alloy nanoparticles showed good peroxidase-mimicking catalytic activity. Meanwhile, using various common ions and substances as controls, the high selectivity in H 2 O 2 detection of the synthesized Au-Cu alloy nanoparticles was demonstrated.

Characterization of Au-Cu Alloy Nanoparticles
The Au-Cu alloy nanoparticles were synthesized by coreducing Au and Cu precursors using L-ascorbic acid (AA) in the presence of poly(ethyleneimine) (PEI) solution in an aqueous phase system. The solution color changed from chartreuse to maroon after adding AA, which indicated the formation of nanoparticles. In this synthesis process, PEI served as a surfactant for the formation of nanosized particles and as a stabilizing agent to prevent the oxidation of Cu species in the nanoparticles [41,42]. By varying the amount of Au and Cu precursors, the molar ratio between Au and Cu in the nanoparticles was controlled from 1/0.1 (Au 1 Cu 0.1 ) to 1/2 (Au 1 Cu 2 ), as determined by inductively coupled plasma (ICP) ( Table S1). The transmission electron microscopy (TEM) images of the nanoparticles depicted in Figure 1a-e revealed that the synthesized nanoparticles had a quasispherical morphology and an average size of around 62.4 nm for Au 1 Cu 0.1 , 63.2 nm for Au 1 Cu 0.2 , 64.8 nm for Au 1 Cu 0.5 , 79.5 nm for Au 1 Cu 1 , and 101.3 nm for Au 1 Cu 2 ( Figure S1). The elemental distribution of the nanoparticles was also analyzed by energy-dispersive X-ray spectrometry (EDS) mapping, which indicated that Au and Cu were well dispersed in the Au-Cu alloy nanoparticles ( Figure S2). Figure 1f shows the powder X-ray diffraction (XRD) patterns of the nanoparticles with different Au/Cu molar rations, which indicate that all the nanoparticles had an alloy structure based on Au (Joint Committee on Powder Diffraction Standards (JCPDS) file card no. 04-0784) rather than Cu (JCPDS file card no. 04-0836). In the XRD patterns, all XRD peaks exhibited most likely were due to the insertion of small Cu atoms in the Au crystal lattice, and no XRD peaks of Cu or CuO were observed. [43] In addition, the UV-VIS absorption spectrum exhibited the characteristic absorption of Au-Cu alloy nanoparticles with different Au/Cu molar ratio in the UV region ( Figure S3) These observations demonstrated that the Au-Cu nanoparticles formed an alloy structure and were well stabilized in the aqueous solution, and that no aggregation occurred.

