Sekhar Deep

Biswas

Paraskevi Gaki

Elisabete Cruz

Da Silva

Antoine Combes

Andreas Reisch

Pascal Didier

Andrey S Klymchenko
email: andrey.klymchenko@unistra.fr

Long-range energy transfer between dye-loaded nanoparticles: observation and amplified detection of nucleic acids

Keywords: fluorescent nanomaterials, dye-loaded polymeric nanoparticles, long-range Förster resonance energy transfer, nucleic acid detection, signal amplification

Förster resonance energy transfer (FRET) is essential in optical materials for light-harvesting, photovoltaics and biosensing, but its operating range is fundamentally limited by the Förster radius of ~5 nm. Here, FRET between fluorescent organic nanoparticles (NPs) is studied for the first time in order to break this limit. The donor and acceptor NPs are built from charged hydrophobic polymers loaded with cationic dyes and bulky hydrophobic counterions. Their surface is functionalized with DNA in order to control surface-to-surface distance. It is found that the FRET efficiency does not follow the canonic Förster law, reaching 0.70 and 0.45 values for NP-NP distance of 15 and 20 nm, respectively. This corresponds to the FRET efficiency decay as power four of the surface-to-surface NP-NP distance. Based on this longdistance FRET, a DNA nanoprobe is developed, where a target DNA fragment, encoding cancer marker survivin, brings together donor and acceptor NPs at ~15 nm distance. In this nanoprobe, a single molecular recognition results in unprecedented color switch for >5000 dyes, yielding a simple and fast assay with 18 attomoles limit of detection. Breaking the Förster distance limit for ultrabright NPs opens the route to advanced optical nanomaterials for amplified FRET-based sensing of biomolecules.

Introduction

Electronic energy transfer is a ubiquitous phenomenon in nature involving light-matter interactions. It is a primary step of photosynthesis, a process responsible for life on our planet, converting solar light energy into chemical energy. [1] It also plays a central role in synthetic optical nanomaterials with applications ranging from photovoltaics and artificial photosynthesis, [2] to chemical and biological sensing. [3] These processes are generally described by Förster resonance energy transfer (FRET) formalism, which assumes that the efficiency of energy transfer decays as the power six of the distance. FRET is one of the most popular approaches for development of functional optical materials, in particular fluorescent probes. [3a, 3c, 4] In a typical FRET-based probe, molecular recognition changes the donoracceptor distance, which leads to a two-color response of the probe, suitable for ratiometric detection of the analyte. [3b, 4] The key problem of current molecular probes based on FRET is their insufficient sensitivity within the nanomolar range. This is caused by physically limited brightness of molecular emitters, defined as a product of extinction coefficient and quantum yield, typically below 300,000 M -1 cm -1 . [5] Detection of disease markers, such as proteins and nucleic acids, requires sensitivity at least in the picomolar range, which is largely below the capacity of dye-based FRET sensors. Therefore, current methods of molecular diagnostics rely on enzymatic amplification schemes such as ELISA [6] and PCR. [7] Emerging amplification approaches include enzymatic [8] and enzyme-free [9] molecular amplification schemes, as well as nanotechnology-driven signal amplification approaches, involving plasmonic [10] and light-harvesting [11] nanomaterials.

Fluorescent nanoparticles (NPs) are particularly promising alternative to organic dyes in the design of FRET sensors, because their brightness can be >100-fold higher, leading to greatly improved signal to background ratio and sensitivity. [12] The prominent examples of luminescent NPs as FRET partners in sensing and imaging applications include semiconductor quantum dots (QDs), [3b] dye-loaded silica NPs, [13] conjugated polymer NPs, [14] aggregation-induced emission NPs [15] and dye-loaded polymeric NPs. [16] However, the general problem of fluorescent NPs is their limited FRET efficiency to a single acceptor, because their size is generally larger than the Förster radius. For example, in the case of QDs, multiple acceptors per particle are needed to achieve efficient FRET in biosensors. [17] The second problem is that biosensors for detection of biomolecules frequently need to operate at the distances comparable or longer than the Förster radius, which makes FRET-based approach inefficient. Therefore, significant efforts were done to design nanomaterials that can overcome the Förster distance limit. In particular, advanced materials were developed to achieve light confinement on the nanoscale, such as whispering gallery mode microcavities [18] zero-mode waveguides, [19] hyperbolic metamaterials [20] and plasmonic nanopores, [21] allowing long-range energy transfer, sometimes reaching 100 nm distances. Alternatively, excitation energy transfer (EET) in conjugated polymers [14] and dye-loaded nanoparticles, [16a] allows energy migration within the donor particle that reaches the acceptor over much longer distances than the Förster radius. An attractive approach is to explore energy transfer between donor and acceptor NPs. Previous works showed that the use of gold nanoparticles as energy acceptor with a molecular donor dye resulted in the phenomenon of surface energy transfer (SET), which displayed a decay as a power four of distance, allowing energy transfer well beyond 10 nm. [22] Similar long-range energy transfer was reported for quantum dots as the energy donors and gold NPs as the acceptors. [23] However, gold NPs are not emissive and thus cannot be used in ratiometric assays. Semiconductor QDs were also exploited as FRET acceptors, [24] but their fundamental limitation is associated to their much stronger absorbance at shorter wavelengths, so their FRET systems suffer from strong direct acceptor excitation. To address the problems above, we considered dye-loaded polymeric NPs as both energy donors and acceptors. Our previous works showed that polymer design allowed tuning the size of NPs in the desired range between 10 and 50 nm, [25] while the use of dyes with bulky counterions enabled dye encapsulation with minimized aggregationcaused quenching, yielding bright fluorescent NPs. [26] These NPs are particularly suitable as FRET donors, because at high dye loading, the encapsulated dyes undergo ultrafast excitation energy transfer (EET), which was evidenced by nearly-zero steady-state anisotropy and fluorescence anisotropy decay on the fs time scale. [16a] Ultrafast EET results in highly efficient FRET from thousands of donor dyes to a single acceptor. [16a] This phenomenon of giant light harvesting was successfully applied to the development of nanoprobes for amplified detection of nucleic acids. [11c, 16b] However, to achieve efficient FRET in these biosensors, the acceptor molecules had to be placed in direct contact with the polymer surface (Figure 1), [11c, 16b] which is not possible in many other biosensing schemes. On the other hand, dye-loaded NPs are expected to be efficient energy acceptors, because their large number of dyes generates high molar absorption coefficient, while their much larger size compared to molecular acceptors could generate phenomena similar to SET with gold NPs as acceptors. Moreover, excitation energy migration within acceptor dyes could provide additional boost to the energy transfer, because the acceptor dyes closer to the donor NP could rapidly transfer energy to more distant dyes within the acceptor NP. However, so far, dye-loaded polymeric NPs have not been explored as donor-acceptor pairs. This could open a unique opportunity to generate FRET nanoprobes operating well beyond the Förster radius and featuring high sensitivity to the biomolecular targets.

Here, we designed dye-loaded polymeric NPs as FRET donors and acceptors and studied energy transfer between them using DNA duplexes to control particle-particle distance. We found that the energy transfer between donor and acceptor NPs can reach 0.45 for ~20 nm distance between their surfaces (~50 nm between the particle centers), which is much higher than the Förster radius (~6.35 nm). Analysis of the distance dependence suggested that NP-NP energy transfer efficiency decays with the fourth power of the particle-particle distance. The observed long-distance FRET can be described by a physical model with a singlemolecule donor and a sphere with surface located acceptors or gold nanoparticle. However, in contrast to these systems, our donor is >1000 times brighter (than organic dyes as donors) and its acceptor is emissive (in contrast to non-emissive gold NPs), which is crucial for constructing biosensing materials. Finally, we applied our NP-NP energy transfer system for detection of a nucleic acid fragment, encoding a cancer marker. Despite the long distance between NPs in the sandwich DNA complex (~15 nm), we achieved efficient FRET and subpicomolar sensitivity to the target nucleic acid, using a fast and simple protocol with robust ratiometric detection.

Results and discussion

Our donor NPs are composed of hydrophobic poly(methyl methacrylate) (PMMA)-or poly(ethyl methacrylate) (PEMA)-based polymer loaded with the rhodamine based dye salt R18/F5-TPB (Figure 1). Previously, we showed that bulky hydrophobic counterion (F5-TPB) enables high loading of cationic dye R18 in the polymer NPs with minimal aggregationcaused quenching and ultrafast excitation energy migration. [16a, 26b] The latter favors efficient transfer energy to one acceptor located at the surface (Figure 1). [11c] Here, to achieve longrange FRET, we replaced the acceptor dye molecule with a dye-loaded polymeric NP. For this purpose, we designed a derivative of ATTO647, because, according to our previous work, it was an efficient FRET acceptor dye for R18/F5-TPB-loaded NPs, displaying remarkably high photostability. [11c] We synthesized a hydrophobic dye ATTO647-C18 and paired it with the bulky counterion F5-TPB to form acceptor NPs (Figure 1). Two types of NP-NP complexes were prepared. First one is a donor-acceptor NP-NP complex by direct contact of oppositely charged particles, which implies zero distance between the NP surfaces. Second, donor and acceptor NPs functionalized with DNA were hybridized to control NP-NP surface-to-surface distances ranging from 6.8 to 20.4 nm (Figure 1). show the light-harvesting process toward FRET acceptor. In case of ET between NPs, the distances between NPs were varied from 0 to 20 nm using complementary DNA strands. Chemical structures of the rhodamine derivative R18 with its bulky counterion F5-TPB (in donor NPs) and Atto-647N derivative Atto647-C18 with F5-TPB counterion (in acceptor NPs) are also shown.

