Caterina Severi

Nina Melnychuk

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

Smartphone-assisted detection of nucleic acids by light-harvesting FRET-based nanoprobe

Keywords: Smartphone-based detection, Detection of nucleic acids, Fluorescence microscopy, Polymeric nanoparticles, Nanoantenna, Point-of-care diagnostics

Point-of-care assays for optical detection of biomolecular markers attract growing attention, because of their capacity to provide rapid and inexpensive diagnostics of cancer and infectious diseases. Here, we designed a nanoprobe for detection of nucleic acids compatible with a smartphone RGB camera. It is based on light-harvesting polymeric nanoparticles (NPs) encapsulating green fluorescent donor dyes that undergo efficient Förster Resonance Energy Transfer (FRET) to red fluorescent acceptor hybridized at the particle surface. Green-emitting NPs are based on rhodamine 110 and 6G dyes paired with bulky hydrophobic counterions, which prevent dye selfquenching and ensure efficient energy transfer. Their surface is functionalized with a capture DNA sequence for cancer marker survivin, hybridized with a short oligonucleotide bearing FRET acceptor ATTO647N. Obtained 40nm poly(methyl methacrylate)-based NP probe, encapsulating octadecyl rhodamine 6G dyes with tetrakis(perfluoro-tert-butoxy)aluminate counterions (~6000 dyes per NP), and bearing 65 acceptors, shows efficient FRET with >20 % quantum yield and a signal amplification (antenna effect) of 25. It exhibits ratiometric response to the target DNA by FRET acceptor displacement and enables DNA detection in solution by fluorescence spectroscopy (limit of detection 3 pM) and on surfaces at the single-particle level using two-color fluorescence microscopy. Using a smartphone RGB camera, the nanoprobe response can be readily detected at 10 pM target in true color and in red-to-green ratio images. Thus, our FRET-based nanoparticle biosensor enables detection of nucleic acid targets using a smartphone coupled to an appropriate optical setup, opening the way to simple and inexpensive point-of-care assays.

Introduction

Point-of-care (PoC) diagnostics is a forefront research direction devoted to greatly accelerate early diagnostics of diseases (Yang et al., 2019). Ideally, point-of-care testing devices should be inexpensive, portable and easy to use while still being sensitive and selective towards the target species. A great deal of efforts has been devoted to the construction of the electrochemical biosensors, which match these requirements (Chen and Chatterjee, 2013;Dakshayini et al., 2019;[START_REF] Roy | Modernization of Biosensing Strategies for the Development of Lab-on-Chip Integrated Systems[END_REF]Shetti et al., 2019). Smartphones are exceptional candidates in the biosensor development due to their almost universal prevalence (Zhang and Liu, 2016). Fluorescence smartphonebased detection systems were developed for a variety of scopes, such as: blood analysis (Breslauer et al., 2009), detection of viruses (Wei et al., 2013), bacteria (Zhu et al., 2012), cells (Breslauer et al., 2009), proteins (Coskun et al., 2013), nucleic acids (Wei et al., 2014;Xu et al., 2020) and DNA sequencing (Kühnemund et al., 2017). However, smartphone cameras are generally less sensitive than the devices commonly used in research, featuring sensitivity down to single-molecule detection (Kanchi et al., 2018). Sensitivity is a vital quality of point-of-care devices, in order to permit the detection of very low concentration of analyte, thus allowing early diagnosis of diseases.

The inherent limitation of fluorescence-based assays is the limited brightness of fluorescent dyes (Grimm et al., 2015). This has led to the development of many amplification strategies for which single molecular recognition event triggers a response equivalent to hundreds of fluorescent dyes [START_REF] Scrimin | [END_REF]. This amplification can be achieved by increasing the number of target molecules, as in polymerase chain reaction (PCR) and methods based on enzymatic amplification or hybridization chain reactions (Ali et al., 2014;Choi et al., 2010). An alternative approach is direct amplification of the fluorescence signal from the dye, which is attractive for PoC applications owing to its simplicity. In this respect, three systems have already been established: (i) plasmonic nanostructures, which amplify the fluorescence of single dyes located at a precisely controlled distance (Acuna et al., 2012;Wei et al., 2017); (ii) light-harvesting with conjugated polymers (Jiang and McNeill, 2017), in which a large number of πconjugated aromatic units efficiently transfer the excitation energy to one energy acceptor (Rochat and Swager, 2013) and (iii) light-harvesting with dye-loaded fluorescent polymeric nanoparticles [START_REF] Trofymchuk | [END_REF].

Previously, we developed dye-loaded polymeric NPs encapsulating hundreds of rhodamine dyes paired with a bulky hydrophobic counterion (Reisch et al., 2014;Reisch and Klymchenko, 2016). The use of the bulky counterion ensures efficient dye encapsulation without leakage in biological media, prevents aggregation-caused quenching of dyes at high loading and at the same time assembles dyes inside the polymeric matrix with relatively short spacing, inducing a collective behavior of dyes inside the nanoparticle (Andreiuk et al., 2019;Reisch et al., 2017). This collective behavior was exploited to design a giant light-harvesting nanoantenna that undergoes efficient Förster Resonance Energy Transfer (FRET) from ~10 000 donor dyes to very few acceptors inside the nanoparticle, yielding an amplification of the acceptor signal of ~1000 times [START_REF] Trofymchuk | [END_REF]. These nanoantennes were then functionalized with DNA yielding nanoprobes for nucleic acids, stable in physiological conditions and operating by FRET with strand displacement mechanism (Melnychuk and Klymchenko, 2018). The obtained nanoprobes, being ~100 times brighter than QDot-605, produced a donor/acceptor ratio response to single hybridization events (Melnychuk et al., 2020).

