Oligonucleotide-functionalized hydrophobic polymer nanoparticles

Abstract

The present invention concerns an oligonucleotide-functionalized hydrophobic polymer nanoparticle and method of its preparation. Said nanoparticle is a dye-loaded polymeric nanoparticle, and being functionalized by: (a) target-specific oligonucleotides, and/or (b) non-specific oligonucleotides.

Claims

1. A dye-loaded polymeric nanoparticle comprising: a hydrophobic polymer chain, said chains bearing at least one moiety of formula (I), ##STR00035## wherein: A represents a spacer chosen from: —(CH.sub.2).sub.m—, —(CH.sub.2).sub.pNH—CO(CH.sub.2).sub.q—, —(CH.sub.2).sub.pCO—NH(CH.sub.2).sub.q—, —(CH.sub.2).sub.pO(CH.sub.2).sub.q—, —(CH.sub.2).sub.pNH(CH.sub.2).sub.qO—, —(CH.sub.2).sub.mCO—, —CO(CH.sub.2).sub.m—, each of m, p and q represent independently from each other an integer chosen from 0 to 8, preferably an integer chosen from 0 to 5, more preferably an integer chosen from 0 to 3, D represents a group chosen from: ##STR00036## wherein R′ represents a hydrogen, a halogen, a (C1-C8)alkyl, a cyclo(C3-C7)alkyl eventually monosubstituted, a monocyclic non-aromatic heterocyclic group eventually monosubstituted, or a monocyclic aromatic group eventually monosubstituted, E represents Y—NR.sub.a, where R.sub.a is H or an (C1-C8)alkyl, X, Y, Z are identical or different and each represent independently of the other a spacer chosen from —(CH.sub.2).sub.r—, —(CH.sub.2—CH.sub.2—O).sub.r, —(CH.sub.2—CH.sub.2—NH).sub.r—, —(CH.sub.2).sub.sNH—CO(CH.sub.2).sub.t—, —(CH.sub.2).sub.sCO—NH(CH.sub.2).sub.t—, wherein r, s and t represent independently from each other an integer chosen from 0 to 8, preferably an integer chosen from 0 to 3, n represents an integer chosen from 1 to 10, in particular 1 to 3 and wherein at least one of m, p, q, r, s and t have a value different from 0, when D represents ##STR00037## E can be absent; said moiety of formula (I) being bound to a carboxyl group of the polymer and being situated on the surface of the nanoparticle and energy donors formed by a salt of at least one donor dye and bulky fluorinated anion, said energy donors being encapsulated in the hydrophobic polymer.

2. The dye-loaded polymeric nanoparticle according to claim 1, wherein the chain of the hydrophobic polymer bears at least a moiety of formula (Ia): ##STR00038## wherein x, y and z represent each an integer chosen from 0 to 8, preferably an integer chosen from 0 to 5, more preferably an integer chosen from 0 to 3.

3. The dye-loaded polymeric nanoparticle according to claim 1, wherein said hydrophobic polymer chain is chosen from polymethacrylates, aliphatic polyesters and polystyrenes, or derivatives thereof.

4. The dye-loaded polymeric nanoparticle according to claim 1, wherein the donor dye is chosen from a rhodamine derivative or a cyanine derivative.

Description

FIGURES

(1) FIG. 1: Concept of oligonucleotide-functionalized hydrophobic polymer nanoparticle for amplified detection of nucleic acids.

(2) FIG. 2: Scheme of synthesis of oligonucleotide-functionalized nanoparticles of the present invention. It includes (i) conjugation of polymer bearing carboxyl groups and azide groups; (ii) nanoprecipitation of the polymer, (iii) DNA attachment to the surface of dye-loaded NPs, hybridization with acceptor probe, target detection.

(3) FIG. 3: Size of NPs by dynamic light scattering. Polymers used for nanoprecipitation: PMMMA-MA modified with 3-aminopropylazide (designed as control); commercial PMMA-MA; AspN3, Asp2N3 and Asp3N3—PMMA-MA bearing the motif AspN3, Asp2N3 or Asp3N3 (designed as AspN3, Asp2N3, Asp3N3).

(4) FIGS. 4a and 4b: FIG. 4a shows hydrodynamic diameter of NPs (by DLS) prepared at low and high concentrations. FIG. 4b shows absorption spectra of NPs showing that the new protocol can increase sample concentration manifold.

