# Quantification of Unknown Nanoscale Biomolecules Using the Average-Weight-Difference Method
## 3 Results and Discussion  
In a normal condition, the \(\upbeta\)-glucan polysaccharide, which has a triple-stranded structure, can be dissolved in DMSO, and its structure changes to a single-stranded form as the hydrophobic and hydrogen bonds are broken. When it is returned to pH 7, it has the characteristic of returning to its original triple-stranded structure (this process is called the renaturation process) [17]. In this renaturation process, ultrasound treatment can be used to produce \(\upbeta\)-glucan self-assembled nanoparticles of 800 to 900 nm (Figure 1) [18]. To load the drug into these nanoparticles, drugs such as INH and DOX are added to the process of \(\upbeta\)-glucan returning from the single-stranded to the triple-stranded structure. During the renaturation process, the drug can aggregate with \(\upbeta\)-glucan and be loaded into nanoparticles (Figure 1). To confirm the loading of the drug in the \(\upbeta\)-glucan particle, surface characterization was carried out by FTIR analysis. The INH amide bond, which was not found in a neat GluNP at 1625 cm\({}^{-1}\), was found in GluNP/INH, indicating that the drug was successfully loaded in GluNP. A standard curve of absorbance and a simple linear relationship between the various concentrations were established based on the range of wavelengths with the maximum absorbance or fluorescence intensity for the sample to be identified. It can be relatively easily quantified by inversely calculating the concentration versus absorbance or fluorescence intensity for an unknown sample. In this study, the UV/Vis absorbance of INH at different concentrations was also confirmed to use this method. In the case of INH, when the concentration was higher than the specific concentration, broadening of the absorbance peak appeared and measurement was impossible. Therefore, it is difficult to select a suitable absorption band for separating one component (e.g., INH) from the mixture (Glu/INH). The highest absorbance peak was observed at 264 nm for the lowest concentration of Glu/INH (Figure 2A) [19]. On the other hand, GluNP had a broad absorption band of 260 to 300 nm, which was superimposed on the absorption band of INH, and there was a limit to the quantitative analysis of individual components (Figure 2B). Figure 1: Characterization of \(\beta\)-glucan nanoparticles (GluNP). (**A–C**) Scanning electron microscopy (SEM) images of GluNP and drug-loaded GluNP: (**A**) Control; Scale bar = 2 μm, (**B**) isoniazid-loaded \(\beta\)-glucan nanoparticles (GluNP/INH); Scale bar = 10 μm, (**C**) GluNP/doxorubicin (DOX); Scale bar = 3 μm, (**D**) Fourier transform infrared spectroscopy (FTIR) analysis of synthesized GluNP/INH, (**E**) lyophilized GluNP samples (left; control-GluNP, middle; GluNP/INH, right; GluNP/DOX). Figure 2: UV/Vis absorbance of \(\beta\)-glucan nanoparticles (GluNP), isoniazid (INH) and isoniazid-loaded \(\beta\)-glucan nanoparticles (GluNP/INH) dissolved in deionized water. (**A**) Aspects of INH absorbance at different concentrations. For higher concentrations above 0.10 mg/mL, it forms a broad band gradually and has a maximum absorbance of INH at 0.10 mg/mL to 264 nm. (**B**) Comparison of absorbance of GluNP and GluNP/INH. It is difficult to quantify INH since the spectra of GluNP and INH overlap. In this study, the quantitative determination of unknown samples using absorbance or fluorescence analysis could not be employed. It is difficult to quantify the INH by absorbance measurement due to the overlap of their peaks with those of Glu, which is a drug carrier of this study. Also, since INH has no fluorescence peaks, it is not possible to quantify them through fluorescence measurements. Therefore, the quantitative analysis of unknown samples was attempted by applying the weight analysis method. As shown in Figure 3, drug-loaded and empty particles were synthesized under the same conditions. After final centrifugation, the target components were precipitated and lyophilized to weigh the two experimental groups precisely.