Extreme Enhancement of Single-Molecule Fluorescence from

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Extreme Enhancement of Single-Molecule Fluorescence from Porphyrins Induced by Gold Nanodimer Antennas Alexandra P. Francisco, David Botequim, Duarte Miguel de França Teixeira Prazeres, Vanda Vaz Serra, Silvia Marília Brito Costa, César Antonio Tonicha Laia, and Pedro M. R. Paulo J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b00373 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 13, 2019

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The Journal of Physical Chemistry Letters

Extreme Enhancement of Single-Molecule Fluorescence from Porphyrins Induced by Gold Nanodimer Antennas Alexandra P. Francisco, † David Botequim, ‡,§ Duarte M. F. Prazeres,§ Vanda V. Serra, ‡ Sílvia M. B. Costa,‡ César A. T. Laia*,† and Pedro M. R. Paulo*, ‡ ‡

Centro de Química Estrutural, Instituto Superior Técnico, and § iBB-Institute for

Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal †

LAQV@REQUIMTE, Chemistry Department, Faculty of Science and

Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal

AUTHOR INFORMATION Corresponding Author: [email protected], [email protected]

ACS Paragon Plus Environment

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Abstract

Porphyrins are typically weak emitters, which challenges their optical detection by singlemolecule fluorescence microscopy. In this contribution, we explore the enhancement effect of gold nanodimer antennas on the fluorescence of porphyrins in order to enable their single-molecule optical detection. Four meso-substituted free-base porphyrins were evaluated: two cationic, one neutral and one anionic porphyrin. The gold nanodimer antennas are able to enhance the emission from these porphyrins by a factor of 105 to 106 of increase in the maximum detected photon rates. This extreme enhancement is due to the combination of an antenna effect on the excitation rate that is estimated to be above 104-fold and an emission efficiency that corresponds to an increase of 2 to 10 times in the porphyrin’s fluorescence quantum-yield.

1200

R

R

R HN

N

R

800

R=

0

R=

N

NH

1200

TMPyP 1000

Counts/ms

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TTMAPP 1000

N CH3

600

800 R= 600

400 TPP 200

400 TPPS 200

R= 0 1.3 1.4 1.5 1.6 1.7 1

Time (s)

CH3 N CH3 CH3

(Iev - Ibg ) SO310 100 1000

No. events

Fluo. Enh.= (Iev - Ibg) / I0 > 105 |E|2 / |E0 |2

104 103 102

k

E

101 100

KEYWORDS Gold Nanoparticles; Fluorescence; Optical Antennas; Plasmonics; Porphyrin; Single-Molecule.


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The research interest in porphyrins, and related chromophores, is strongly motivated by their role in natural photosynthesis.1,2 Inspired by nature, porphyrins and their derivatives have been extensively used as building blocks for artificial light harvesting systems.3 Some of these approaches seek to mimic the supramolecular organization of chromophores in natural systems using, for instance, dendrimers or lipid vesicles,4-8 while others combine porphyrins with photoactive materials as a route to new or improved functionalities.

9-12

For instance, the

conjugation of porphyrin derivatives with metal nanoparticles has been employed as a strategy to create multifunctional nanocomposites for solar cells,13-15 optical sensors,16-18 biomedical applications,19-24 nanophotonics and molecular electronics.25-28 In the examples above, there is a significant role of plasmon-molecule interactions in the nanocomposite systems through its effects on the porphyrin’s fluorescence or other photochemical processes. Most of the studies reported so far on plasmon-enhanced fluorescence of porphyrins, or related compounds, are from multi-chromophore systems. These include studies on core-shell assemblies composed of metal nanoparticles coated by a layer of porphyrins directly linked to its surface or entrapped in a silica layer.29-34

From a different perspective, plasmon-enhanced

fluorescence has also been studied on light-harvesting complexes from natural systems that instead of porphyrins comprise chlorophyll pigments.35-42 The collective behavior of the multiple chromophores in these systems, such as exciton coupling or energy transfer, bring additional complexity to the evaluation of antenna effects. Therefore, a clear evaluation of plasmon-enhanced fluorescence on individual chromophores would be desirable for a rational design of nanocomposite systems that incorporate porphyrins and it would allow to assess the limits of emission enhancement on a weak emitting molecule.