In a single-molecule FRET detection, highly sensitive monochromatic cameras are used with an image splitting module (Roy et al., 2008), which is a highly costly and difficult to handle considering point of care devices. Modern consumer grade cameras are comprised of a color filter array for red, green and blue light (RGB) overlaid on an active pixel sensor array based on a complementary metal-oxide semiconductor (CMOS) light sensor [START_REF] Takayanagi | CMOS Image Sensor[END_REF]. These cameras allow the simultaneous detection of three spectral channels and can provide a rapid and simple detection of color change in FRET nanoprobes (Wang et al., 2011). FRET detection assays with the RGB camera of a smartphone is an emerging field (Noor and Krull, 2014;Petryayeva and Algar, 2015;Yan et al., 2018). However, smartphone-based FRET detection at the single nanoparticle level has not been achieved so far, which would open the opportunity to ultrasensitive point-of-care detection. Light-harvesting dye-loaded polymeric NPs, owing to their exceptional brightness, offer this opportunity; but in these nanoprobes both donor and acceptor emit in the red region (Melnychuk et al., 2020;Melnychuk and Klymchenko, 2018), which make these probes not compatible with common RGB cameras.

In this work, we redesigned these light-harvesting nanoprobes for nucleic acids to make them compatible with RGB detection. We selected a donor-acceptor FRET pairs with emissions compatible with the red and green channels of the RGB camera and designed nanoprobe for a nucleic acid fragment encoding survivin, an important anti-apoptotic cancer marker (Altieri, 2003;Stobiecka et al., 2019). We found that the optimized nanoprobe provides a ratiometric response to the target nucleic acid with limit of detection in solution of 3 pM. Remarkably, our nanoprobes, immobilized on glass surface, enabled detection of nucleic acids at the single-particle level, using RGB microscopy camera as well as a smartphone camera. We show that our biosensor system can work with an inexpensive consumer-grade RGB camera, using low excitation power densities. These results open the route to very simple smartphone-based biosensing devices for point-of-care detection of nucleic acid markers of diseases.

Results and discussions

Design of RGB compatible FRET-based nanoprobes

The design of the nanoparticle probe is based on DNA-functionalized nanoantenna, which serves as a FRET donor for a FRET acceptor conjugated to a short oligonucleotide hybridized at the particles surface (Fig. 1A,B). Then, hybridization with the target nucleotide displaces the acceptor leading to FRET turn off and thus a color switch (Melnychuk and Klymchenko, 2018;Melnychuk et al., 2020). To obtain a system compatible with RGB detection, it is necessary to select a FRET pair where emission of the donor and the acceptor is compatible with two distinct RGB channels with minimal crosstalk (Fig. 1C,D). To this end, we designed as the FRET donors the green-emitting octadecyl ester of Rhodamine 110 (R110-C18) and the yellow-emitting octadecyl ester of Rhodamine 6G (R6G-C18). Both of them emit in the green channel region of the RGB systems (Figure 1D and2A). They were synthesized by esterification of corresponding acid form of the dyes with octadecanol in acidic conditions. ATTO 647N (Fig. 3B) was chosen as the FRET acceptor because its absorption overlaps well with the emission of the donor dyes, thus ensuring an efficient FRET, and its emission is compatible with the RGB red channel (Figure 1D).

Fig. 1. DNA nanoprobes for smartphone-based RGB detection of nucleic acids. (A) Light-harvesting in the nanoantenna particles: donor-donor excitation energy transfer inside NPs is followed by FRET to the acceptor at the NP surface. (B) Nanoprobe design: the green donors inside the NP transfer part of their excitation energy towards FRET acceptor at the surface, making the fluorescence from the nanoprobe yellow-orange. In presence of the target sequence the sequence bearing the FRET acceptor is displaced, turning off FRET and switching the emission color to green. (C) Smartphone-based data analysis workflow: an RGB image is taken and the ratio between the red and green channel is evaluated; this ratio is a function of target concentration. (D) Comparison between the spectral response (quantum efficiency) of the RGB channels of the color camera (Nikon DS-Fi3) with the emission spectra in ethanol of the dyes selected for the nanoprobe design.

Donor NPs preparation

Polymeric NPs were prepared based on a polymer derivative of poly(methyl methacrylate-co-methacrylic acid (PMMA-MA, 1.6% methacrylic acid) (Fig. 2A) bearing azide groups (PMMA-AspN3) as was previously described (Melnychuk and Klymchenko, 2018) and loaded with donor dye paired with a bulky hydrophobic counterion. The role of the counterion is to insulate the dye molecules in order to prevent their aggregation-caused quenching (ACQ) (Reisch et al., 2014) and to ensure their efficient encapsulation without dye leakage in biological environment (Andreiuk et al., 2017b). Moreover, the presence of counterion creates a supramolecular organization of dyes and counterions which produce an ultrafast excitation energy migration allow efficient FRET from thousands of donor dyes to few acceptors [START_REF] Trofymchuk | [END_REF]. We chose to test two different counterions: one based on a fluorinated tetraphenylborate (F12) and tetrakis(perfluoro-tert-butoxy)aluminate (F9-Al) (Krossing, 2001), because of their already established effectiveness (Andreiuk et al., 2017a(Andreiuk et al., , 2017b) (Fig. 2A).

