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FRET signal: (A) donor and acceptor individual thin films, (B) donor−acceptor mixture thin film, and (C) pure donor, acceptor, and donor−acceptor mixture fluorescence spectra.
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Adhesion is caused by molecular interactions that only take place if the surfaces are in nanoscale contact (NSC); i.e., the distance between the surfaces is in the range of 0.1-0.4 nm. However, there are several difficulties measuring the NSC between surfaces, mainly because regions that appear to be in full contact at low magnification may show no...
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... exciting them at the same excitation wavelength, the energy transfer can be observed. 40 Figure 2 shows a basic experiment to demonstrate FRET between a pair of one donor and one acceptor dye uniformly distributed in pHema thin films. First, their emission spectra are collected for the individual dyes (a) and then for a mixture of the dyes (b). ...
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... their emission spectra are collected for the individual dyes (a) and then for a mixture of the dyes (b). Energy transfer between the dyes, i.e., a FRET signal, is identified when, in comparison to the spectra of the pure dyes, in the mixture, the intensity of the donor dye is dropping (left downward arrow) and the intensity of the acceptor dye is increasing (right arrow up from I A to I AD ; see Figure 2C). Please note that the energy transfer between the dyes (in this case, FTSC and DCCH) can only take place between donor and acceptor molecules closer than 2R 0 < 10.2 nm, which is obviously the case in the mixture ( Figure 2B). ...
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... transfer between the dyes, i.e., a FRET signal, is identified when, in comparison to the spectra of the pure dyes, in the mixture, the intensity of the donor dye is dropping (left downward arrow) and the intensity of the acceptor dye is increasing (right arrow up from I A to I AD ; see Figure 2C). Please note that the energy transfer between the dyes (in this case, FTSC and DCCH) can only take place between donor and acceptor molecules closer than 2R 0 < 10.2 nm, which is obviously the case in the mixture ( Figure 2B). ...
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... thin films consisted of pHema and were produced by doctor blading. The dyes were mixed into pHema ( Figures 2 and 4) at an equivalent molar concentration of 1.5 mM. The thickness (1.5 ± 0.1 μm) and low roughness (0.04 ± 0.01 μm) of the thin films show a uniform distribution of the dyes, which is of extreme importance for a correct FRET result. ...
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... fluorescence spectra ( Figure 5A) of the pure donor and acceptor thin films ( Figure 2A) show the DCCH emission and FTSC excitation spectra overlapping area, which is necessary for FRET. 31,44 The spectra were measured on thin films bonded in the configuration shown in panels A and B of Figure 4. ...
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... individual thin films of the pure donor, pure acceptor ( Figure 2A), and a donor−acceptor (D−A) mixture ( Figure 2B) are investigated as a positive control. In the D−A thin film both donor and acceptor dye are mixed, leading to short distances between the molecules in the thin-film polymeric matrix. ...
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... individual thin films of the pure donor, pure acceptor ( Figure 2A), and a donor−acceptor (D−A) mixture ( Figure 2B) are investigated as a positive control. In the D−A thin film both donor and acceptor dye are mixed, leading to short distances between the molecules in the thin-film polymeric matrix. ...
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... the D−A mixture thin film represents the maximum value of FRET efficiency that the system can reach, at this dye concentration. Figure 2C exhibits the fluorescence spectra where a strong FRET signal can be observed (drop in the donor intensity and an increase of the acceptor signal; see arrows). The FRET efficiency in acceptor sensitization measured for this system was FRETeff = 30.5%. ...
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... Spectroscopy. Spectra measurements of the individual and bonded thin films (Figures 2 and 4) were recorded using a spectra fluorophotometer RF-5301PC (Shimadzu, Kyoto, Japan), at an excitation wavelength of 440 nm in a 45°/45° configuration, as demonstrated in Figure 2B. ...
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... Spectroscopy. Spectra measurements of the individual and bonded thin films (Figures 2 and 4) were recorded using a spectra fluorophotometer RF-5301PC (Shimadzu, Kyoto, Japan), at an excitation wavelength of 440 nm in a 45°/45° configuration, as demonstrated in Figure 2B. ...
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... efficiency (%) was calculated by the acceptor sensitization method 1 (eq 3). It is the ratio of the acceptor spectral intensity peak value in the presence (I AD ) and absence (I A ) of the donor ( Figure 2C). To achieve appropriate FRET efficiency results, the direct luminescence of I A is subtracted from I AD and multiplied by the correct luminescence ratio of the acceptor and donor molar attenuation coefficients (ε A and ε D ) at the excitation wavelength used for the FRET experiments ( Figure 5B). ...
Citations
... In this way, we built a simple but highly ordered system, with Förster resonance energy transfer (FRET). Simões et al. recently showed the application of a similar idea, with the assembly of two thin films composed of simple organic fluorophores for the quantification of nanoscale contacts [22]. FRET networks have been proposed for broadening the possible utilization of FRET-based biosensors [23]. ...
