# Fluorescence Masking Based Multifunctional Quantum Dots+
## 3 Results and Discussion
### Testing Protein-Protein Interaction  
#### 3.4.1 Checking the Sensitivity of the Assay  
In Figure 5, it is shown that HSP90\(\alpha\) masked the fluorescence intensity of the Sav-QDs by binding on the biotinylated anti HSP90\(\alpha\) in a dose-dependent manner and presented an EC\({}_{50}\) value of ~43 nM. These data thus indicate that Sav-QDs can be used as a microarray-based sensor for detecting the presence of a specific antigen qualitatively and quantitatively.  
Figure 4: Concentration-dependent masking is fitted in a dose-responsive manner. Maximum fluorescence intensity is counted as 100%, which is 0.05 nM and 5 nM HSP90\(\alpha\) in graphs (**A**,**B**), respectively. The concentration of the Sav-QDs was 100 nM. In graph (**A**), masking of Sav-QDs was achieved as a function of biotin-ATP which was kept constant at 500 nM and varying only HSP90\(\alpha\) concentration with and without radicicol. IC\({}_{50}\) value of the graph with 1 uM of radicicol is 92.06 nM and without radicicol is 27.27 nM. Additionally, in graph(**B**), there is a varying Hsp90\(\alpha\) concentration with ten times the streptavidin concentration with and without radicicol. IC\({}_{50}\) values of graphs with radicicol is 396.7 nM and without radicicol is 52.21 nM. Parameters of the dose-response fitting for graph (**A**), A1 = 0.05, A2 = 5000, \(p\) = 1 log X = 1.964 (1 uM of radicicol), 1.436 (no radicicol) X = 92.06 (1 uM of radicicol), 27.27 (no radicicol). Parameters of the dose–response fitting for graph (**B**), A1 = 5, A2 = 1000, \(p\) = 1 log X = 2.598 (1 uM of radicicol), 1.718 (no radicicol), X = 396.7 (1 uM of radicicol), 52.21 (no radicicol).  
This was done to calculate the sensitivity, LoD, and LoQ of the assay and further apply the principle to detect HSP90\(\alpha\) in complex biological samples such as HeLa cell lysate. Here, 50 nM of Sav-QDs spots were used instead of 100 nM because, in this case, the masking of the fluorescence was not as high as biotin-ATP and Hsp90\(\alpha\). It was assumed that a bigger antibody size was causing steric hindrance, hence not many of them attaching to Sav-QDs as compared to biotin-ATP; in the end, lesser HSP90\(\alpha\) binds. A 50 nM concentration of Sav-QDs has a lower fluorescence than 100 nM, providing a better analysis in a concentration-dependent manner for the biotin antibody and HSP90\(\alpha\). To obtain a linear relationship between the protein concentration and fluorescence masking, a concentration range of 1-100 nM was chosen. As it was seen in the dose-response curve (Figure 3) with biotin-ATP and HSP90\(\alpha\), concentrations above 100 nM and below 1 nM were in the saturation range. From the graph, in Figure 6, K\({}_{\text{SV}}\) is evaluated to be 0.06784 nM\({}^{-1}\) with linear data regression (\(\pi^{2}\) = 0.97). The LoD and LoQ of the assay are 5.18 nM (0.45 \(\mu\)g/mL) and 15.2 nM (15.62 \(\mu\)g/mL). These values are nearby the EC\({}_{50}\) value of the biotin-ATP and HSP90\(\alpha\) binding assay.  
Figure 5: Masking of Sav-QDs as a function of biotin-HSP90 antibody and HSP90\(\alpha\) concentration. Concentration-dependent masking fitted in a dose-responsive manner with an EC\({}_{50}\) value of 43.96 nM. Maximum fluorescence intensity is counted as 100%, which is 0.1 nM of biotin antibody and Hsp90\(\alpha\). Concentration of the Sav-QDs was 50 nM. Parameters of the dose–response fitting, A1 = 00.1 nM, A2 = 500 nM, slope = \(-\)1.467, log X = 1.643, X = 43.96.