Dye-loaded fluorescent polymeric nanoparticles as nano-antenna

Abstract

Dye-loaded fluorescent polymeric nanoparticles working as light-harvesting nano-antenna, which efficiently transfer the excitation energy to acceptor dyes and, therefore, amplifies emission of the latter are provided.

Claims

1. A dye-loaded fluorescent polymeric nanoparticle as nano-antenna, comprising: a) a polymer chosen from: a polymethacrylate or its derivative; a polystyrene or its derivative; an aliphatic polyester or its derivative; or a copolymer of aforementioned polymers with polyethylene glycol (PEG) or with charged monomers; b) an energy donor, which content is from 5 to 50% by weight of the polymer; and c) an energy acceptor, which content from 0.001 to 0.04% by weight of the polymer; said polymer forming a matrix in which from 1000 to 50000 of said energy donor molecules are encapsulated per nanoparticle, wherein the energy donor is a salt of a donor dye with bulky fluorinated anions, said donor dye being chosen from: (i) a rhodamine derivative represented by formula (I) ##STR00007## in which: R1, R2, R3 and R4 are identical or different and each represent a hydrogen or a (C1-C8) alkyl group; R5 is a (C1-C24) alkyl; or (ii) a cyanine derivative represented by formula (II) ##STR00008## in which: n is a integer chosen from 1, 2 or 3; Ra and Rb are identical or different and each represent a (C1-C24) alkyl group; and wherein the energy acceptor is a salt of another cyanine derivative of formula (II) defined above with a counterion, said energy donor and said energy acceptor being different.

2. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the polymer is chosen from polycaprolactone, poly(lactic acid), poly(glycolic acid), poly(Lactide-co-Glycolide), Poly(methyl methacrylate), poly(methyl methacrylate-co-methacrylic acid), and poly (Lactide-co-Glycolide-co-PEG).

3. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the content of energy donor is from 50 to 700 mmol/kg with respect to the total mass of the nanoparticles.

4. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the bulky fluorinated anion is chosen from tetrakis(pentafluorophenyl)borate (F5-TPB), tetrakis[3,5-bis-(trifluoromethyl)phenyl]borate (F6-TPB), tetrakis[3,5-bis-(1,1,1,3,3,3-hexafluoro-2-methoxy-2-propyl)phenyl]borate (F12-TPB), and tetrakis[perfluoro-tert-butoxy]aluminate (F9-A1).

5. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the counterion of the energy acceptor is an inorganic anion chosen from chloride, bromide, iodide, perchlorate, sulfonate, nitrate, tosylate, or an organic anion chosen from tetrakis(pentafluorophenyl)borate (F5-TPB), tetrakis[3,5-bis-(trifluoromethyl)phenyl]borate (F6-TPB), tetrakis[3,5-bis-(1,1,1,3,3,3-hexafluoro-2-methoxy-2-propyl)phenyl]borate (F12-TPB), and tetrakis[perfluoro-tert-butoxy]aluminate (F9-A1).

6. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein: the energy donor is chosen from a salt of rhodamine B octadecyl ester, Cy3, Cy5, Cy3.5, or Cy5.5 with an above-mentioned bulky fluorinated anion; and the energy acceptor is chosen from a salt of Cy5, Cy5.5, Cy7, Cy 7.5 with an above-mentioned anion; said energy donor and said energy acceptor being different.

7. The dye-loaded fluorescent polymeric nanoparticle according to claim 6, wherein the energy donor and the energy acceptor in said nanoparticle form a couple of energy donor/energy acceptor, which is chosen from below table: TABLE-US-00003 Donor/Acceptor couple:   rhodamine B octadecyl ester salt/DiD salt rhodamine B octadecyl ester salt/Cy5.5 salt DiI salt/ DiD salt DiI salt/Cy5.5 salt DiI salt/Cy7 salt DiD salt/Cy7 salt Cy3.5 salt/Cy5.5 salt Cy3.5 salt/Cy7.5 salt Cy5.5 salt/Cy7.5 salt.

8. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the nanoparticle has a diameter of 10 nm to 150 nm.

9. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the ratio between the energy acceptor and energy donor is from 1:100 to 1:50000.

10. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the excitation power density of said fluorescent polymeric nanoparticle is from 1 to 1000 mW/cm2, at 530 nm with up to 50 nm bandwidth.

11. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the energy acceptor is either encapsulated inside the matrix of polymer or linked to or adsorbed on the surface of polymer.

12. The dye-loaded fluorescent polymeric nanoparticle according to claim 1, wherein the surface of said nanoparticle is modified through the adsorption of a polymeric or lipidic amphiphile bearing at least one polyethylene glycol chain or zwitterionic groups, preferably poloxamers, polysorbates, and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol); or covalently modified by polyethylene glycol or zwitterionic groups.