In the first approach, the donor NPs were prepared with hydrophobic PMMA-based polymer bearing negatively charged carboxylates (PMMA-MA) and loaded with R18/F5-TPB dye (Figure 2a). The acceptor NPs were prepared from PEMA-based polymer bearing trimethylammonium cationic groups (PEMA-NMe3) and loaded with Atto647-C18/F5-TPB (Figure 2a). Donor NPs of two different sizes were prepared by the nanoprecipitation of PMMA-MA polymer with R18/F5-TPB dye (50 wt% loaded with respect to the polymer, i.e. 33 wt% to the total particle mass) in phosphate buffer (PB) at pH 7.4 and in phosphate buffered saline (PBS), which yielded NPs of 68 and 113 nm hydrodynamic diameter, respectively, according to DLS (Table S1). According to TEM, the measured diameters were 40 and 66 nm, respectively (D50%-NP40 and D50%-NP66, respectively, Figure S1, Table S1). The size increase in PBS can be explained by the higher salt concentration in this buffer, which favors formation of larger NPs, in line with our earlier studies. [25a] As observed previously, the TEM size, giving the core diameter, was significantly smaller than the hydrodynamic size obtained by DLS. [16a] Therefore, here, the TEM data was systematically used for all calculations related to the particle size. The nanoprecipitation of cationic PEMA-NMe3 in MilliQ water yielded NPs of 14 and 16 nm diameter by TEM for 1 and 30 wt% Atto647-C18/F5-TPB dye loading, respectively (A1%-NP15 and A30%-NP15, respectively, Figure S2, Table S1). The absorption and emission spectra of these two types of acceptor NPs showed minimal variations in the absorption and emission spectra, compared to the Atto647-C18/F5-TPB dye in acetonitrile (Figure S3). Their absorption spectrum overlapped well with the emission spectrum of the donor NPs, which is an important condition for obtaining NP-NP FRET (Figure S3). At both 1 and 30 wt% dye loadings PEMA-NMe3 NPs displayed relatively high fluorescence quantum yields: 0.40 and 0.21, respectively. Thus, relatively high dye loading can be achieved for Atto647-C18/F5-TPB in the polymeric NPs with minimal aggregation-caused quenching effects, which makes the obtained NPs good candidates as emissive FRET acceptors. To check whether the encapsulated Atto647-C18/F5-TPB dyes undergo EET, we studied their fluorescence anisotropy, which is known to drop due to dyedye energy hopping. [26b, 27] We found that steady-state anisotropy decreased dramatically with the dye loading from 0.060±0.001 at 0.1 wt% loading to 0.014±0.004 and 0.006±0.007 for 1 and 30 wt% loading, respectively, being much smaller than the fundamental anisotropy of Atto647N (0.40). [28] Nearly zero fluorescence anisotropy in case of 30 wt% dye loading (the observed value was smaller than the standard error) suggested that the excitation energy redistributed rapidly over the particle, similar to that previously observed for R18/F5-TPB in polymeric NPs. [16a, 26b] Then, to obtain NP-NP donor-acceptor complexes (Figure 2a) the cationic and anionic NPs were mixed together in MilliQ water at 1:2 donor:acceptor ratio and incubated for 1 h at rt before the spectroscopic studies. The fluorescence spectra recorded by exciting the donor NPs (at 530 nm) displayed a strong emission band centered at ~668 nm, corresponding to the acceptor NPs, and only minor contribution of the donor emission (centered at ~585 nm), for both small (40 nm, D50%-NP40) and large (66 nm, D50%-NP66) donor NPs (Figure 2b). The control experiments performed with donor NPs (Control 1: D50%-NP40) excited in the same conditions gave a characteristic single-band donor emission at 580 nm, while the acceptor NPs (Control 2: A30%-NP15) displayed only minor fluorescence due to the direct acceptor excitation at 530 nm (Figure 2b). In the third control experiment, we prepared donor and acceptor NPs using the same PMMA-MA polymer (Figure S4). In this case, both donor and acceptor NPs are negatively charged and, therefore, are not expected to interact. Their mixtures did not show significant acceptor emission (Figure S4), which excluded any contribution of the dye exchange between the donor and acceptor NPs. These observations indicate that, in case of donor and acceptor NPs of opposite charges, the donor-acceptor NP-NP complex was formed, resulting in the efficient FRET between them. To characterize FRET efficiency, we used two approaches. First, the classical approach based on variation of the donor intensity (E = 1-(I/I 0 ), where I and I 0 are donor intensities with and without the acceptor). However, in cases when it was difficult to obtain the same concentration of the donor NPs, empirical FRET ratio parameter (so-called proximity ratio) was used, expressed as E = I A /(I A +I D ), where I A and I D are peak intensities of the acceptor and donor, respectively, in the fluorescence spectrum. [29] The titration of the donor NPs with increased concentration of the acceptor NPs showed that high FRET ratio (>0.97) was achieved already with 1-2 acceptors NPs per donor NP (Figure 2c). The smaller donor NPs (40 nm) showed systematically higher FRET ratio values. According to our previous studies with a single acceptor dye, the smaller size of donor NPs improved FRET efficiency because the propagation length for the donor-donor excitation energy transfer from a randomly excited dye to the particle surface is shorter. [11c] Moreover, compared to the previous works, where we used dye molecules as acceptors, for NPs of similar 40-nm size, 23 acceptors at the surface were needed to achieve FRET efficiency ~0.5, [16b] while in the most optimized NPs with ~0.5 FRET efficiency to a single acceptor, the particle size was only 16 nm. [11c] Here, for ~40 nm donor NPs, we achieved ~0.97 FRET ratio to 1-2 acceptor NP, which is a dramatic improvement in the FRET efficiency.

To understand better the nature of this highly efficient FRET, we studied donor-acceptor NP-NP complex at different loadings of donor (1 and 50 wt%) and acceptor (1 and 30 wt%) dyes. Remarkably, a significant loss in the FRET ratio was observed for NPs with lower donor dye loading (Figure 2d). This effect was observed for different ratios of donor:acceptor NPs (varied concentration of acceptor NPs, Figures 2d andS5). According to our previous fluorescence anisotropy data for the donor NPs [16a] , at low dye loading the EET is inefficient, while at high dye loading, ultrafast EET takes place within the encapsulated dyes. Thus, donor-donor EET can explain at least in part the high FRET efficiency. The decrease in the acceptor loading also decreased FRET ratio for both types of donor NPs (Figures 2d andS5), which indicates that the large number of acceptors in the acceptor NPs is also crucial to achieve a high FRET efficiency. Then, to increase the spacing between donor and acceptor NPs, we functionalized them with complementary DNA oligonucleotides (Figure 3a): A20 and T20 oligonucleotides were grafted to donor and acceptor NPs, respectively. Their hybridization is expected to yield a duplex that can ensure surface-to-surface particle spacing of 6.8 nm. The DNAfunctionalization is based on our previously developed methodology, [16b] which provides high density of grafted DNA and thus proper NP-NP spacing without letting NPs to collapse (Figure 3a). Both donor and acceptor NPs were built from PEMA-AspN3 polymer, allowing preparation of NPs functionalized with azide groups. In case of donor NPs, PEMA-AspN3 polymer and R18/F5-TPB (at 50 wt% loading) were nanoprecipitated in PB at pH 7.4 with 30 mM NaCl, yielding NPs of 64 nm dimeter according to DLS and 42 nm according to TEM (Figure 3b,c, Table S1). The increase in the dye loading from 1 to 50 wt% (vs polymer) in these NPs did not produce significant changes in the absorption spectra and they matched well with the excitation spectra (Figure S6). Fluorescence quantum yield decreased with dye loading, but still remained high (0.44) even at 50 wt% vs polymer (Table S2). The average fluorescence lifetime followed the same trend as the QYs with some decease at 50 wt% loading (Figure S8, Table S2), in line with an earlier work on PMMA-based NPs. [16a] Thus, at high loading, the dyes remain highly emissive with spectroscopic properties close to the molecular form. For 50 wt% dye loading with respect to the polymer, 42-nm particle is expected to contain 5600 dyes per particle, making it highly fluorescent. Moreover, at this high dye loading, PEMA-AspN3 NPs exhibited nearly zero fluorescence anisotropy suggesting ultrafast EET within the encapsulated dyes, important for efficient FRET to a single acceptor. [11c] The acceptor NPs were prepared from PEMA-AspN3 with Atto647-C18/F5-TPB dye (30 wt% dye loading) by nanoprecipitation in PB at pH 7.4 without additional salt. This condition yielded smaller acceptor NPs: 26 nm diameter according to TEM (Figure 3b,c, Table S1), which corresponds to ~800 acceptor dyes per NP. Their QY remained good (0.19), similar to that observed before the cationic PEMA-based NPs. Both donor and acceptor PEMA-AspN3 NPs were sufficiently photostable, as continuous irradiation under standard excitation power density of the fluorometer resulted in rather small changes in the emission intensity over 30 min (Figure S8).

The obtained donor (D50%-NP40) and acceptor (A30%-NP25) NPs bearing azide groups at the surface were then coupled with corresponding DNA oligonucleotides bearing reactive DBCO group: A20-DBCO and T20-DBCO, respectively. Mixing A20-functionalized donor NPs with T20-functionalized acceptor NPs is expected to yield the DNA duplex, where the surfaces of NPs are separated by a distance of ~6.8 nm, close to the Förster radius for the couple of parent dyes rhodamine B and Atto647N (6.35 nm [30] ). The mixtures of donor and acceptor NPs with varied concentrations of the acceptor NPs were incubated to achieve hybridization of their complementary DNA strands and the FRET spectra were recorded. Already at a 1:1 ratio of the donor NPs to the acceptor NPs, we observed a nearly complete loss of the donor emission together with appearance of the acceptor emission corresponding to a FRET efficiency of 0.97 (Figure 3d,e). Further increase in the acceptor concentration provided only a minor increase in the FRET efficiency, which reached 0.99. In contrast, a control experiment, where both donor and acceptor NPs were functionalized with A20 oligonucleotide, gave almost negligible FRET signal (Figure 3f). The latter suggests that the contribution of non-specific interactions within A20-functionalized NPs, exchange of encapsulated dyes and direct excitation of the acceptor can be excluded, so that the observed efficient FRET originates from specific hybridization of donor A20-NPs and acceptor T20-NPs. One should note that here we observed comparable FRET efficiency to that for the direct contact between NPs (see above). Given that the distance between surfaces of the donor and acceptor NPs is close to the Förster radius for the Rhodamine-Atto647 pair (6.35 nm), the observed FRET efficiency close to 100% is clearly much higher than the efficiency predicted by the Förster model for a single donor-single acceptor pair. The donor-acceptor NP complexes were also visualized by fluorescence microscopy (Figure S9). The control donor NPs showed mainly emission at the donor channel, while in the presence of acceptor NPs, the donor channel intensity dropped, and the acceptor emission became dominant. As a result, in the merged images, the color of NPs switched from green to red, which confirmed formation of FRET complexes of NPs. Presence of more intense spots in these images may indicate presence of larger aggregates of donor and acceptor NPs. In the view of this unexpectedly high FRET efficiency, we next tuned the surface-to-surface distance between NPs using other hybridization schemes. To increase the distance nearly twice (to 14.9 nm), we used a sandwich hybridization scheme, where 23 nt oligonucleotide on the donor NP and 21 nt oligonucleotide on the acceptor NP were hybridized through a linking sequence of 45 nt complementary to these two oligonucleotides (44 bp length for the two duplexes, Figure 4a). Then, we also designed a pair of complementary sequences of 60 nt, which is expected to further increase the NP-NP surface-to-surface distance, corresponding to the duplex length of 20.4 nm. We incubated the donor and acceptor NPs and then measured the FRET spectra and corresponding FRET efficiency. For both NP-NP distances studied, the increase in the concentration of the acceptor NPs resulted in rapid increase in the FRET efficiency, which reached relatively stable values starting from concentrations corresponding to 1:2 ratio of the donor NPs to acceptor NPs (Figures 4b andS10). When all NP-NP distances were compared for the 1:2 ratio, the FRET efficiency was similarly high for 0 and 6.8 nm (0.96-0.97) and then decreased for longer NP-NP distances, reaching values of 0.70 and 0.45 at 14.9 and 20.4 nm, respectively. We also studied timeresolved emission decays of the donor NPs in the 1:2 donor-acceptor complexes at different NP-NP distances and obtained very similar values of FRET efficiency as those in the steadystate measurements (Figure 4c and S11, S12). (c) FRET efficiencies between these NPs measured using both steady-state (black squares) and time-resolved (green triangles) spectroscopy as a function of their surface-to-surface distance. The magenta points correspond to the FRET efficiencies calculated using the numerical model described in the Materials and Methods section. The blue curve corresponds to the analytical models developed previously for lipidic systems. [31] All errors are s.d.m. (n .