Controlled junctions of proteins and nanomaterials offer multiple potential applications in the further construction of nanobiodevices. One of the possible junction types is a set of sequential monolayers of various components deposited on a given substrate. The advantage of such an organization is its high sensitivity, resulting from a huge surface covered by molecules or particles. What is more, the molecules/particles adsorbed on a substrate might be easier to handle than the assay in a cuvette. For further application, there should be crosstalk between monolayers; this is defined by the type of individuals forming a complex system. Here, we are studying, using mainly confocal microscopy and FLIM imaging, crosstalk through resonance energy transfer. The sequential monolayers of fluorescent proteins and CdTe quantum dots were deposited on a convenient substrate, a polyvinylidene difluoride membrane. First, we found that the degree of coverage is lower in the second monolayer. Hence, by manipulating the order of deposition, we obtained a system with a varied yield of resonance energy transfer with a donor excess or an acceptor excess. For a deeper understanding of the energy transfer and its limitations in this system influencing the assay pursuit, we utilized Monte Carlo computation. We found that, indeed, the distance between the monolayers, as well as the degree of coverage, is crucial. With the results of the simulation, we might estimate the relative degree of coverage in our sequential monolayers. We also found that in quantum-dots/protein-composed systems, the yield is stronger than predicted by Monte Carlo simulation. Hence, there should be protein reorientation on the nanoparticle surface, leading to such an effect. Finally, we showed that the yield of resonance energy transfer may be modulated by the external application of poly-L-lysines. These chemicals influenced QD fluorescence but not protein fluorescence and might be used, therefore, as a trigger or a switch in nanobiodevices employing those types of sequential monolayers.
... Recently, it has been proposed to utilize FRET as a direct and nondestructive quantitative method to evaluate the degree of NSC [13,20], which can be applied to all kinds of research where adhesion or NSC are playing a role, for instance soft matter, lubricants, adhesives, fiber reinforced materials, fibrous materials, porous materials, or optical sensors for contact between soft interfaces. ...
... The FRET distance detection range relies on the Förster Radius (R 0 ) of the dye system. Thus, between donor and acceptor labelled surfaces in NSC within 0.5 R 0 -2 R 0 , a non-radiative transfer of energy occurs from the donor to the acceptor molecules, and a FRET signal can be identified [20,21]. Fig. 1 shows a schematic FRET spectroscopy experiment of the fluorescence emission spectra, collected for the dye surfaces alone (Fig. 1A) and at NSC (Fig. 1B) using the same excitation wavelength. ...
... Recently we have shown that the NSC measured with FRET can be related to the adhesion force between soft materials [20]. The study demonstrated that FRET is a suitable technique to quantify the degree of NSC on large -i.e., mm to cm-scalemeasurement areas and for the correlation between FRET and adhesion in bonded surfaces, of any nature and/or roughness, on statistically representative size scales [20]. ...
... The FRET distance detection range relies on the Förster Radius (R0) of the dye system. Thus, between donor and acceptor labelled surfaces in NSC within 0.5R0-2R0, a nonradiative transfer of energy occurs between from the donor to the acceptor molecules, and a FRET signal can be identified [12,18]. Figure 1 shows a classic FRET spectroscopy experiment of the fluorescence emission spectra, collected for the dye surfaces alone ( Figure 1A The FRET signal ( Figure 1C) can be noticed when comparing the spectra of the individual dye surfaces with the NSC donor-acceptor surfaces spectrum, donor intensity decreases, and acceptor intensity increases (from IA to IAD). ...
... Only very recently it has been shown that the NSC measured with FRET can be related to the adhesion force between soft materials [18]. The study revealed that for thin films pressed under different loads, the FRETeff and adhesion force increase with the applied pressure, caused by the correspondent increase of the degree of NSC [18]. ...
... Only very recently it has been shown that the NSC measured with FRET can be related to the adhesion force between soft materials [18]. The study revealed that for thin films pressed under different loads, the FRETeff and adhesion force increase with the applied pressure, caused by the correspondent increase of the degree of NSC [18]. Thus, FRET provides a suitable technique to quantify the degree of NSC on large -i.e. ...
Interfacial adhesion is caused by intermolecular forces that only occur between surfaces at nano-scale contact (NSC) i.e., 0.1-0.4nm. To evaluate NSC and its influence on adhesion, F\"orster resonance energy transfer (FRET) spectroscopy has been used. FRET is a technique capable to measure nanometric distances between surfaces by taking advantage of the interaction amid some specific fluorescence molecules, named donor and acceptor. The F\"orster radius (R0) of the FRET pair indicates the distance detection range (0.5R0-2R0) of the system and, must be selected considering the final purpose of each study. Here, we propose a new FRET pair: 7-Amino-4-methyl-cumarin (C120) and 5(6)-Carboxy-2',7'-dichlor-fluorescein (CDCF) with high quantum yield (QY, QYC120=0.91 and QYCDCF=0.64) and a distance range of 0.6-2.2nm (0.1 mM) specifically developed to measure NSC between soft surfaces. For this, polymeric thin films were bonded using different loads, from 1.5 to 150 bar, to create different degrees of NSC, analyzed by FRET spectroscopy, and later pulled apart to measure their interfacial separation energy (adhesion force). Our experiments showed that NSC increases with the applied pressure in the bonded thin films, leading to higher FRET intensity and adhesion force/separation energy. Thus, we have validated a new FRET pair, suitable to measure the degree of NSC between surfaces and establish a linear relationship between FRET and adhesion force; which can be of interest for any type of study with soft materials interfaces that include NSC and its influence on adhesion, as sealants, adhesives or sensors.