13. A method for producing a dye-loaded fluorescent polymeric nanoparticle according to claim 1, with an energy acceptor and an energy donor encapsulated inside the nanoparticle, comprising: (i) preparing a water-miscible solvent solution of a polymer as defined in claim 1 containing; a. from 0.001 to 0.04% by weight of the polymer of the polymer of an energy acceptor defined in claim 1; and b. from 5 to 50% by weight of the polymer of an energy donor defined in claim 1; and (ii) nanoprecipitating said water-miscible solvent solution of polymer in a basic, neutral or weakly acidic aqueous buffer to obtain said polymer based fluorescent nanoparticles.

14. A method for producing a dye-loaded fluorescent polymeric nanoparticles according to claim 1 with an energy acceptor absorbed at the nanoparticle surface and an energy donor encapsulated inside the nanoparticle, comprising: (i) preparing a water-miscible solvent solution of polymer as defined in claim 1 at 0.1-5 mg/ml containing from 5 to 50% by weight of the polymer of an energy donor; (ii) nanoprecipitating said water-miscible solvent solution of polymer in a basic, neutral or weakly acidic aqueous buffer to obtain a nanoparticle which encapsulates the energy donor; and (iii) adding a water-miscible solvent solution of acceptor to above aqueous buffer containing nanoparticles to a final concentration from 0.001 to 0.04% by weight of the polymer.

15. A method for detecting single biomolecules in vitro or in vivo, the method comprising contacting a biomolecule in vitro or in vivo with the dye-loaded fluorescent polymeric nanoparticle of claim 1.

16. A method for in vitro fluorescent detection of a biomolecular marker of a disease in a sample, with amplification due to nanoparticle antenna effect, comprising the step of: contacting the dye-loaded fluorescent polymeric nanoparticle according to claim 1 with said sample; illuminating at power densities equivalent to ambient sunlight conditions; and detecting the donor and amplified acceptor fluorescence emission.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention is further illustrated by following figures and examples.

FIGURES

(2) FIG. 1 shows the concept of a nanoparticle of the invention. (a) Chemical structures of the donor dye rhodamine B octadecyl ester (R18) and its counterion tetrakis(pentafluorophenyl)borate (F5-TPB) and of the acceptor dye cyanine 5 DiD. (b) Short-range ordering of R18 cations (represented by an oval) by the F5-TPB counterion (represented by a triangle) inside the PMMA-MA matrix prevents dye aggregation and leads to short interfluorophore distance and ultrafast EET with subsequent FRET to a single acceptor molecule (represented by a circle). (c) Schematic presentation of the giant light harvesting nano-antenna concept inside polymer NPs.

(3) FIG. 2 shows spectroscopic characterization of the nanoparticles of the invention. (a) Steady-state fluorescence anisotropy and quantum yield of the encapsulated energy donors (R18/F5-TPB) as a function of their loading in PMMA-MA NPs (44 nm by TEM). (b) Schematic presentation of fluorescence anisotropy loss due to EET within randomly oriented fluorophores (c) Anisotropy decay measured at 580 nm with a 60-fs probe beam for NPs loaded at 1 and 30 wt % of donor dyes. (d) Emission spectra of FRET NPs loaded with different amounts of the acceptor dye (DiD) while keeping the same amount of the donor (30 wt %). The emission intensity was normalized to the same absorbance of the donor. (e) Experimental FRET efficiency for NPs of the invention with varied donor but constant acceptor (0.004 wt %) concentration and the calculated one assuming no EET within donor dyes. (f) Amplification factor of the acceptor emission (antenna effect) measured from the excitation spectra, its estimated values based on the observed FRET efficiency and theoretical estimation assuming no EET. Error bars in (a, e, f) represent the standard error of the mean (s.e.m., n=3).

(4) FIG. 3 shows size and brightness of individual nanoparticles of the invention. (a) TEM images and size histograms of NPs containing 30 wt % R18/F5-TPB prepared with different pH of buffer. Scale bar is 50 nm (b) 3D representation of wide-field fluorescence microscopy images of these NPs under illumination of a 532 nm laser with power 0.1 W/cm.sup.2.

(5) FIG. 4 shows effect of size of the nanoparticles of the invention on their performance. Fluorescence spectra (a), FRET efficiency and antenna effect (b) of PMMA-MA FRET NPs of different size loaded with 30 wt % of R18/F5-TPB and DiD. Donor/Acceptor ratios were 1000:1 or 10000:1. Error bars represent the standard error of the mean (n=3). Sample marked “No EET” represents theoretical calculations in the absence of exciton diffusion within donor dyes. (c) Spectra of NPs45 loaded with 30 wt % of R18/F5-TPB upon addition of acceptor molecule Cy5C2. (d) Amplification of acceptor emission (antenna effect) of Cy5C2 adsorbed on the surface of NPs of different sizes loaded with 30 wt % of donor at an acceptor concentration corresponding to a donor/acceptor ratio of 1000:1. Error bars represent s.e.m. (n=3).

