Green synthesis and stabilization of earthworm-like gold nanostructure and quasi-spherical shape using Caesalpinia sappan Linn. extract

2.1 Materials

Tetrachloroauric (III) acid or hydrogen tetrachloroaurate (III) (HAuCl₄·4H₂O) was obtained from Anhui Jin’Ao chemical (Anhui Jin’Ao Chemical Co., Ltd., Anhui, China). Sodium hydroxide was supplied by Labscan (RCI Labscan Limited, Samutsakorn, Thailand). All chemicals were used without further purification. Dried heartwood chips of CS were purchased from a local market in Chiangmai, Thailand.

2.2 Preparation of CS extract

CS heartwood chips were reduced to small flakes in a Moulinex blender and 50 g of heartwood flakes were boiled for 3 h at 80°C with deionized water. The ratio of heartwood flakes to deionized water was 1 g:10 ml. After boiling for 3 h, the solution was lyophilized to obtain red extracted powder.

2.3 AuNP synthesis

The green synthesis of AuNPs with CS extract as a reducing and stabilizing agent was performed as follows: hydrogen tetrachloroaurate (III) 180 μl, 0.025 wt% Au was added to a 25 ml of CS solution. The mixture was adjusted until the pH became 7 by using 0.1 M NaOH. The concentrations of CS (0.004 wt%, 0.01 wt%, 0.02 wt%, 0.04 wt%, 0.12 wt%, 0.20 wt%) were tested for the green synthesis of AuNPs. The reaction mixture was stirred overnight (12 h) on a hotplate at room temperature followed by heating the colloidal solution at 90°C for 1 h until AuNPs formation was observed by the appropriate color change. The experiment was repeated at least five times in each condition to confirm the reproducibility of the experiment.

2.4 Characterization

UV-visible (UV-vis) absorption measurements were performed using a UV-vis spectrophotometer (Jusco, V-530, Jasco International Co., Ltd., Tokyo, Japan) in dual beam mode. Fourier-transform infrared (FTIR) data were collected using an FTIR spectrophotometer (Nicolet 6700, Thermo Electron Corporation, MD, USA) with Omnic software (Thermo Fisher Scientific). A laser particle sizer (Malvern Zetasizer Nano ZS, Malvern Instruments Limited, Worcestershire, UK) equipped with an He-Ne laser at 633 nm, 4 mW was used to determine particle size and zeta potential at 25°C by dynamic light scattering (DLS) in back scattering mode. An X-ray diffractometer (Rigaku Miniflex 600 X-Ray diffractometer using Cu Kα X-ray radiation, Rigaku Corporation, Tokyo, Japan) was used to obtain the diffraction patterns of the AuNPs on a glass slide. A transmission electron microscope (TEM, Philips Tecnai 12, FEI Company, OR, USA) was used to investigate the size and morphology of AuNPs.

3.1 Spectroscopic measurements

UV-vis spectroscopy was used to observe the formation and stabilization of AuNPs in aqueous solution containing CS extract. Figure 2 illustrates the UV-vis spectra of AuNPs reduced by 0.004 wt%, 0.01 wt%, 0.02 wt%, 0.04 wt%, 0.12 wt% and 0.20 wt% of CS. It can be observed that the absorbance peaks appeared around λ_max=520–530 nm for the concentration range of 0.004–0.04 wt% of CS extract, which is characteristic of small NPs formation. When the CS extract concentration was increased (from 0.004 wt% to 0.04 wt%), the absorbance intensity increased. However, at high concentrations of 0.12 wt% and 0.2 wt% CS extract, no absorbance peak due to AuNPS was evident, indicating that AuNPS were not formed. The reason for this could be due to the large excess of functional groups of the CS extract at high concentrations that overwhelmingly compete with the gold ions for the reduction process. This result indicates the formation of AuNPs due to the excitation of the surface plasmon vibrations in the AuNPs at the optimized concentration range of 0.004 wt% to 0.04 wt%. The plasmon bands can appear between 520 nm and 570 nm depending on the size and shape of the AuNPs, the concentration of gold chloride precursor, solvent type and reaction temperature [38], [39]. In addition, the formation of AuNPs can be observed by a change in color, turning red/violet after the reduction completed. Figure 3 shows the color of AuNPs colloidal solutions after the reduction completed. From (D) to (G), the colloidal solutions turned to dark purple, red, dark red and purple red for the CS concentrations of 0.004 wt%, 0.01 wt%, 0.02 wt% and 0.04 wt%, respectively, indicating the formation of AuNPs in colloidal solution. Noticeably, the orange/reddish-orange color of CS extract was not observed in any AuNPs colloidal solution after reduction. Furthermore, a comparison was made with (A) CS extract 0.01 wt%, (B) CS extract 0.01 wt% pH 7 and (C) HAuCl₄·4H₂O pH7. It can be observed that the CS extract was yellow and then turned to orange after the pH was adjusted to 7, while HAuCl₄·4H₂O at pH 7 was very pale yellow. Thus, it was found that the optimized concentration for AuNPs synthesis using CS was in the range of 0.004 wt% to 0.04 wt%. The stability of AuNPs colloidal solutions was subjected to testing for a whole month by doing UV-vis measurements every week. Figure 4A–D show the UV-vis spectra of AuNPs at 0.004 wt%, 0.01 wt%, 0.02 wt% and 0.04 wt% CS for 4 weeks. Figure 4A–D represent the 1st, 2nd, 3rd and 4th week measurements, respectively. Good UV-vis spectra were observed during the 4 weeks and are in good agreement with the stability. These results show the efficiency of CS extract in stabilizing AuNPs over an extended period of time. No agglomeration was observed during the first few weeks, while small aggregates were found in the 4th week or thereafter.