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In this contribution, we have investigated the enhancement effect on the single-molecule fluorescence from four selected porphyrin molecules using gold nanodimer antennas. This enabled us to characterize the antenna effect directly on porphyrin molecules using an approach that is free from averaging or local concentration artifacts. We have found impressive emission enhancement factors that correspond to an increase by 105 up to 106-fold in the detected photon rates from porphyrin emission in the presence of the gold nanodimer antennas. The nanodimer antennas were prepared from gold spherical particles with a diameter of 80 nm that were assembled into dimers by a DNA-directed approach. We have previously investigated the antenna effect of similar gold nanodimers on Atto-655 dye with remarkable maximum enhancements of almost 4000-fold in emission increase.43 The enhancement factors here reported for weakly emitting porphyrins are even larger, but most importantly the enhancement effect has enabled their single molecule fluorescence detection. The porphyrins investigated in this study are all meso-substituted free-base porphyrins (Fig. 1a). In order to explore different charge interactions with the gold nanodimers, we have selected two cationic porphyrins, TMPyP and TTMAP, one neutral, TPP, and one anionic, TPPS. The absorption spectra show the characteristic four Q-bands (Fig. 1b and Fig. S1 of SI). The subset of two vibronic bands at the lower energy side correspond to the S0-S1 transition, which is a quasiforbidden transition.44 Therefore, the extinction coefficient el of Q-bands and the emission quantum yield ffl0 of porphyrins are typically low. For instance, the porphyrins used in this study have values of el around 103 M-1cm-1 at the excitation wavelength (639 nm) and values of ffl0 of about 10% (Table S1 of SI). Only TMPyP has a slightly lower ffl0 of 5% due to mixing of S1 with a CT-state involving the N-methyl pyridinium substituents and the central porphyrin ring, as postulated in Ref. 45.


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a

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Figure 1. a) Chemical structure of the four porphyrins investigated for plasmon-enhanced fluorescence; b) Absorption and emission spectrum of TMPyP in aqueous solution (blue and magenta, respectively), and photoluminescence spectrum of an individual gold nanodimer immobilized on glass (orange); c) gel electrophoresis used for separation of gold nanoparticles: first and second gel lanes are for gold nanoparticles functionalized with only one type of thiolated ss-DNA oligonucleotides, and the third gel lane is for a 1:1 mixture of gold nanoparticles functionalized with complementary sequences for their assembly through DNA hybridization; d) histogram of gap sizes measured from TEM images of gold nanodimers; e), f), and g) TEM images of gel fractions identified in image c with the same letters showing dimers, trimers and higher


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aggregates of gold nanoparticles obtained by DNA-directed assembly – the bar scale corresponds to 200 nm.

The gold nanodimers were prepared by a DNA-directed assembly approach, which was inspired from the examples of DNA nanotechnology reported for the fabrication of metal nanostructures.4648

Briefly, two samples of gold nanoparticles were coated in separate with complementary,

thiolated ss-DNA oligonucleotides. After washing the excess of thiolated chains, the two samples were mixed together under salt conditions to promote DNA hybridization. This resulted in particle assembly during an overnight reaction time. The mixture of aggregated particles was then purified by gel electrophoresis (Fig. 1c). The samples functionalized with only one type of ss-DNA display a single band in the gel (left and middle gel lanes). Hybridized samples, on the contrary, produce a series of bands corresponding to the separation of particle fractions of single, dimer, trimer and larger aggregates, respectively from bottom to top (right lane with labels “e” to “g”). The composition of bands extracted from the gel in terms of the number of particles assembled was confirmed by performing analysis by transmission electron microscopy (TEM). The photoluminescence spectrum of gold nanodimers is composed of two bands from the hybridized plasmon modes that are optically active (Fig. 1b). The plasmon band appearing at a longer resonance wavelength results from coupling of individual plasmon oscillations along the longitudinal interparticle axis and gives rise to large nearfield enhancements within the particle gap that result in strong antenna effects. This plasmon mode is spectrally overlapped with the porphyrins’ absorption and emission which enables the simultaneous electronic excitation of molecule and plasmon, and the resonant plasmonic enhancement of molecular emission.