(6) FIG. 5 shows single-particle evaluation of performance of the nanoparticles of the invention. (a) Wide-field fluorescence microscopy images of NPs. Right panel represents images of NPs60 containing 30 wt % R18/F5-TPB without acceptor and left ones are those with ˜1.5 Cy5 (DiD) acceptors per NP. The illumination at 532 nm was set to a laser power density of 1 W/cm.sup.2. Both channels are represented at the same intensity scale. (b) 3D representation of wide-field TIRF images of acceptor emission from NPs60 nano-antenna containing ˜1.5 Cy5 acceptors per NP under the illumination at 532 nm with laser power density of 0.1 W/cm.sup.2, under the direct excitation of acceptor at 642 nm with laser power density of 100 W/cm.sup.2 and of QD655 under illumination at 532 nm with laser power 0.1 W/cm.sup.2. In all cases integration time was 30.53 ms. (c) Amplification factor of acceptor emission calculated by eq. 1 for antennas of different size with 1-2 acceptors per NP. (d) Representative single particle trace excited at 532 nm with a power density of 1 mW/cm.sup.2. (e) Scheme of experimental setup using excitation by external light source that mimics direct sunlight. (f) Donor and acceptor channels of single particle microscopy under sunlight excitation mimics using NPs60 nano-antennas containing ˜1.5 Cy5 per NP before and after 5 min illumination, and of NPs without acceptor under the same conditions. (g) Single particle traces at the acceptor channel for the nano-antennas without and with Cy5 acceptor.

(7) FIG. 6(A) shows fluorescence spectra of PMMA nanoparticles containing ˜50 wt % (with respect to the polymer) of DiI cyanine dye salt with F12-TPB counterion (energy donor) and DiD cyanine dye salt with F12-TPB counterion (energy acceptor) at different acceptor/donor ratios. Excitation wavelength was 520 nm.

(8) FIG. 6(B) shows antenna effect calculated from the excitation spectra of PMMA-MA nanoparticles containing ˜50 wt % (with respect to the polymer) of DiI cyanine dye salt with F12-TPB counterion (energy donor) and DiD cyanine dye salt with F12-TPB counterion (energy acceptor) at different acceptor/donor ratios.

DETAILED DESCRIPTION

Materials and Methods

(9) Materials. Poly (methyl methacrylate-co-methacrylic acid) (PMMA-MA, 1.3% methacrylic acid, Mn ˜15000, Mw ˜34000), acetonitrile (anhydrous, 99.8%), rhodamine B octadecyl ester perchlorate (>98.0%), lithium tetrakis (pentafluorophenyl)borate ethyl etherate were purchased from Sigma-Aldrich. DiD oil (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine Perchlorate) (Cy5) was purchased from Life-Technologies. Sodium phosphate monobasic (>99.0%, Sigma-Aldrich) and sodium phosphate dibasic dihydrate (>99.0%, Sigma-Aldrich) were used to prepare 20 mM phosphate buffers at pH 5.8-9.0. Milli-Q water (Millipore) was used in all experiments.
Synthesis. Rhodamine B Octadecyl Ester Tetrakis(penta-fluorophenyl)borate (R18/F5) was synthesized by ion exchange and purified by column chromatography as described by Holzmeister et al. (Holzmeister, P.; Acuna, G. P.; Grohmann, D.; Tinnefeld, P. Chemical Society Reviews 2014, 43, (4), 1014-1028). 1,1′-Diethyl-3,3,3′,3′-tetramethylindodicarbocyanine iodide (Cy5-C2) was synthesized as described by Pisoni et al. (Pisoni D. S.; Todeschini L.; Borges A. C. A.; Petzhold C. L.; Rodembusch F. S.; Campo L. F.; J. Org. Chem. 2014, 79, 5511.)
Nanoparticle Preparation. Stock solutions of the polymer in acetonitrile were prepared at a concentration 2 mg mL.sup.−1 containing different amount of R18/F5-TPB (0.1 to 30 wt % relative to the polymer). 50 μL of the polymer solutions were then added quickly using a micropipette and under shaking (Thermomixer comfort, Eppendorf, 1000 rpm) to 450 μL of 20 mM phosphate at 21° C. The particle solution was then quickly diluted 5-fold with the phosphate buffer 20 mM, pH7.4. For preparation of FRET nanoparticles, different concentrations of DiD (from 0.001 wt % to 0.04 wt % relative to the polymer) were added to the acetonitrile solution of polymer containing desired concentration of R18/F5-TPB, and the particles were prepared as described above. Preparation of NPs of different size was achieved by varying the pH of phosphate buffer at the first dilution.
Nanoparticle characterization. Measurements for the determination of the size of nanoparticles were performed on a Zetasizer Nano ZSP (Malvern Instruments S.A.). The mean value of the diameter of the size distribution per volume was used for analysis. Absorption spectra were recorded on a Cary 4000 scan UV-visible spectrophotometer (Varian), excitation and emission spectra were recorded on a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon) equipped with a thermostated cell compartment. For standard recording of fluorescence spectra, the excitation wavelength was set to 530 nm. The fluorescence spectra were corrected for detector response and lamp fluctuations. To calculate FRET efficiency based on fluorescence spectra, a classical equation was used:

