## 3 Results and Discussion
### The QD/Protein Monolayers Are Better with the QD Layer Deposited First
This change was accompanied by a spectral change corresponding to a significant reduction in the resonance energy transfer yield (Figure S6) and complete or partial restoration of the amplitudes and \(\tau\) values found for the control protein monolayer (Table S5). The mechanism of this quenching is most probably related to the interaction of the NH2 groups of PLL with a  
Figure 7: FRET efficiencies are calculated for the varied distance between the first and second layers of a composite relative. (**A**) An mBanana as an acceptor or (**B**) an mCherry as an acceptor. QDs were, respectively, a first layer (filled data marker) or a second layer (empty data marker). Calculations performed form maximum layer occupancies. negatively charged QD surface, resulting in the neutralization of a surface, which might influence the overall behavior of QDs. What is important here, for a QD530-mCherry assembly in solution, which we used to test the effect of PLLs on the nonimmobilized system, PLL also reduced the FRET yield. Therefore, PLL might modify the energy transfer between QDs and proteins by (i) decreasing the fluorescence yield of QDs, (ii) changing a protein-QD orientation, and (iii) changing the distance between the QDs and protein layer. The third possibility might be the effect of PLL intercalation and might result in the releasing of the protein monolayer. The last option was excluded by checking for the fluorescence of the liquid collected from the sample surface after PLL application. Recently, a formation of nanoplexes, combining fluorescent ZnS QD and Fe3O4 nanoparticles in a PLL matrix, was described [45]. The authors showed the decrease in ZnS luminescence intensity in the final combination, however, without elaborating the actual mechanism for this change. Figure 8: The fluorescence lifetime of fluorescent proteins is increased in the presence of a QD monolayer. The fluorescence lifetime of an mBanana (**A**) or an mCherry (**B**) immobilized onto PVDF membrane with or without the presence of the first layer of QDs. The excitation was by 471 nm laser, and emission signal was collected at 612 nm (mCherry) or 551 nm (mBanana). Particular values of decays and amplitudes are presented in Supplementary Table 55. Statistical significance of differences (\(p\)-values) is presented in figures as * (\(p<0.5\)) and ** (\(p<0.01\)). Red * represent the difference of the last group (QD570-based monolayers) versus mCherry only (first bar). As poly-L-lysine might be found in several length variants, we tested if the size of this polymer influences the observed effects on QD fluorescence and resonance energy transfer yield. We compared the effect of the set of available poly-L-lysines, starting from a small peptide, penta-Lys, via 1-5 kDa PLL, 15-30 kDa PLL, and 84 kDa PLL, together with the described-above 150-300 kDa PLL. We found (Figure S8) that the PLL effects are much stronger for their bigger versions and correlate directly with PLL's ability to quench QD fluorescence (Figure S9). Similar results were found for a QD530-mCherry assembly analyzed in solution (see Figure S7). Interestingly, we found that smaller PLL may more efficiently quench QD510 monolayer fluorescence but not QD570 monolayer fluorescence (Figure S9). For PLLs which were not properly stored and resulted in huge aggregates (with a diameter of about 1 \(\upmu\)m, as verified by DLS; not shown), the quenching efficiency was also reduced. None of the tested PLLs can be removed from the applied monolayers by buffer washing, indicating that the binding, although possibly electrostatic, is strong. Therefore, the application of PLL may work only as a one-way off-switch, without the possibility of reverting the effect to \"ON\" and restoring the basic state of a nanobiodevice. One may consider PLL use for time-gating barcoding, similar to fluorescence lifetime tuning, realized by a varied donor/acceptor ratio [24].