(5) FIG. 5: Fluorescence spectra of NPs at cold condition (line represented by —), room temperature (line represented by ⋅—⋅—), or 40° C. (line represented by ⋅⋅⋅⋅⋅) of conjugation with nucleic acids. To NP-Asp-N3 was added 10 μM of SurC and after 16 h of reaction hybridized with 10 μM of acceptor probe Cy5-Flare. Purification was performed using 20 mM phosphate buffer.

(6) FIGS. 6a-6g show characterization of oligonucleotide-functionalized nanoparticles.

(7) FIG. 6a: Size by DLS of non-modified NPs (NP), NPs only bearing azide groups (NP-N.sub.3), NPs bearing the motif Asp-N.sub.3 (NP-Asp-N.sub.3), NPs bearing Asp-N.sub.3 prepared at high concentration (NP-Asp-N.sub.3-Conc), NPs bearing the motif Asp-N.sub.3 and target-specific oligonucleotide (NP-SurC), NPs bearing the motif Asp-N.sub.3, target-specific oligonucleotide and non-specific oligonucleotide (NP-SurC-T20), the latter after 2 months storage (NP-SurC-T20, 2 months).

(8) FIG. 6b: Absorption of non-modified NPs (NP), of NPs bearing the motif Asp-N.sub.3, target-specific oligonucleotide and acceptor probe Cy5-Flare (NP-SurC/Cy5), of NPs bearing the motif Asp-N.sub.3, target-specific oligonucleotide, non-specific oligonucleotide and acceptor probe Cy5-Flare (NP-SurC-T20/Cy5).

(9) FIG. 6c: fluorescence spectra of NPs bearing the motif Asp-N.sub.3, target-specific oligonucleotide and non-specific oligonucleotide (NP-SurC-T20), and of NPs bearing the motif Asp-N.sub.3, target-specific oligonucleotide, non-specific oligonucleotide and acceptor probe Cy5-Flare (NP-SurC-T20/Cy5).

(10) FIGS. 6d and 6e: TEM images of non-modified NPs and corresponding size distribution statistics.

(11) FIGS. 6f and 6g: TEM images of oligonucleotide-functionalized NPs and corresponding size distribution statistics.

(12) FIG. 7: Fluorescence emission spectra of oligonucleotide-functionalized NPs immediately after preparation and after 2 months storage in the dark at 4° C. The spectra are normalized at the short-wavelength band.

(13) FIGS. 8a-8d show response of oligonucleotide-functionalized NPs to the target.

(14) FIG. 8a: Fluorescence spectra of oligonucleotide-functionalized NPs with concentration of Cy5-Flare 20 pM after keeping overnight at 4° C. without Target (Control) and with Target (100 pM).

(15) FIG. 8b: Fluorescence spectra of oligonucleotide-functionalized NPs after incubation with target at different concentrations: 0 (Control), 20, 50, 100 and 200 pM.

(16) FIG. 8c: A/D ratio values of oligonucleotide-functionalized NPs after incubation with target at different concentrations: 0 (Control), 20, 50, 100 and 200 pM.

(17) FIG. 8d: A/D ratio response of the probe (100 pM Cy5-Flare) to the target (500 pM) in different biological media FIGS. 9a-9f: Single-particle imaging of immobilized oligonucleotide-functionalized NPs.

(18) FIG. 9a: The oligonucleotide-functionalized NPs on a BSA-biotin-neutravidin-biotin-A20 surface is shown. The hybridization with surface occurs due to formation of A20-biotin and T20-NP double strand.

(19) FIG. 9b: Single particle total intensity distribution of donor in NP-T20.

(20) FIG. 9c: Sum of FRET-donor and FRET-acceptor in oligonucleotide-functionalized NPs (nanoprobes).

(21) FIG. 9d: Wild-field microscopy images of the nanoprobes at the acceptor, donor and merged channels (both channels are represented at the same intensity scale). The excitation was at 550 nm with excitation power 0.2 mW at the sample level. Integration time was 200 ms.

(22) FIG. 9e: Direct excitation of the acceptor (650 nm with laser power 10 mW and the integration time 200 ms).

(23) FIG. 9f: Signal amplification (antenna effect) of oligonucleotide-functionalized NPs at the single-particle level presented as a distribution histogram. At least 1500 NPs were analysed.

