3.1.2. Effect of pH

The role of pH on the formation of GNS was illustrated by UV–Vis absorption spectra (Figure 3) and TEM images (Figure 4). There was a rise of SPR absorbance and intensity of SPR, as pH 1.0 was adjusted to pH 1.5 and reached the highest value, 865 nm of absorbance and 0.49 of intensity. The SPR and intensity of SPR significantly declined when pH was adjusted to 2.0, 2.5, and finally 3.0. The SPR dropped to the lowest peak 833 nm, while intensity went down to 0.04, following pH adjustment (Table 3). As per the UV–Vis spectra and TEM examinations, the effect of both size of core and branches of multi-branched particles on SPR absorbance was shown. Adjusting pH from 1.0 to 2.0, the SPR absorbance spectra shifted to the NIR region and SPR intensity declined from 0.54 to 0.16. These were contributed by an increase of size of core and prolongated branches (Table 4). At pH 1.0, the GNS formed were short, multi-branch particles with 53.44 ± 8.30 nm average core and 36.23 ± 8.84 nm branches (Figure 4a). At pH adjusted to 1.5, the prepared GNS particles had elongated branches of 45.23 ± 10.03 nm (Figure 4a) and a core diameter of 59.32 ± 8.08 nm. Meanwhile, when the pH increased to 2.0, and the branches of GNS were short and unsharp (Figure 4b). At pH 3.0, a just, non-star shape were formed (few branches particles) and aggregation of the core of nanoparticles was observed from Figure 4c. Both TEM images and decrease in SPR intensity indicated that there was a greater contribution to the adsorption spectrum because of the core aggregation than the surface plasmon resonance from multibranches, at basic pH. The influence of pH to formation mechanism of gold nanoparticles could be explained by the relation between pH and HQ, as a reducing agent [37]. HQ can reduce Au3+ ions to Au<sup>0</sup> atoms by electrons produced in the oxidation/reduction process. Oxidation/reduction is a reversible process at equilibrium. At high acidic conditions (pH below 1.0), HQ could not reduce Au3+ to Au atoms, in the absence of gold seeds, because there were not any produced electrons. Adjusted to pH 1.5, few gold seeds appeared due to rapid reduced Au3+ ions. Furthermore, the reversible reaction promoted some electrons that reduced Au3+ to Au<sup>+</sup> ions. Under protection of CS, Au<sup>+</sup> attached to the gold seeds, and grew in anisotropic directions to form multi-branched particles. However, at pH towards basic condition (pH upper 2.0), the process became irreversible and was driven to the right. This promoted numerous electrons and uncontrolled gold nanoparticles were synthesized.

**Figure 3.** Absorption spectra of GNS prepared in different pH: (**a**) 1.0, (**b**) 1.5, (**c**) 2.0, (**d**) 2.5 and (**e**) 3.0.

**Figure 4.** TEM images of GNS prepared in different pH: (**a**) 1.5, (**b**) 2.0, and (**c**) 3.0.


**Table 3.** The influence of pH to surface plasmon resonance and intensity absorbance.

**Table 4.** The influence of pH to morphology, average core and branches of GNS.


#### 3.1.3. Effect of Hydroquinone

Different volumes of HQ 0.1 M were adjusted to investigate the effect of HQ for preparation of GNS. The absorption spectra of GNS prepared in a variety of HQ volume are displayed in Figure 5. There was an increase of intensity of SPR, from 0.40 to 1.16, and the SPR absorbance also shifted to 708 nm, as 1.0 to 2.0 mL of HQ volume was added (Table 5). The intensity of SPR declined rapidly to 0.67 and 611 nm of SPR absorbance, when amounts of 2.0 mL to 3.0 mL HQ 0.1 M were added, respectively. TEM micrographs in Figure 6 revealed morphology of three samples of GNS. At low volume of HQ (2.0 mL or less), GNS exhibited short multi-branched particles of 19.58 ± 5.19 nm, with an average core of 51.22 ± 6.67 nm. The 2.0 mL HQ volume resulted in long, sharp branches of GNS. The average branched length was 76.11 ± 14.23 nm and the core was 64.85 ± 6.79 nm. However, the branches of GNS were shorter when the HQ volume increased to 3.0 mL. This resulted in 55.15 ± 10.68 nm average core and 49.85 ± 7.42 nm in branched length (Table 6). Additionally, TEM revealed to the interaction of core particles. The influence of volume of HQ 0.1 M could be explained as follows. Gold seeds were first formed, then Au3+ ions were reduced to Au+. Then, the Au+ ions attached to the gold seeds and grew in anisotropic directions to form multi-branched particles. At low HQ, the electrons produced by the reversibility of HQ was enough to promote seed-particles, meanwhile small amounts of Au+ ions appeared. Therefore, the short multi -branched particles were synthesized. At high HQ, not only seed particles formed but also all Au3+ changed to Au+ ions, resulting in the formation of long and sharp multi-branched particles. However, if the HQ was too high, many electrons were promoted, which led to an uncontrolled reaction [38].