The two donor dyes were paired with the two counterions, using ion exchange method (Reisch et al., 2014), for a total of four ion pairs. Then, NPs were prepared by nanoprecipitation of the polymer with these four pairs at three different loadings (10, 120 and 250 mM with respect to the polymer). Charged carboxylate group in the polymer ensures controlled formation of the small particles during nanoprecipitation due to electrostatic repulsion (Reisch et al., 2018(Reisch et al., , 2017)). The as-prepared samples were characterized by dynamic light scattering (DLS) and optical spectroscopy. Hydrodynamic diameters of NPs remained relatively stable, in the range of 35-50 nm, independently of dye loading and ion pair (Table S1 of the Supplementary Materials). The increase in the dye loading resulted in some broadening of the absorption and emission spectra, indicating the effect of dye aggregation in the polymer matrix. These changes were accompanied by blue shifts in the absorption spectra and red shifts in the emission spectra, which were more pronounced for the R6G-C18 dyes (Figure S1 of the Supplementary Materials). For all ion pairs the fluorescence quantum yields (QY) decreased with the loading due to some ACQ. Nevertheless, R6G-C18/F12 loaded NPs displayed relatively high QY (Fig. 2C), around 20% at 250 mM dye loading (i.e. ~6000 dyes per particle) and the emission band for all dye salts remained relatively narrow (Fig. 2D).

FRET-based DNA nanoprobes preparation

Ion pair R110-C18/F12 was excluded from FRET studies, because of its too low quantum yield (Fig. 2C). For the remaining ion pairs, NPs loaded with 250 mM and different quantities of FRET acceptor (DiD, Figure S3, Supplementary Material) were prepared by co-precipitating donor and acceptor dyes together and their FRET properties were studied (Figures S4-S6, Supplementary Material). It was found that both R6G-C18/F12 and R6G-C18/F9-Al performed better in terms of FRET efficiency than R110-C18/F9-Al (Figure S7, Supplementary Material). Moreover, antenna effect (AE), which is a value indicating the magnitude of acceptor amplification by the nanoantenna particle measured by the excitation spectrum [START_REF] Trofymchuk | [END_REF]Woller et al., 2013), was much higher R6G-C18/F12 and R6G-C18/F9-Al NPs compared to R110-C18/F9-Al NPs. Therefore, we selected R6G-C18 salts for fabrication of FRET-based DNA nanoprobes, bearing the hybridized FRET acceptor (TCS-ATTO647N, which has similar spectral properties to DiD) at the NP surface. After nanoprecipitation (Fig. 2B) azide-bearing NPs were functionalized by clicking oligonucleotide sequences bearing dibenzocyclooctyne (DBCO) reactive group, and subsequently annealed with an acceptor-bearing complimentary sequence (TCS-ATTO647N) (Fig. 3A), following a previously described methodology (Melnychuk and Klymchenko, 2018). To control the amount of capture sequence (SurC) on NPs surface, SurC-DBCO was added to NPs at different concentrations (0.5 µM, 1.5 µM and 3 µM) together with a non-coding DNA-DBCO (T20), while keeping constant the total amount of added DNA-DBCO (23 µM). Then, the obtained DNA-functionalized NPs were hybridized with TCS-ATTO647N and purified by ultrafiltration. The amount of hybridized TCS-ATTO647N on the surface of the nanoparticles was quantified by absorption spectroscopy (Fig. S8 of the Supplementary Materials). For each SurC concentration the number of TCS-ATTO647N acceptors per NP was, respectively, 5, 30 and 65 acceptors per particle. For both tested donor dye salts, the increase in the number of acceptors per NP increased relative intensity of the acceptor emission, accompanied by a small decrease in the donor emission (Figure 3C and S9A, Supplementary Material). These spectral changes indicate that FRET takes place from donor NPs to ATTO647N acceptor at the surface. FRET efficiency, expressed as the empirical FRET ratio A/(A+D), increased with the increase of the number of acceptors. The FRET ratio was slightly higher for R6G-C18/F9-Al (Figure 3D). The total QY value of donor and acceptor also increased in the presence of acceptor, especially in the case R6G-C18/F9-Al NPs, where QY increased from 0.11 ± 0.01 for NPs without acceptor, to 0.212 ± 0.002 for those with 65 acceptors per particle (Fig. 3E). This QY enhancement could be due to the fact that a part of the excitation energy transferred to the acceptor derives from the non-emissive self-quenched donor dyes, thus producing a de-quenching effect on the whole system, as shown for other NPs (Genovese et al., 2013). For both donor dye salts (R6G-C18/F12 and R6G-C18/F9-Al) the antenna effect increased with decrease in the number of acceptors per NP. R6G-C18/F9-Al NPs performed slightly better than R6G-C18/F12 NPs, with its AE ranging from 25 till 34 for number of acceptors per NP varied from 65 till 5. The latter means that the signal of the acceptor is amplified 25-34 times due to the lightharvesting effect of the nanoantenna, where a large number of donor dyes inside nanoparticle (~6000 per 43-nm particle) undergo efficient FRET to a few acceptors at the surface. R6G-C18/F9-Al NPs were selected for the experiments on the DNA target detection. Taking into account the number of encapsulated dyes (n), extinction coefficient of Rhodamine 6G (116,000 M -1 cm -1 ) and their quantum yield (QY), we can estimate the brightness of R6G-C18/F9-Al nanoprobe: B = n ×  × QY = 6000 × 116,000 cm -1 M -1 × 0.212 = 1.48 × 10 8 M -1 cm -1 . This value is equivalent of ~2000 high-performance dyes with typical extinction coefficient of 150,000 M -1 cm -1 and QY of 0.5 (B = 7.5× 10 4 M -1 cm -1 ) and of ~135 Qdots-605 (B = 1.1 x 10 6 M -1 cm -1 , excitation at 488 nm, assuming QY = 1).