(24) FIG. 10: Response to the target at the single particle level. (a) Wild-field microscopy images using GEMINI setup of the oligonucleotide-functionalized NPs (first row), after target addition and 30 min incubation at room temperature (second row) and control NP-T20 (third row). All channels are represented at the same intensity scale. The excitation was at 550 nm (excitation power was 0.20 mW, integration time was 200 ms). (b) Corresponding distribution histograms of relative FRET for nanoprobe, nanoprobe with target (1 nM) and control NP-T20.

EXAMPLES

(25) 1. Materials and Methods

(26) Materials

(27) Chemical compounds: Poly (methyl methacrylate-co-methacrylic acid) (PMMA-MA, 1.6% methacrylic acid, Mn ˜15000, Mw ˜34000), 3-chloropropanamine hydrochloride (98%), rhodamine B octadecyl ester perchlorate (>98.0%), lithium tetrakis (pentafluorophenyl)borate ethyl etherate, N,N-dimethylformamide (anhydrous, 98%), N,N-Diisopropylethylamine (≥99%), acetonitrile (anhydrous, 99.8%), dichloromethane (anhydrous, ≥99.8%), 1-Hydroxybenzotriazole (≥97%), BSA-biotin, Amicon Centrifugal filters (0.5 mL, 100K) were purchased from Sigma-Aldrich. Citric acid monohydrate (≥99.5%), sodium azide (99%), sodium iodide (≥99.5%) and trifluoroacetic acid (99%) were purchased from Alfa Aesar. Fmoc-Asp(OtBu)—OH was purchased from Activotec. HBTU was purchased from ChemPep Inc. Neutravidin and LabTek chambers (Borosilicate cover glass, eight wells) were purchased from Thermo Scientific. Lyophilized single strand DNA sequences were purchased from IBA, dissolved in Milli-Q water, aliquoted and stored at −20° C. for further experiments.

(28) 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 7.4. For saline buffer sodium chloride (≥99%, Sigma Aldrich) 30 mM and magnesium chloride (≥98%, Sigma Aldrich) 12 mM was added to 20 mM phosphate buffer and pH was adjusted with sodium hydroxide 1N solution. Milli-Q water (Millipore) was used in all experiments. For immobilization protocol DPBS (without Ca.sup.2+ and Mg.sup.2+) was purchased from Lonza.

(29) Synthesis of the Linker

(30) Fmoc-Asp-Cl tert-butyl 3-[(3-chloropropyl) carbamoyl]-3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino] propanoate—Fmoc-Asp(OtBu)—OH (1 eq 6 mmol 2.47 g), HBTU (1.2 eq 7.2 mmol 2.73 g) and HOBt (1.3 eq 7.8 mmol 1.05 g) were dissolved in 30 ml of anhydrous N,N-dimethylformamide. After complete dissolution the N,N-diisopropylethylamine (3 eq 18 mmol 2.97 mL) was added to the stirring mixture under argon at room temperature and in 15 min 3-chloropropanamine hydrochloride (1 eq 6 mmol 0.78 g) was added. The reaction was stirred for 24 h and completion of the reaction was checked by TLC (DCM/MeOH 98/2). The solvent was evaporated under reduced pressure, the residue was diluted with water and precipitate was collected. The crude product was washed with solution of citric acid, followed by solution of sodium bicarbonate. After the crude product was purified by column chromatography (DCM/MeOH 98/2), the product was obtained as pale-yellow solid (2.3 g 80% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.46 (s, 9H), 1.98 (quin, J=6.30 Hz, 2H), 2.60 (dd, J=16.75, 6.24 Hz, 1H), 2.95 (br d, J=15.16 Hz, 1H), 3.45 (m, 2H), 3.56 (br t, J=6.24 Hz, 2H), 4.23 (m, 1H), 4.36-4.60 (m, 3H), 5.93 (br s, 1H), 6.59 (br s, 1H), 7.27-7.36 (m, 2H), 7.42 (t, J=7.50 Hz, 2H), 7.60 (d, J=7.34 Hz, 2H), 7.78 (d, J=7.58 Hz, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ, ppm: 28.00, 31.99, 37.09, 37.43, 42.23, 47.20, 51.19, 67.14, 81.95, 120.06, 120.08, 121.01, 124.98, 125.00, 127.09, 127.12, 127.81, 141.35, 141.38, 143.65, 143.70, 156.10, 170.71, 171.27. HR/LC/MS for C.sub.26H.sub.31ClN.sub.2O.sub.5 m/z (M+) calc 487.19, found 487.19706.

