## 3 Results and Discussion
### Analyses of Size Distribution and Zeta Potential
showed that capping proteins with molecular weights between 25 and 66 kDa are responsible for the surface capping of AuNPs produced by _Bacillus subtilis_[25]. In our study, FTIR results confirmed the presence of the proteins on the surface of AuNPs. At the natural pH, the acidic amino acids are aspartate (Asp) and glutamate (Glu). The pI values for Asp and Glu are 2.77 and 3.22, respectively [45]. We hypothesized that the capping agents in our experiment had a high content of those two amino acids, which were responsible for the negative charge of AuNPs. At pH 2, these amino acids would have a positive charge and, thus, we expected to obtain positively charged AuNPs. However, we did not confirm that hypothesis. We believe that there are other molecules in addition to the proteins (confirmed by the FTIR technique) that form capping agents on the surface of AuNPs. It was recommended to use more precise techniques, such as X-ray photoelectron spectroscopy (XPS) or nuclear magnetic resonance (NMR), for the detection of these non-protein molecules [46; 47]. In the future, we plan to analyze the surface of AuNPs using those approaches and reveal the composition of the capping agent formed during our AuNP production. In summary, while using a biological method to produce nanoparticles is safe, fast, inexpensive, and environmentally friendly, it does not offer a wide range of nanoparticle charge manipulation (from negative to positive). In our previous research, we conjugated chemotherapeutic drugs directly to the biologically prepared AuNPs by electrostatic interactions [24; 26]. The interaction was based on the effector molecule used. A minor modification to the cargo can make a significant difference in the interaction with AuNPs. For example, dsDNA or dsRNA molecules could not attach to the non-biological AuNPs with the negative surface charge [48]. However, this was not the case for ssDNA [48] and ssRNA [49] molecules. Those molecules possessed negative charge due to the fact of their phosphate groups, preventing an ionic bond to the negatively charged AuNPs. Nevertheless, the conjugation occurred via electrostatic interactions, as those nucleic acids, like the polar molecules, adsorbed to the negatively charged AuNPs via their nitrogen bases [48; 49]. Using polyethyleneimine (PEI) [42] or cetyltrimethylammonium bromide (CTAB) [43] is another way to change the surface charge of the nanoparticles. These molecules have been successfully used to obtain positively charged non-biological AuNPs. Considering that the main advantage of the biologically prepared AuNPs is the avoidance of additional chemicals, we prefer to avoid such compounds if possible. Based on the results of TEM (Figures 4, 5 and 10-12 and Tables 1 and 2) and the average size distribution (hydrodynamic diameter) (Figure 14), the sizes of the nanoparticles measured by TEM were smaller than those obtained by DLS. According to [PERSON] et al., the digital images from TEM differ from DLS [33]. On this basis, the results of these two different techniques are complementary and aligned. In this study, we used three different techniques (i.e., TEM, SEM, and DLS) to determine the dimensions of the AuNPs. According to [PERSON] et al., although all three methods were able to characterize the AuNPs, SEM and DLS were the least suitable for small AuNPs, and TEM was able to measure very small particles [32].