Figure 2:

UV-visible spectra of different colloidal solutions of gold nanoparticles (AuNPs) reduced and stabilized with Caesalpinia sappan Linn. (CS) extract; CS concentrations vary from 0.004 wt% to 0.20 wt%.

Figure 3:

From left to right; (A) Caesalpinia sappan Linn. (CS) 0.01 wt%, (B) CS 0.01 wt% pH 7, (C) HAuCl₄·4H₂O pH 7, (D) gold nanoparticles (AuNPs)/CS 0.004 wt%, (E) AuNPs/CS 0.01 wt%, (F) AuNPs/CS 0.02 wt% and (G) AuNPs/CS 0.04 wt%.

Figure 4:

(A)–(D) represents UV-visible spectra of different colloidal solutions of gold nanoparticles (AuNPs) reduced and stabilized with Caesalpinia sappan Linn. (CS) extract from week 1 to week 4.

FTIR spectra were obtained from the pure CS extract and dried powder isolated from AuNPs colloidal solutions (Figure 5). FTIR spectra of CS extract show a broad peak at around 3400 cm⁻¹, attributable to the -OH functional group (H-bond stretching). Some other peaks at 1615 cm⁻¹, 1507 cm⁻¹ and 1456 cm⁻¹ were observed which correspond to C=C stretching vibrations in the aromatic ring. The remaining three others at 1256 cm⁻¹, 1114 cm⁻¹ and 1035 cm⁻¹ were also seen, which seem to correspond to C-O stretching vibration (=C-O-C), C-O-C stretching vibration and O-H deformation vibrations or C-O stretching vibrations (=C-O-H). These results agree with the FTIR spectra of brazilin reported by de Oliveira and co-workers [34]. Furthermore, the high similarity between CS extract and AuNPs reduced by CS indicated that the same compounds existed in all samples. As the concentration of CS extract was increased, clear peaks in the spectra were revealed. Despite the similarity between the CS extract and AuNPs spectra, marked shifts at 3400 cm⁻¹ and 1615 cm⁻¹ were also observed, related to the adsorption of CS extract constituents on the AuNPs surface.

Figure 5:

Typical Fourier-transform infrared spectra of Caesalpinia sappan Linn. (CS) extract and gold nanoparticles obtained using the CS extract.

As we discussed previously, the chemical constituents found in CS heartwood are xanthone, coumarin, chalcones, flavones, homoisoflavonoids and especially brazilin. These chemical constituents are phenolic compounds which are composed of mainly -OH functional groups that are responsible for the reduction of AuNPs by donating electrons to the gold ions. For example, brazilin, which possesses 4 hydroxyl groups in the molecular structure, can donate four electrons per molecule in the reduction process. Moreover, the hydroxyl functional groups can act as stabilizing moieties by electrostatic stabilization due to their negative charge, as confirmed by the negative zeta potential in the next section.

3.2 Zeta potential and size distribution

DLS was used to determine the size of AuNPs synthesized by the reduction of gold ions by the CS extract. It was found that AuNPs obtained from 0.004 wt% and 0.04 wt% CS concentrations show three different mean sizes for each condition (20±6 nm, 223±128 nm, 4135±1044 nm, and 41±15 nm, 213±92 nm, 4483±876 nm, respectively), while at 0.01 wt% and 0.02 wt% of CS, there were only two different mean sizes for each condition (16±4 nm, 110±37 nm, and 14±3 nm, 48±8 nm, respectively), as shown in Table 1. The largest mean particle sizes of 4135±1044 nm and 4483±876 nm for 0.004 wt% and 0.04 wt%, respectively, may be attributed to the agglomeration of the earthworm-like AuNPs structure and/or AuNPs clusters. Obviously, it can be seen that when the CS concentration increases, the sizes of AuNPs decrease. The smallest size of AuNPs was obtained from the use of a CS concentration of 0.02 wt%. However, a higher concentration of CS extract may not be advantageous to produce AuNPs in this system, due to the formation of bigger particles. These results correspond to previous work published by Kim and co-workers [40] reporting the concentration effect of reducing agents (caffeic acid) on green synthesis of AuNPs. The sizes of AuNPs decreased when the concentration of caffeic acid increased, due to the adsorption and stabilizing effect of caffeic acid. However, the highest concentration of caffeic acid produced the larger size of AuNPs.