(31) Asp-N.sub.3—Fmoc-Asp-C (1 eq 3 mmol 1.5 g), sodium azide (5 eq 15.4 mmol 1 g) and sodium iodide (1 eq 3 mmol 0.47 g) were dissolved in anhydrous N,N-dimethylformamide (20 ml) and stirred under Ar overnight at 60° C. After the solvent was evaporated, the residue was extracted with water/DCM and washed twice with brine. The crude product was purified by column chromatography (DCM/MeOH 94/6). By LCMS and NMR was observed Fmoc deprotected Asp-N3 as pale-yellow oil (460 mg 55% yield). .sup.1H NMR (400 MHz, CDCl3) δ ppm: 1.46 (s, 9H), 1.65 (m, 2H), 1.81 (quin, J=6.72 Hz, 2H) 2.57 (dd, J=16.63, 8.07 Hz, 1H) 2.8-2.85 (m, 1H) 3.29-3.44 (m, 4H) 3.63 (dd, J=8.07, 3.67 Hz, 1H), 7.57 (br s, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ ppm 28.08, 28.89, 36.65, 40.52, 49.20, 52.02, 81.15, 171.24, 173.71. HR/LC/MS for C.sub.11H.sub.21N.sub.5O.sub.3 m/z (M+) calc 272.17, found 272.17153.

(32) Polymer

(33) PM-Asp-N3-Boc. The PMMA-MA (1 eq of COOH groups, 0.06 mmol, 400 mg) was dissolved in anhydrous N,N-dimethylformamide (5 ml). To this solution HBTU (3 eq, 0.183 mmol, 70 mg), HOBt (4 eq, 0.24 mmol, 33 mg) and N,N-diisopropylethylamine (10 eq 0.61 mmol 0.1 mL) was added. The mixture was stirred for 15 min and after Asp-N3-Boc (3 eq 0.183 mmol 50 mg) was added. The reaction was stirred overnight at room temperature under argon. The solvent was evaporated under reduced pressure and residue was dissolved in minimum of acetonitrile and precipitate with methanol. The precipitate was washed with methanol, redissolved in acetonitrile and reprecipitated twice in methanol. After drying under a vacuum, product was obtained as a white solid—220 mg, yield 55%. .sup.1H NMR (400 MHz, CDCl.sub.3) δ, ppm: 0.85 (br s, 2H), 1.02 (br s, 1H), 1.18-1.29 (m, 1H), 1.46 (br s, 1H), 1.65 (br s, 1H), 1.74-2.11 (m, 2H), 3.60 (br s, 3H). (Degree of modification 82% calculated from BOC signal in NMR spectra).

(34) PM-Asp-N.sub.3—PM-Asp-N.sub.3-Boc (200 mg) was dissolved in anhydrous dichloromethane (5 mL) and 2 mL of trifluoroacetic acid was added. The mixture was stirred vigorously for 3 hours. Then solvents were evaporated under reduced pressure. To the residue the acetonitrile was added and evaporation was repeated several times until the absence of the trifluoroacetic acid. Then the product was precipitated in methanol and filtrated. After drying under a vacuum, the product was obtained as a white solid—160 mg, yield 80%. .sup.1H NMR (400 MHz, CDCl.sub.3) δ, ppm: 0.78-1.11 (m, 2H), 0.94-1.11 (m, 1H), 1.24 (br s, 1H), 1.45 (br s, 1H), 1.72-2.15 (m, 2H), 3.61 (br s, 3H).

(35) Rhodamine B octadecyl ester trakis(penta-fluorophenyl)borate (R18/F5) was synthesized by ion exchange and purified by column chromatography as described previously. (Reisch, A. et al. Nat. Commun. 5, 4089 (2014))

(36) Nanoparticle Preparation. Stock solution of the polymers in acetonitrile was prepared at a concentration of 1 or 2 mg ml.sup.−1 containing R18/F5-TPB (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, 1100 rpm) to 450 μL of 20 mM phosphate buffer. The particle solution was then quickly diluted 5-fold with the same buffer. For preparation of NPs functionalized with DNA the protocol was slightly different (see below).

