## 3 Results and Discussion
### The QD/Protein Monolayers Are Better with the QD Layer Deposited First  
Monolayers can also be formed from nanomaterials, such as gold nanoparticles [37] and colloidal quantum dots (QDs). Here, we tested if CdTe QDs, applied on a substrate in the form of the monolayer, can efficiently transfer energy to a fluorescent protein monolayer. Due to the spectral characteristics of QDs (broad absorption spectra and a lack of selectivity of excitation), they were used as donors only. The ability of resonance energy transfer from QDs to other fluorophores in solution is already well documented [37; 38; 39; 40]. The acceptor, an mBanana or an mCherry, was selected due to spectral overlaps. We also noted that the order of the monolayer deposition influences the FRET efficiency; however, the effect was less pronounced than in the case of FP-FP sequential monolayers. We noted that the transfer was most effective if QDs were the first monolayer. For example, in the preliminary tests, a QD510-mBanana monolayer resulted in a 0.2 FRET yield, while for an mBanana-QD510, the yield was 0.07 only. Even more significant differences were noted for the mCherry-QD550 pair (see results shown in Figure S2). In the actual experiment (Figure 5), we compared four versions of sequential monolayers with the QD layer being the first. We noted that, depending on the composition, the yield varied from 0.2 to 0.65. Similar variations were found when the monolayers were analyzed using FLIM methodology (the yield was between 0.25 for the QD510-mBanana pair and 0.95 for the QD550-mCherry pair; see Figure S2). Together with a donor fluorescence lifetime decrease, the acceptor fluorescence lifetime increases (see Figures S7 and S9). The change is dependent of the change in amplitudes and significant decreases in all QD \(\tau\) components (Table S4). To understand the obtained difference in the FRET efficiency, we again used MC computation to simulate the FRET energy transfer between QDs and FPs. Figure 6 summarizes the results. As expected, the transfer yield increases with the relative concentration of a second layer. With an mBanana second layer (Figure 6A), the yield linearly reaches 0.12 for the pair with a QD510 and 0.25 with a QD530. With an mCherry second layer (Figure 6B), the maximal yield was about 0.25 for a QD510 and close to 0.6 for the other tested QDs. Compared with  
Figure 4: Mean fluorescence lifetimes of fluorescent proteins as measured in the FLIM experiment. Samples on the PVDF membrane were excited with 485 nm, and a time-resolved fluorescence image was recorded through a monochromator set at band range 505–535 nm (GFP, **(A)**) or 545–615 nm (mBanana, **(B)**). Decay curves were extracted as means averaged for each image pixel and fitted to a two-exponential decay equation. Particular values of decays and amplitudes are presented in Supplementary Table S3. Statistical significance of differences (\(p\)-values) is presented in figures as * (\(p<0.5\)) and **(\(p<0.01\))**. the experimental data, one may conclude that monolayers prepared with QDs as the first layer allow very effective resonance energy transfer. In the case of the mBanana-QD510 pair, the calculated yield is even higher than predicted from the computation, which suggests conformation changes in the proteins or the better-than-expected adaptation of proteins and smallest tested QDs. In a computation with proteins as the first layer, for all coverage variants of a second layer, the yield was almost maximum. The computation here is not in line with the experiments, where much lower efficiencies were found. All this led to the conclusion that the binding of a protein on the QD surface might be promoted, most probably by HisTag interaction with the QD surface. Therefore, we addressed the question of the FRET yield change with a variation in the distance between the QD layer and the protein layer. Figure 7A,B show the computation results. As expected, the efficiency strongly decreases with the distance increase.