# Fluorescence Masking Based Multifunctional Quantum Dots+
## 4 Conclusions  
With this technique, we developed a miniaturized and multifunctional assay of Sav-QD for HSP90\(\alpha\) detection. Sav-QDs were spotted on the NC surface to measure the fluorescence masking by HSP90\(\alpha\) interaction with biotinylated ATP or antibodies, which were attached to Sav-QD through a biotin-streptavidin interaction. It is shown that the binding of the molecules to QDs caused a reduction in the fluorescence. A protein-ligand interaction was observed in a dose-dependent manner using biotin-ATP and HSP90\(\alpha\) binding. The inhibitory effect of radiciol was also observed on the binding of ATP and HSP90\(\alpha\); although, a further optimization of the assay is required to remove of the unspecific binding of the proteins and to reveal the complete inhibitory effects of the compounds. In the future, an assay can be further applied to test natural compounds with inhibitory effects for specific protein-ligand interactions. The presence of HSP90\(\alpha\) was detected using a protein-protein interaction. Biotinylated antibodies attached to Sav-QDs interacted with HSP90\(\alpha\) in a dose-dependent manner, revealing LoD and LoQ in the nano molar range, which was also nearby the EC\({}_{50}\) value of a biotinylated ATP-HSP90\(\alpha\) interaction. This was further applied to detect HSP90\(\alpha\) in a complex biological sample such as HeLa cell lysate. An antibody and protein interaction in a purified form and in HeLa cell was confirmed with the fluorescence measurement in the solution and the presence of HSP90\(\alpha\) in HeLa cell lysate was confirmed with Western blot. When the traditional assay system was used in a reversed way, i.e., the dye was spotted on the NC surface, and then protein was added for attachment and masking of the signal, it did not work. The reasons could be the unstable attachment of the dye to NC, which was washed away during blocking and incubation with proteins, and the fluorescence of the dye was not high enough for the masking effect to be detected. Tiny volumes in the range of 150 picolitres for one spot were used in this assay; it can be suggested that with 50 \(\mu\)L of QD solution, a large number of microarrays can be prepared. Another advantage is the flexible application of the system as a protein function assay for drug screening and a protein detection assay for antigen presence detection. With optimization, HSP90\(\alpha\) can be used as a biomarker for monitoring its presence in blood, oropharyngeal, and nasopharyngeal swabs, providing an invasive, non-invasive, and facile method of sample testing. Figure 10: Fluorescence microscopy measurements showed a decrease in fluorescence when a comparison was made between the attachment of biotinylated antibodies to Sav-QDs and the attachment of HSP90\(\alpha\) present in HeLa cell lysate to these biotinylated antibodies. Confirming the overall binding of HSP90\(\alpha\) present in a complex biological sample such as HeLa cell lysate to biotinylated antibodies attached to Sav-QDs. Fluorescence is plotted against samples used for attachment of HSP90\(\alpha\) or biotinylated antibodies to Sav-QDs present in PBS buffer. In an earlier work, it was found that QDs as dyes in assay format were based on their attachment to the target protein from the top, in which either ligands or proteins were spotted on a solid surface. In this work, we have shown a different approach by spotting the quantum dots on a solid surface by a contactless printing technique. This has miniaturized the assay to a great degree as QDs were spotted in a picolitre volume. The non-contact feature protects the nitrocellulose membrane from being destroyed during printing, thus QDs are attached to it strongly. The single format of the assay is multifunctional as we can detect protein-ligand and protein-protein interactions as well as the effects of inhibitory compounds. With these two features, the assay has the potential to develop into a cheap and compact point-of-care sensor for detecting proteins in multiple ways in biological samples.