(37) NP-Probe synthesis. 100 μL of the polymer solution in acetonitrile (2 mg ml.sup.−1 with 30 wt % R18/F5-TPB relative to the polymer) were then added quickly using a micropipette and under shaking (Thermomixer comfort, Eppendorf, 1,100 rpm) to 900 μL of 20 mM phosphate buffer, pH7.4 at 21° C. After the residues of acetonitrile was evaporated. Aliquots of DNA were added to 300 μL of nanoparticles. The reaction was mixed and kept overnight at 40° C. on Thermomixer without shaking protected from light. Then the reaction was cooled down to room temperature. For annealing with Flare-Cy5, the aliquot of Flare-Cy5 in ratio 1:1 with SurC was added and mixture was heated for 70° C. in water bath for 3 mins. To complete hybridization the reaction was cooled down to room temperature and kept in the dark for 2 hours. Then the mixture was diluted with 20 mM phosphate buffer containing 12 mM MgCl.sub.2 and NaCl 30 mM to 4 mL and purified by centrifugation using centrifuge filters (Amicon, 0.5 ml, 100 k) on 1000 g at 20° C. for 2 min. The procedure of centrifugation was repeated 5 times to remove the non-reacted oligonucleotides. The obtained NP-probes in volume 1 mL were kept in the dark at 4° C.

(38) 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). 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: E.sub.FRET=1−I.sub.D-A/I.sub.D, where I.sub.D is the integral donor intensity and I.sub.D-A is the integral intensity of the donor in the presence of the acceptor.

(39) Amplification factor of the acceptor emission (antenna effect, AE) was 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 690 nm:.sup.14

(40) $\mathrm{AE}=\frac{I_{D-\mathrm{FRET}}^{\mathrm{ex}}-I_D^{\mathrm{ex}}*\frac{I_{D-\mathrm{FRET}}^{\mathrm{em}}}{I_D^{\mathrm{em}}}}{I_{A-\mathrm{FRET}}^{\mathrm{ex}}-I_A^{\mathrm{ex}}}$
Where I.sub.D-FRET.sup.ex and I.sub.A-FRET.sup.ex are the maximal excitation intensities of donor and acceptor in NP-Probe, 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 NP-SurC-T20; I.sub.D-FRET.sup.em and I.sub.D.sup.em maximum emission intensity of donor for NP-Probe and NP-SurC-T20, respectively.

(41) Quantum yields of donor in NPs were calculated using Rhodamine 101 in methanol as a reference (QY=1.0) with an absorbance of 0.01 at 530 nm. (Karstens, T. & Kobs, K. J. Phys. Chem. 84, 1871-1872 (1980)). QYs of an acceptor were measured using DiD in methanol (QY=0.33) as a reference. (Texier, I. et al. J. Biomd. Opt. 14, 054005 (2009)).

(42) Transmission Electron Microscopy (TEM)

(43) Carbon-coated copper-rhodium electron microscopy grids with a 300 mesh (Euromedex, France) were surface treated with a glow discharge in amylamine atmosphere (0.45 mbar, 4-4.5 mA, 22 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 onto the grids and left for 2 min. The grids were then treated for 1 min with a 2% uranyl acetate solution for staining. They were 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.

(44) Fluorescence Microscopy.

(45) Immobilization of nanoparticles in LabTek chamber was performed according to Protocol (Jurgen J Schmied et al, Nature Protocols, Vol. 9, NO 6, 2014): The LabTek chamber was washed 3 times with DPBS followed by incubation with 200 μl of BSA-Biotin (0.5 mg ml.sup.−1 in DBPS) for 5 min. Then BSA-biotin solution was removed and the chamber was washed 3 times with 500 μl of DPBS. After the chamber was incubated with 200 μL of neutravidin solution (0.5 mg ml.sup.−1 in DBPS) for 5 min and washed 3 times with 500 μL of DPBS. Then the camber was incubated with 200 μL of 1 μM solution of A20-biotin in DPBS for 5 min and washed 3 times with 20 mM phosphate buffer containing 12 mM MgCl.sub.2 and NaCl 30 mM. Then NP-Probe solution was deposed with proper concentration to achieve desired density and incubated for 1 hour at room temperature in the dark. Before measurements the chamber was washed 2 times with 20 mM phosphate buffer containing 12 mM MgCl.sub.2 and NaCl 30 mM and covered with 200 μL of the same buffer.