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In this work, we present fluorescence enhancement results obtained only when using dimers as optical antennas, even though larger aggregates can produce similar enhancement effects. Due to the role of the interparticle gap as a plasmonic hot-spot, the gap size as measured from TEM images was characterized (Fig. 1d). Most of the dimers analyzed had gap sizes between 2 and 4 nm. Our simulations using the method of discrete dipole approximation indicate that the maximum emission enhancement, theoretically, should occur in this gap range (Tables S3-S6 of the SI). The antenna effect of the gold dimer particles on the porphyrins’ emission was evaluated by confocal fluorescence microscopy (Fig. 2a). Firstly, the dimer particles were immobilized on a glass coverslip at low surface density, so that individual dimers are detected within an area of 20 µm ´ 20 µm. For control purposes, the emission spectrum and time trace of each individual dimer later used for fluorescence enhancement were first measured in water (or DMSO) to check for spurious emission from impurities and for estimating the gap separation (Fig. S2 of the SI). Next, the dimer particles were immersed in a dilute solution of porphyrin (10 nM) and the fluorescence enhancement was measured from selected dimer particles as diffusing porphyrin molecules enter the detection volume and stochastically interact with one dimer particle. These interactions result in strong fluorescence bursts that show up in the intensity time traces of dimer particles only in the presence of porphyrin molecules (Fig. 2d-i). Such strong emission is attributed to the antenna effect of gold dimer particles on the emission of porphyrin fluorescence.


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Figure 2. a) Scheme of fluorescence measurements showing an individual gold nanodimer within the confocal volume and detail of a porphyrin molecule (green square) diffusing through the gap hot-spot; b) Intensity time trace (left) showing an event of extreme fluorescence enhancement for TMPyP porphyrin, and the respective histogram of emission intensity (right) indicating the event’s average intensity and standard deviation (Iev, sev) and the same parameters for background intensity (Ibg, sbg); c) Emission intensity (IN) measured in the absence of gold nanodimers (non-enhanced) for the several porphyrins studied (blue – TMPyP, cyan – TTMAPP, green – TPP, and red – TPPS) at different concentrations (Cporph) plotted against the estimated number of porphyrin molecules (N) occupying the confocal volume (Veff); d) and e) Intensity time traces showing several enhancement events for TMPyP and TTMAPP, respectively; f), g), h) and i) Examples of events of extreme fluorescence enhancement for the several porphyrins studied – the number indicated inside is the enhancement factor calculated for that event.


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The brightness of the porphyrins studied here does not allow for direct single-molecule detection at the experimental conditions of the intensity traces shown in Figure 2. However, the enhancement effect provided by the gold dimer particles makes it possible to clearly detect the emission from single porphyrins. The magnitude of this effect in the emission from cationic porphyrins TMPyP and TTMAPP is so strong that it gives rise to single step “on-off” events with bright emission levels that are several orders of magnitude larger than background signal (Fig. 2g,h). The strongest events most likely result from a porphyrin molecule interacting with the plasmonic hot-spot at the dimer gap, as illustrated in the zoom-in of Figure 2a. The reduced volume of the gap region, that is estimated to be approximately 106-fold smaller than the confocal volume, makes it relatively rare to have one dye molecule in the gap at nanomolar concentrations. Indeed, it takes several seconds to observe strong fluorescence bursts, as seen in the intensity traces of Figure 2d,e, because of the low porphyrin concentration used (10 nM). For porphyrins TPP and TPPS, that are neutral or negatively charged, the frequency of bursts detected is much lower than for TMPyP or TTMAPP, because dimer particles are negatively charged due to their DNA-coated surface. The several events of enhanced emission during the course of a time trace display variable intensities and durations due to the stochastic interaction of porphyrin molecules with dimer particles. This is expected because the porphyrin molecule, as it diffuses close to the dimer surface, probes local plasmon fields that vary so steeply, particularly in the gap hot-spot, that the antenna effect is also changing considerably.43 In order to make quantitative comparisons of fluorescence enhancement between different systems it was used an approach commonly known as “cherrypicking” method.53 It consists of picking the strongest emission event from an intensity trace that samples a time interval long enough in order to assume that this event corresponds to one of the dye molecules probing the region of highest enhancement at the plasmonic hot-spot. Thus, the top