Detection of target DNA sequence

All the following experiments were performed on the R6G-C18/F9-Al-loaded NP-probe with 65 acceptors per NP (NP-probe-65). In the nanoprobe design, DNA target sequence, encoding survivin fragment (20mer ssDNA), displaces the TCS-ATTO647N acceptor from NP surface, thus turning off the FRET signal (Fig. 1B). Indeed, after incubation with the target, the signal corresponding to the acceptor at 662 nm decreased. This confirmed that the FRET acceptor was hybridized at the NP surface instead of just being nonspecifically absorbed, so that the target effectively displaced the TCS-ATTO647N and turned off the FRET. This result is in line with our previous FRETbased probes operating in the red region (Melnychuk et al., 2020;Melnychuk and Klymchenko, 2018). To optimize sensitivity of our assay, we diluted the NPs suspension to 10 pM of the acceptor (corresponding to ~0.15 pM of NPs) and incubated it (6 h, rt) with the DNA target at different concentrations (3-200 pM). We observed that, with increase in the target concentration, the acceptor relative intensity decreased gradually with some recovery of donor fluorescence (Figure S11), showing almost linear change of the FRET ratio vs target concentration (Fig. 4). The limit of detection and the limit of quantitation (LOD and LOQ, defined as, respectively, 3 and 10 times the ratio between the standard error of the control without target and the slope of the linear regression (Analytical Methods Committee, 1987) (see Table S2, Supplementary Material) for this assay were 3 and 9 pM, respectively.

To study the response of NP-probe-65 at the single-particle level, it was immobilized on the glass surface using a sandwich of biotinylated BSA, neutravidin and biotinylated A20, followed by a hybridization of the nanoprobe (Fig. 5A) (Melnychuk and Klymchenko, 2018). Immobilized NP-probe-65 was visualized via fluorescence microscopy using both a sCMOS (Hamamatsu Orca Flash 4.0) camera equipped with a beam splitter (W-VIEW GEMINI) and an RGB camera (Nikon, DS-Fi3). The beam splitter at 640 nm allowed us to separately detect donor an acceptor channels and then to construct merged and ratio images. The increase in the target concentration decreased gradually the intensity in the acceptor channel, so that on the merged images the particles turned from orange to green (Fig. 5B). In the ratio images drastic change of NPs color towards blue (low acceptor/donor ratio) was observed, in line with the spectroscopic data in solution (Fig. 4). A histogram of the ratio distribution shows that ratio of the NP-probe-65 initially centered at ~0.6 shifted gradually to ~0.4 and ~0.2 for 10 and 1000 pM of the target, respectively (Fig. 5D), indicating that this method could in principle allow quantification of the target. On the other hand, for RGB color camera, merged images shoed only small color change from orange to yellow with addition of the 1000 pM DNA target. The differences in performance between the sCMOS camera and the RGB camera could be due to two reasons: (i) sCMOS has an higher sensitivity and dynamic range to the RGB sensor and (ii) the use of the beam splitter with a dichroic at 640 nm provides efficient separation of donor and acceptor signals of NP-probe-65 than green and red channels of the RGB sensor. Nevertheless, the ratio images for the RGB camera (Fig. 5C) showed clear changes in the red/green ratio in response to the target (color change from orange to blue), which was confirmed by the ratio histogram (Fig. 5D). Finally, we challenged our NP-probe-65 biosensor by imaging it on glass surface using a cellphone camera (iPhone SE, Backlit Sony Exmor RS 12 MP, 1.22 μm, f/2.2 aperture). The detection setup was simply constructed by replacing the Nikon RGB camera with a smartphone together with an eyepiece (10x) at the exit of the microscope (Fig. 6A). The estimated pixel size in our images was 0.526 µm/px (see Supplementary Materials 1.3.8). Using a special smartphone application that allows controlling key camera parameters (ISO, exposure time, aperture), we acquired images where individual particles could be readily identified. Remarkably, the true-color images recorded with a smartphone could directly show the change in the color of NP-probe-65 in the presence of 10 pM target. The color change from orange to yellow-green corresponded to loss of FRET in NP-probe-65, expected for the strand displacement induced by the DNA target (Fig. 6B). Red/green channel ratio images revealed drastic changes in the ratio from red pseudo-color to green-yellow, green-blue and blue for 10, 100 and 1000 pM target, respectively (Fig. 6C). The ratio histograms confirmed the gradual change in the red/green ratio, and distribution histograms looked better separated compared to Nikon RGB camera (Fig. 6D vs 5D). The major response was observed already at 10 pM target, probably because at this concentration corresponded to ~10-fold access with respect to the FRET acceptor (TCS-ATTO647N). Thus, the ratiometric response of FRET-based nanoprobe to target DNA can be readily recorded with a smartphone camera using a standard epi-fluorescence optical microscope.