These high values of FRET efficiencies between NPs are clearly above the values expected from the Förster theory for classical single donor-single acceptor pair. Indeed, assuming that FRET takes place from surface to surface of NPs, FRET efficiency for 6.8 nm spacing would be <0.5 according to the Förster law (for the Förster radius of 6.35 nm), whereas in our case, it is ~0.97. Moreover, for 14.9 and 20.4 nm distance, the Förster theory predicts nearly zero FRET efficiency, while we observe values of 0.70 and 0.45, respectively. In addition, if we calculate the distance between the centers of NPs, the NP distances would be 26.0, 40.6, 48.7 and 54.2 nm, which would lead to nearly zero FRET efficiency for all our NP-NP couples, including that with the direct contact without DNA. As a consequence, the decrease in the transfer efficiency in case of NP-NP FRET does not follow a power of six law, as it would be the case for single donor-single acceptor pair (Figure 4c). This deviation from the standard Förster model has already been observed for FRET between a dye and gold NPs, where the energy transfer efficiencies decrease with distance to the fourth power. [22] Remarkably, the distance dependence in our system also scales with the fourth power of the distance (Figure 4c), although the materials used here are completely different from those in the previous report. To account for the energy transfer between a single donor and a single acceptor, Förster formalism uses the point-dipole approximation. However, when the acceptors are homogeneously distributed over a surface, one has to integrate the FRET rate between each donor-acceptor pairs over 2 dimensions giving rise to a power four dependence with respect to the distance, as previously demonstrated. [32] In particular, the fourth power distance dependence was described with an analytical model proposed earlier to account for the energy transfer between donors and acceptors embedded in lipid bilayers. [31] A numerical model based on this approach was adapted to our system. In our approach, the FRET efficiency was calculated as a function of the distance between a single donor and a halfsphere with homogenously distributed acceptors at the surface (Figure S13). In this case, the donor can transfer its excitation energy to each individual acceptor with a rate given by the Förster theory (k FRET r -6 , Figure S13). The steady-state fluorescence intensities of the donor and acceptors are determined by solving a set of coupled differential equations (see Materials and Methods). We found that the increase in the number of acceptors per surface of 20 nm half-sphere increased the deviation from the Förster law towards longer distances (Figure S14). However, to match the experimental observations the number of acceptors homogeneously distributed over the surface of the half-sphere must be ~10,000 (Figure 4c and S13), far above the real number of acceptor dyes loaded within our acceptor NPs. Indeed, the 26-nm acceptor NP encapsulates ~800 dyes (i.e. only ~400 dyes in a half-sphere) and they are distributed all over the volume of the particle, so that the effective number of acceptors at its surface is even lower. Thus, even though some previous reports and the numerical model could match the current experimental observations, our NP-NP FRET system presents unprecedented features. First, the donor is not a point emitter, but a nanoparticle containing thousands of dyes within a radius far beyond the Förster distance. Second, our acceptor NP requires much less dyes than the theoretical prediction and they can be distributed all over the particle volume. This unique behavior could be explained by ultrafast excitation energy transfer within both donor and acceptor NPs. Indeed, our previous works showed that due to ultrafast EET within the encapsulated dyes, [16a] the excitation energy propagates within the donor NPs beyond the Förster radius and ensures efficient FRET from hundreds encapsulated dyes to a single acceptor attached to the NP surface. [11c] Therefore, the donor NP can be considered as a single emitter that can transfer energy from the NP surface (Figure 1). On the other hand, in the acceptor NP, the dyes located closer to the surface are coupled by fast EET with the all-other encapsulated dyes, as evidenced by the anisotropy data (see above). Therefore, after initial transfer to the NPs surface, the excitation energy can rapidly redistribute over other parts of NPs (Figure 1), which should boost the performance of the acceptor NPs. Thus, the numerical model with multiple acceptors combined with the ultrafast EET within both donor and acceptor NPs could explain why in our system the energy transfer This article is protected by copyright. All rights reserved. 13

is essentially surface-to-surface processes characterized by high efficiency at long NP-NP distances, which, to the best of our knowledge, has not been reported yet for particle-particle FRET.

The observed phenomenon of long-distance NP-NP FRET is of outmost importance for biosensing applications. Here, based on it, we developed an assay for nucleic acid sensing. We took our DNA sandwich system, where the energy transfer between NPs is induced in the presence of the 45 nt linking oligonucleotide (Figure 4a). The latter is our "target" oligonucleotide, which corresponds to a fragment of mRNA of survivin, a key anti-apoptotic protein overexpressed in cancer cells, serving as important cancer biomarker. [33] In this assay, the mixture of the donor and acceptor NPs without the target oligonucleotide is not expected to undergo FRET, while the presence of the target should induce formation of the donoracceptor pair (with 14.9 nm spacing) that undergoes FRET (Figure 5a). However, using Atto647-C18 as acceptor led to a small contribution of the direct excitation of the acceptor present in excess, so that even without target DNA, some background signal from the acceptor emission was observed (Figure S15). Therefore, we changed the acceptor to Cy5based dye DiD with bulky counterion F5-TPB, which was shown to exhibit efficient encapsulation and bright emission inside polymeric NPs. [34] The acceptor NPs were formulated exactly as for the Atto647-C18 NPs, with 30 wt% dye loading (vs polymer), yielding NPs of 17 nm size by TEM (Figure S16, Table S1), which were further functionalized with corresponding oligonucleotides. In this case, at the donor-acceptor NP-NP ratio 1:2, no signs of the acceptor emission were observed (Figure 5b), which implies negligible direct excitation of acceptor and minimal non-specific interaction between the donor and acceptor DNA-functionalized NPs. The addition of increasing concentrations of the target DNA led to a gradual increase in the relative intensity of the acceptor emission centered at ~670 nm. Thus, as expected, the target DNA triggers the formation of donoracceptor NP-NP pair and turns on the acceptor emission. The FRET ratio showed gradual growth with increase in the target DNA concentration in total RNA extract from cells (Figure 5c), which suggests that this approach allows detection of specific oligonucleotides. Estimation of a limit of detection (LOD), based on three standard deviations without the target, gave a value of 0.36 pM. Considering that the detection volume was 50 µL, the LOD expressed in absolute amount of the oligonucleotide was 18 attomol. The obtained LOD values are significantly lower compared to those we previously observed for our FRET system with a single dye acceptor (1-5 pM). [11c, 35] Moreover, in this case, the detection process is much faster: 1h instead of 6h incubation.

Multiple reasons can explain the much better performance of the present FRET system. First, this is a turn-on system where the target induces the appearance of FRET signal, in contrast to that in the previous DNA strand-displacement assays, which are based on the loss of FRET. [11c, 16b] Second, all capture nucleic acids attached to the surface of NPs are complementary to the target, whereas in the previous assays the capture oligonucleotides corresponded to a small fraction mixed with the excess of non-coding (A20) oligonucleotides. [11c, 16b] This implies much higher effective concentration of the capture This article is protected by copyright. All rights reserved. 14

DNA and, therefore, faster kinetics of hybridization (i.e. less incubation time) for the new assay. It is clear that this NP-NP FRET detection assay can be further optimized for improving its sensitivity and it can be further applied for specific biomedical applications, which will be a subject of our future studies. Here, we provide the first proof-of-concept of exploiting long-range energy transfer between NPs for the biomolecular sensing. It is important to stress that in this case the single molecular recognition event (i.e. hybridization of the target sequence) induces formation of a donor-acceptor NP-NP pair that switches the emission of >5000 dyes inside donor particle to the emission of the acceptor particle with comparable intensity (Figure 5), owing to efficient energy transfer over 15-nm surface-tosurface distance. Previous reports of efficient energy transfer from a large ensembles of emitters to a single acceptor, for instance using dye-loaded polymeric NPs [11c, 16a] and upconversion NPs, [36] were limited to short distances with acceptors placed directly on the particle surface. To the best of our knowledge, this is the first report showing that one molecular recognition event can trigger efficient energy transfer from so large dye ensemble to an acceptor beyond Forster distance, which enables unpresented signal amplification in the fluorescence sensing.

