## 3 Results and Discussion
### The QD/Protein Monolayers Are Better with the QD Layer Deposited First
With zero distance, the efficiency is as predicted in 100% coverage. Hence, the most possible explanation for the observed experimental results of QD510-mBanana remains the reorientation and slight conformational changes in proteins. The same effect might be present in other QD-protein pairs but masked by less efficient transfer. Figure 5: Fluorescence lifetimes of QDs immobilized onto PVDF membrane. The excitation was by 405 nm laser, and emission signal was collected at QD emission maxima. Fluorescence lifetime values are amplitude-weighted averages of three exponential decays. Particular values of decays and amplitudes are presented in Supplementary Table S4. Statistical significance of differences (\(p\)-values) is presented in figures as ** (\(p\) < 0.01). Figure 6: FRET efficiencies are calculated for varied relative concentrations of QDs as a first layer (filled data marker) or a second layer (empty data marker). (**A**) An mBanana as an acceptor or (**B**) an mCherry as an acceptor. In a Quest for the FRET Yield Modification in QD-Protein Monolayers--The Effect of Specific Bleaching and Externally Added Poly-L-Lysines  
FRET-based assemblies are broadly designed to be used as sensors with multiple applications in chemistry, biological sciences, and medicine [16, 41, 42]. The idea of FRET networks for sensing was recently proposed as the natural development and broadening of a probe potential [23]. There, QDs are considered a platform, which can bring together various fluorophores. The sequential monolayers we elaborated on might be considered one of the variants of networks, however, simplified to HomoFRET and FRET in pairs. However, due to the monolayer geometry, the energy transfer may not be simply restricted to exactly one possible acceptor. This point is illustrated in the geometry design chapter of the Methods section of this paper. To be able to apply FRET-based elements as nanodevices, one needs to have a way to further modify the energy transfer yield, obtaining switches and logic gates [23]. In a formed sequential monolayer, an established FRET yield is found, depending on the factors described in the previous paragraphs. We, therefore, tested the change in this yield through the application of two external factors: laser light, with the possibility to bleach an acceptor, and a chemical factor, poly-L-lysine (PLL). The bleach might be applied in the study of protein-protein sequential layers. The bleaching may translate to a reduction in the number of available donors, which was already proved as crucial for the efficient tuning of the intensity and fluorescence lifetime of multihybrid nanoparticles [24]. The bleaching was realized in combination with the FLIM experiment. The small square was illuminated by high laser power at the wavelength specific for a given protein absorption. With QDs and their broad absorption spectrum, it is hard to select the proper wavelength for bleaching. Additionally, QDs are relatively resistant to photobleaching. A procedure applied on an FP-FP sequential monolayer resulted in a significant decrease in a bleached protein intensity (compare Figure S3). Although the drop of the acceptor intensity was significant, the analysis of fluorescence lifetime showed that the donor fluorescence lifetime length was only partially restored (Figure S4). As mentioned, the bleaching procedure is not easily applied to samples containing QDs. However, we were able to find another option; in some preliminary testing, we found that PLL was strongly quenching QD emission while not influencing the fluorescence properties of proteins (see Figure S5). In the literature, PLLs are shown as nanoparticle covers; however, only those that are nonfluorescent, like iron oxide nanoparticles [43] or graphene sheets [44]. When PLL was applied to an assembled sequential monolayer, the fluorescence lifetime of an acceptor fluorescent protein was restored to a value close to the value measured for a free fluorescent protein monolayer (Figure 8).