**Figure 5.** Absorption spectra of GNS prepared in various HQ 0.1 M volume—(**a**) 1.0 mL, (**b**) 1.5 mL, (**c**) 2.0 mL, (**d**) 2.5 mL and (**e**) 3.0 mL.

**1.0 mL HQ 0.1 M 2.0 mL HQ 0.1 M 3.0 mL HQ 0.1 M** 

**Figure 6.** TEM images of GNS prepared in various HQ 0.1 M volume—(**a**) 1.0 mL, (**b**) 2.0 mL, and (**c**) 3.0 mL.


**Table 6.** The influence of HQ on morphology, average core and branches of GNS.

*3.2. Ultrasound Combined Surfactant-Free Preparation of GNS*

3.2.1. Effect of Sonication Amplitude

UV–Vis results (Figure 7) and TEM analysis (Figure 8) were used to study the effect of amplitude sonication on the morphology and size of GNS synthesized in permanent time sonication, for 5 min. It was noticeable that the intensity of SPR of sonicated GNS was more intent than without the sonication handled sample. The intensity of SPR increased from 0.26 to 0.53, while the SPR absorbance rose from 831 nm to 877 nm, due to the changing amplitude from 20 to 60 μm (Table 7). Adjusting the amplitude to 80 μm, the branches of multi-branched particles shortened dramatically to 27.88 ± 5.83 nm, while the core diameter increased to 73.10 ± 24.66 nm. In addition, the aggregation of multi-

branched particles was revealed due to the interaction of the spherical core with each other. Additionally, both SPR absorbance and intensity of SPR still had a high value, 874 nm and 1.14, respectively. This could be assigned to the greater contribution of the UV–Vis spectrum because of aggregation rather than the formation of surface plasmon resonance branches. The intensity of SPR significantly declined to 0.80, when amplitude was adjusted to d 100 μm, and the SPR absorbance fell to 690 nm. Synthesized GNS in 60 μm amplitude sonication have long and sharp multi-branches, whereas in prepared GNS in higher amplitude, the branches were short and un-harp (Table 8). Sonication power is the electrical energy supplied to the probe sonicator and transformed into mechanical energy. This is executed by exciting the piezoelectric crystals moving in the longitudinal direction, where the mechanical energy result in the probe vibrating up and down [39]. The stronger amplitude applied, the higher is the acoustic energy produced. When suitable amplitude sonication was applied, the reduction of Au<sup>+</sup> to Au<sup>0</sup> were accelerated, which resulted in higher reaction yields than without sonication-assisted sample, in the same time reaction. Nevertheless, an overly strong amplitude generated a high acoustic energy that led to uncontrolled rapid reduction, so the overall isotropic small particles reaction dominated the over multi-branched anisotropic.

**Figure 7.** Absorption spectra of GNS prepared in different amplitudes—(**a**) 0 μm, (**b**) 20 μm, (**c**) 40 μm, (**d**) 60 μm, (**e**) 80 μm and (**f**) 100 μm.

**Figure 8.** TEM images of GNS prepared in different amplitudes—(**a**) 60 μm, (**b**) 80 μm and (**c**) 100 μm.


**Table 7.** The influence of amplitude to surface plasmon resonance and intensity absorbance.

**Table 8.** The influence of amplitude sonication to morphology, average core and branches of GNS.


#### 3.2.2. Effect of Sonication Time

Figure 9 (the UV–Vis results) and Figure 10 (TEM images) show the influence of time sonication on the morphology and size of GNS prepared with a constant amplitude 50 μm. Both intensity and SPR absorbance increased when a longer sonication time was applied, and it reached the highest value at 6 min. Nevertheless, both decreased rapidly, despite a prolonged time sonication. Table 9 indicates that the intensity from 0.32 rose to 1.15 and SPR absorbance from 831 nm shifted to 833 nm, with an elongated time sonication of 6 min. The SPR absorbance shifted to the NIR region because both the size of core and the branched length increased. Synthesized GNS at 4 min exhibited an average branched length of 31.32 ± 7.62 nm and a core diameter of 52.02 ± 9.95 nm, that from 6 min had branches of 65.01 ± 11.39 nm and an average core of 75.34 ± 18.37. Intensity of SPR dropped to 0.44, while the SPR shifted significantly down to 630 nm. The time sonication of 8 min resulted in GNS with short and unsharp branches, besides, there was a rise of core diameter of multi-branched particles due to the interaction of each particle. Meanwhile, longer time sonication obtained gold nanoparticles that were short rod particles (Table 10). It is possible that the influence of time sonication might be based on cavitation bubbles. Sonication for a prolonged period that generated more cavitation bubbles led to an uncontrolled and rapid reduction, due to higher acoustic energy. As a result, the gold nanoparticles had a smaller core, and fewer and shorter branches were formed [40].