(46) Single particle measurements were performed in the epi-fluorescence mode using Nikon Ti-E inverted microscope with an objective (Apo TIRF 100X , oil, NA 1.49, Nikon). A 550 nm a 650 nm light emitting diodes were used to excite the samples. The 550 nm light power was set to 0.2 mW. For direct excitation of acceptor Cy5, the 650 nm light was used with a power of 10 mW. The fluorescence signal was recorded with a Hamamatsu Orca Flash 4 camera. The exposure time was set to 200 ms per image frame. To enable two color images W-VIEW GEMINI image splitting optics were used with the following filter set: dichroic 640 nm (Semrock FF640-FDi01-25×36), 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 3 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. The amplification factor of acceptor emission at a single particle level was determined using:

(47) $\mathrm{AF}=\frac{I_A^{550nm}}{I_A^{650\mathrm{nm}}}\times \frac{P^{650nm}}{P^{550nm}}$
Where I.sub.A.sup.550 nm and I.sub.A.sup.650 nm are mean intensities of acceptors under excitation at 550 and 650 nm, respectively, and P.sup.650 nm and P.sup.550 nm are laser powers corresponding wavelength.

(48) 2. Experimental Results

(49) 2.1 NPs Synthesis and DNA Modification

(50) Nanoparticles were obtained by nanoprecipitation. In case of control polymer bearing azide group without carboxyl group, large aggregates were formed (FIG. 3). By contrast, polymers bearing the motif AspN3 can produce the particles with the size of 32-36 nm. Remarkably, with the increase of number of carboxyl groups on the surface the size of NPs was decreasing.

(51) For the effective reaction between DNA and the dye-loaded NPs, it's necessary to achieve micromolar concentrations of the NPs functional groups. The increase of azide concentration can be calculated by the increase of dye donor encapsulated in the NPs, since dye donor content is about 30 wt. % of that of polymer. Common protocol of nanoprecipitation usually includes 50 times dilution from initial stock solution of polymer (1 g/L) with the dye in CH.sub.3CN. This procedure allows obtaining 30-40 nm NPs with total concentration of reactive azide groups at 0.6-1.3 μM. To increase the concentration of functional groups, the protocol of nanoprecipitation is modified. First, the concentration of the polymer in acetonitrile is increased to (2 mg/ml), while keeping dye content at 30 wt. % with respect to polymer. Then, 100 μl of this acetonitrile stock solution was added to phosphate buffer (900 μL), followed by evaporation of the acetonitrile from the mixture. DLS data suggests that the size of NPs only slightly increased for NPs obtained by the new method and evaporation of acetonitrile did not influence significantly the particle size. Moreover, according to absorption spectroscopy, new protocol allowed increasing the concentration of dye-loaded NPs around 9-fold (FIG. 4).

(52) For the attachment of DNA to NPs cupper-free click reaction is carried out between DNA modified with DBCO and NPs containing azide groups on the surface. As DNA sequence, 20 mer encoding survivin (SurC), which is an important cancer marker, is used. The reaction of the concentrated NPs and Sur-C was realized at 4, 20 and 40° C. for 18 h. To monitor the reaction, the reaction mixture was annealed with complementary Flare-Cy5, which is shorter strand (12NA) labelled with Cy5 (FRET acceptor). NPs were loaded with RhC18-F5 (FRET-donor). The excess of DNA was removed by repeating (5 times) filtration through 100 kDa filters. The efficiency of purification was monitored by absorbance. Fluorescence spectra revealed that at 4 and 20° C. the reaction is not occurred, while at 40° C. it was observed a clear FRET signal from particle to hybridized DNA, indicating successful conjugation reaction (FIG. 5). The evidence for grafting was also provided by absorption spectroscopy: absorption of the acceptor-flare was clearly observed in the purified samples, in contrast to negative control, where NPs without azide group were mixed with Sur DNA. However, in PBS buffer containing 12 mM Mg.sup.2+ ions (required for formation of stable duplexes), NPs-DNA conjugates showed relatively large size (FIG. 6a), probably because of partial aggregation of the obtained NPs in high-salt conditions.