(10) $E_{\mathrm{FRET}}=1-\frac{I_{D-A}}{I_D},$
where I.sub.D is the integral donor intensity and I.sub.D-A the integral intensity of the donor in the presence of the acceptor. Measurement of fluorescence anisotropy was performed at 20° C. with a Fluorolog spectrofluorometer (Horiba Jobin Yvon). Excitation wavelength was set to 530 nm and detection to 585 nm. Each measurement of the anisotropy value corresponds to an average over 10 subsequent measurements of 0.1 s each. The anisotropy value (r) was expressed as r=(I.sub.∥−I.sub.⊥)/(I.sub.∥+2I.sub.⊥). To measure the anisotropy value of Cy5-C2 the excitation wavelength was 620 nm and emission wavelength 655 nm. For standard recording of excitation spectra, the emission wavelength was set to 700 nm. These spectra were corrected for the lamp intensity. Amplification factor of the acceptor emission (antenna effect, AE) was then expressed as the ratio of the maximal excitation intensity of the donor to that of the acceptor with correction from the emission of the donor dyes at 700 nm:

(11) $\mathrm{AE}=\frac{I_{D-\mathrm{FRET}}^{\mathrm{ex}}-I_D^{\mathrm{ex}}*f}{I_{A-\mathrm{FRET}}^{\mathrm{ex}}}$
Where I.sub.D-FRET.sup.ex and I.sub.A-FRET.sup.ex are the maximal excitation intensities of donor and acceptor in FRET NPs, respectively; I.sub.D.sup.ex and I.sub.A.sup.ex are the excitation intensities at the wavelengths of excitation maximum of donor and acceptor in NPs without acceptors, respectively; f is the correction factor calculated as the ratio of maximum emission intensity of donor for FRET NPs to that for NPs without acceptor dyes:

(12) $\left(f=\frac{I_{D-\mathrm{FRET}}^{\mathrm{em}}}{I_D^{\mathrm{em}}}\right).$

(13) The value of the antenna effect was also estimated based on the FRET efficiency using the following equation: AE=(n.sub.D×ε.sub.D×E)/(n.sub.A×ε.sub.A), where n.sub.D and n.sub.A are the numbers of donors and acceptors, respectively, per particle, ε.sub.D and ε.sub.A are the extinction coefficients of donors and acceptors, respectively, and E is the FRET efficiency. QYs of NPs were calculated using Rhodamine 101 (QY=1) in ethanol as a reference with an absorbance of 0.01 at 530 nm (Sauer, M.; Hofkens, J.; Enderlein, J., Handbook of Fluorescence Spectroscopy and Imaging: From Ensemble to Single Molecules. Wiley-VCH: 2011). QY of an acceptor molecule DiD in PMMA-MA matrix was calculated for the concentration 0.02 wt % using DiD in methanol (QY=0.33) as a reference (Magidson, V.; Khodjakov, A., Chapter 23-Circumventing Photodamage in Live-Cell Microscopy. In Methods in Cell Biology, Greenfield, S.; David, E. W., Eds. Academic Press: 2013; Vol. Volume 114, pp 545-560).

(14) Time-resolved anisotropy. For the time-resolved anisotropy measurements, we used an amplified Ti: sapphire laser that produces ultrashort pulses (100 fs) at a repetition rate of 100 kHz. 60 fs linearly polarized pulses centered at 520 nm (Δλ=12 nm) were obtained by means of an optical parametric amplifier (OPA). The pump's power density was around 120 W cm.sup.−2 for all the measurements. Ultrashort continuum probe pulses were generated in a sapphire crystal (500-800 nm). The normalized differential transmission of a 60 fs (

(15) $(\frac{\Delta T}{T}=\frac{I_t^p-I_t^0-I_{\mathrm{fl}}^p}{I_t^0}$
where I.sub.t.sup.p and I.sub.t.sup.0 are the intensities of the transmitted probe with and without pump and I.sub.fl.sup.p is the fluorescence intensity generated by the pump) linearly polarized probe (parallel or perpendicular to the pump polarization) centered around 580 nm (Δλ=13 nm) was measured as a function of the pump-probe delay by using a monochromator coupled to a liquid nitrogen cooled CCD (Princeton Instrument). The anisotropy decay was calculated using the following relation

