# Quantification of Unknown Nanoscale Biomolecules Using the Average-Weight-Difference Method
## 3 Results and Discussion
The average-weight-difference method was developed to calculate the average difference between the two, through sufficient experimental repetition to determine the amount of the drug that is loaded in the carrier nanoparticles. To quantify the amount of INH successfully loaded in GluNP in this study, the average-weight-difference method was used, as shown in Figure 4. In the experiment, we first weighed a 1.5-mL microtube, and after the synthesis was completed for two experimental groups of drug-free test material (GluNP, Figure 4B) and drug-loaded test material (GluNP/INH, Figure 4A), they were completely dried through lyophilization. For each experiment, GluNP/INH and GluNP samples were synthesized three times. After the weight of the samples was precisely measured, the weight of the compound in the tube (GluNP/INH or GluNP) was determined by subtracting the weight of the tube from the experimental group. The content of unknown drug in the drug delivery carrier was calculated by the following equations (Equations (1)-(3)). The weighing process was repeated to reduce measurement error and ensure reliability (Figure 4). Figure 3: Spectrophotometric method for quantitative analysis of microscopic samples. **(A)** Schematic description of employing the average-weight-difference method for the analyses of drug molecules loaded onto Glu particles. **(B)** Schematic introduction of UV/Vis absorption spectrometry analysis for unknown samples. **(C)** Schematic introduction of fluorescence spectrometry analysis for unknown samples. **(D)** Quantitative analysis of unknown samples according to each method and comparison with average-weight method. Glu/INH total content, _Ai_ (mg)  
= (Initial test tube weight before reaction + Glu/INH sample weight after freeze drying) (1)  
= Initial test tube weight before reaction  
Glu total content, _Bi_ (mg)  
= (Initial test tube weight before reaction + Glu sample weight after freeze drying) (2)  
- Initial test tube weight before reaction  
\[\text{INHaveragecontent}\left(\text{mg}\right)=[\sum_{i=1}^{n}(Ai-Bi)]/\text{n }\left(\text{n}=3\right) \tag{3}\]  
Six repeated experiments were carried out to confirm the experimental results. The amount of INH in the Glu drug delivery system was 0.1731 mg with a deviation of 6.7%. The environmental factors such as humidity, temperature, or static electricity during the weight measurement may lead to the loss of sample, which may lead to errors in calculating the average weight difference between the sample loaded with the drug and the original sample. Therefore, this average weight difference  
Figure 4: Determination of INH amount loaded onto \(\beta\)-glucan nanoparticles by average-weight-difference method. (**A**) Multiple weighing results of Glu/INH. (**B**) Multiple weighing results of empty nanocarriers, \(\beta\)-glucan nanoparticles. (**C**) Confirmation of the average weight values of INH through 6 repeated experiments. (**D**) The mean value of each experimental group (Number of repeated experiments = 6). method can be used to estimate the loaded amount of the drug relatively easily at a low cost, but it is also necessary to consider measures to minimize the preceding environmental factors. We sought to confirm the reliability of the quantitative analysis of the average-weight-difference method to overcome the difficulty of measuring the amount of INH loaded in the GluNP. DOX has relatively high absorbance and fluorescence peaks, so weight determination based on this is relatively easy. Therefore, we compared the difference between the weight obtained from the absorbance or fluorescence measurement and the result obtained by the average-weight-difference method after loading DOX into GluNP. When the DOX that was loaded into GluNP was diluted to a concentration of 250, 125, 62.5, 31.25, or 15.62 \(\upmu\)g/mL and its UV/Vis absorption was measured, peaks were observed in the wavelengths ranging from 231 to 235 nm, 250 to 254 nm, and at approximately 481 nm.