# Quantification of Unknown Nanoscale Biomolecules Using the Average-Weight-Difference Method
###### Abstract  
In order to quantify the amount of drug molecules in drug delivery systems, analytical techniques such as high-performance liquid chromatography are used due to their accuracy and reliability. However, the instruments required for such techniques are expensive and not available in all laboratories. Therefore, in this study, we introduce a method that can be a relatively inexpensive and easy to perform drug analysis in almost any laboratory set-up. We have devised the \"average-weight-difference method\" within the limits of existing spectral analyses. By employing this method, we quantitatively analyzed the amount of isoniazid or doxorubicin molecules loaded onto \(\beta\)-glucan nanoparticles. This proved to be a relatively simple and reliable method and can be used to estimate the amount of nanoscale biomolecules before their analysis through expensive equipment in an environment where the instruments are not readily available.  
quantitative analysis; \(\beta\)-glucan; isoniazid; doxorubicin; UV/Vis spectrometry; fluorescence spectrometry +
Footnote †: journal: Applied Physics Letters  
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mixture depending on the material, and simultaneous quantification is often difficult in this case. Thus, it is necessary to separate and purify only the target component, and it is easy to cause loss and error of the sample in this process [10; 11; 12]. Furthermore, if the analyte is not fluorescent, it cannot be analyzed with a fluorescence spectrophotometer. On the other hand, prior to the development of these analytical methods, researchers have attempted quantification by weight analysis because it is faster and less labor-intensive. Gravimetric analysis can be used to determine the amount of material to be analyzed by sedimenting, drying, and weighing the target analyte [13].  
In this study, drug delivery nanoparticles (GluNP) were prepared from \(\beta\)-glucan polysaccharides reported to have immunostimulatory activity. It was confirmed through SEM and FT-IR analysis that it succeeded in loading non-fluorescent isoniazid (INH), a therapeutic agent for pulmonary tuberculosis. However, as described above, since the absorption bands of UV/Vis absorption spectra between synthesized GluNP/INH \(\beta\)-glucan and isoniazid overlap, quantitative analysis through basic instrumental analysis has been limited.  
As a method for solving these problems, in this study, the degree of drug loading was calculated by averaging the weight difference between the drug-loaded test group and the untreated control group under the same conditions. We have devised the \"average-weight-difference method\", which allows a simple and quick quantitative analysis at a relatively low cost. The target drug was replaced with doxorubicin (DOX), a fluorescent anticancer drug, and the result of the measurement with the average-weight-difference method was compared with that of UV/Vis spectrophotometry and fluorescence spectrophotometry. In the case of DOX, the UV/Vis absorption peak of DOX alone was observed at 481 nm, and compared to isoniazid, it was easier to quantitate DOX by assigning its UV/Vis absorption peak after loading DOX on the \(\beta\)-glucan nanoparticle. This approach may be useful for pre-screening quantitative assays before using expensive equipment for the exact quantification of nanoscale biomolecules in drug delivery applications.