Highly Biocompatible Hemoglobin-Stabilized Gold Nanoparticles for an Enhanced Catalytic Reduction of 4-Nitrophenol

Abstract

4-nitrophenol (4-NP) is a frequently encountered toxic phenolic organic pollutant in water. It is important to develop a simple method to treat 4-NP. Small and monodispersed gold nanoparticles often have good catalytic performance of 4-NP. Hemoglobin (Hb) is a kind of common and important protein in organisms. Herein, highly biocompatible bovine hemoglobin-stabilized gold nanoparticles (Au_n-Hb NPs) were synthesized using hemoglobin as a biological template. Then, the size, zeta potential, and composition of Au_n-Hb NPs were investigated by transmission electron microscopy, dynamic light scattering, and X-ray photoelectron spectroscopy. The Au_n-Hb NPs with small gold nanoparticles of about 1.4–2.4 nm had good catalytic capabilities in reducing 4-NP to form 4-aminophenol. Au₂₀-Hb NPs demonstrated superior catalytic efficiency in the reduction of 4-NP when compared to other nanoparticles. Moreover, as-synthesized Au₂₀-Hb NPs exhibited excellent biocompatibility through the MTT experiment. The method of preparation of gold nanoparticles offers one way to prepare metal nanoparticles for good potential catalytic applications of gold nanoparticles.

1. Introduction

In recent years, nitrophenol compounds as water pollutants seriously threaten people’s health and safety [1]. For example, 4-nitrophenol (4-NP) is a very toxic phenolic compound [2]. The concentration of phenolic compounds is strictly controlled in China. Catalytic reduction of 4-NP is one method to eliminate its environmental impact. The resulting compound from the catalytic reduction of 4-NP is known as 4-aminophenol (4-AP) [3]. 4-AP plays a vital role in the manufacturing process of fine chemicals like dyes, pharmaceuticals, and pesticides. The efficiency of converting 4-NP to 4-AP is closely linked to the choice of catalysts. Many nanomaterials such as gold nanoparticles (Au NPs) [4], palladium nanoparticles [5], Cu/Fe nanocomposite [6], and Fe₃O₄@C nanoparticles [7] have been developed to reduce 4-NP. Noble metal nanoparticles with small particle sizes and monodispersed states have good catalytic performance in the 4-NP reduction. However, the noble metal nanoparticles easily aggregate.

The development of nanotechnology in recent years has provided new insights and opportunities for the design of small particle sizes and monodispersed catalysts. Some organic polymers are often used as stabilizers to control the particle size and monodispersed state. There are many options for the use of stabilizers, including polyvinyl pyrrolidone (PVP) and polyethyleneimine (PEI) [5]. Recently, the exploration of an efficient and biocompatible catalyst to treat 4-NP has attracted widespread attention. The use of biocompatible substances is good for overcoming this problem. Many biocompatible molecules have been used to stabilize the noble metal nanoparticles. He et al. [8] employed sericin as the reducing agent for silver ions, as well as the dispersing and stabilizing agent for the composite of sericin–silver nanoparticles, which exhibited favorable size distribution and maintained long-term stability. Nogueira et al. [9] used cashew gum-hydrolyzed collagen, kappa carrageenan-hydrolyzed collagen, and agar-hydrolyzed collagen as effective nanoparticle stabilizers to prepare silver nanoparticles, respectively. San et al. [10] used three proteins: aminopeptidase PepA, serine endoprotease DegP, and Clp protease to prepare platinum nanoparticles. Bitter gourd polysaccharide, elm pod polysaccharide, and lentinan were also used to stabilize noble metal nanoparticles [11,12]. Large-ring cyclodextrins were also used to stabilize gold nanoparticles in an aqueous phase [13,14,15]. However, some proteins and polysaccharides have high prices, which is not good for the preparation of noble metal nanoparticles on a large scale.