**Table 9.** The influence of time sonication to surface plasmon resonance and intensity absorbance.

#### *3.3. Investigation of Interaction between CS and GNS*

The FT–IR of pure CS and CS-coated GNS spectra are shown in Figure 11. In the spectrum of pure CS, there was a band at around 2883 cm−<sup>1</sup> that corresponded to the stretching vibration of the C-H groups. Two peaks located at 1649 cm−<sup>1</sup> and 1324 cm−<sup>1</sup> related to the C=O stretching vibration of amide I and C-N stretching of amide III, respectively [41]. These bands confirmed the N-acetyl groups of CS [42]. The band at 1589 cm−<sup>1</sup> corresponded to the bending vibration of the N-H groups (amide II) [42].

**Figure 9.** Absorption spectra of GNS prepared in different sonication time—(**a**) 0 min, (**b**) 2 min, (**c**) 4 min, (**d**) 6 min, (**e**) 8 min and (**f**) 10 min.

**Figure 10.** TEM images of GNS prepared in different sonication time—(**a**) 6 min, (**b**) 8 min and (**c**) 10 min.



The FT–IR spectrum of CS-capped GNS was the shift of bands observed in pure CS. The C-H stretching shifted to 2880 cm−1, while the bending of N-H bonds moved to 1557 cm<sup>−</sup>1. Additionally, stretching of C=O (amide I) and C-N (amide III) was located at 1642 cm−<sup>1</sup> and 1310 cm−1. These shifts indicated the interaction between the functional groups of CS and GNS [42].

**Figure 11.** FT–IR spectrum of CS and CS-coated GNS.

#### *3.4. The XRD Diagram of GNS*

Figure 12 is the XRD diagram of CS-coated GNS. The recorded pattern exhibited peaks located at 38.0◦, 44.9◦, 65.2◦ and 77.4◦, which correspond to the (111), (200), (220), (311) and (222) planes of the gold face-centered-cubic (fcc) crystalline structure, respectively [43]. It was clear that there was an intense peak located at 38.0◦, which was indexed to the (111) plane. Additionally, a weaker peak for the (200) plane at 65.2◦ and another at 77.3◦ for the (311) plane was observed. Finally, a very weak peak located at 77.3◦ related to the (311) plane.

### *3.5. Cytotoxicity of the CS-Capped GNS*

Biocompatibility is an important property for biomedical applications. MTT assay was used to study the cell compatibility of CS-coated GNS. The cell viability of normal rat fibroblast (NIH/3T3) and normal human fibroblast (BJ-5ta), exposed to various concentrations of multi-branched nanoparticles are shown in Figure 13. The proliferation of both NIH/3T3 and BJ-5ta cell lines treated in various GNS concentration were still around 90%, even at a high concentration of 200 μg/mL, except for BJ-5ta, which had a concentration of over 82%. The results indicate that CS-coated GNS was a good biocompatible agent and is a prospective material for use in biomedical applications. GNS have wide biomedical applications in Raman scattering sensing [44], stem cell tracking [45], bioimaging [46], photothermal treatment [47] and immunotherapy [48].

**Figure 12.** XRD pattern of the CS-coated GNS.

**Figure 13.** Cell viability of NIH/3T3 and BJ-5ta treated with GNS.

#### **4. Conclusions**

This study presents a rapid and green preparation of GNS using the seedless, free surfactant, and ultrasound-assisted method. The GNS particles obtained had long and sharp multi-branches with an average core of 67.85 ± 6.79 nm and a branched length of 76.11 ± 14.23 nm, through an adjusted conditional reaction, such as pH, mass concentration of CS, HQ concentration, as well as amplitude and time sonication. The influences of the conditions above and properties of the prepared GNS were characterized using UV–Vis absorption, FTIR, TEM, XRD, to determine the standard procedure for synthesizing GNS. Furthermore, cytotoxicity of the CS-coated GNS were investigated by the MTT assay on two cell lines, including NIH/3T3 and BJ-5ta. The results indicated that CS-coated GNS was a biocompatible agent, due to its high cell viability. Multi-branched gold nanoparticles are prospective materials not only for biomedical applications but also in cosmetics.

**Author Contributions:** Investigation, G.D.N., K.T.L.T. and T.M.H.; Investigation, writing—original draft preparation, P.T.H.; review and editing, V.Q.L.; writing—review and editing, project administration and funding acquisition, T.V.K.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Saigon Hi-tech Park under grant number 01/2020/HÐNVTX-KCNC-TTRD and the Project of Department of Science and Technology of Ho Chi Minh (118/2019/ HÐ-QPTKHCN).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data is contained within the article.

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

#### **References**

