## 3 Results and Discussion
### Synthesis and Characterization of HB-AuNPs
#### 3.1.1 Effect of Temperature, HBE Concentration, and Time  
The successful synthesis of the HB-AuNPs was indicated by the appearance of a wine red color, which results in a sharp and intense absorption band in a visible region at 500-600 nm [37; 38]. Likewise, the HB-AuNPs had an absorption peak or SPR in the range of 542 to 564 nm for HBE concentrations starting from 0.5 mg/mL at both 25 \({}^{\circ}\)C (Figure 1A) and 70 \({}^{\circ}\)C (Figure 1B) at 0-2 h (T\({}_{0}\)-T\({}_{2}\)). The SPR of the HB-AuNPs stabilized at 542 nm for HB-AuNPs synthesized with 2 and 4 mg/mL (summarized in Table 1). There were also notable broad peaks and red shift in the SPR of 8 mg/mL_HB-AuNPs, an indication that larger and non-uniform NPs were formed at this concentration. The SPR of NPs is reflective of some factors such as their size, shape, refractive index of dispersive medium, and distance of neighboring NPs [39].  
Figure 1: Synthesis of HB-AuNPs using different concentrations of HBE at 25 \({}^{\circ}\)C (**A**) and 70 \({}^{\circ}\)C (**B**). Optical properties of the HB-AuNPs were measured at 0–2 h, designated as T\({}_{0}\)–T\({}_{2}\).  
The time of reaction is one of the important factors that influences the physico-chemical properties of NPs. The rate of HB-AuNPs synthesized with 1 to 8 mg/mL. HBE at an optimal temperature (70 \({}^{\circ}\)C) at different time points is shown in Figure 2. The synthesis of HB-AuNPs was immediate, starting at time zero (T\({}_{0}\)) for the selected HBE concentrations. The absorption peaks increased with an increase in reaction time (T\({}_{0}\)-T\({}_{2}\)), which was most evident in HB-AuNPs synthesized with 1 and 2 mg/mL of HBE (Figure 2A,B). Synthesis of HB-AuNPs was time and concentration dependent, i.e., synthesis with 1 mg/mL of HBE took a longer time compared with the higher HBE concentrations (2-8 mg/mL). The 2 mg/mL_HB-AuNPs did not show a significant variation in SPR with change in time (Figure 2B). However, the peak for 4 mg/mL_HB-AuNPs became broader at T\({}_{2}\), causing a blue shift in SPR (Figure 2C); the same behavior was also observed with 8 mg/mL_HB-AuNPs at both T\({}_{0}\) and T\({}_{1}\) (Figure 2D). The broadening of the peaks suggested that the size and shape of AuNPs has changed, and this was further confirmed by aggregation of 8 mg/mL_HB-AuNPs at T\({}_{2}\). Thus, reaction time and temperature, in addition to other parameters such as the pH of the solution and the reducing agents influences the physical parameters of the NPs. Shorter reaction times mainly produce smaller and spherical sizes, whereas longer times change the nanospheres to other shapes such as nanoprisms [40].  
\begin{table}
\begin{tabular}{c c c c c} \hline
**AuNPs** & **SPR (nm)** &
\begin{tabular}{c} **Hydrodynamic** \\ **Size (d. nm)** \\ \end{tabular} & **PDI** & **ZP (mV)** \\ \hline
1 mg/mL\_HB-AuNPs & 548 & 98.44 & 0.58 & \(-\)18.1 \\
2 mg/mL\_HB-AuNPs & 542 & 63.41 & 0.31 & \(-\)22.7 \\
4 mg/mL\_HB-AuNPs & 542 & 121.67 & 0.39 & \(-\)18.7 \\ \hline \end{tabular}
\end{table}
Table 1: Optical and DLS analysis of the HB-AuNPs synthesized at various HBE concentrations at 70 \({}^{\circ}\)C.  
Figure 2: Effect of reaction time on the synthesis of HB-AuNPs at 70 \({}^{\circ}\)C using 1 mg/mL (**A**), 2 mg/mL (**B**), 4 mg/mL (**C**), and 8 mg/mL (**D**) of HBE. The numbers (1, 2, 4, and 8) represent the concentration of HBE in mg/mL; T\({}_{0}\)–T\({}_{2}\) represent the reaction time from 0–2 h.