Conclusion

In this work, a FRET-based DNA nanoprobe was developed for detection with a smartphone camera. To this end, we designed 40-nm PMMA-based polymeric NPs encapsulating green emitting fluorescent dyes, octadecyl Rhodamines 110 and 6G, paired with bulky counterions for preventing aggregation-caused quenching in the polymer matrix. Their surface was functionalized with DNA complementary to the sequence encoding cancer marker survivin, which is hybridized with the target competitive sequence bearing red emitting FRET acceptor ATTO647N. Among tested dye salts, octadecyl Rhodamine 6G with aluminum-based bulky counterion, showed best FRET characteristics, while the optimal acceptor concentration was 65 molecules per NP. The obtained nanoprobe displayed fluorescence quantum yield >20 % and antenna effect of 25. The nanoprobe showed ratiometric response to the target DNA in solution with a limit of detection of 3 pM. Single-particle fluorescence microscopy of immobilized nanoprobe revealed that both sCMOS camera with a beam splitter and RGB camera can detect the color switch of the FRET nanoprobe in presence of varying concentrations of the DNA target. Finally, our nanoprobe enabled detection of 10-pM DNA target using RGB camera of a smartphone in true color or ratio (red-to-green) imaging at the single-particle level. Thus, high brightness of NPs and signal amplification through light harvesting allowed DNA sensing by a smartphone sensor coupled to an appropriate optical setup. We foresee that the optical part could be greatly simplified, which would open the way to simple and inexpensive smartphonebased point-of-care assays for detection of biomolecular markers of diseases.

Declaration of competing interest

Nina Melnychuk and Andrey S. Klymchenko are co-inventors of a patent application: European patent application n° 18305253.9. The authors declare that they have no other known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authorship Contribution statement

Fig. 2 .

 2 Fig. 2. Design and fluorescence properties of light-harvesting nanoantenna particles (donor NPs). (A) Azidemodified polymer PMMA-AspN3 (left), dyes (upper row) and counterions (lower row) used for the nanoparticles preparation. (B) Nanoparticles preparation scheme: nanoprecipitation from acetonitrile (ACN) to phosphate buffer (PB). (C) Quantum yields of NPs prepared with different dye salts at different loadings. Error bars are standard deviation (n = 3). (D) Fluorescence spectra for the NPs formulated with the four different dye salts at 250 mM loading.

Fig. 3 .

 3 Fig. 3. Preparation, optical properties and antenna effect of DNA nanoprobes. (A) Preparation of nanoprobe: functionalization of dye-loaded nanoparticles with DNA and subsequent annealing with the FRET acceptor bearing oligonucleotide. (B) Structure of the FRET acceptor ATTO647N. (C) Fluorescence spectra, (D) FRET ratio (semiquantitative FRET efficiency), antenna effect, and (E) quantum yield of NPs loaded at 250 mM of R6G-C18 with two different counterions (F12 and F9-Al) with increasing number of ATTO647N acceptors per NP. Error bars are standard deviation (n = 3).

Fig. 4 .

 4 Fig. 4. Ratiometric detection of the DNA target. (A) Fluorescence spectra of NP-probe-65 incubated with increasing concentrations of the DNA target sequence. The spectra were normalized at the donor intensity. (B) FRET ratio as a function of target concentration. (C) Data points in the square shown in (B). Error bars are standard deviation (n = 3).

Fig. 5 .

 5 Fig. 5. Nucleic acid detection by NP-probe-65 on glass surfaces using scientific-grade sCMOS with image splitting (GEMINI) and RGB camera. (A) Surface immobilization of nanoprobe for microscopy experiments. (B) and (C): Detection of DNA target (survivin) at the single-particle level using sCMOS (Hamamatsu Orca Flash 4) with image splitter (B) and RGB (Nikon DS-Fi3) camera (C). Samples were incubated for 3 h (rt) with nucleic acid target at different concentrations. From up to down, fluorescence microscopy images: donor channel, acceptor channel, donor + acceptor merged image, and ratiometric image of the acceptor (red) over the donor (green) channel. Excitation was at 488 nm with irradiance of 2 W cm -2 . (D) Ratio distribution histograms for the corresponding ratiometric images using the two camera types. Scale bars: 5 µm.

Fig. 6 .

 6 Fig. 6. Smartphone-based detection of nucleic acids by NP-probe-65 on glass surface. (A) Smartphone-based detection setup. (B) RGB true-color images acquired with a smartphone (iPhone SE). Images were acquired after 3 h of incubation (rt) with DNA target (survivin) at different concentrations. (C) Ratiometric images of the red over the green channel from the images shown in (B). Excitation was at 488 nm with irradiance of 2 W cm -2 . (D) Ratio distribution histograms for the images shown in (C). Scale bars: 25 µm.

Acknowledgments

This work, CS and NM were supported from European Research Council ERC Consolidator grant BrightSens 648528 and SATT Conectus Alsace grant Nanoantenna. We thank I. Krossing for providing sample of lithium tetrakis(perfluoro-tert-butoxy)aluminate.