Conclusions

Excitation energy transfer is essential in the development of optical materials for lightharvesting, photovoltaic and biosensing applications. In particular, long-range energy transfer in nanomaterials is a route to biosensors with high brightness and sensitivity to the target, important for molecular diagnostics applications. The present work is the first study of energy transfer between two fluorescent organic nanoparticles, which demonstrates that the excitation energy can propagate far beyond the Förster radius. We designed polymeric NPs loaded with donor or acceptor dyes and studied energy transfer between them at different distances controlled by DNA duplexes. We found that the FRET efficiency does not follow the canonic Förster law as a power six of distance, reaching 0.70 and 0.45 for 15 and 20 nm surface-to-surface distances between NPs. FRET efficiency in our NP-NP systems decays as a power four of the distance, which was previously reported for a single dye emitter as the donor and a gold NP as the acceptor. [22] Based on the previous works on the lipid bilayers, [31] we developed a numerical model for FRET from a single dye emitter to a half-sphere with surface-located acceptors. However, it suggested that the power four distance dependence can be only achieved when the number of acceptors on the surface of NPs is much higher than we actually use in our acceptor NPs (10,000 dyes vs 400 actually used for a half-sphere). Thus, our NP-NP FRET system presents a number of unprecedented features. First, its donor contains >5000 dyes and, thus, it is much brighter than a single dye emitter, owing to strong light-harvesting capacity of this dye ensemble and high fluorescence quantum yield. Second, its acceptor is an emissive nanoparticle (unlike gold NP) and it requires much less dyes distributed all over NP, unlike the numerical model above. These unique features can be explained by ultrafast excitation energy migration (EET) within dyes in both donor and acceptor NPs, which ensures that the energy transfer between NPs takes place from the surface of the donor NP to the surface of the acceptor NP. Indeed, EET brings the excitation energy to the surface of the donor NP and then, after energy transfer to the surface of the acceptor NP, the EET can further spread it to the acceptor dyes all over the particle. On the other hand, in comparison to existing advanced materials that allow light confinement at the nanoscale, [18][19][20][21] our long-distance FRET nanomaterials feature simplicity and flexibility. Indeed, our system is essentially based on functionalized donor and acceptor polymeric NPs and their FRET response can be measured both in solution and on surfaces using a variety of fluorescence techniques.

As a proof-of-concept, we applied this NP-NP FRET system for the detection of a DNA fragment encoding cancer marker survivin. The donor and acceptor NPs were functionalized with sequences complementary to the two parts of this fragment, so that they could get together in a sandwich-like complex at a distance of ~15 nm. We found that the DNAfunctionalized donor and acceptor NPs did not interact by themselves, whereas presence of the 45 nt DNA target triggered a ratiometric response, observed as an increase in the FRET signal vs the donor signal. Titration experiments with the DNA target revealed a limit of detection of 0.36 pM, which corresponded to 18 attomoles in the absolute amount. In comparison to the previous FRET probes based on these NPs as donors and single dye acceptors, [11c] much higher sensitivity was achieved for significantly shorter incubation times.

Owing to the high FRET efficiency beyond the Förster radius in this NP-NP system, a single molecular recognition of the target leads to response of >5000 dyes with color switch from donor to acceptor emission. This is an outstanding amplification that has not been achieved for any kind of optical biosensing material to date. Moreover, the color switching will enable robust ratiometric detection of the target using relatively simple detection setups, including portable fluorometers and plate readers. On the other hand, the remaining challenges of this long-distance FRET system, based exclusively on organic NPs, are potential aggregation of NPs, their photostability and multiplexing capabilities. Finally, given such long energy transfer distances, our NP-NP FRET system could be adapted to other detection schemes including those using antibodies. This work is currently in progress and will be reported in due course.

Materials and Methods

Materials

PMMA-MA (1.6% methacrylic acid, M n' 15,000; M w' 34,000), acetonitrile (anhydrous, spectroscopic grade, 99.8%), rhodamine B octadecyl ester perchlorate (>98.0%), potassium tetrakis(pentafluorophenyl)borate, HEPES sodium salt, potassium chloride (KCl), magnesium chloride (MgCl 2 ), potassium carbonate and octadecyl iodide were purchased from Sigma-Aldrich. 1,1'-dioctadecyl-3,3,3',3'-tetramethyllinododicarbocyanine perchlorate (DiD) and LabTek chambers (Borosilicate cover glass, eight wells) were purchased from ThermoFisher Scientific and Atto647N carboxylic acid were purchased from ATTO-TEC. Sodium phosphate dibasic dihydrade (>99.0%) and sodium phosphate monobasic (>99.0%), both were purchased from Sigma-Aldrich and used to prepare 20 mM phosphate buffers at pH 7.4 and pH 9.2. Dulbecco's phosphate buffered saline were purchased from Dominique Dutscher. Amicon Centrifugal filters (0.5mL, 100kD) were purchased from Sigma-Aldrich. For saline buffer, sodium chloride (≥99%, Sigma Aldrich) 30 mM was added to 20 mM phosphate buffer and pH was adjusted to pH 7.4 with sodium hydroxide 1M solution. Milli-Q water (Millipore) was used in all the experiments. Protein LoBind Eppendorfs were purchased from Eppendorf.

Nucleic acids sequences

Lyophilized single strand DNA sequences were purchased from Biomers GmbH, dissolved in Milli-Q water, aliquoted and stored at -20 °C for further experiments. The oligonucleotide sequences used in this study are shown below.

DNA: A20-DBCO, 5'-AAA AAA AAA AAA AAA AAA AA -(DBCO)-3'. This article is protected by copyright. All rights reserved.

The NPs were prepared by nanoprecipitation method describe previously. [25b, 26b] For preparation of PMMA-MA NPs (anionic NPs), the stock solutions of PMMA-MA in acetonitrile (2 mg/mL) were prepared containing different amount of R18/F5-TPB (1 and 50 wt% relative to the polymer weight; i.e. 50 wt% corresponds to 1:2 dye to polymer ratio). Then, 50 µL of these stock solutions were diluted quickly using micropipette to 500 µL of 20 mM phosphate buffer (PB) at pH 7.4, r.t under vigorous shaking (Thermomixer in 1,150 rpm). Next, these NP suspensions were again quickly diluted to 1 mL with the same PB (20 mM, pH 7.4) at r.t. For the larger-sized NPs, Dulbecco's Phosphate Buffered Saline (PBS) was used only at the first dilution step. For the smaller-sized NPs, phosphate buffer at pH 9.2 was used only at the first dilution step.

For the preparation of cationic NPs, different amounts of Atto647-C18/F5-TPB in acetonitrile (1 and 30 wt% relative to the polymer weight) were added to the polymer (PEMA-NMe 3 1%) solution (2 mg/mL) in acetonitrile. Then, 50 µL of these stock solutions were diluted quickly using micropipette to 500 µL of Milli-Q water, r.t under vigorous shaking (Thermomixer in 1,150 rpm). Next, these NP suspensions were again quickly diluted to 1 mL with the same Milli-Q water at r.t.

PEMA-AspN 3 NPs were obtained by nanoprecipitation with 20 mM PB at pH 7.4 with 30 mM of NaCl: 50 μL of the polymer solution in acetonitrile (2 mg/mL with a required wt% of R18/F5-TPB relative to the polymer i.e. from 0.1, 1, 5 and 50 wt% for R18/F5-TPB and 30 wt% of Atto647-C18/F5-TPB or DiD/F5-TPB) were then added quickly using a micropipette to 450 μL of PB (with 30mM NaCl for R18/F5-TPB NPs and without NaCl for Atto647-C18/F5-TPB or DiD/F5-TPB NPs) under shaking with Thermomixer (1,150 rpm). Next, these NP suspensions were again quickly diluted to 1 mL with the same phosphate buffer respectively, at r.t. Then, the residues of acetonitrile were evaporated under reduced pressure.

Synthesis of DNA-modified NPs

Aliquots of A20-DBCO, T20-DBCO, Sur1-DBCO, Sur2-DBCO, 60nt1-DBCO and 60nt2-DBCO (20 μM) were added to 100 μL of corresponding nanoparticles. Then, they were mixed carefully, microcentrifuged and kept overnight (18 h) at 40 °C in incubator chamber without shaking protected from light. After the mixtures were cooled down to r.t. Then, these reaction mixtures were purified by centrifugation using centrifuge filters (Amicon, 0.5 mL, 100 kDa) on 1000 rcf at 20 °C for 5 min. The procedure of centrifugation was repeated 4 times to remove the non-reacted oligonucleotides. The obtained nanoprobes in a volume of 0.5 mL were kept in the dark at 4 °C.

Characterization of NPs

The hydrodynamic diameters of the NPs were measured using dynamic light scattering (DLS) on a Zetasizer Nano ZSP (Malvern Instruments). The mean values were obtained from three sets of results based on volume distribution analysis. Transmission electron microscopy (TEM) was performed to further characterize the NPs. Carbon-coated copper-rhodium electron microscopy grids with a 300 mesh (Euromedex, France) were surface treated with a glow discharge in amylamine atmosphere (0.5 mbar, 4 -5 mA, 30 s) in an Elmo glow discharge system (Cordouan Technologies, France). Then, 5 μL of the solution of NPs at 0.04 g/L were deposited on the grids and left for 5 min. The grids were then treated for 1 min with a 2% uranyl acetate solution for staining. After that, the experiments were performed on a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100 kV. Images were analyzed using Fiji software.

Absorption spectra were recorded on a Cary 5000 scan UV-vis spectrophotometer (Varian); and emission spectra and fluorescence anisotropy were recorded on a FS5 spectrofluorometer (Edinburg Instruments) equipped with thermostated compartment. The fluorescence spectra the NPs were recorded at the excitation wavelength of 530 nm (for R18/F5-TPB NPs) and 620 nm (DiD/F5-TPB and Atto647-C18/F5-TPB NPs). The QYs of the NPs were measured using rhodamine 101 (QY=1.00) [38] (for R18/F5-TPB NPs) and DiD in methanol as a reference (QY=0.33) (for DiD/F5-TPB and Atto647-C18/F5-TPB NPs). [39] For photostability experiments, 50 µL of the donor or acceptor NPs (50 pM concentration of NPs) were placed in 3×3 mm black quartz cuvette and kept under continuous light illumination 530 or 640 nm, respectively, with power density 0.2 mW/cm 2 . Their fluorescence was measured at 580 or 660 nm, respectively.

Estimation of the concentration of DNA-modified NPs

We calculated the volume of the NPs using the diameter measured by TEM. Then, we estimated how many dye molecules are there per nanoparticle. By the loading of the dye into the NPs (30 wt% or 50 wt%), we estimated the number of moles of the dye present in the unit volume of a particle. These values are multiplied with Avogadro's number to estimate the number of dye molecules per particle (Table S1). Then, based on the absorption spectrum of NPs and known molar extinction coefficient of dyes (125,000 M -1 cm -1 for R18, 150,000 M -1 cm -1 for Atto647-C18, and 250,000 M -1 cm -1 for DiD), we estimated the concentration of NPs in solution as

This concentration of NPs was used for all the particle-particle FRET experiments.