Bovine hemoglobin is a kind of natural protein found in the blood of bovine. Bovine hemoglobin has an important impact on the survival of the organism. Hemoglobin is a protein composed of four subunits, each consisting of a globin molecule and a heme group. The globin molecule is made up of a chain of amino acids folded into a specific three-dimensional structure, while the heme group contains an iron ion coordinated within a porphyrin ring [16,17]. Bovine hemoglobin functions in the transport of oxygen and carbon dioxide, as well as in regulating the blood’s acid–base equilibrium. The bovine hemoglobin extracted from bovine blood is a good ideal metal stabilizer because of its affordable price, good stability, and environmental friendliness. In addition, Au NPs, as a kind of noble metal nanoparticles, are stable in their zero-valence state. Au NPs find extensive application across a spectrum of catalytic processes; they have good catalytic capabilities in the reduction of 4-NP.

In this study, in order to reduce the pollution caused by nitrophenol compounds in the environment, bovine hemoglobin was used to stabilize gold nanoparticles (Scheme 1). We prepare bovine hemoglobin-stabilized gold nanoparticles (Au_n-Hb NPs) with small particle sizes by a facile method. The prepared gold nanoparticles inside Au_n-Hb NPs had a huge specific surface area originating from their small particle size, which was very beneficial for catalyzing 4-NP. Moreover, the biocompatibility and stability of the gold nanoparticles had been greatly improved with the assistance of bovine hemoglobin. Thus, Au_n-Hb NPs demonstrated effective catalysis in the reduction of 4-NP. Overall, we prepared highly biocompatible bovine hemoglobin-stabilized gold nanoparticles for catalytic reduction of 4-NP. The method in this report is good for the development of catalysts in the degradation of nitrophenol compounds.

2. Results and Discussion

2.1. UV-Vis Spectra Analysis

The bovine hemoglobin solution and HAuCl₄ solution were mixed together for 15 min. Then, the HAuCl₄ solution was reduced by reducing agent NaBH₄, and bovine hemoglobin was used to stabilize the generated gold nanoparticles. The samples of the bovine hemoglobin solution, HAuCl₄ solution, and Au_n-Hb NP solution were measured by a UV-Vis spectrometer with a wavelength from 200 to 600 nm. As shown in Figure 1a, the bovine hemoglobin solution and HAuCl₄ solution had characteristic peaks at 406 nm and 306 nm, respectively. After the preparation of Au_n-Hb NPs, the characteristic peaks at 306 nm for the HAuCl₄ solution disappeared, and the characteristic peaks at 406 nm for the bovine hemoglobin solution were greatly reduced. These results indicated that AuCl₄^- was completely reduced to form Au NPs, and Au NPs were complexed within bovine hemoglobin. In addition, the absorbance of the Au_n-Hb NPs solution gradually increased with a n-value from 20 to 40, indicating that the amount of Au NPs in the Au_n-Hb NPs continued to increase. The color of the Au_n-Hb NP (n = 20, 30, 40) solution gradually deepened based on the same concentration of bovine hemoglobin, as shown in Figure 1b. Taken together, all the results suggested that we successfully prepared Au_n-Hb NPs.

2.2. TEM Observation

The size of nanomaterials plays a crucial role in determining their catalytic efficiency. Herein, the particle size and dispersion condition of Au_n-Hb NPs were determined by TEM. Figure 2 shows TEM images of Au_n-Hb NPs prepared using bovine hemoglobin with different HAuCl₄ contents. TEM images indicated that Au_n-Hb NPs (n = 20, 30, 40) had highly dispersed states. The sizes of Au_n-Hb NPs were 1.4 ± 0.6 nm for Au₂₀-Hb NPs, 2.1 ± 0.6 nm for Au₃₀-Hb NPs, and 2.4 ± 0.8 nm for Au₄₀-Hb NPs, respectively. The specific surface area of Au NPs inside Au₂₀-Hb NPs, Au₃₀-Hb NPs, and Au₄₀-Hb NPs was 4.3 nm⁻¹, 2.9 nm⁻¹, and 2.5 nm⁻¹, respectively. The Au NPs inside Au_n-Hb NPs have small and monodisperse states. It can also be seen that the molar ratio of HAuCl₄ to hemoglobin affects the size of Au NPs. The average diameter of Au NPs inside Au_n-Hb NPs increases with the molar ratio of HAuCl₄ to bovine hemoglobin, and the shape of Au NPs remains basically unchanged. Thus, Au_n-Hb NPs were characterized by their small size and uniform size distribution, which is beneficial to their catalytic performance.