Caterina Severi: most of the experimental work, data analysis, paper writing. Nina Melnychuk: synthesis of the polymer and some dyes, help with nanoprobe synthesis, manuscript reviewing. Andrey S. Klymchenko: conceptualization, supervision of the project, help with data analysis and fluorescence microscopy, paper writing.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org


Dr Nina Melnychuk

Sylvie Egloff

Dr Anne Runser

Dr Andreas Reisch

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

Light-harvesting nanoparticle probes for FRET-based detection of oligonucleotides with single-molecule sensitivity

Keywords: light-harvesting nanoantenna, fluorescent nanoparticles, Förster resonance energy transfer, singlemolecule detection, nucleic acids biosensor

Controlling emission of bright luminescent nanoparticles by a single molecular recognition event remains an ultimate challenge in the design of ultrasensitive probes for biomolecules. It would require an efficient Förster resonance energy transfer (FRET) from the nanoparticle to a single acceptor at its surface, which is not realized to date. Here, we developed 20-nm light-harvesting nanoantenna particles, built of a tailor-made hydrophobic charged polymer poly(ethyl methacrylate-co-methacrylic acid), encapsulating ~1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87-fold brighter than quantum dots QDots605 in single-particle microscopy (with 550-nm excitation), these DNAfunctionalized nanoparticles exhibit >50% total FRET efficiency to a single hybridized FRET acceptor, a highly photostable carbopyronine dye (ATTO665). The obtained FRET nanoprobes enable singlemolecule detection of short DNA and RNA, encoding a cancer marker, and imaging single hybridization events by an epi-fluorescence microscope with ultralow excitation irradiance close to ambient sunlight.

Introduction

Detection of biomolecular targets, such as nucleic acids, with single-molecule sensitivity is an ultimate goal in biosensing and biomedical diagnostics. [1] In optical detection of single molecules, the brightness of organic dyes is limited by molar absorption coefficients below 300,000 M -1 cm -1 and quantum yields below unity. [2] Therefore, the use of very high excitation powers (~1 kW cm -2 ) and minimized detection volumes [1a, 3] is required in order to achieve good signal-to-background ratio. Plasmonic nanomaterials can address this problem by enhancing emission of single dyes in the hot spots, [1h, 4] in particular between two gold nanoparticles with strictly respected distances. [3b] This sophisticated system based on DNA origami enabled up to 100-300-fold signal amplification of single molecules. [3b, 5] However, so far, the reported biosensor for nucleic acids based on this concept can amplify the signal by 7-fold on the average. [6] A promising amplification approach is to use bright luminescent nanoparticles (NPs) [7] as energy donors that pump excitation energy to a single dye molecule by Förster Resonance Energy Transfer (FRET). [8] However, nanoparticles are generally not efficient FRET donors, because their sizes are beyond the FRET operating range between 1 and 10 nm, defined by Förster radius (R0). [9] Indeed, in case of quantum dots (QDots), 10-50 acceptor molecules are needed to ensure efficient FRET. [10] Similar large number of acceptors are also needed to ensure FRET from upconverting NPs (UCNPs) [11] and dye-doped silica NPs. [12] One way to go beyond the Förster radius is to use light-harvesting nanomaterials [13] where the strongly coupled energy donors communicate through excitation energy migration, [14] which allows transporting the energy through long distances up to the FRET acceptor. Previous works showed that energy migration can improve FRET efficiency in both dye-doped silica NPs [12b] and UCNPs. [15] Moreover, in conjugated polymers, [16] this approach enabled 25-100-fold signal amplification, although no singlemolecule FRET-based detection was reported. New possibilities appeared with recently introduced giant light-harvesting nanoantenna, [8a] based on dye-loaded polymeric NPs. [17] In these NPs, dyes (R18, Scheme 1A) are loaded into polymer matrix at high concentration using bulky hydrophobic counterions (F5-TPB) that prevent dye self-quenching [18] and leaching (leakage) in biological media, including cells. [18c, 18d] Importantly, the high dye loading ensured short inter-fluorophore distance (~1 nm) controlled by the counterion, which enabled ultrafast dye-dye excitation energy migration on a time scale < 30 fs. [8a] In this system the excitation energy can freely migrate through the whole particle within the fluorescence lifetime until it reaches a donor close to the acceptor leading to FRET (Scheme 1A). Therefore, the energy can be transferred beyond R0 from multiple donors to a single acceptor, providing a basis for signal amplification. Indeed, in our nanoantenna, ~10000 energy donors transferred energy to a single acceptor inside 60-nm particle (R0 = 6.1 nm for R18 / cyanine 5 couple) leading to unprecedented signal amplification of >1000-fold. [8a] More recently, we functionalized 40 nm nanoantenna with nucleic acids and reported the first DNAbased biosensor featuring exceptional brightness of 3300 encapsulated R18 dyes and 75-fold signal amplification. [19] However, to achieve efficient FRET in these NPs, 23 acceptor dyes at the particle surface were needed, so that this system was still far from being able to detect single nucleic acid molecules. To the best of our knowledge, no luminescent NPs with a diameter above 10 nm are capable to undergo efficient FRET (i.e. >50%) to a single acceptor dye at the particle surface. Herein, we overcame the problem of inefficient FRET in nanoparticle-based biosensors and developed a light-harvesting nanoantenna of 20 nm size, encapsulating ~1000 dyes with 52% fluorescence quantum yield (QY), which undergoes FRET with 51% efficiency to a single acceptor (ATTO665) at the DNAfunctionalized surface. This new nanomaterial enabled preparation nanoprobes capable to switch their color in response to a single copy of RNA and DNA molecules and monitor in real time single hybridization events using a basic epi-fluorescence microscope with low excitation power density (irradiance) close to that of ambient sunlight.