Particle-particle complexes for FRET studies

For direct contact FRET experiment, the aliquots of Atto647-C18/F5-TPB PMMA-NMe 3 (A1%-NP15 or A30%-NP15) NPs at indicated concentration were added to R18/F5-TPB This article is protected by copyright. All rights reserved. 20

PMMA-MA (D1%-NP40, D50%-NP40 or D50%-NP66) NPs (10 pM) in PBS to the final volume of 50 μL in LoBind Eppendorfs. Then the mixture was kept for 1h in the Thermomixer (300 rpm) protected from light at r.t. Finally, steady state spectra were recorded after diluting the mixtures to 500 μL.

For FRET between DNA-modified NPs, the aliquots of Atto647-C18/F5-TPB PEMA-AspN 3 (A30%-NP25-N3) NPs, bearing T20 DNA or 60nt2 DNA, at indicated concentration were added to R18/F5-TPB PEMA-AspN 3 (D50%-NP40-N3) NPs (8 pM), bearing, respectively, A20 DNA or 60nt1 DNA, in HEPES (Mis-B, see below) buffer to the final volume of 50 μL in LoBind Eppendorfs. Then, the mixture was kept for 65 min at 50 °C protected from light.

After the mixtures were cooled down to r.t. Finally, steady state spectra were recorded after diluting the mixtures to 500 μL. Mis-B buffer contained 12 mM of HEPES buffer along with 120 mM of KCl and 0.4 mM of MgCl 2 at pH 7.4.

For FRET between DNA-modified NPs (with 44 b.p.), the aliquots of Atto647-C18/F5-TPB PEMA-AspN 3 (A30%-NP25-N3) NPs, bearing Sur2 DNA, were added to R18/F5-TPB PEMA-AspN 3 (D50%-NP40-N3) NPs (8 pM), bearing Sur1 DNA, and 500 pM of 45 nt (target) DNA sequence (Sur-target) in HEPES (Mis-B) buffer to the final volume of 50 μL in LoBind Eppendorfs. Then, the mixture was kept for 65 min at 50 °C protected from light. After the mixtures were cooled down to r.t. Finally, steady state spectra were recorded after diluting the mixtures to 500 μL.

Estimation of limit of detection (LOD)

For LOD calculation, 8 pM of donor NPs D50%-NP40-N3, bearing Sur1 DNA were mixed with 16 pM of acceptor NPs A30%-NP17-N3 (prepared with DiD/F5-TPB dye), bearing Sur2 DNA, in 50 μL of HEPES (Mis-B) buffer containing 0.01 g/L RNA extract (HeLa) in LoBind Eppendorfs. After different concentrations of 45 nt DNA target (Sur-target) were added to the mixture. Then, the mixture was kept for 1h at 50 °C protected from light. After the mixtures were cooled down to r.t. Then, the steady state spectra were recorded after diluting these mixtures to 100 μL, using a micro-cuvette. These experiments were performed thrice to construct the calibration curve (Figure 5). The intermediate dilution of nucleic acid target was done in Milli-Q water containing 0.01mg/mL Tween 80. RNA extraction from HeLa cells was performed as previously described.

Fluorescence lifetime measurements

Time-resolved fluorescence measurements were performed using time-correlated singlephoton counting (TCSPC) technique. Excitation pulses at 532 nm were generated by a supercontinuum laser (NKT Photonics SuperK Extreme) with 10 MHz repetition rate. The fluorescence decays were collected at 585 nm, using a polarizer set at magic angle and a 16 mm band-pass monochromator (Jobin Yvon). The single-photon events were detected with a micro-channel plate photomultiplier R3809U Hamamatsu, coupled to a pulse pre-amplifier HFAC (Becker-Hickl GmbH) and recorded on a time-correlated single photon counting board SPC-130 (Becker-Hickl GmbH). The instrumental response function (IRF) recorded with a polished aluminum reflector showed a full-width at half-maximum of 70 ps for 532 nm excitation.

Time-resolved exponential decays were fitted by using the global fit procedure of Igor Pro (Wavemetrics). The fitting function was a sum of exponential decays (up to 4 components) convolved with a normalized Gaussian curve of standard deviation σ standing for the temporal IRF and a Heavy side function. All emission decays were fitted using a weighting that corresponds to the standard deviation of the photon number squared root.

Calculation of FRET Efficiency and FRET ratio

FRET efficiencies were calculated using the following equation

Where E is FRET efficiency, is fluorescence intensity donor in presence of acceptor and is fluorescence intensity donor without acceptor; is average lifetime of donor in presence of acceptor and is average lifetime of donor without acceptor.

FRET ratio was calculated using the following equation

Where A is the fluorescence intensity of acceptor and D is the fluorescence intensity of donor of same NPs.

Distance dependent FRET efficiency data fitting

Distance dependent FRET efficiency data were fitted by using the Igor Pro (Wavemetrics).

For FRET efficiencies (E) between single donor and single acceptor system the classical FRET formula was used:

where r is the distance between donor and acceptor chromophores and R is the characteristic distance (the Förster distance or Förster radius) with a 50% transfer efficiency. [40] To account for our experimental observations, a numerical simulation was used to calculate the FRET efficiencies as a function of the distance between a single donor and the surface of a sphere where the acceptors are homogenously distributed on its surface. In this case, the donor can transfer its excitation energy to each individual acceptor at a rate given by the Förster theory (k FRET r -6 ). The steady state intensities of the donor and acceptor NP were determined by the solving the following set of differential equations:

{ ( ) ( )
Where k exc is the excitation rate of the donor dye, in this model we used a 10 ps pulsed gaussian excitation ( ) ). and are the fluorescence lifetimes of the donor (4 ns) and acceptor (2,4 ns) respectively. D 0 and D 1 are the ground and first excited state of donor, A n stands for the first excited state of acceptors. The energy transfer rates are calculated using the following equation:

( )
Where R 0 is the Förster radius (6.35 nm), the fluorescence lifetimes of the donor (4 ns) and the distance between the donor and the n th localized on the surface of the acceptor NP. The distances were calculated by using a spherical reference frame.

corresponds to the number of acceptor dyes localized at the distance equal to (i.e. considering the surface density of acceptor dyes).

The FRET efficiency for a given configuration was calculated using the ratio of the total intensity emitted by the acceptors and the total intensity emitted by the acceptors plus the donor:

∑ ∫ ∑ ∫ ∫

The radiative rates have been estimated from the fluorescence lifetimes of the dyes and their quantum yield. In the simulation, we considered up to 10 distances and the number of acceptors dyes was changed to account for the experimental FRET values (Figure 4c).

For surface energy transfer efficiencies, the following formula was used: where d is the distance between donor and acceptor and D is the distance at which SET efficiency decreases to 50% (i.e., equal probability of energy transfer and spontaneous emission). [22] 4.11.

Single-particle measurements

For single particle FRET measurements, the LabTek chambers were incubated with 1M KOH solution followed by extensive washing with Milli-Q water. Then, 100 µL of 0.5 mg/mL BSA-biotin solution in autoclaved Milli-Q water were added in each chamber of the LabTek. After 20 min of incubation the chamber was washed 3 times with PBS and then was incubated with 100 µL of 0.5mg/mL neutravidin solution in PBS for 15 min. Following that, the chamber was cleaned thoroughly with PBS, and then, it was incubated with 200 µL of 1 µM solution of T20-biotin in PBS for 20 min and subsequently washed with PBS. After that, 100 µL of the D-A20-NP40/A-T20-NP25 complexes were added and incubated for 45 min followed by cleaning with PBS and finally the surface of each chamber was fully covered by PBS.

Single-particle measurements were performed in the epi-fluorescence mode using Nikon Ti-E inverted microscope with a 60x objective (Apo TIRF, oil, NA 1.49, Nikon). The excitation was provided by light emitting diodes (SpectraX, Lumencor) at 550 nm for R18/F5-TPB and 640 nm for Atto647-C18/F5-TPB. To have multi-channel image detection, corresponding to R18/F5-TPB and Atto647-C18/F5-TPB signals, different filters were used (600±50nm for R18/F5-TPB and 705±72nm for Atto647-C18/F5-TPB. Single-particle analysis was performed using the Fiji software.

Figure 1 .

 1 Figure 1. Schematic representation of energy transfer (ET) from a dye-loaded nanoparticle to a single acceptor or another dye-loaded nanoparticle. The dye-loaded nanoparticles functionalized with oligonucleotides are schematically presented. Black arrows schematically

Figure 2 .

 2 Figure 2. FRET between donor and acceptor NPs in direct contact. (a) Schematic representation of anionic and cationic NPs loaded with donor (R18/F5-TPB) and acceptor (Atto647-C18/F5-TPB) dyes respectively, and the chemical formula of the polymers used for the experiment. (b) Steady-state fluorescence spectra of small (40 nm, A50%-NP40) or large (66 nm, D50%-NP66) donor NPs (50 wt% of R18/F5-TPB dye with respect to the polymer) in direct contact with 15 nm acceptor NPs (A30%-NP15, 30 wt% of Atto647-C18/F5-TPB dye with respect to the polymer). For this experiment, the ratio between the donor NP concentration (10 pM) and acceptor NP concentration (20 pM) was 1:2. Controls: 40-nm donor NPs without acceptor NPs (Control 1) and acceptor NPs without donor NPs (Control 2, intensity multiplied 10-fold for visibility), measured at 530 nm (donor) excitation. PBS buffer was systematically used. (c) FRET ratio for the large or small donor NPs (concentration of donor NPs was 10 pM in both cases) measured by varying concentration of the acceptor NPs. (d) FRET ratio of donor (40 nm) and acceptor (15 nm) NPs in direct contact as a function of dye loading (donor NPs: 50 wt% and 1 wt% of R18/F5-TPB dye with respect to the polymer; acceptor NPs: 30 wt% and 1 wt% of Atto647-C18/F5-TPB dye with respect to the polymer). The donor NPs concentration was 10 pM and the acceptor NPs concentrations were 20, 40, 100 pM. All errors are s.d.m. (n .

Figure 3 .