(53) 2.2 Optimization of Sensor and Characterisation of NPs with DNA

(54) To achieve the highest optical amplification through a light-harvesting mechanism, it need to ensure high donor/acceptor ratio in the nanoprobe of the invention. This means that the minimal amount of DNA/acceptor-flare should be grafted to the particle surface, but the number of grafted acceptors should be large enough to ensure efficient FRET. On the other hand, large number of nucleic acids should improve colloidal stability of NPs in biological media, which is supported by a recent report for NPs built from ring-opening metathesis block copolymers..sup.13 Therefore, to ensure controlled small amount of coding nucleic acids and particle stability, the reaction of NPs with a mixture of coding (SurC) and non-coding DNA (T20) was performed. Addition of T20 (20 μM) to the SurC (3 μM) did not inhibit the grafting of SurC to NPs surface, as evinced by absorption spectroscopy (FIG. 6b). Indeed, in comparison to NPs reacted with SurC (3 μM) without T20, only a slight decrease in the absorbance of acceptor-Flare was observed. Fluorescence spectra showed strong emission of the FRET acceptor hybridized on SurC for both type of samples (see FIG. 6 for NPs modified with SurC and T20), which confirmed successful grafting of SurC to NPs in both cases. Remarkably, particles containing T20 remained small in high-salt buffer (PBS) with Mg.sup.2+ ions and there size did not change even after 2 month incubation in this medium (FIG. 6a). Moreover, the emission spectrum of this NP-DNA conjugate, showing both FRET donor and acceptor bands, remained practically invariant for this 2 month period (FIG. 7). These results suggest that excess of non-coding DNA is essential to provide stability to the polymer NPs. Electron microscopy also confirmed that conjugation with nucleic acids preserved the spherical shape and monodispercity of NPs, while their size increased only by ca 5 nm.

(55) The further studies were focused on SurC/T20 NPs, exhibiting most promising properties. The synthesis of these NPs was repeated four times showing good reproducibility of their size and spectroscopic properties (Table 1). Based on absorption data and the particle size from TEM we could estimate that 23±3 (s.e.m. n=4) acceptor-flare units were grafted per particle containing 3200±400 donor dyes. Remarkably, FRET efficiency in this system was 60±6%, indicating that 138±20 donor dyes inside nanoparticle transfer 60% of their energy to a single acceptor located at the NPs surface. This is highly efficient light-harvesting phenomena, which should result in the amplification of the acceptor emission (antenna effect). The antenna effect can be directly measured as a ratio of the donor to acceptor excitation intensity obtained from the excitation spectra recorded at the emission wavelength of the acceptor..sup.15 The obtained antenna effect was 58±1 with remarkably high reproducibility for all four preparations (Table 1), showing that excitation though NP donor (nano-antenna) amplifies acceptor emission 58-fold. This large signal amplification phenomenon should be of key importance for the next step on detection of the target nucleic acids.

(56) TABLE-US-00001 TABLE 1 Size, composition and light-harvesting properties of the NPs-DNA conjugate prepared four times..sup.a Size d. FRET nm, by Ratio A per efficiency, Sample DLS.sup.c D/A NP % AE 1 71.8 ± 2.5 143.54 20.1 42.31 58.23 2 60.2 ± 2.3 116.87 25.7 66.85 56.7 3 61.4 ± 3.1 189.64 15.8 65.08 58.19 4   52 ± 1.3 101.3 29.6 66.75 57.28 Average.sup.b   61 ± 4   138 ± 20 23 ± 3 60 ± 6 58 ± 1 .sup.aStatistics by volume was used in DLS data; Ratio D/A is the donor acceptor ratio; A per NP is the number of acceptors per particle; AE is antenna effect. .sup.bAvarage values values are shown together with standard error of the mean. .sup.cDespite variations in the DLS data, the average size by TEM was relatively stable (40 ± 10 nm), where the error is the full width at half maximum of the size distribution (number of particles analysed >500).