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emission event should give information about the maximum attainable antenna effect, which is characteristic of a particular emitter-antenna system. The top enhancements recorded may then be used to compare results from different systems, or from experiment and simulations, as detailed further ahead. The top enhancement is determined as an enhancement factor between the emitter’s brightness modified by the antenna effect relatively to that of the emitter alone. Experimentally, it is calculated from the intensity of the strongest emission event normalized to the average intensity of the emitter in the absence of the antenna. In Figure 2b, the calculation of the enhancement factor is exemplified for the event histogram on the right side, firstly, by subtracting the average intensity of the bright level (Iev) from the background signal (Ibg) and, then, dividing by the average nonenhanced emission (I0) of the porphyrin to yield the enhancement factor, F/F0 = (Iev – Ibg) / I0. The experimental uncertainties of these intensities (sev, sbg and s0) were used to estimate the error of the calculated enhancement factors (see details in SI). The non-enhanced emission (I0) of each porphyrin was determined from the average intensities (IN) measured in a dilution series under the same conditions as those of the enhancement experiments (Fig. 2c). The intensity values were plotted against the occupation number of the confocal volume or, more precisely, the effective volume (Veff) that was calibrated by an FCS experiment using Atto-655 as a reference dye. Thus, the line slopes from Figure 2c are the detected photon rates per molecule, i.e. I0, for the porphyrins studied here (see also Table S2 of SI). We have also made an independent determination of I0 based on the comparison with Atto-655 dye, which yielded comparable values, thus, validating the approaches employed for I0 determination.


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The top enhancements for the events shown in Figures 2f-i are indicated there by the number inside the figure. The typical values measured range from 105 to 106 more photons detected during these enhanced emission events than those from the emission of the same porphyrin alone. This extraordinary enhancement effect could have been anticipated from the fact that these porphyrins are weak emitters and that plasmonic gap antennas are very efficient for fluorescence enhancement.43,49-52 Nevertheless, the enhancement factors found for porphyrins are among the largest reported so far, and these results are a practical demonstration of a common argument used for investigating plasmon-enhanced fluorescence, which is the possibility to detect singlemolecule fluorescence from weak emitters. Figure 3a shows the top enhancements for the several porphyrins measured from a large collection of dimer particles. It is clear that TMPyP affords the largest top enhancements with values around 106, followed by TTMAPP, and then TPP and TPPS with enhancement values closer to 105. The average value from each data set, which is indicated in Figure 3a by the horizontal lines, also shows the same trend. It should be noticed that the intrinsic quantum-yield of TMPyP (5%) is about half of that from the other porphyrins (> 10%), which allows for a larger margin for fluorescence enhancement and, thus, it can bias the comparison of antenna effects. It was proposed in Ref. 51 that, instead of comparing directly enhancement factors (F/F0), the following figure-of-merit should be employed: FOMenh= F/F0 ´ ffl0. The calculated values of FOMenh are shown in Figure 3b, and compared in this way there is only a marginal difference between the enhancement effect of TMPyP and TTMAPP. The FOMenh values for TPP and TPPS are again lower than for cationic porphyrins, but this could be the consequence from a poor sampling of the plasmonic hot-spot in the case of the neutral and anionic porphyrins.


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a

b

1E+07 107

Fluo. Enh. , F/F0 Fluo. Enh. , F/F0

1E+05 106

FOMenh

1E+06 106

1E+04 105

1E+05 105

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1E+04 104

TMPyP 1 2 TPP 3 4 TPPS TTMAPP

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0 56.9

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TMPyP 1 2 TPP 3 4 TPPS TTMAPP

Figure 3. a) Fluorescence enhancement factors (F/F0) for porphyrin emission modified by gold nanodimer antennas determined from the top event of each time trace sampled from several dimers (symbols) – solid horizontal lines show the average enhancement factor from the respective data set, and open horizontal lines are the top value calculated for fluorescence enhancement from our simulations; b) Figure-of-merit for fluorescence enhancement calculated from the average enhancement factor multiplied by the fluorescence quantum-yield of each porphyrin, i.e. FOMenh= (F/F0) ´ ffl0 – open bars show the maximum estimated value from our simulations; c) Example of an intensity time trace and frequency histogram showing the count of extreme enhancement events (red asterisks) using the median level (Q2) between a minimum threshold for enhanced emission (Ibg + 6sbg) and top emission from the most intense event (Imax); d) Number of extreme enhancement events (NQ2) measured for each porphyrin - the bar length stretches from minimum to maximum number of events from the set of dimers sampled and the white line is the average value.