2.3. XPS Analysis

XPS analysis was utilized for further examination of the composition and valence state of the synthesized Au₂₀-Hb NPs. As shown in Figure 3a, the binding energies of about 531.3, 399.7, and 284.6 eV were indexed to O 1s, N 1s, and C 1s, respectively. These elements were derived from bovine hemoglobin. There is little Fe 2p_3/2 in the XPS survey spectrum of Au₂₀-Hb NPs. To further determine the gold valence, the peaks of Au 4f_7/2 and Au 4f_5/2 were observed at 83.92 and 87.64 eV in the high-resolution XPS spectrum of the Au 4f region, as shown in Figure 3b, respectively. The gap between the two peaks is 3.72 eV. Consistent with zero-valent Au, the XPS binding energy of Au 4f_7/2 confirmed the presence of Au (0) based on the peaks observed [13,18]. It is important to mention that the spectrum lacked the typical characteristic peaks associated with Au(III), suggesting a thorough reduction of Au(III) to Au(0) during the preparation reaction. The completed reduction of Au(III) is due to an excess of reducing agent NaBH₄. Gold nanoparticles were visible-light-induced synthesized using Lantana camara flower extract, which has good antibacterial activity. The gap between Au 4f_7/2 and Au 4f_5/2 is also 3.7 eV [19]. The XPS survey spectra manifest that the surface elements of Au₂₀-Hb NPs were derived from bovine hemoglobin and HAuCl₄. Thus, the XPS measurements confirm the existence of zero-valent gold nanoparticles, which were stabilized by bovine hemoglobin.

2.4. Stability in Solution

Basically, the size and dispersion state of Au NPs in an aqueous solution are the key factors for their catalytic activity. In order to evaluate the states of Au_n-Hb NPs, DLS was employed to measure the hydrodynamic size and zeta potential of Au_n-Hb NPs. As depicted in Figure 4a, the hydrodynamic size of bovine hemoglobin was 6.5 nm, and the hydrodynamic sizes of Au_n-Hb NPs (n = 20, 30, 40) were 7.5 nm, 8.7 nm, and 10.1 nm, respectively. It can be seen that Au_n-Hb NPs had a small hydrodynamic size. The hydrodynamic size of Au_n-Hb NPs also increases with the increasing amount of HAuCl₄ added. It should be noted that the hydrodynamic sizes of Au_n-Hb NPs were larger than the corresponding particle sizes shown in Figure 2. This was due to the different ways of preparing the samples. The Au_n-Hb NPs solution was deposited onto a carbon-coated copper grid and left to air dry overnight in preparation for the TEM technique, while the sample solution of Au_n-Hb NPs was measured in a hydration state for the DLS method. In addition, as we all know, the zeta potential plays a pivotal role in determining the strength of the mutual repulsion or attraction between nanoparticles in solution. The bigger of absolute value of the zeta potential of the nanoparticles, the more stable the nanoparticles in the solution. That is, a high zeta potential is good for resisting the aggregation of nanoparticles. In this experiment, the zeta potential of Au_n-Hb NPs (n = 20, 30, 40) was −1.0 mV for Au₂₀-Hb NPs, −21.3 mV for Au₃₀-Hb NPs, and −23.2 mV for Au₄₀-Hb NPs, as shown in Figure 4b. It shows that as the content of Au element in Au_n-Hb NPs increases, the absolute value of the zeta potential of Au_n-Hb NPs gets bigger. The pH values of Au ₂₀-Hb NPs, Au ₃₀-Hb NPs, and Au₄₀-Hb NPs were different. This should be the reason that the volume of the HAuCl₄ solution and NaBH₄ solution increased, which led to the pH of the mixture solution being slightly increased in the process of Au_n-Hb NP preparation. More carboxy groups of Au_n-Hb NPs were deprotonated with the increasing pH. Moreover, Au_n-Hb NPs remained stable without precipitation for at least four days, as shown in Figure 4c, while Au₂₀, Au₃₀, and Au₄₀ without bovine hemoglobin easily aggregated into large particles and formed precipitates, as shown in Figure 4d. The difference in Figure 4c,d proved that the bovine hemoglobin had a positive influence on preventing self-aggregation of Au NPs during preparation and storage. The hydrophilic primary amine groups and carboxyl groups on the outside of the molecule keep bovine hemoglobin in a highly water-soluble state, which is conducive to maintaining its structural stability and exerting its functional properties. Hydrophilic bovine hemoglobin is good for the stability of Au_n-Hb NPs. The main contributing factors to the exceptional stability of Au_n-Hb NPs are electrostatic repulsion and steric hindrance.