H 2 O 2 Detection
In previous reports, the peroxidase-like catalytic properties of Au-based alloy nanoparticles have been explored by different detection strategies. In the present work, we performed the catalytic activity analysis of the prepared Au-Cu alloy nanoparticles by colorimetric method using TMB as a chromogenic substrate [44]. As previous reported, the catalytic activity of enzyme-mimicking nanoparticles is dependent on pH and temperature. Thus, the catalytic activity of the Au-Cu nanoparticles was investigated under different pH and temperature conditions. The reaction solution pH-and temperature-dependent curves are shown in Figures 2 and 3. The results show that the highest absorbance intensities were at pH = 4 and room temperature, respectively. Accordingly, under these conditions, we confirmed that there was no reaction in the absence of the Au-Cu alloy nanoparticles ( Figure 4). When the reaction was conducted in the presence of the Au-Cu alloy nanoparticles, the intensity of the absorbance peak at around 652 nm increased, which is characteristic of TMBox, and indicated that the synthesized Au-Cu alloy nanoparticles possessed catalytic activity in TMB oxidation by generating OH radicals from H 2 O 2 , thereby causing the color change.
Monitoring of the UV-VIS absorption-peak evolution of the TMB reaction solutions containing Au 1 Cu 0.1 , Au 1 Cu 0.2 , Au 1 Cu 0.5 , Au 1 Cu 1 , and Au 1 Cu 2 showed that the UV-VIS absorbance intensity increased with the H 2 O 2 concentrations (Figure 5a-e and Figure S4). Wide detection ranges from 1 µM to 10 mM was observed for all Au-Cu alloy nanoparticles. According to the equation LOD = 3δ/k [45], where δ is the standard deviation of 10 replicate measurements of absorbance of the blank signal (absorbance of TMB solution without H 2 O 2 ), and k is the slope of the calibration curve, the LODs for H 2 O 2 were calculated to be 0.609 µM for Au 1 Cu 0.1 , 0.508 µM for Au 1 Cu 0.2 , 0.274 µM for Au 1 Cu 0.5 , 0.178 µM for Au 1 Cu 1 , and 0.141 µM for Au 1 Cu 2 . The lowest LOD of Au 1 Cu 2 may be related to the increase in Cu content, since many reports have demonstrated that not only Au, but also Cu + nanomaterials possess peroxidase activity [46]. In addition, a comparison with other nanomaterials exhibiting activity for H 2 O 2 detection demonstrated that the present Au-Cu alloy nanoparticles had low LOD and a wide detection range (Table 1).
Catalysts 2021, 11, x FOR PEER REVIEW 4 of 1 Figure 2. The pH-dependent response curve for H2O2 detection using the as-prepared Au-Cu alloy nanoparticles incubated at room temperature. The error bars represent the standard deviation of three measurements.     Monitoring of the UV-VIS absorption-peak evolution of the TMB reaction s containing Au1Cu0.1, Au1Cu0.2, Au1Cu0.5, Au1Cu1, and Au1Cu2 showed that the UV sorbance intensity increased with the H2O2 concentrations (Figures 5a-e and S4 detection ranges from 1 μM to 10 mM was observed for all Au-Cu alloy nanop According to the equation LOD = 3δ/k [45], where δ is the standard deviation of 10 measurements of absorbance of the blank signal (absorbance of TMB solution H2O2), and k is the slope of the calibration curve, the LODs for H2O2 were calculat 0.609 μM for Au1Cu0.1, 0.508 μM for Au1Cu0.2, 0.274 μM for Au1Cu0.5, 0.178 μM for and 0.141 μM for Au1Cu2. The lowest LOD of Au1Cu2 may be related to the increa content, since many reports have demonstrated that not only Au, but also Cu + nano als possess peroxidase activity [46]. In addition, a comparison with other nanom exhibiting activity for H2O2 detection demonstrated that the present Au-Cu allo particles had low LOD and a wide detection range (Table 1).   To gain more insight into the catalytic properties of the Au-Cu alloy nanoparticles, we also prepared single Au nanoparticles and Cu nanoparticles by coreduction ( Figure S5) and detected their catalytic activity under the same conditions, as shown in Figure 5f,g and Figure S6. The LOD of the Au nanoparticles and Cu nanoparticles were 1.97 µM and 4.55 µM, respectively, in the linear range of 10-100 µM. Therefore, it can be concluded that the Au-Cu alloy nanoparticles had a wider detection range and a lower LOD than the single derivatives (Figure 5h), indicating that the formation of the Au-Cu alloy is beneficial for peroxidase-like activity.

Selectivity Analysis
The catalytic performance of enzymes is determined by their selectivity and sensitivity for the detection of target substrates; therefore, the selectivity of enzyme mimetics is worth investigating. In this study, to determine the detection selectivity of the Au-Cu alloy nanoparticles for H 2 O 2 , we performed control experiments using various common ions (Na + , K + , Ca 2+ , Mg 2+ , PO 4 3− , and Cl − ) and other substances (AA, glucose, and urea). These ions and substances can be usually found in human plasma and might influence the catalytic activity of the Au-Cu alloy nanoparticles. As shown in Figure 6, these controls generated negligible UV-VIS absorbance compared with H 2 O 2 in the colorimetric reaction, which is indicative of the high selectivity of the Au-Cu alloy nanoparticles for H 2 O 2 detection. In contrast, the tested ions, ascorbic acid, glucose, and urea, cannot directly generate • OH radicals from H 2 O 2 for TMB oxidation. As for glucose, the Au-Cu alloy nanoparticles may also exhibit peroxidase-mimicking activity in the presence of glucose oxidase (GO x ), because the GOx can decompose glucose to generate H 2 O 2 , thereby causing the colorimetric reaction in the presence of Au-Cu alloy nanoparticles. Therefore, we performed the comparison of absorbance intensity between TMB solution in the absence and in the presence of GO x by the colorimetric method, as shown in Figure 7. The results showed enhanced absorbance intensity in the presence of GO x , indicating that glucose detection can be performed in the presence of GO x .

Glucose Detection and Selectivity Analysis
Glucose oxidase (GOx) can promote the catalytic oxidation of glucose to produce H2O2. Therefore, the Au-Cu-based colorimetric method can be used for glucose detection in the presence of GOx. The typical UV-VIS absorption spectra of the TMB reaction solutions and glucose-concentration response curve for Au-Cu are shown in Figures 8 and S7. The prepared Au-Cu alloy nanoparticles showed the lowest detection limit of 2.6 × 10 -7 mol/L in the linear range of 2 to 10 μM for glucose detection, which was superior to that of several previously reported artificial enzymes (Table 2). Furthermore, the selectivity of glucose detection was investigated via control experiments. Several typical common glucose homologues (sucrose, lactose, maltose, and mannitol) and glucose were comparatively analyzed, and the results are presented in Figure 9. The absorption intensity was clearly higher in the presence of glucose than those of the control samples, indicating that the proposed Au-Cu based system exhibited high selectivity for glucose detection.