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Another remarkable result for TMPyP and TTMAPP is that the experimental top enhancements are several times larger than the maximum enhancement factor calculated from our simulations (solid and open horizontal lines in Fig. 3a, and solid and open bars in Fig. 3b). This result is intriguing because enhancement factors predicted from simulations are typically larger than the ones determined experimentally. In the simulations, the dye-particle configuration is fixed to the position that should afford the largest enhancement effects. The fluorescent molecule is approximated to a point-like dipole sitting in the middle of the gap with a colinear orientation regarding the interparticle axis (see SI). In the experiment, the porphyrin molecule is not fixed within the gap and, thus, its translational or rotational motion would average over several dyeparticle configurations that do not correspond to the ideal position for largest enhancement. The opposite trend observed here between experiment and simulation suggests that some additional effect can be at play in the emission enhancement of porphyrins that is not taken into consideration in the theoretical framework of our simulations. In particular, the approximation of the porphyrin to a point-like dipole may be over simplistic, and we briefly speculate that some specific interaction of the porphyrin macrocycle with the local environment in the gap could partially lift the quasiforbidden character of the molecule’s optical transition, thus, explaining the additional enhancement effect. A more detailed calculation that explicitly describes the electronic structure of the porphyrin in the local plasmon field of the gap could offer deeper insights and allow us to check the validity of this hypothesis. Other model adjustments that could improve the comparison with experimental results would consist of: i) including the glass substrate and a surrounding layer to simulate the DNA coating on the particles, and ; ii) simulate some rough features on the particle’s shape instead of a smooth surface.


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The intensity time traces were also analyzed in terms of the frequency of events showing extraordinary enhancements. For this purpose, a minimum threshold for enhanced emission (Ibg + 6sbg) and the maximum intensity of the strongest event (Imax) were established, as shown in Figure 3c. Then, it was defined that extraordinary emission events were those reaching an intensity above the median level (Q2), e.g. as the ones indicated by red asterisks in the example of Figure 3c. The burst events with lower intensities (i.e. count rates below Q2) are attributed to diffusion trajectories that randomly cross the gap at peripheral regions where lower enhancements are expected. The number of events with extraordinary emission enhancement (NQ2) were counted in each time trace and the collected results are shown in Figure 3d. It is evident that time traces for TMPyP show more events of extraordinary emission that those for other porphyrins. This result suggests that TMPyP interacts more often with the gap hot-spot in dimer particles. As previously noticed, TMPyP and TTMAPP are tetra-cationic porphyrins, which could explain a stronger gap interaction with the negatively charged DNA linkers of particles in the dimers. The number of events observed for TPP and TPPS are much lower than for the cationic porphyrins. Also, the enhancement effects in TPP and TPPS are slightly weaker, which lead us to hypothesize that a lower number of events may result from an insufficient probing of the hot-spot during the time trace interval. Nevertheless, TPP and TPPS show remarkable top enhancements of 105, even if their FOMenh are slightly lower than those for cationic porphyrins. The confocal images show that plasmon-enhanced emission from porphyrins is clearly distinguishable from the photoluminescence of individual dimers, but only at porphyrin concentrations in the micromolar range (Fig. 4a). When comparing images of the same area in the absence of TMPyP and immersed in a solution of 10 nM there is barely any difference, because at low concentration the porphyrin molecules rarely interact with dimer particles (left and middle


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images). Upon increasing porphyrin concentration to 1 µM, the same spots become more intense due to plasmon-enhanced emission from the porphyrin (right image). The contribution from transient events of extreme fluorescence enhancement add some flickering to the diffractionlimited spots during the image scan. Plasmon-enhanced emission is also characterized by reduced emission lifetimes due to the acceleration in the radiative and non-radiative decay rates of the emitter. The lifetime reduction is more pronounced for larger enhancement effects. The extreme enhancement effect of porphyrins’ emission is supposed to reduce their emission lifetime to subpicosecond times (Tables S3 to S6 of the SI). The fluorescence decay integrated over the image for TMPyP at 1 µM shows that there is an ultrafast decay component with a similar profile to the instrument time response (box I in Fig. 4b). The long decay component has a decay time of 5 ns that agrees with the known lifetime for non-enhanced TMPyP emission (box II in Fig. 4b). Upon re-plotting the image filtered by a time-gate selecting only the short decay component, it is recovered a similar image to that of enhanced emission (image I in Fig. 4b). On the other hand, applying a time gate for the long decay component, shows that non-enhanced emission is spread throughout the image, as it results from porphyrin molecules diffusing in solution giving a background fluorescence decay (image II in Fig. 4b). Another characteristic of plasmon-enhanced emission from anisotropic particles is that it can be polarized according to one of the particle axes even if excitation light is non-polarized. In the case of the longitudinally hybridized mode of dimer particles, the enhanced emission is polarized along the interparticle axis. This characteristic can be observed when resolving the polarization of enhanced emission because the interparticle axis of the dimer is fixed on the glass surface (Fig. 4c).