2.5. Catalytic Performance

The catalytic performance of the synthesized Au_n-Hb NPs was quantitatively assessed by their ability to facilitate the reduction of 4-NP to 4-AP in the presence of an excess of NaBH₄ as the reducing agent. A time-dependent absorbance change in a mixed solution was monitored by UV-Vis spectra. As depicted in Figure 5a, the absorption peak observed at 317 nm corresponded to the pure 4-NP aqueous solution. Upon the introduction of NaBH₄, the absorption peak of 4-NP at 317 nm underwent a shift to 400 nm, demonstrating the formation of 4-nitrophenolate ions in the alkaline condition. The solution underwent a color alteration from transparent to intense yellow. Au₂₀-Hb NPs were picked up for further catalytic research. After the addition of the Au₂₀-Hb NPs, the absorption peak intensity of 4-nitrophenolate ions at 400 nm dramatically decreased, indicating the consumption of 4-nitrophenolate ions and the generation of 4-AP, as shown in Figure 5b. Moreover, the 4-NP conversion rate reached 98% after 21 min for Au₂₀-Hb NPs in Figure 5c, and the resulting solution became colorless. As depicted in Figure 5d, ln(C_t/C₀) versus reaction time (t) for the different amounts of Au₂₀-Hb NPs is linear. The catalytic performance of Au₂₀-Hb NPs was improved with increasing amounts of Au₂₀-Hb NPs. Furthermore, the catalytic reaction conformed to a pseudo-first-order kinetic equation in the presence of an excess of NaBH₄. (Equation (2)). Ct represents the concentration of 4-NP at time t, while C₀ denotes the initial concentration of 4-NP at t = 0, k_app is the rate constant (s⁻¹), and t is time.

$$\ln \frac{{\mathrm{C}}_{\mathrm{t}}}{{\mathrm{C}}_0}=\ln \frac{{\mathrm{A}}_{\mathrm{t}}}{{\mathrm{A}}_0}=-{\mathrm{k}}_{\mathrm{a}\mathrm{p}\mathrm{p}}\mathrm{t}$$

(1)

The k_app value is also linearly dependent on the amount of Au₂₀-Hb NPs in Figure 5e. It has been shown that the k_app value is related to mass transfer resistance, size of gold nanoparticles, temperature, and 4-NP, NaBH₄ [20], or catalyst concentration [21]. The k_app value increases with increasing temperature, NaBH₄, or catalyst concentration [20]. It has been reported that k_app decreases with increasing 4-NP concentration and increasing mass transfer resistance [20]. Increasing mass transfer resistance limits substrate contacts with active metal sites on the catalyst. Here, the low k_app value of Au₂₀-Hb NPs should be due to their low concentration. In order to compare the effect of the n value on the catalytic activity of Au_n-Hb NPs, the same amount of Au_n-Hb NPs was added in the catalytic reduction process of 4-NP, where the hemoglobin concentration was the same. Figure 5f illustrates the linear relationship between ln(C_t/C₀) and time (t) during the catalytic reduction of Au_n-Hb NPs. As the number of gold nanoparticles on each hemoglobin molecule increased, the corresponding catalytic activity also increased. Au_n-Hb NPs exhibited a good catalytic reduction behavior, indicating that Au_n-Hb NPs were effective catalysts for the reduction of 4-NP.