(16) $\frac{\left(\frac{\Delta T_{//}}{T_{//}}-\frac{\Delta T_{\perp }}{T_{\perp }}\right)}{\left(\frac{\Delta T_{//}}{T_{//}}+2\frac{\Delta T_{\perp }}{T_{\perp }}\right)},$
where // and ⊥ denotes for a probe beam with a linear polarization parallel and perpendicular with respect to the linear polarization of the pump beam. The decay curves were fitted with a bi-exponential function and the analysis took into account the laser pulse duration.
Transmission electron microscopy (TEM). Five microliters of the particles solution were deposited onto carbon-coated copper-rhodium electron microscopy grids that were used either as obtained or following an air or amylamine glow-discharge. The grids were then treated for 1 min with a 2% uranyl acetate solution for staining. They were then observed with a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100 kV. Areas covered with nanoparticles of interest were recorded at different magnifications on a Peltier cooled CCD camera (Model 794, Gatan, Pleasanton, Calif.). Image analysis was performed using the Fiji software.
Fluorescence Microscopy. For single particle fluorescence microscopy measurements, the NPs were immobilized on glass surfaces on which a polyethylenimine (PEI) layer was initially adsorbed. The solutions of NPs were diluted 5000, 2000, 1000 and 500 times for NPs30, NPs45, NPs60, NPs65 and NPs110 correspondingly. 400 μL of these solutions per cm.sup.2 were then brought in contact with the PEI covered glass for 15 min, followed by extensive rinsing with Milli-Q-water. The surfaces were left in Milli-Q water during microscopy.

(17) Single particle measurements were performed in the TIRF (Total Internal Reflection Fluorescence) mode on a homemade wide-field setup based on an Olympus IX-71 inverted microscope with a high-numerical aperture (NA) TIRF objective (Apo TIRF 100×, oil, NA 1.49, Olympus). A 532 nm diode laser (Cobolt Samba 100) and a 642 nm diode laser (Spectra-Physics Excelsior 635) were used to excite the samples. The 532 nm laser intensity was set to 1 mW/cm.sup.2-100 mW/cm.sup.2 by using a polarizer and a half-wave plate (532 nm). For direct excitation of acceptor Cy5, the 642 nm laser was used with intensity 0.1 kW/cm.sup.2. The fluorescence signal was recorded with an EMCCD (ImagEM Hamamatsu) (0.106 μm pixel size) using an open source Micro-Manager software. The exposure time was set to 30.53 msec per image frame. To enable two channel images W-VIEW GEMINI image splitting optics were used with the following filter set: dichroic 640 nm (Semrock FF640-FDi01-25×36), bandpass filters 593±20 nm(Semrock FF01-593/40-25) and 685±20 nm (Semrock FF02-685/40-25)) were used to image R18/F5-TPB and Cy5, respectively. Single particle analysis was performed using the Fiji software: particle locations were detected through a Fiji routine applied to a projection (maximum intensity) of 1000 frames. After the automatic background subtraction, the mean intensities of circular regions of interest with a diameter of 8 pixels around the found particle locations were then measured. At least three image sequences (245 pixel×245 pixel) per condition were analyzed with, on average, 500-700 particles per sample.

(18) Microscopy mimicking ambient sunlight excitation. The sunlight power density (24 mW cm.sup.−2) was recorded at midday on 19 Oct. 2016, Strasbourg region, using Handheld Laser Power Meter, 1917-R and Semrock band-pass filter 527 nm (50 nm bandwidth). The artificial white light mimicking sunlight was provided by a Cold light source from Zeiss, type KL 1500 LCD. The sample was illuminated from the top ˜2 cm from the divergent light source output through the same 527-nm filter, which corresponded to 15 mW cm.sup.−2 power density at the sample. Single-molecule imaging was done using Nikon Ti-E inverted microscope using CFI Plan Apo 20× air (NA=0.75) and CFI Plan Apo 60× oil (NA=1.4) objectives and Hamamatsu Orca Flash 4 camera. Donor channel was recorded through a 600-nm band-pass filter (50 nm bandwidth, Semrock), while the acceptor channel used 647-nm long-pass filter (Semrock). Data were recorded and analyzed using NIS Elements and Fiji software, respectively.