 3 Figure 3. FRET between donor and acceptor NPs at distance of 20 base pairs (6.8 nm). (a) Schematic representation of A20 and T20 DNA-modified NPs loaded with donor (R18/F5-TPB) and acceptor (Atto647-C18/F5-TPB) dyes, respectively. (b) TEM images of individual NPs (containing 50 wt% of R18/F5-TPB dye with respect to the polymer for donor NPs and 30 wt% of Atto647-C18/F5-TPB dye with respect to the polymer for acceptor NPs) before their modification with DNA. The donor NPs were prepared in phosphate buffer with 30 mM

  This article is protected by copyright. All rights reserved. 10NaCl (pH 7.4) and the acceptor NPs were prepared in phosphate buffer (pH 7.4) without NaCl. ~200 NPs were analyzed per condition. Scale bars: 100 nm. (c) size distributions of the donor and acceptor NPs. (d) Steady-state fluorescence spectra of A20-modified donor NPs (40 nm) with varied concentration of T20-modified acceptor NPs (25 nm). (e) FRET efficiencies for donor NPs (40 nm) with varied concentration of the acceptor NPs (25 nm). The donor NPs concentration was 8 pM. All errors are s.d.m. (n ≥ 3). (f) Control experiment for non-interacting NPs: fluorescence spectra of A20-modified donor NPs (40 nm) with 40 nM A20-modified acceptor NPs (25 nm). The donor NPs concentration was 8 pM. HEPES (Mis-B) buffer was systematically used. Excitation wavelength was 530 nm.

Figure 4 .

 4 Figure 4. FRET between donor and acceptor NPs at different distances. (a) Schematic representation of DNA-modified NPs loaded with donor (R18/F5-TPB) and acceptor (Atto647-C18/F5-TPB) dyes. The distance between donor and acceptor NPs were controlled by the length of DNA duplexes. (b) FRET efficiencies between DNA-modified NPs (containing 50 wt% of R18/F5-TPB dye with respect to the polymer for the 40 nm donor NPs and 30 wt% of Atto647-C18/F5-TPB dye for the 25 nm acceptor NPs) at different distances (20, 44 and 60 b.p. corresponding to 6.8, 14.9 and 20.4 nm). The case of direct NP-NP contact (0 nm) is also shown. The donor NP concentration was 8 pM. HEPES (Mis-B) buffer was systematically used. All errors are s.d.m. (n. (c) FRET efficiencies between these NPs measured using both steady-state (black squares) and time-resolved (green triangles) spectroscopy as a function of their surface-to-surface distance. The magenta points correspond to the FRET efficiencies calculated using the numerical model described in the Materials and Methods section. The blue curve corresponds to the analytical models developed previously for lipidic systems.[31] All errors are s.d.m. (n .

Figure 5 .

 5 Figure 5. Response of the NP-NP FRET assay to the nucleic acid target. (a) Schematic representation of DNA-modified NPs loaded with donor (50 wt% of R18/F5-TPB) and acceptor (30 wt% of DiD/F5-TPB) dyes at surface-to-surface distance of 14.9 nm. The target DNA is represented in red color. Normalized steady-state fluorescence spectra (b) and corresponding FRET ratios (c) for the donor (8 pM) and acceptor (16 pM) NPs with varied concentration of the nucleic acid target. HEPES (Mis-B) buffer containing total RNA extract from cells (0.01 g/L) was used. All errors are s.d.m. (n ≥ 3).

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Acknowledgements

This work was supported by Agence nationale de la recherche (ANR) grant ANR-19-CE09-0006, the European Research Council ERC Consolidator Grant BrightSens 648525 and ERC Proof of Concept Grant AmpliFISH 899928. We thank Tanushree Gupta for advices with nanoparticles preparation and Corinne Crucifix from the FRISBI platform for her help with the electron microscopy.

Synthesis

Rhodamine B octadecyl ester tetrakis(pentafluorophenyl)borate (R18/F5-TPB) was synthesized by ion exchange and purified by column chromatography as explained earlier. [26b] Synthesis of DiD/F5-TPB was also performed using ion exchange and purification by column chromatography as described previously. [34] Synthesis of PEMA-AspN3 was performed using peptide coupling method as described previously. [11c] Synthesis of PMMA-NMe 3 1% was described previously. [37] 4.3.1. Synthesis of octadecyl A647N (Atto647-C18) Atto647N carboxylic acid (2 mg, 2.7 µmol) was dissolved in dry dimethylformamide (DMF) then octadecyl iodide (10 mg, 27 µmol) and potassium carbonate (5 mg, 37 µmol) were added to it. The reaction mixture was heated at 60 C overnight in the dark. Then, it was cooled and concentrated under reduced pressure to remove DMF and re-dissolved in dichloromethane (DCM) then washed thoroughly with brine. Then the DCM part was concentrated under reduced pressure and then purified with preparatory thin layer chromatography and dried over argon atmosphere. Final yield was 87% (2.13 mg) This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

Conflicts of interests

ASK and AR deposited a patent protecting this technology and they are co-founders of BrightSens Diagnostics SAS. This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved. 26

TOC graphic

Breaking FRET limit: operating range of FRET is fundamentally limited by the Förster radius of ~5 nm. The study on dye-loaded fluorescent polymeric nanoparticles connected by DNA duplexes of different lengths reveals efficient energy transfer far beyond the Förster radius, following powerfour distance dependence. This phenomenon is applied for amplified detection of a target nucleic acid with attomole sensitivity.


Background and Objectives

Photophobia is a sensory disturbance provoked by light. Little is known about the association between photophobia and dementia with Lewy bodies (DLB). In this study, we aimed to identify the frequency and the neural basis of photophobia in prodromal and mild DLB.

Methods

One hundred thirteen DLB patients, 53 Alzheimer's disease (AD) patients, 20 AD and DLB patients, 31 patients with other neurocognitive diseases -including prodromal and mild demented patients -, and 31 healthy elderly controls were included in this case-control study. Photophobia was systematically looked for and compared between groups. Among a selection of 77 DLB patients, we used voxel-based morphometry (VBM) to compare those with and those without photophobia (gray matter volume; SPM12, XjView and Matlab R2021b software).

Results

The frequency of photophobia was higher in the DLB group (47.3%) than in the other groups (P=.002). The photophobia questionnaire score was higher in the DLB group than in the AD group (P=.001). Comparison between DLB patients with and those without photophobia showed decreased gray matter in the photophobia subgroup, in the right precentral cortex, in the eyelid motor region of Penfield's homunculus (P=.007, FWE corrected).

Conclusion

Photophobia is a quite frequent symptom of prodromal and mild DLB. The neural basis of photophobia in DLB involves the right precentral cortex, which could have a role in the decrease of cerebral excitability, but also the motricity of the eyelids.

Introduction

Dementia with Lewy bodies (DLB) is a frequent neurocognitive disease with key features such as hallucinations, cognitive and alertness fluctuations, REM sleep behavior disorder (RBD), and parkinsonism. [START_REF] Mckeith | Research criteria for the diagnosis of prodromal dementia with Lewy bodies[END_REF] In addition to these key characteristics, other symptoms have been described, such as depression and anxiety at the behavioral level and constipation and orthostatic hypotension at the neurovegetative level. [START_REF] Van De Beek | Characterization of symptoms and determinants of disease burden in dementia with Lewy bodies: DEvELOP design and baseline results[END_REF] Recently, in the MEMENTO cohort study, we described new symptoms in the prodromal stage of DLB, such as rhinorrhea, sicca syndrome, and photophobia; 3 in this cohort, photophobia was present in 36.3% of patients. Photophobia is a sensory disturbance provoked by light, and known since 1856 to be associated with the nervous system and in particular the trigeminal nerve. [START_REF] Castorani | Mémoire sur la photophobie[END_REF] Photophobia was first associated with ophthalmological diseases, such as lesions of the cornea, and subsequently with neurologic and neurogeriatric conditions, such as migraine, [START_REF] Chong | Interictal photosensitivity associates with altered brain structure in patients with episodic migraine[END_REF] blepharospasm, [START_REF] Wu | Effect of light on blinking in patients with idiopathic isolated blepharospasm[END_REF] and progressive supranuclear palsy (PSP). [START_REF] Nath | Clinical features and natural history of progressive supranuclear palsy: a clinical cohort study[END_REF] Photophobia, present in 43 to 100% of PSP patients, [START_REF] Baschieri | Autonomic dysfunction in progressive supranuclear palsy[END_REF] appears to cause a reduction in quality of life, with an impact on outdoor activities. [START_REF] Mohanty | Clinical implications of photophobia in progressive supranuclear palsy[END_REF] Photophobia has been very rarely studied in other neurocognitive diseases. In a study of 6 patients with Alzheimer's disease and 6 with Parkinson's disease, photophobia was described in all patients. [START_REF] Bargagli | Neurophthalmologic and Orthoptic Ambulatory Assessments Reveal Ocular and Visual Changes in Patients With Early Alzheimer and Parkinson's Disease[END_REF] In a larger study of 525 PD patients, the proportion of photophobia is not precisely described but visual symptoms present in 63 patients (12%) included photophobia. [START_REF] Barrell | High-Order Visual Processing, Visual Symptoms, and Visual Hallucinations: A Possible Symptomatic Progression of Parkinson's Disease[END_REF] The neural basis of photophobia is unclear. Recently, activation of the posterior thalamus by optogenetic techniques in mice has induced photophobic behaviour. [START_REF] Sowers | Stimulation of Posterior Thalamic Nuclei Induces Photophobic Behavior in Mice[END_REF] The thalamus has already been described as affected in DLB both functionally, [START_REF] Kanemoto | Thalamic pulvinar metabolism, sleep disturbances, and hallucinations in dementia with Lewy bodies: Positron emission tomography and actigraphy study[END_REF][START_REF] Chabran | Changes in gray matter volume and functional connectivity in dementia with Lewy bodies compared to Alzheimer's disease and normal aging: implications for fluctuations[END_REF] and structurally, [START_REF] Watson | Does attentional dysfunction and thalamic atrophy predict decline in dementia with Lewy bodies?[END_REF][START_REF] Kantarci | Longitudinal atrophy in prodromal dementia with Lewy bodies points to cholinergic degeneration[END_REF] and in particular in the pulvinar. [START_REF] Erskine | Specific patterns of neuronal loss in the pulvinar nucleus in dementia with lewy bodies[END_REF] We hypothesized that DLB patients could have photophobia frequently and that it could be related to thalamic atrophy. Thus, our goal was to clarify the frequency of photophobia in DLB compared to other neurocognitive diseases, such as Alzheimer's disease (AD), and to better understand the neural basis of photophobia using voxel-based morphometry (VBM).