Results and Discussion

Development of this single-molecule biosensor requires boosting FRET performance of our dye-loaded NPs. To this end, we need to design a polymer capable to assemble maximum number of dyes into a particle of optimal small size, while ensuring their efficient emission and excitation energy migration (Scheme 1A). Moreover, we need to find robust FRET acceptor able to collect energy from the whole particle without rapid photobleaching. Then, based on a previously developed DNA-functionalization strategy, [19] the donor NPs should be modified with only a single FRET acceptor through a nucleic acid sensing unit. In our biosensor, azide-functionalized dye-loaded polymeric NPs are modified with two different oligonucleotide sequences (Scheme 1B): (i) excess of non-coding T20 to ensure NPs stability and (ii) a statistically one copy per particle of specific sequence (SurC), encoding a fragment of survivin, an important cancer marker. [20] The latter is hybridized with a shorter complementary DNA sequence (target competitive sequence, TCS) bearing the acceptor, which serves in the FRET displacement assay to detect the survivin DNA/RNA target (Scheme 1B). First, we searched for the best FRET acceptor, using previously developed dye-loaded donor NPs based on polymer PMMA-AspN3-1.6% (obtained from PMMA-MA with 1.6% of methacrylic acid). [19] Among ten tested acceptor dyes (Figure 1A) conjugated to DNA TCS, only positively charged and neutral (zwitterionic) cyanines (Cy5 and Cy5.5) and carbopyronines (ATTO647N and its close analogue ATTO665 with undisclosed structure), were found to provide a strong FRET signal after hybridization in the DNA nanoprobe. This signal was observed as an intense emission band around 650-750 nm compared to the FRET donor band of R18 dye around 590 nm (Figure 1B andS1). The number of hybridized acceptors per particle was also larger for these dyes (Table S1), probably because their duplexes were more stable than those bearing strongly anionic dyes. Then, we tested the fluorescence response of the obtained four nanoprobes to the target DNA sequence encoding survivin fragment. The three nanoprobes based on Cy5, ATTO647N and ATTO665 showed complete replacement of the acceptor, resulting in loss of the acceptor emission (Figure S2). By contrast, Cy5.5-based acceptor showed residual FRET in the presence of the DNA target, showing that it remains non-specifically bound to the particle (Figure S3). We also compared photostability of the four nanoprobes in solution under laser irradiation (Figure S4). Remarkably, nanoprobes built of Cy5 or Cy5.5 dyes showed fast decay of the FRET acceptor signal, expressed as A/(A+D) ratio (Figure S4), whereas the signal from ATTO647N and ATTO665 was far more stable (half-life for ATTO665 and Cy5 was 3500 and 98 s, respectively). The significantly higher photostability of ATTO-based FRET acceptors was also confirmed at a singleparticle level (half-life for ATTO665 and Cy5 was 128 and 3.7 s, respectively), using fluorescence microscopy of nanoprobes immobilized on the glass surface (Figure 1C, S5, S6). The observed much higher photostability for ATTO665 and ATTO647N is probably due to their more rigid carbopyronine scaffold [21] compared to flexible Cy5 dyes prone to photoinduced reactions. [22] The obtained results suggest that, in comparison to Cy5, ATTO665 as FRET acceptor should provide ~35-fold more photons before photobleaching, which is important for construction of the single-molecule FRET nanoprobe. After finding the best acceptor (ATTO665), we turned our attention to nanoantenna particle as the FRET donor. The primary approach to improve FRET donor efficiency of a particle together with its light-harvesting capacity is to minimize its size while keeping largest possible number of encapsulated donor dyes. Our earlier studies showed that smaller polymer NPs can be obtained by increasing the pH during nanoprecipitation [8a] or increasing the charge on the polymer. [23] In the first approach, we performed nanoprecipitation of PMMA-AspN3-1.6% at pH 9, which yielded NPs of 30 nm (Figure S7 A-B). However, the obtained DNAfunctionalized nanoprobes required >8 acceptors per particle to achieve efficient FRET (Figure S7 C-D), so further decrease in the size was needed. Therefore, we increased the fraction of methacrylic acid units in PMMA-MA to 5 mol%, and further converted it into azide-carboxylate-bearing polymer PMMA-AspN3-5% (Scheme 1). Moreover, to compensate the increase in the fraction of polar carboxylates in the polymer, we replaced its methyl methacrylate unit with more hydrophobic ethyl methacrylate (PEMA-AspN3 polymer, Scheme 1, obtained from PEMA-MA with 1.6% of methacrylic acid). Nanoprecipitation of these polymers yielded dye-loaded NPs of 16 and 18 nm for PMMA-AspN3-5% and PEMA-AspN3, respectively (Figure 2 A-B). Strikingly, PEMA-AspN3 NPs outdid PMMA-AspN3-5% NPs in all tested properties: (i) better size homogeneity according to TEM (Figure 1 A-B); (ii) higher fluorescence quantum yield (54 vs 31% at 30 wt% RhB/F5-TPB loading); (iii) 2.7-fold higher single particle brightness and (iv) much lower blinking and higher photostability (Figure 2 C-D, S8, Table S2). Thus, combining higher fraction of carboxylic groups with higher hydrophobicity of PEMA matrix enabled us to prepare very small dye-loaded NPs with drastically improved morphology and optical properties. As the particle brightness and the nanoantenna performance are directly linked to the dye loading, we varied it from 0 to 100 wt% with respect to the PEMA-AspN3 polymer. Even though QY values remained high (40-60%) for all loadings tested (Table S3), above 50 wt% the NPs showed lower single-particle photostability (Figure S9), probably linked to larger fraction of photo-induced dark states. [18b] Therefore, we selected 50 wt% as the optimal dye loading, exhibiting QY of 52%. In addition, increase in the dye loading till 50 wt% led to >100-fold drop in the fluorescence anisotropy (Figure S10), which suggested strong coupling of closely packed dyes inside NPs and efficient excitation energy migration within donor dyes independently from the acceptor. [14, 18a, 18b] This process should ensure long-distance excitation energy transport up to acceptor leading to an overall FRET beyond the Förster radius (Scheme 1A). [8a] Next, in order to prepare the nanoprobes, we functionalized dyeloaded PEMA-AspN3 with oligonucleotides. It was important to increase the concentration of oligonucleotide-DBCO in order to ensure stability of these small DNA-NPs (Figure S11). In the obtained FRET nanoprobes we varied the number of hybridized TCS-ATTO665 acceptors from 0 to 3 (Figure S12) and found that FRET signal was sensitive to the addition of a single acceptor (Figure 2 E-F, S12). Indeed, the nanoprobe with a single acceptor exhibited remarkably high total FRET efficiency (51%), which implies efficient energy transfer from 980 donors inside the 20-nm particle to one acceptor at its surface, i.e. far beyond the Fӧrster radius (R0 = 6.7 nm, estimated for R18 and ATTO665 in our system, Table S3). The nanoprobe also showed high total fluorescence quantum yield (46%), while the amplification of acceptor emission (antenna effect) was 209 ± 16. This outstanding performance was highly reproducible in 5 independent preparations (Table S4). The obtained nanoprobes showed strong ratiometric response to the target survivinencoding DNA and RNA sequences, where the target detection was associated with the loss of FRET signal (Figure 2G, S13). At 10-pM nanoprobe concentration, both DNA and RNA targets could be quantified at low pM concentrations (limit of detection was around 2 pM, estimated as three times of the standard deviations from the control, Figure 2H). In this case, the sensitivity is limited by slow oligonucleotide hybridization at these low concentrations. Incubation of the nanoprobe with the target DNA containing 1, 2 and 3 mismatches revealed high sequence specificity of the probe (Figure 2G). Moreover, the nanoprobe was found compatible with human serum. Indeed, the presence of 10% serum in phosphate buffer did not affect FRET signal of the nanoprobe and its response to the DNA target was only slightly decreased (Figure S14). These results, in line with the first generation DNA nanoprobe, [19] indicate that (i) both the donors and the acceptor remained within the nanoprobe without signs of leaching to serum and (ii) the strand displacement at the particle surface is weakly affected by the complex protein mixture.