(19) Result

(20) Spectroscopic Characterization of the NPs of the Invention

(21) According to the theory, when the energy migrates within dyes randomly distributed inside a rigid polymer matrix, the fluorescence anisotropy should decrease (FIG. 2b). The results of FIG. 2a show that, as expected, an increase in the dye loading produces a fast drop in the fluorescence anisotropy values. By the way, the fluorescence quantum yields (QY) of the obtained NPs decreased with the donor loading but remained remarkably high (˜30%) even at 30 wt % loading (FIG. 2a). This result corroborates with relatively small changes in the absorption spectra, where the short-wavelength shoulder, an indicator of donor aggregation, showed minimal increase. Moreover, the emission spectra of NPs of the invention displays only a small red shift without broadening. At 30 wt % loading the average distance between encapsulated dyes should be ˜1 nm, which is far below the Förster radius of homo-FRET transfer for R18/F5-TPB at this loading (4.6 nm). Therefore, an efficient EET could take place, explaining this loss in the anisotropy. To understand better the EET process, femtosecond anisotropy decay studies are performed by using a pump-probe technique. For low dye loading (1 wt % of R18/F5-TPB), the anisotropy remained stable during the first 10 ps, indicating that EET in this system should be very slow (FIG. 2c). In sharp contrast, at high dye loading (30 wt %) the anisotropy decayed to zero already within 1 ps (FIG. 2c). The bi-exponential fit revealed 30 fs (45%) and 600 fs (55%) components, the former being limited by the resolution of the used setup (60 fs pulse width). This means that the EET process is exceptionally fast and thus can involve thousands of dyes (between 2.7×10.sup.3 and 5.3×10.sup.4) within their emission lifetime (˜1.6 ns).

(22) Further, a FRET acceptor is introduced into the NPs by nano-precipitating polymer together with donors and accepter in phosphate buffer. The chosen acceptor in this experience is the lipophilic cyanine 5 derivative DiD. It is a perfect energy acceptor for rhodamine B with very good spectral overlap, and its two hydrophobic octadecyl chains should ensure efficient encapsulation inside polymer matrix. Being encapsulated at 0.02 wt %, DiD displayed a high fluorescence quantum yield of 77±4%, so that the nanoparticle of the invention is tested on a highly emissive acceptor. For NPs containing 30 wt % of donor, an increase in the acceptor concentration resulted in a rapid growth of acceptor emission, accompanied by a drop of the donor emission, indicating a FRET process (FIG. 2d). Remarkably, efficient FRET (28±5%) is already observed for 0.004 wt % of the acceptor (with respect to the mass of the polymer), which corresponds to 1.2 acceptors per 44-nm particle. According to Poisson distribution this acceptor loading ensures that >67% of NPs contain at least one acceptor. Thus, in these NPs a single acceptor could produce FRET from ˜5900 donors through distance of >20 nm (NPs radius) that is much larger than the Förster radius.

(23) To understand better the role of exciton diffusion in this efficient FRET process, NPs are prepared with a constant concentration of the acceptor (0.004 wt %) and varied the concentration of the donor (0.1-30 wt %). Theoretical prediction, assuming no donor-donor communication (no EET), suggested negligibly low FRET efficiency (4-6%) for 0.004 wt % acceptor independently from the donor loading (FIG. 2e). By contrast, the results of the invention show that the increase in the donor loading produces a significant growth in the FRET efficiency (FIG. 2e) and an increase in the acceptor relative intensity. Together with fluorescence anisotropy data, this efficient FRET is clearly a result of fast EET that delivers energy from thousands of donors to a single acceptor (FIG. 2a-c).

(24) Owing to the efficient FRET, NPs of the invention behave like light-harvesting nano-antenna. To quantify the antenna effect (AE), the excitation spectra of the donor and acceptor at the emission wavelength of the acceptor (700 nm) are recorded. AE is measured as the ratio of the maximal excitation intensity of the donor to that of the acceptor with correction from the emission of the donor dyes in the acceptor channel, as described in Materials and Methods.

(25) At low loading of the donor, no antenna effect was observed (AE˜1), whereas at higher donor loadings, AE increased rapidly reaching 560 for 44-nm NPs at 30 wt % (FIG. 2f). These values correlated well with the antenna effect calculated as AE=(nDεDE)/(nAεA), where nD and nA are the numbers of donors and acceptors, respectively, per particle, εD and εA are the extinction coefficients of donors and acceptors, respectively, and E is the FRET efficiency (FIG. 2e). By contrast, theoretical estimations assuming no EET gave AE values much below the experimental ones (3-6 fold lower), emphasizing the importance of the communication within donor dyes for obtaining an efficient antenna.