Participants and Methods

Study Design and Participants

AlphaLewyMA is a longitudinal study, whose aim is to search for biomarkers of DLB, taking place in the tertiary memory clinic named "Centre Mémoire de Ressources et de Recherche" (CM2R) of Strasbourg and Colmar, France. [START_REF] Bousiges | Differential diagnostic value of total alpha-synuclein assay in the cerebrospinal fluid between Alzheimer's disease and dementia with Lewy bodies from the prodromal stage[END_REF] All patients enrolled in the AlphaLewyMA study had been referred to experienced neurologists and geriatricians of the CM2R for cognitive or behavioral complaints by their specialist, their local memory clinic, or their general practitioner. Healthy elderly controls (HC) were recruited among friends and relatives of the patients or via the listing of controls of the local clinical investigation center. Patients and HC underwent the following: a standardized screening protocol including medical history, various questionnaires including one on photophobia, physical and neurological examinations, neuropsychological tests including Mini-Mental State Examination (MMSE), brain MRI, and blood and CSF collection. Features of parkinsonism were evaluated using the Unified Parkinson's Disease Rating Scale (UPDRS III): akinesia, rigidity, and tremor at rest (rated from 0 for no symptoms to 4 for severe symptoms). Fluctuations were assessed with the Mayo Clinic fluctuations scale. [START_REF] Ferman | DLB fluctuations specific features that reliably differentiate DLB from AD and normal aging[END_REF] The Parkinson's disease-associated psychotic symptoms questionnaire was used to evaluate the presence of hallucinations. [START_REF] Fenelon | The changing face of Parkinson's disease-associated psychosis: a cross-sectional study based on the new NINDS-NIMH criteria[END_REF] RBD was evaluated using a sleep questionnaire on RBD from the publication by Gjerstad et al., [START_REF] Gjerstad | Occurrence and clinical correlates of REM sleep behaviour disorder in patients with Parkinson's disease over time[END_REF] simplified into 2 questions for the patient and the caregiver: one concerning movements during sleep, the second concerning vivid dreams and nightmares. Photophobia was assessed with a 3-level questionnaire (0=normal sensitivity to light, 1=occasional photophobia or with only certain types of light, 2=permanent photophobia or with all types of light). Data were collected at 3 time points, each year during the 2-year follow-up. We considered photophobia to be present when patients responded positively to the questionnaire about photophobia at least once out of the 3 visits. A systematic questioning on new health problems is carried out every year, including ophthalmic diseases, in the aim to avoid bias about photophobia due to eye diseases.

Patients with prodromal DLB or DLB dementia were selected according to McKeith's criteria. [START_REF] Mckeith | Research criteria for the diagnosis of prodromal dementia with Lewy bodies[END_REF][START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: Fourth consensus report of the DLB Consortium[END_REF] Patients with prodromal AD or AD dementia were selected according to Dubois' criteria. [START_REF] Dubois | Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS-ADRDA criteria[END_REF] Patients were considered to have DLB and AD if they met both the Dubois criteria and the McKeith criteria concurrently. For example, a patient with memory storage disorders, a CSF in favor of AD, and two of the four clinical criteria for DLB was considered to have both DLB and AD. A total of 247 participants were included in this study: 110 DLB patients (DLB group), 57 AD patients (AD group), 19 DLB and AD patients (DLB+AD group), 30 diseased control patients (DC group), and 31 HC (HC group). The DC group consisted of patients originally included in the study with cognitive disorders as found in AD and DLB, who, after a follow-up in the study, were found to have neither AD nor DLB. The DC group had various diagnoses, defined according to international criteria. All participants provided written informed consent for the study, in accordance with the Declaration of Helsinki, and the study was approved by the Ethics Committee of East France (IV).

Neuroimaging Study

We used VBM to investigate the neuroanatomical correlates of photophobia in 77 of the 110 DLB patients. These 77 patients were selected because they had participated in all 3 visits during the 2 years. Presence of photophobia at any time point was scored 1 and its absence was scored 0, using the photophobia questionnaire (see above). Each of these patients had undergone a high-resolution anatomical MRI scan at inclusion. T1-weighted three-dimensional anatomical images were obtained using a 3T MRI scanner (Verio 32-channel Tim Siemens scanner; Siemens, Erlangen, Germany) using a volumetric magnetization-prepared rapid acquisition with gradient echo (MPRAGE) sequence (FOV = 256 × 256 mm 2 , image matrix = 256 × 256, slice thickness = 1 mm, repetition time = 1900 ms, echo time = 2.52 ms, flip angle = 9°). VBM analyses included image preprocessing and statistical analyses. These steps were carried out using the SPM12 software package (Wellcome Department of Imaging Neuroscience, London; http://www.fil.ion.ucl.ac.uk/) running on Matlab R2021b (MathWorks, Natick, MA). Anatomical MR images were spatially preprocessed using standard procedures. [START_REF] Botzung | Pay attention to the basal ganglia: a volumetric study in early dementia with Lewy bodies[END_REF] All T1-weighted structural images were first segmented, bias-corrected, and spatially normalized to the Montreal Neurological Institute (MNI) space using an extension of the unified segmentation procedure that includes six classes of tissue. The DARTEL registration toolbox was then used to build a study-specific template and to bring into alignment all the segmentation images. The VBM analysis was done on modulated gray matter (GM) images; that is, the GM value in each voxel was multiplied by the Jacobian determinant derived from the spatial normalization. This procedure preserves the total amount of GM from the original images. These modulated GM images were smoothed with a Gaussian kernel (FWHM, 8 mm). Analyses compared DLB with photophobia and DLB without photophobia in terms of GM volume (SPM12 and XjView), including age and total intracranial volume as covariates, and thresholded at p <0.05 corrected (family-wise error, FWE).

Statistical Analysis

The Statistical Package for Social Sciences software (SPSS ver. 27.0.0.0, http://www-01.ibm.com/software/analytics/spss/) was used for further statistical evaluation as required. Where appropriate, differences in demographic and clinical data were assessed using parametric (ANOVA, ttests) and nonparametric tests (Kruskall-Wallis H, Mann-Whitney U). Post-hoc analyses employed Tukey and Mann-Whitney U for ANOVA and Kruskall-Wallis tests respectively. For categorical measures, χ 2 tests were applied. For each test statistic, a probability value of <0.05 was regarded as significant.

Results

Clinical results

A detailed description of the 5 groups is given in Table 1. The 5 groups were comparable in terms of educational level, gender, and handedness. The DC and HC groups were younger than the AD and DLB+AD groups; only the HC group was younger than the DLB group (F=6.653, P<.001). The DC and HC groups had higher MMSE scores than the AD and DLB+AD groups; only the HC group had a higher MMSE score than the DLB group (F=61.781, P<.001). The DLB and DLB+AD groups had higher rigidity UPDRS III scores than the AD and HC groups (F=51.150, P<.001). The DLB and DLB+AD groups had higher akinesia UPDRS III scores than the AD and HC groups, and the DLB group had a higher score than the DC group (F=54.148, P<.001). The DLB groups had a higher tremor UPDRS III scores than the AD group (F=9.876, P<.043). The Parkinson's disease-associated psychotic symptoms questionnaire score was higher in the DLB group when compared to the AD and HC groups and was higher in the DC group when compared to the AD group (H=42.335, P<.001). The Mayo Clinic Fluctuations Scale score was higher in the DLB group than in the AD, HC, and DC groups. The RBD sleep questionnaire score was higher in the DLB group compared to the AD group but not when compared to the other groups (H=22.780, P<.001). The number of prodromal DLB was of 77, prodromal AD of 29, prodromal DLB+AD of 5. FP-CIT SPECT (DAT-scan) has been done to 45 prodromal and mild DLB patients: we found dopamine transporter loss in 31 patients. The frequency of photophobia was higher in the DLB group (47.3%) than in the other groups (AD group, 19.3%; DLB+AD group 15.8%; DC group 30.0%; and HC group 35.5%; P=.002). The photophobia questionnaire score was higher in the DLB group than in the AD group (P=.001) but was not higher than in the DLB+AD group (P=.060), the DC group, and the HC group (Table 1). We determined the reliability of our questionnaire by applying Cronbach's alpha for the 3 years: the alpha was .741. The diagnosis of patient in the DC group with photophobia status is given in the supplementary table. During the 2-year follow-up, in the DLB group, 7 patients had ophthalmic disease: 3 patients had no photophobia (2 cataract surgeries and one retinal detachment), 4 patients had photophobia (one ectropion, 2 aged-related macular degeneration -AMD-and one chronic glaucoma); in the AD group, 5 patients had ophthalmic disease: 3 without photophobia (One AMD, one chronic conjunctivitis, one chronic glaucoma) and 2 with photophobia (one bilateral blindness, one chronic uveitis); in the DLB+AD groups no patient developed eye disease; in the HC group, three patients had ophthalmic disease all with photophobia (one angle closure glaucoma, one AMD , one unilateral blindness); and in the DC group, one patient with photophobia developed an ophthalmic sarcoidosis and one without photophobia had bilateral AMD. No keratitis, iritis, blepharitis, or blepharospasm was noted whatever the group during the 2-year follow-up.

Neuroimaging Results

Of the 77 DLB patients with brain MRI, 44% have photophobia. Comparison of the DLB subgroup with photophobia and the DLB subgroup without photophobia showed decreased GM in the subgroup with photophobia, in the right precentral cortex (Brodmann area 9), in the eyelid motor region of Penfield's homunculus (P=.007, FWE corrected, T=5.34, k=90 for the peak level, and P=.006, FEW corrected for the cluster level). No other area was found with this correction. Figure 1 shows the precise localization of the precentral GM decrease.