Many groups reported the catalytic reduction of 4-NP by metal nanoparticles treated with NaBH₄ [22,23,24,25]. For a comparative evaluation of catalytic efficacy against alternative catalysts, calculations were performed for the normalized rate constant (k_nor = k_app/n_Au) and turnover frequency (TOF) of Au_n-Hb NPs. TOF is quantified as the ratio of the number of molecules generated by the reducing species 4-NP to the moles of catalytically active sites per hour when the 4-NP conversion achieves 90%. Table 1 shows the calculated k_app and TOF comparison of Au₂₀-Hb NPs in conjunction with catalysts mentioned in previous studies. Here, the TOF of Au₂₀-Hb NPs was determined to be 6768 h⁻¹, which was much higher than those of Au/graphene (12 h⁻¹), Au NPs (94 h⁻¹), GO@NH₂-Au NPs (595 h⁻¹), Au₁₀-LP (6053 h⁻¹), and Au/Fe₂O₃@HAP (241.3 h⁻¹). The k_nor of Au₂₀-Hb NPs was 3.32 × 10⁴ s⁻¹mmol⁻¹, which was also much higher than those of GO@NH₂-Au NPs (5.85 × 10² s⁻¹mmol⁻¹), Au₁₀-LP (1.31 × 10³ s⁻¹mmol⁻¹), Au/Fe₂O₃@HAP (1.27 × 10³ s⁻¹mmol⁻¹), Au NPs/AOBC (2.98 × 10³ s⁻¹mmol⁻¹), and Cu-Au BNSs (2.01 × 10⁴ s⁻¹mmol⁻¹). Thus, Au₂₀-Hb NPs had superior activity in the catalytic reduction reaction of 4-NP. It should be noted that the size of the AuNPs inside Au_n-Hb NPs is smaller than most Au NPs in Table 1. The advantages of 4-NPs can be attributed to good stability and the small size of gold nanoparticles. It is well known that smaller Au NPs should have more active sites and higher catalytic activity. The specific surface area of Au₂₀-Hb NPs is quite high, and the elevated catalytic efficiency can primarily be attributed to the considerable specific surface area or high active site content for catalytic reactions on the gold nanoparticle surface. Gold nanoparticles that are small in size can also be stabilized by ligands. However, robust ligands may hinder the functioning of active surface sites, ultimately reducing catalytic ability. The Au NPs without ligands will precipitate for a long time, which also largely reduces their catalytic activity.

The reaction rate is very slow in kinetics without Au₂₀-Hb NPs. It can be concluded that Au₂₀-Hb NPs were effective catalysts for the reduction of 4-NP. The bovine hemoglobin acted as an excellent stabilizer for Au NPs. The absorption of BH₄⁻ onto Au nanoparticles potentially provides Au−H species, which play a role in facilitating the transfer of all four electrons [1,26]. Many research groups have documented the mechanism involved in the catalytic reduction of 4-NP using noble metal nanoparticles under the conditions of NaBH₄ [26,27,28]. It is widely acknowledged that the catalytic reaction of 4-NP follows Langmuir–Hinshelwood kinetics. Ballauff et al. [29] proposed a detailed mechanism of the catalytic reaction. All compounds rapidly achieve equilibrium between adsorption and desorption. 4-NP is first rapidly converted to the stable intermediate (4-Hx). The concentration of 4-Hx remains approximately constant without catalysts. The intermediate 4-Hx is further reduced to form 4-AP after the addition of catalysts, which is the rate-determining step in the kinetic process [30].