(26) Effect of the Size of the Nanoparticles on Their Performance

(27) The influence of the size of 30 wt % dye-loaded NPs on the nanoparticles performance is further investigated. Due to the strong effect of polymer charge on the size of obtained NPs, the pH of the phosphate buffer used in nanoprecipitation are varied, which could change the protonation state of the carboxylate in PMMA-MA polymer. Based on DLS measurements, a decrease in pH from 9.0 and 5.8 produces an increase in NP size from 30 to 230 nm, while preserving a good polydispersity (Table 2). TEM confirmed the increase in NPs size with decrease in pH of buffer, but revealed that the sizes of NPs were smaller, in the range of 30-67 nm for pH range from 9.0 to 6.5. However, for pH 5.8 aggregates of >100 nm size are observed (FIG. 3a).

(28) TABLE-US-00002 TABLE 2 Hydrodynamic diameter and spectroscopic properties of PMMA-MA NPs encapsulating 30 wt % of R18/F5-TPB prepared at varied pH. Size, Size, DLS TEM SPB.sup.g SPB.sup.h Sample pH.sup.a (nm).sup.b (nm).sup.c QY.sup.e Anisotropy Donors/NP.sup.f estim. exp. NPs30 9.0 36 ± 1 29 ± 1 0.31 ± 0.04 0.001 1700 1 1 NPs45 7.4 67 ± 2 44 ± 2 0.30 ± 0.03 0.0028 5900 3 5 NPs60 6.7 105 ± 5  58 ± 2 0.28 ± 0.03 0.0025 13000 7 12 NPs67 6.5 144 ± 6  63 ± 4 0.27 ± 0.03 0.0024 17000 9 20 NPs107 5.8 231 ± 16 113 ± 6  0.29 ± 0.03 0.0029 99000 54 31 .sup.aAfter preparation, NPs were diluted in pH 7.4 buffer. .sup.bStatistics by volume was used in DLS measurements (error is s.e.m., n = 5 PDI (polydispersity index) values were 0.1-0.2 for all NPs. .sup.cMean and standard error of the mean (s.e.m.) are calculated from 80-500 NPs in two independent preparations. .sup.dMean ± s.e.m. fluorescence lifetime (n = 3). .sup.eQuantum yield ± s.e.m. of NPs without acceptor molecules (n = 5). .sup.fEstimation is based on NPs size measured by TEM. .sup.gEstimated single particle brightness normalized to NPs30 based on QY and size of NPs measured by TEM. .sup.hNormalized experimental single particle brightness of NPs under illumination of a 532-nm laser with 0.1 W cm.sup.−2 power density.

(29) Independently of the size, the QY of 30 wt % dye-loaded NPs remained high (0.27-0.31, Table 2). Moreover, according to wide-field fluorescence microscopy of NPs immobilized on the surface, the single particle brightness increased with size (FIG. 3b), so that the largest NPs (NPs110) were 31 times as bright as the smallest ones (NPs30) (Table 2). The experimental brightness correlates well with the estimations based on QY and the size of NPs measured by TEM (Table 2). Finally, the anisotropy values are close to zero for all particles sizes (Table 2), indicating that EET is efficient in all these systems.

(30) Using different pH of phosphate buffer, nanoparticles of different size containing 30 wt % of R18/F5-TPB donors with varied amount of acceptor corresponding to donor/acceptor ratio 1000 and 10000 (0.02 and 0.002 wt %, respectively) are prepared. For both ratios, an increase in the NP size increases the contribution of the acceptor emission (FIG. 4a) and the FRET efficiency (FIG. 4b). Thus, for the same donor/acceptor ratio, the larger antennas transfer the energy more efficiently to the acceptor. The antenna effect also increases with NP size, especially for NPs with donor/acceptor ratio of 10000 (FIG. 4b). Remarkably, for NPs60 and NPs65 (containing 1.3 and 1.7 acceptors per NP, respectively) the antenna effect reached 910±120 and 1150±150, respectively (FIG. 4b). It is also verified whether the nano-antennas of the invention can amplify fluorescence of acceptor dye directly at the particle surface, using a less hydrophobic analogue of DiD, Cy5-C2. According to fluorescence anisotropy data, Cy5-C2 at 15 nM concentration binds the NPs of the invention at the used concentrations (0.04 g/L). Being bound to NPs it exhibited good quantum yield (28±4%). In case of 45 nm NPs loaded at 30 wt % with R8/F5-TPB, a strong FRET with nearly equal intensity of acceptor and donor bands was observed for a donor-acceptor ratio of 1000 (FIG. 4c). Remarkably, based on the excitation spectra the antenna effect for NPs of different sizes was systematically above 200 (FIG. 4d). Thus, nano-antennas of the invention can strongly amplify the acceptor emission also at their surface, although the antenna effect is weaker than for the acceptor inside NPs. The amplification of the acceptor emission at the surface is of key importance for obtaining FRET-based fluorescent probes for detection of biomolecules.