Glucose Detection and Selectivity Analysis
Glucose oxidase (GOx) can promote the catalytic oxidation of glucose to produce H 2 O 2 . Therefore, the Au-Cu-based colorimetric method can be used for glucose detection in the presence of GOx. The typical UV-VIS absorption spectra of the TMB reaction solutions and glucose-concentration response curve for Au-Cu are shown in Figure 8 and Figure S7. The prepared Au-Cu alloy nanoparticles showed the lowest detection limit of 2.6 × 10 -7 mol/L in the linear range of 2 to 10 µM for glucose detection, which was superior to that of several previously reported artificial enzymes (Table 2). Furthermore, the selectivity of glucose detection was investigated via control experiments. Several typical common glucose homologues (sucrose, lactose, maltose, and mannitol) and glucose were comparatively analyzed, and the results are presented in Figure 9. The absorption intensity was clearly higher in the presence of glucose than those of the control samples, indicating that the proposed Au-Cu based system exhibited high selectivity for glucose detection.

Characterization
A D8 Advance X-ray diffractometer (XRD) was used for the crystal-structure characterization of the Au-Cu alloy nanoparticles. For the morphological characterization, transmission electron microscopy (TEM) was performed using a JEM-2100F, JEOL microscope. The energy-dispersive X-ray spectrometry (EDS) analysis was performed using a JEM-2100F microscope operated at 200 kV for the elemental analysis. The UV-VIS absorption spectra were acquired using a Cary 60 UV-VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). An inductively coupled plasma spectrometer (Direct Reading Echelle ICP, LEEMAN, Hudson, NH, USA) was used to determine the Au/Cu molar ratio in the nanoparticles.

Colorimetric Detection of H 2 O 2 Using Au-Cu Alloy Nanoparticles as Peroxidase Mimetics
The colorimetric detection process was performed as follows. A total of 30 µL of an aqueous dispersion containing Au-Cu alloy nanoparticles (0.4 mg/mL), 200 µL of TMB solution (2.5 mM), and 200 µL of different concentrations of H 2 O 2 were mixed with 1.5 mL of acetate buffer (20 mM, pH 4.0), and the mixture was incubated at room temperature for 120 min. After the reaction, the solution was subjected to UV-VIS absorption spectroscopy analysis.

Selectivity of H 2 O 2 Detection Using Au-Cu Alloy Nanoparticles as Peroxidase Mimetics
An analysis of the selectivity of the H 2 O 2 detection by the Au-Cu alloy nanoparticles was performed through the following process. A total of 30 µL of an aqueous dispersion containing Au-Cu alloy nanoparticles (0.4 mg/mL), 200 µL of TMB solution (2.5 mM), and 200 µL of H 2 O 2 (10 mM) was added into 1.5 mL of acetate buffer (20 mM, pH 4.0). The resulting solution was then incubated at room temperature for 120 min. For the control experiments under the same conditions, the same number of common ions (Na + , K + , Ca 2+ , Mg 2+ , PO 4 3− , Cl − ) or substances (AA, glucose, urea) as that of H 2 O 2 was added to the reaction system.

Colorimetric Detection and Selectivity Analysis of Glucose Using Au-Cu Nanoparticles as Peroxidase Mimetics
Glucose detection was performed as follows. First, 100 µL of glucose oxidase (GOx) (0.0032 g/mL) and 100 µL of the glucose solution (various concentrations) prepared in the HEPES buffer solution (pH 6.5) were incubated at 37 • C for 30 min. Subsequently, 1.5 mL of the NaOAc buffer solution (pH 4), 100 µL of Au-Cu alloy nanoparticles (0.0004 g/mL), and 200 µL of the TMB (1 mM) solution were added to the above solution. The mixture was incubated at room temperature for 120 min and was further used for performing the absorption spectroscopy measurement. As for the selectivity analysis, the glucose homologues (sucrose, lactose, maltose, and mannitol) were used in control experiments.

Conclusions
In this paper, we synthesized five types of Au-Cu alloy nanoparticles with different Au/Cu ratios by a facile coreduction method. The synthesized Au-Cu alloy nanoparticles were able to act as peroxidase mimetics for H 2 O 2 and glucose colorimetric detection with a LOD of 0.141 µM and 0.26 µM. Furthermore, it was demonstrated that both the catalytic activity and selectivity of Au-based nanocatalysts were enhanced by mixing Au and Cu into alloy catalysts with specific properties. We expect that the developed method can be extended to prepare other noble-metal-based alloy nanocatalysts.
Author Contributions: Conceptualization, synthesis, characterization, catalytic activity assay, and writing-original draft preparation, C.L.; supervision, methodology, further data analysis, and writing-review and editing, S.H.I. and T.Y. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.