The surface charge property of AuNPs can be characterized in terms of zeta potential. The zeta potential is a crucial indicator showing the stability of colloidal dispersions/solution. The magnitude of the zeta potential is used to indicate the electrostatic interaction (repulsive or attractive) between charged particles. In the case where attractive forces exceed the repulsive forces, the value of the zeta potential is low, and may be close to zero, resulting in the particles becoming unstable and likely to aggregate or collapse. In contrast, if repulsive forces exceed attractive forces, the magnitude of the zeta potential is high, resulting in good dispersibility from stronger electrostatic repulsion. The mean zeta potential values of AuNPs stabilized by CS extract at CS concentrations of 0.004 wt%, 0.01 wt%, 0.02 wt% and 0.04 wt% (Table 1) are −23.1±5.8 mV, −11.5±8.7 mV, −14.9±5.9 mV and −17.7±8.5 mV, respectively. The mean zeta potential values are in the range of −10 mV to −25 mV, indicating a medium stability for the AuNPs in colloidal solution. As we discussed previously, aggregates or sediments can be observed in AuNPs colloidal solution after a period of time. However, the aggregates or sediments can be redispersed by sonication of AuNPs colloidal solution prior to use. Furthermore, the dried powder isolated from AuNPs colloidal solution can be redispersed in water by sonication, thus indicating the advantage of using CS extract as a reducing and stabilizing agent for AuNPs preparation.

3.3 X-ray diffraction

The crystalline nature of AuNPs synthesized by CS extract was further characterized by using X-ray diffraction (XRD) analysis. The XRD patterns of the synthesized AuNPs are shown in Figure 6A–D. Four diffraction peaks were observed at 2θ values of around 38°, 45°, 64°, and 77° corresponding to the (111), (200), (220), and (311) reflections of metallic crystalline gold, respectively (International Centre for Diffraction Data, ICDD No. 4–0783). XRD patterns of the synthesized AuNPs show a strong diffraction peak corresponding to the (111) facet, while the three others were less intense. These results suggest the predominance of (111) plane orientation of AuNPs. However, nearly identical diffraction peaks corresponding to the (111) and (200) facet of AuNPs at the concentration of 0.004 wt% were observed, showing the predominance of (111) and (200) facet of AuNPs under this synthesis condition. However, the diffraction peaks corresponding to the (200) facet show splitting (especially observed in Figure 6D). This suggests structural defects in the AuNPs that were sustained during synthesis [41].

Figure 6:

Powder X-ray diffraction pattern for gold nanoparticles (AuNPs) prepared with Caesalpinia sappan Linn. (CS) extract. The CS concentrations are (A) 0.004 wt% CS, (B) 0.01 wt% CS, (C) 0.02 wt% CS and (D) 0.04 wt% CS.

3.4 TEM morphology

TEM images of AuNPs prepared with the concentration of 0.004 wt% CS extract show agglomeration of the particles with a cluster size of approximately 200 nm. The seed of AuNPs seems to grow continuously to become an earthworm-like structure (Figure 7A and B). When the concentration of CS extract reached 0.01 wt%, the growth of AuNPs was limited. Only quasi-spherical and short length earthworm-like structures can be observed. The small size of AuNPs starting from 5 nm with clusters of more than 20 nm can be measured by TEM (Figure 7C and D). When the CS concentration reached 0.02 wt%, no more earthworm-like structures were observed at this stage; only AuNPs with a completely quasi-spherical shape with a size range between 5 nm and 10 nm (Figure 7E and F) were obtained. This seems to be the best optimized condition to obtain the smallest size of AuNPs prepared with CS. Finally, on addition of CS to 0.04 wt% concentration, some AuNPs seem to start growing again. The combination of both quasi-spherical and earthworm-like structures was observed (Figure 7G and H). Noticeably, the spherical shape was observed predominantly compared with the short earthworm structure. The sizes of NPs/clusters were approximately in the range of 10–200 nm.

Figure 7:

Transmission electron microscopy (TEM) images of gold nanoparticles (AuNPs) prepared with different Caesalpinia sappan Linn. (CS) concentrations: (A) and (B) 0.004 wt% CS, (C) and (D) 0.01 wt% CS, (E) and (F) 0.02 wt% CS, and (G) and (H) 0.04 wt% CS. Earthworm-like structures are observed at 0.004 wt% and 0.04 wt% CS, while the smallest AuNPs with spherical shape were obtained at 0.02 wt% CS.

However, AuNPs size/cluster determined by DLS and TEM are different due to the enveloped state of particles in media which gives a hydrodynamic diameter, while TEM gives particle size in the dry state [42], [43]. The size reduction of AuNPs when CS extract was added to the system can be explained in terms of the strong interaction between the protective polyphenolic molecules and the surface of AuNPs that prevents the growth of AuNPs [44]. Furthermore, the increase in the concentration of CS extract can increase the number of nucleation sites resulting in smaller particles, while the low concentration of CS extract produces less nucleation sites, leading to more reduction and large particles formation [45]. However, the final CS concentration of 0.4 wt% produced both large quasi-spherical and earthworm-like structures again. This suggests that too high a CS concentration is not suitable for synthesizing small size AuNPs in this system.