(57) 2.3 Detection of Target Nucleic Acids

(58) As a target, a DNA sequence (20 NA) corresponding to a part of survivin gene was used. First, the target at 100 pM concentration was mixed with nanoprobe bearing 20 pM flare (Cy5) (corresponds to 0.87 pM nanoprobe concentration) and incubated at 4° C. for 24 h. It was found that in the presence of the excess of the target DNA, nanoprobe totally lost FRET signal, in contrast the control sample without the target (FIG. 8a). This result shows that, as expected, the target sequence replaced flare-Cy5 at NPs surface, thus stopping FRET. To make an estimation of the limit of detection, NPs were further diluted to flare concentration of 10 pM and added increasing concentrations of the target. Remarkably, the relative intensity of the acceptor gradually decreased (FIG. 8b,c), indicating that the nanoprobe operates well in the studied concentration range (0-200 pM). Based on the obtained concentration dependence the estimated LOD was 5 pM. This remarkably low LOD was achieved for a standard fluorometer and it can be much lower when a dedicated detection setup is used. Then, the nanoprobe was verified whether it is operational in different biological media. In all studied media, namely phosphate buffered saline (PBS), PBS with bovine serum albumin, Opti-MEM (cell culture medium without serum) and DMEM with serum (full cell medium), the probe showed strong ratiometric response to the target: decrease in the FRET signal in the presence of the target. This is a very important result, because it shows that the probe is compatible with highly complex media containing variety of biomolecules, including proteins. Moreover, the nanoprobe of the invention detected successfully the target also in the extract of RNA from cells, showing that it is highly specific to unique nucleic acid sequence in the presence of large variety of other sequences.

(59) 2.4 Evaluation of Nanoprobe at the Single-Particle Level

(60) The ultimate test for the performance of the nanoprobe is to verify whether it can operate at the level of single particle. To this end the glass surface was modified with A20 sequence, which are complementary to that of non-coding sequence of the nanoprobe of the present invention. Then, addition of the nanoprobe at 100 pM of Cy5-flare concentration resulted in sufficiently good coverage of the surface. One should notice remarkable homogeneity of the total fluorescence intensity the nanoprobes of the invention at the glass surface, suggesting that the deposition was done successfully without any probe aggregation (FIG. 9c). To evaluate FRET signal at the single particle level, the images of NPs were recorded simultaneously at the green (donor) and acceptor (red) channels. It can be seen in the overlay that the NP-probe showed similar intensities at the donor and acceptor channels as most of NPs appear in yellow. By contrast, control NPs bearing only T20 (NP-T20) showed signal only in the donor channel. These results provide clear evidence that, after immobilization of the surface, the NP-probe of the invention preserved strong FRET, as it was observed in the spectroscopy measurements. Then, to evaluate antenna effect at the single-particle level, emission of the acceptor excited to the nanoprobe (at 550 nm) was compared with its direct excitation at 640 nm. Remarkably, 50-fold higher excitation power density at 640 nm was required to achieve emission intensity comparable to that excited at 550 nm. Quantitative image analysis revealed that the amplification of the acceptor emission thorough the NP-probe of the invention is 75±25. This result is in line with the antenna effect measured using excitation spectra in the cuvette. It is to be noted that, this is the first report, where this high amplification is reported at the single particle level or a biosensor. Previous report that used QD as a FRET donor did not exploit antenna effect because large number of acceptors (˜50) should absorb light at least as good as a single QD. The only report on the amplification at the single particle level was reported very recently for a plasmonic biosensor using DNA origami, where the average amplification was 7.3..sup.10

(61) Finally, the response of the nanoprobe of the invention to the target at the single particle level was tested (FIG. 10). Immobilized NPs were incubated with excess of the target (1 nM) for 30 min, followed by their two-colour detection. It is clear that after incubation with the target the emission in the red channel strongly decreases, as it was previously observed by fluorescence spectroscopy in the cuvette. The relative FRET efficiency, expressed as A/(A+D), decreased from 0.4 down to 0.1 (FIG. 10), reaching values close to that for the control NPs without FRET acceptor (NP-T20). These results constitute a clear demonstration that the nanoprobe of the invention can report on the presence of target nucleic acids at the single particle level. This implies that ˜23 hybridization events at the surface of the nanoprobe (corresponding to the number of flares per NPs) resulted in the color switch of a nano-emitter exhibiting the brightness of >3000 rhodamine dyes. This unique phenomenon has two importance consequences. Firsts, this nearly 6-fold change in the intensity ratio at the two emission channels for ˜23 hybridization events implies that, at the single particle level, the nanoprobe could readily detect just a few copies of the nucleic acid target. Moreover, due to the signal amplification produced by light harvesting from >3000 fluorescent dyes, the target nucleic acids could be detected at very low illumination power (˜1 W/cm.sup.2) of LED in an epi-fluorescence mode, which is ˜100 fold-lower that required in the single-molecule detection measurements. The use of low power significantly decreases the background noise and makes it possible detection of a few copies of DNA using relatively weak and inexpensive light sources and relatively simple imaging setup.

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