It is expected that the catalytic reaction facilitated by Au₂₀-Hb NPs also adheres to Langmuir–Hinshelwood kinetics. All the components in this reaction quickly reach an adsorption/desorption equilibrium on the surface of gold nanoparticles of Au₂₀-Hb NPs. The rate-determining step is the formation of 4-AP, which occurred only on the surface of the gold nanoparticles.

Table 1. Comparative analysis of k_nor and TOF values for Au₂₀-Hb NPs with other catalysts.

Catalyst	Au Size (nm)	kapp (×10−3 s−1)	knor (s−1 mmol−1)	TOF (h−1)	Ref.
Au20-Hb	1.4	3.32	3.32 × 104	6768	This work
Au/graphene	14.6	3.17	6.25	12	[31]
Au NPs	80	7.42	1.46 × 102	94	[23]
GO@NH2-Au NPs	14	35.6	5.85 × 102	595	[32]
Au10-LP	7.8	4.65	1.31 × 103	6053	[33]
Au/Fe2O3@HAP	10	7.12	1.27 × 103	241.3	[34]
Au NPs/AOBC	10.6	4.47	2.98 × 103	1198	[35]
Cu-Au BNSs	78	30.2	2.01 × 104	536.4	[36]

Table 1. Comparative analysis of k_nor and TOF values for Au₂₀-Hb NPs with other catalysts.

Catalyst	Au Size (nm)	kapp (×10−3 s−1)	knor (s−1 mmol−1)	TOF (h−1)	Ref.
Au20-Hb	1.4	3.32	3.32 × 104	6768	This work
Au/graphene	14.6	3.17	6.25	12	[31]
Au NPs	80	7.42	1.46 × 102	94	[23]
GO@NH2-Au NPs	14	35.6	5.85 × 102	595	[32]
Au10-LP	7.8	4.65	1.31 × 103	6053	[33]
Au/Fe2O3@HAP	10	7.12	1.27 × 103	241.3	[34]
Au NPs/AOBC	10.6	4.47	2.98 × 103	1198	[35]
Cu-Au BNSs	78	30.2	2.01 × 104	536.4	[36]

2.6. Biocompatibility

The catalysts have good catalytic ability with regard to the degradation of organic pollutants, and they also should be biocompatible with our body. MTT is a widely accepted method to measure the cytotoxicity of nanomaterials [37]. Bovine hemoglobin is a kind of biocompatible biomolecule in the body. Here, the MTT assay was employed to determine the cytotoxicity of bovine hemoglobin and Au₂₀-Hb NPs against HeLa cells and A549 cells. Cell viability exceeded 90% when the concentration of Au₂₀-Hb NPs and bovine hemoglobin was lower than 200 μg/mL, as illustrated in Figure 6. Thus, Au₂₀-Hb NPs and bovine hemoglobin were biocompatible towards cells. The good biocompatibility should be due to biocompatible bovine hemoglobin. Bovine hemoglobin has the function of transporting nutrients and oxygen and emitting carbon dioxide in the body. Compared with highly toxic PEI, Au₂₀-Hb NPs can be applied well in bio-related catalysis.

3. Materials and Methods

3.1. Materials

Bovine hemoglobin (Hb) from bovine blood, chloroauric acid (HAuCl₄), sodium borohydride (NaBH₄), 4-nitrophenol (4-NP), and dimethyl sulfoxide (DMSO) were bought from Aladdin. HeLa cells and A549 cells were purchased from the China Center for Typical Culture Collection.

3.2. Synthesis of Au_n-Hb NPs

A total of 32 mg of bovine hemoglobin was dissolved in 10 mL of deionized water to obtain 0.05 mM of the bovine hemoglobin solution. Then, 2 mM of the HAuCl₄ solution at different volumes (200 μL, 300 μL, 400 μL) was mixed with 400 μL of the prepared bovine hemoglobin solution, respectively. The molar ratio of HAuCl₄ to bovine hemoglobin was 20:1, 30:1, and 40:1, respectively. The mixed solution was kept at 25 °C in a constant temperature mixer (600 rpm) for 15 min. The freshly prepared NaBH₄ (1 mg/mL) with an equal volume to the HAuCl₄ solution was added rapidly, respectively. The solution underwent a color change from yellow to dark red, suggesting the formation of Au_n-Hb NPs (n = 20, 30, 40). The sample was stored at 4 °C for further experiments.