Discussion

This study confirms that photophobia exists in DLB, that it correlates clinically with the presence of hallucinations, and that structurally the difference between DLB with and without photophobia is in the precentral cortex. We showed that photophobia is quite a frequent neurosensory symptom in DLB, since it was present in 47.3% of patients. We had previously demonstrated in the MEMENTO cohort with prodromal DLB patients that the frequency of photophobia was slightly lower at 36.3%. [START_REF] Blanc | Prodromal characteristics of dementia with Lewy bodies: baseline results of the MEMENTO memory clinics nationwide cohort[END_REF] This difference may be explained by the fact that the present study included patients with mild cognitive impairment (MCI) and mild dementia, whereas the previous study included patients with subjective cognitive complaints and MCI, thus overall at an earlier stage. Moreover, photophobia is less common in AD: in the present study, the frequency was 19.3%. Many studies suggest that photophobia in the context of migraine is characterized by diffuse associative visual cortex abnormalities, possibly linked to thalamic structures. [START_REF] Puledda | Imaging the Visual Network in the Migraine Spectrum[END_REF] In the same way, the neural basis of visual hallucinations in DLB almost systematically includes the associative visual cortex and frequently includes the cuneus. [START_REF] Pezzoli | Structural and Functional Neuroimaging of Visual Hallucinations in Lewy Body Disease: A Systematic Literature Review[END_REF][START_REF] Heitz | Neural correlates of visual hallucinations in dementia with Lewy bodies[END_REF] The main hypothesis is that visual areas are deficient and send false information (bottom-up phenomenon), and the information is not recognized as abnormal because of a deficiency also of the frontal lobe (top-down phenomenon). [START_REF] Heitz | Neural correlates of visual hallucinations in dementia with Lewy bodies[END_REF] Thus the visual system, including the occipital cortex, could be the link between visual hallucinations and photophobia. However, the comparison of the two DLB subgroups with and without photophobia did not find a difference in the visual cortex but in the right precentral cortex. Several mechanisms could explain photophobia in DLB. The first one would be an impairment of the trigeminovascular system, as described in migraine. [START_REF] Noseda | A neural mechanism for exacerbation of headache by light[END_REF] The second could be related to a possible blepharospasm secondary to DLB. The third one could be related to the eye directly. Indeed, photophobia is frequently associated with disorders of the eye such as iritis, uveitis, and blepharitis. [START_REF] Burstein | Neurobiology of Photophobia[END_REF] No such problem was identified in the DLB group, but one uveitis in the AD group, and one in the DC group (sarcoidosis) during the two-year follow-up. We found a proportion of photophobia of 35.5% in the elderly controls. In young subjects, using a question on gene by light, the proportion is also 35% according to a study on student without migraine. [START_REF] Mulleners | Self-reported photophobic symptoms in migraineurs and controls are reliable and predict diagnostic category accurately[END_REF] The lower proportion of photophobia in the DLB+AD group may be explained by the greater cognitive impairment (although not significant), or by a lesser awareness of the disorders (anosognosia or anososodiaphoria), or by the fact that the association of the two diseases may modify the symptomatology, rather on the side of the disappearance of symptoms that are frequent in the healthy subject than on the side of the appearance of a symptom.

The trigeminovascular system is a complex system with first-order thalamus neurons located in the ventral posteromedial nucleus (VPM) that project to different parts of the cortex, including the primary and secondary sensory cortex and the insula. [START_REF] Qin | Disrupted functional connectivity between sub-regions in the sensorimotor areas and cortex in migraine without aura[END_REF][START_REF] Noseda | Cortical Projections of Functionally Identified Thalamic Trigeminovascular Neurons: Implications for Migraine Headache and Its Associated Symptoms[END_REF] These projections (the so-called pain matrix) are more likely to play a role in the perception of pain, and particularly the perception of a headache in migraine. [START_REF] Noseda | Cortical Projections of Functionally Identified Thalamic Trigeminovascular Neurons: Implications for Migraine Headache and Its Associated Symptoms[END_REF] Noseda et al. identified, in the posterior thalamus of rats, units of neurons that responded to the stimulation of the dura and were in the majority of cases also photosensitive. [START_REF] Noseda | A neural mechanism for exacerbation of headache by light[END_REF] The cortical projections of dura/light-sensitive neurons include the somatosensory cortex, the visual cortex, but also the primary and secondary motor cortex. [START_REF] Noseda | A neural mechanism for exacerbation of headache by light[END_REF] These cortex are part of the precentral cortex. This raises the question of a link between this motor system and the photophobia of DLB patients. Normally, the precentral cortex exhibits 20-Hz oscillatory activity that is essential for setting up proper levels of intracortical and thalamic inhibition. [START_REF] Noseda | Cortical Projections of Functionally Identified Thalamic Trigeminovascular Neurons: Implications for Migraine Headache and Its Associated Symptoms[END_REF] In the context of DLB patients with photophobia, the decreased GM volume in the right precentral cortex could decrease the ability of this precentral cortex to govern cortical excitability and the activity of descending modulatory pathways. These differences between photophobia and non-photophobia have also been found in migraine patients in the premonitory phase: patients with photophobia had hyperactivation of the right precentral cortex. [START_REF] Maniyar | Photic hypersensitivity in the premonitory phase of migraine -a positron emission tomography study[END_REF] Photophobia can also be considered as an autonomic nervous system symptom as it is the case in Parkinson's disease, multiple system atrophy or PSP. [START_REF] Reimann | Comprehensive autonomic assessment does not differentiate between Parkinson's disease, multiple system atrophy and progressive supranuclear palsy[END_REF] It is then often associated with a pupillomotor dysfunction. [START_REF] Baschieri | Autonomic dysfunction in progressive supranuclear palsy[END_REF] We described this autonomic involvement in DLB in a previous article, where we showed that DLB patients also had other early neurovegetative disorders: rhinorrhea, sicca syndrome and constipation. [START_REF] Blanc | Prodromal characteristics of dementia with Lewy bodies: baseline results of the MEMENTO memory clinics nationwide cohort[END_REF] The precentral region on the right that we detected as being decreased in GM volume corresponds to the motor activity of the face and particularly the eyes and eyelids according to Penfield's homunculus. [START_REF] Penfield | Somatic motor and sensory representation in the cerebral cortex of man as studied by electrical stimulation[END_REF] Photophobia in DLB could therefore be related to a motor dysfunction of the face. The patients would blink less or less well, which would promote the entry of light into the orbit and therefore a discomfort.

Conclusion

This study confirmed the existence of photophobia in almost half of the prodromal and mild DLB patients. The neural basis of photophobia in DLB involves the precentral cortex, which could have a role in the motricity of the eyelids, and the decrease of cerebral excitability. The next step will be to evaluate brain MRI microstructural and functional changes in DLB patients, to determine more precisely the brain areas involved in photophobia.

Figure 1 .

 1 Figure 1. Comparison between prodromal and mild dementia with Lewy bodies patients with and without photophobia, showing decreased gray matter concentration in the right precentral cortex in the photophobia subgroup. Sagittal, axial, and coronal view. FWE corrected threshold p<0.05, including age and total intracranial volume as nuisance covariates.

Table 1 . Clinical and Demographic Features of Dementia with Lewy bodies patients, Alzheimer's Disease Patients, Dementia with Lewy Bodies and Alzheimer's Disease Patients, Diseased Controls, and Healthy Controls

 1 Scores on the Mayo Fluctuation Scale (/4). d Mean (standard deviation, number of patients tested). d according to Scheltens et al., JNNP, 1992. f Tukey post-hoc test for ANOVA (F), Kruskall-Wallis post-hoc test on SPSS (H) AD=Alzheimer's disease; DC=diseased controls; DLB=dementia with Lewy bodies; HC=healthy controls; MMSE=Mini-Mental State Examination; N=number; RBD= rapid eye movement sleep behavior disorder; UPDRS=Unified Parkinson's Disease Rating Scale. 14681331, ja, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15829 by Cochrane France, Wiley Online Library on [17/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

			DLB N=110	AD N=57	DLB+AD N=19	DC N=30	N=31 HC	test, P Statistic	Post hoc f
	Age, years a		71.4 (9.3)	74.5 (8.4)	74.8 (8.5)	67.3 (9.4)	66.0 (9.0)	F=6.653, P<.001*	HC<DLB, AD and
									DLB+AD
									DC< AD
									and
									DLB+AD
	Education a, b		11.9 (4.3)	12.0 (3.9)	11.7 (2.9)	12.0 (3.3)	12.7 (2.1)	H=1.994, P=.737
	Sex (F/M)		58/52	30/27	9/10	17/13	18/13	χ 2 =.711, P=.950
	Handedness (R/L/A)		94/9/1	49/6/1	18/0	25/3/1	29/2	χ 2 =4.645, P=.795
	MMSE score (/30) a		25.3 (3.9)	24.1 (3.3)	23.2 (3.9)	27.0 (2.8)	28.8 (1.2)	H=61.781 , P<.001*	HC> DLB+AD, AD, and
									DLB
									DC>
									DLB+AD,
									AD
	Photophobia N (%)		52 (47.3)	11 (19.3)	3 (15.8)	9 (30.0)	11 (35.5)	χ 2 =16.91 2, P=.002*
	Photophobia questionnair	0/1/2	58/25/27	46/9/2	16/2/1	21/3/6	20/9/2	, P<.001* H=19.061	DLB>AD
	e
	Parkinsonis m	Rigidity 0/1/2/3/4	36/59/6/1/0	34/4/1/0/0	6/6/1/0/0	16/6/1/1/ 0	30/1/0/0/0	H=51.150 , P<.001*	DLB>HC and AD DLB+AD>H
									C and AD
		Akinesia 0/1/2/3/4	39/52/11/0/0 35/3/0/0/0	7/7/1/0/0	18/3/3/0/ 0	30/1/0/0/0	H=54.148 , P<.001*	DLB>HC, AD and DC DLB+AD>H
									C and AD
		Tremor 0/1/2/3/4	75/21/0/0/0	35/2/0/0/0	11/4/0/0/ 0	21/2/0/0/ 0	27/2/0/0/0	H=9.876, P<.043*

DLB>AD Hallucination s (/10) a 1.5 (1.9) 0.3 (0.8) 0.4 (0.7) 1.3 (2.0) 0.1 (0.3) H=42.335 , P<.001* DLB>HC and AD; DC>AD Fluctuations c 0/1/2/3/4 a Mean (standard deviation). b Education: years from primary school. c

14681331, ja, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15829 by Cochrane France, Wiley Online Library on [17/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

This study was funded by Appel à Projet Interne (API) of the University Hospital of Strasbourg, France Alzheimer, Fondation Université de Strasbourg, and Projet Hospitalier de Recherche Clinique (PHRC) inter-régional (IDRCB 2012-A00992-41).

Data availability statement: data available on request due to privacy/ethical restriction