## 4 Conclusions  
The materials based on sequential monolayers have a great potential in the further building of nanobiodevices. Their building blocks might be of different origins and properties; also, the substrates for monolayer deposition may vary. Here, we obtained and characterized sequential monolayers of fluorescent proteins and colloidal quantum dots deposited on a PVDF membrane. In this application, there is a possibility of resonance energy transfer between the monolayers. We showed that depending on the order of deposition, we may limit the availability of donors or availability of acceptors, hence modulating the FRET yield. Additionally, we explored the possibility of gating a FRET signal by bleaching with high light power or with externally added poly-L-lysines. The last option came out as the most interesting--we showed that PLLs selectively quench quantum dots while not affecting fluorescent proteins. This results in restoring the fluorescent protein signal to a \"non-FRET\" situation. We found also, that--although the effect is irreversible--the size of the PLL is a factor modulating the observed effects, and various PLLs might be used for tuning the system. For a deeper understanding of the observed changes, we performed a Monte Carlo simulation of the studied systems. With this method, we proved the foregoing observation that relative acceptor concentration is crucial. This point will be explored in further studies, also taking into consideration redox-based interactions. This is important as QDs were proved to be not only resonance energy donors but also electron donors [47; 48]. We also found that the effects of PLL application are not only distance-related but also include the reorientation of proteins within the monolayer. Our data make possible the precise building and prediction of the behavior of sequential monolayers, which is crucial for their further utility. The first step, we believe, should be the implementation of sequential monolayers in microfluidic systems; this would benefit a high active surface on a relatively small device area, resulting in its high functional yield. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app132111917/s1](https://www.mdpi.com/article/10.3390/app132111917/s1)]([https://www.mdpi.com/article/10.3390/app132111917/s1](https://www.mdpi.com/article/10.3390/app132111917/s1)), Table S1: FRET-related characteristics of possible donor-acceptor pairs used in the study. Tables S2-S5: Particular components of decays, shown as averages in Figures 3-5 and 8. Figure 3: FRET efficiencies calculated for the varied relative concentration of donor proteins. Figure S2: Fluorescence lifetimes of QD510 and QD550 immobilized onto PVDF membrane. Figure S3: Representative images obtained during bleaching experiment. Figure S4: Mean lifetime values of donor proteins in bleaching experiment calculated from FLIM images. Figure S5: Emission spectra of QD+mBanana/mCherry samples before and after incubation with 0.1% PLL. Figure S6: PLL effects on a QD530-mCherry FRET pair in solution changes with the PLL size. Figure S7: PLL affects FRET efficiency in QD530-FP monolayers, as evidenced by the change in fluorescence lifetime. Figure S8: Fluorescence lifetime of QDs forming monolayer is shortened by externally applied PLLs. Figure S9: Fluorescence lifetime of FP in QD-FP sequential monolayers is restored by externally applied PLLs. Figure S10: Volume-weighted distribution of hydrodynamic diameters of PLLs obtained by DLS measurements. Conceptualization, [PERSON], [PERSON] and [PERSON]-S.; methodology, [PERSON] and [PERSON]-S.; software, [PERSON] and [PERSON]-S.; investigation, [PERSON], [PERSON] and [PERSON]-S.; writing--original draft preparation, review and editing, [PERSON], [PERSON] and [PERSON]-S.; funding acquisition, [PERSON] and [PERSON]. All authors have read and agreed to the published version of the manuscript. This research was financed by the National Science Centre, Poland, under Sonata Bis grant No. UMO-2016/22/E/NZ1/00673.