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Counts 300

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Time (ns)

Figure 4. a) Confocal microscope images of gold nanodimers immobilized on glass in the absence of porphyrin (left) and immersed in aqueous solutions of TMPyP with 10 nM and 1 µM (middle and right, respectively); b) Fluorescence decays integrated over the images shown in (a) – the orange boxes define two time-gates used to re-plot the image for TMPyP concentration of 1 µM that are labeled as images I and II on the right side; c) Example of an intensity time trace showing the enhanced fluorescence of TMPyP analyzed according to two mutually orthogonal polarizations, labeled as (||) and (^) – the images on the right shows the gold nanodimer from which the enhanced-fluorescence time trace was collected; d) Fluorescence decays of TMPyP and TTMAPP in aqueous solution (non-enhanced emission) and the correlation function calculated from their emission (inset); e) Fluorescence decays of enhanced emission from TMPyP and TTMAPP (blue and cyan, respectively) and photoluminescence decay from the gold nanodimers used for fluorescence enhancement (light and dark orange curves) – the inset shows the respective correlation functions.


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The enabling of single-molecule fluorescence detection from weak emitters using plasmonic nanoantennas is exemplified in Figures 4d,e. The direct measurement of emission from TMPyP or TTMAPP in solution at a nanomolar concentration affords decay curves with characteristic emission lifetimes from these porphyrins, but the intensity correlation curves show only noise (inset of Fig. 4d). On the other hand, the plasmon-modified emission of the same porphyrins by a dimer particle show ultrafast decays due to the extreme decay rate acceleration, but the intensity correlation curves now reveal the dynamics from porphyrin interactions within the gap hot-spot (inset Fig. 4e). In the systems studied, the porphyrins were free in solution and transiently interact with the dimers on the surface. The complex dynamics from these interactions, which probably involve multiple events of binding-unbinding with the DNA linkers on the gap hot-spot, are beyond the scope of this study and have been reported in the literature. 54,55 In conclusion, the possibility of detecting fluorescence emission from porphyrins at singlemolecule level opens up the possibility of using these molecules as fluorescent labels in either biophysical studies or biosensing applications, in order to probe molecular interaction events at low detection limits. The enhancement effect reported here is unprecedented as it shows that an emission increase by factors of 105 to 106-fold may be accomplished for porphyrin molecules. It further explores the boundaries of large fluorescence enhancements that are possible by combining a weak emitter with a powerful and well-matched gold nanodimer antenna.

Supporting Information. Experimental details. Absorption and fluorescence spectra of porphyrins in solution. Photoluminescence spectra of individual gold nanodimers. Determination of the non-enhanced fluorescence intensities of porphyrins. Error estimation of fluorescence enhancement factors. Details on the DDA simulations.


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AUTHOR INFORMATION Notes Any additional relevant notes should be placed here. The authors declare no competing financial interests. Acknowledgements Authors gratefully acknowledge financial support from Fundação para a Ciência e a Tecnologia, FCT through national projects (REEQ/115/QUI/2005, PTDC/CTM-NAN/2700/2012, and PTDC/QEQ-QIN/3007/2014). The authors would like to thank the Associate Laboratory for Green Chemistry- LAQV financed by national funds from FCT/MCTES (UID/QUI/50006/2019). They would also like to thank the Institute for Bioengineering and Biosciences and Centro de Química Estrutural financed by FCT/MCTES (UID/BIO/04565/2019 and UID/QUI/00100/2019, respectively). DB acknowledges a PhD grant from BIOTECnico Program (PD/BD/113630/2015). PMRP acknowledges a Post-Doc grant from FCT (SFRH/BPD/111906/2015).


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Extreme Enhancement of Single-Molecule Fluorescence from

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