3.3. Characterization of Au_n-Hb NPs

The UV-Vis spectra of hemoglobin and the Au_n-Hb NP solution were acquired by a UV-vis spectrophotometer (TU-1810PC). The hydrodynamic size and zeta potential of Au_n-Hb NPs were measured three times using a dynamic light scattering (DLS) measurement from Zetasizer (Nano ZS90). The software for Nano ZS90 is Zetasizer version 7.11. X-ray photoelectron spectroscopy (XPS) spectra were performed on a Thermo Scientific EACALAB 250Xi. The sample for XPS was dialyzed and lyophilized. The size and shape of Au_n-Hb NPs were analyzed using transmission electron microscopy (TEM, HT 7700). Au_n-Hb NPs were immobilized onto a carbon-coated copper grid and left to dry overnight prior to measurement. The software used was Nano Measurer version 1.2, which counted Au nanoparticles to determine the size distribution.

3.4. Catalytic Performance of Au_n-Hb NPs

Au_n-Hb NPs were employed to catalytically reduce 4-NP, following a procedure based on prior reports with slight alterations.

(1) The merging of a 4-NP aqueous solution (0.1 mM, 2 mL) and freshly prepared NaBH₄ (0.5 M, 1 mL) occurred in a quartz cuvette at room temperature. Then, Au₂₀-Hb NPs (100 μL) were added into the mixture solution, with UV-Vis spectra monitoring the reaction solution every 3 min.

(2) In situ 4-NP reduction by NaBH₄ occurreed by mixing the 4-NP (0.1 mM, 2 mL) aqueous solution and fresh NaBH₄ (0.5 M, 1 mL) in a quartz cuvette. Then, Au_n-Hb NPs (100–400 μL) were added. In situ measurement of the absorbance at 403 nm in a mixed solution was conducted using a UV-Vis spectrophotometer.

3.5. MTT Assay

The cytotoxicity of HeLa cells and A549 cells were inoculated into 96-well culture plates (1 × 10⁴ cells/well). After 24 h, the DMEM medium supplemented with 10% fetal bovine serum was exchanged with a fresh DMEM medium containing the samples (hemoglobin and Au₂₀-Hb NPs) from 5 to 200 µg/mL. After 24 h, the cells were treated with a new 100 µL DMEM medium containing 500 µg/mL MTT. After 4 h, the MTT solution was aspirated and substituted with 150 µL of DMSO. The absorbance (A) value at 490 nm was measured with a microplate reader. Cell viability was assessed using Formula (1). A_sample means the absorbance value of the sample, and A_control means the absorbance value of the control group.

$$\mathrm{Cell}\mathrm{viability}(\%)=\frac{{\mathrm{A}}_{\mathrm{sample}}}{{\mathrm{A}}_{\mathrm{control}}}\times 100$$

(2)

4. Conclusions

In conclusion, biocompatible and stable Au_n-Hb NPs were prepared by a simple method. Bovine hemoglobin played an important role in stabilizing Au NPs. The Au NPs were well-dispersed with a small size of about 1.4–2.4 nm. Au_n-Hb NPs exhibited good stability for at least four days. In addition, Au_n-Hb NPs were good catalysts for the catalytic reduction of hazardous 4-NP. The catalytic kinetics follow the pseudo-first-order kinetic equation. The TOF and k_nor of Au₂₀-Hb NPs were 6768 h⁻¹ and 3.32 × 10⁴, which were much higher than those of other catalysts. More importantly, Au_n-Hb NPs exhibited no cytotoxicity towards cells. The prepared catalysts will have a good aspect in the treatment of phenolic pollutants.