(31) Single-Particle Evaluation of Nanoparticles Performance

(32) To carry out this evaluation, the nanoparticles of invention are immobilized on the glass surface and imaged them by wide-field TIRF microscopy. The control NPs without acceptors appear as bright spots at the donor channel and as dim spots at the acceptor channel. In the presence of ˜1.3 acceptors/nanoparticle, the emission in the acceptor channel becomes comparable or brighter than that in the donor channel (FIG. 5a). This result shows that inside the nanoparticles, the emission of 1-2 acceptors is comparable to the emission of thousands of donor dyes.

(33) To quantify the amplification of acceptor emission under the microscope, the acceptor intensity under excitation through FRET at 532 nm is compared to that obtained by direct excitation with a 642 nm laser. Remarkably, to obtain similar acceptor intensities, the excitation through nanoparticles NPs60 at 532 nm required ˜1000-fold lower laser power than direct excitation of the acceptor at 642 nm (FIG. 5b). The amplification factor (AF) of acceptor emission at the single particle level was determined as:

(34) $\begin{array}{cc}\mathrm{AF}=\frac{I_A^{532\mathrm{nm}}}{I_A^{642\mathrm{nm}}}\times \frac{P^{642\mathrm{nm}}}{P^{532\mathrm{nm}}}& \left(1\right)\end{array}$
where I.sub.A.sup.532 nm and I.sub.A.sup.642 nm are the mean intensities of acceptors under excitation at 532 and 642 nm, respectively, and P.sup.532 nm and p.sup.642 nm are laser powers at the corresponding wavelengths. Using equation (1), it is found that the amplification factor increased with the particle size (FIG. 5c). For NPs60, we obtained a ˜1040±100-fold amplification factor, which is in good agreement with the antenna effect measured from the excitation spectra. Owing to this giant amplification factor, the brightness of 1-2 acceptors inside NPs60 is 25-times higher than that of a QD655 excited at 532 nm with the same power (FIG. 5b). This is an exceptional performance of single Cy5 dyes, taking into account that the extinction coefficient of QD655 at 532 nm is 2.4×10.sup.6 M.sup.−1cm.sup.−1 (data from the provider) and QY is close to unity.

(35) Finally, using the nanoparticle of invention NPs60, we can observe single molecule events, such as one-step bleaching of the Cy5 (DiD) acceptor at extremely low laser powers of 1 mW/cm.sup.2 (FIG. 5d), which is accompanied by one step growth of the donor emission. This opposite behavior of donor and acceptor is typically observed in single molecule FRET measurements using one donor and acceptor dyes, but here FRET takes place between 15000 donors and 1-2 acceptors. Moreover, previous reports on single-molecule detection were systematically based on light power densities of 10-5000 W/cm.sup.2. In the present invention, similar single molecule traces are observed at 1-10 mW/cm.sup.2, reaching the values of the ambient sunlight.

(36) Therefore, whether the NPs of the invention enable detection of single molecules using a simple microscopy setup is tested by the excitation provided by directly shining light on the sample at powers equivalent to sunlight. The measured power of direct sunlight (at midday, 19 Oct. 2016, Strasbourg) through the excitation filter 527/50 nm was 24 mW cm.sup.−2. In the used setup, an artificial white light source providing 15 mW cm.sup.−2 through the same filter is used. The fluorescence of immobilized NPs60 nanoparticles with ˜1.5 Cy5 dyes is collected using either 20× air or 60× oil immersion objective and detected using sCMOS camera (FIG. 5e). These NPs displayed significant acceptor emission in contrast to control NPs without acceptor (FIG. 5f). Moreover, after 5-min illumination with an artificial light source, the former NPs lost the acceptor emission, probably due to acceptor bleaching, and became similar to the control NPs. Strikingly, using 20× air objective, it is able to record in the acceptor channel one-step bleaching events, corresponding to single Cy5 dye molecules (FIG. 5g). By contrast, the control NPs display much lower intensity at the acceptor channel without abrupt bleaching steps.

(37) Cyanine dyes were also tested as energy donors in PMMA-based nanoparticles of ˜45 nm diameter. Following the same protocol as for R18/F5-TPB, we prepared PMMA-MA NPs containing ˜50 wt % with respect to polymer mass (˜130 mmol/kg of total particle mass) of DiI cyanine dye salt with F12-TPB counterion as energy donor. DiD salt with F12-TPB was used as energy acceptor co-encapsulated with the energy donor at different molar ratios. It was found that FRET between DiI and DiD was efficient even at very low acceptor/donor ratio 1/2000 (FIG. 6A), indicating very efficient light-harvesting processes from donors to a single acceptor. Based on the excitation spectra, the measured antenna effect increased with decrease in the acceptor/donor ratio, reaching values close to 800 (FIG. 6B). The result is similar to that observed for R18/F5-TPB as energy donor, which shows that cyanine dyes can also be used to preparation of light-harvesting nanoantenna.