Figure 3. Characterization of nanoprobes at the single particle level. A) Scheme of PEMA-nanoprobe immobilization on a glass surface modified with BSA-biotin, neutravidin, and A20-biotin and B) Wide-field fluorescence microscopy of the immobilized NPs: donor, antenna-amplified acceptor and merged acceptor and donor Without (above) and with (under) TCS-ATTO665. Excitation wavelength was 550 nm with power density 0.24 W cm -2 . Signals from Donor and FRET-Acceptor were recorded at <640 and >640 nm, respectively. C) Wide-field fluorescence microscopy of the immobilized Qdot-605 excited at 550 nm with power density 15.4 W cm - 2 . To obtain comparable signals excitation power density for QDot-605 was 65-fold higher than that for PEMA-T20. D) Wide-field fluorescence microscopy of PEMA nanoparticles at direct excitation of acceptor at 640 nm with power density 27.7 W cm -2 . Integration time was 400 ms. Histogram of single-particle intensity distribution for E) NP-PEMA-T20 and Qdot-605 and F) acceptor through direct excitation and nanoantenna energy transfer. Smaller graph represents a distribution histogram of amplification of FRET-Acceptor emission (antenna effect) by the nanoprobe at the single-particle level. At least 2000 NPs were analyzed in each case. Examples of single-particle intensity curves of FRET nanoprobe (G) excited at 550 nm with power density 0.24 W cm -2 and recorded at donor and acceptor channels and (H) direct excitation of acceptor at 640 nm on TIRF setup with power density 24.7 W cm -2 . 50 wt% R18/F5-TPB dye loading was systematically used.

Then, our DNA-functionalized NPs were studied at the singleparticle level. PEMA-AspN3 NPs bearing only T20 were immobilized on A20-modified glass surface (Figure 3A) and compared with commercial Qdot-605 under the wide-field microscope at 550 nm excitation. We found that these 20-nm NPs were 87-fold brighter than Qdot-605 measured at the same conditions (Figure 3 A-C, E). This outstanding brightness for such a small particle size is explained by the high QY (52%) of 980 encapsulated rhodamine B derivatives, giving a theoretical brightness (number of dyes × extinction coefficient × QY) of 5.1 x 10 7 vs 5.8 x 10 5 M -1 cm -1 for Qdot-605.

FRET-based nanoprobes bearing a single acceptor showed emission in both donor (<640 nm) and acceptor (>640 nm) channels, whereas NPs without acceptor showed signal in the donor channel only (Figure 3B). Remarkably, the signal of acceptor excited through the nanoantenna (Acceptor-FRET) was 257 ± 30-fold stronger than that obtained by direct excitation (Acceptor-direct) (Figure 3D,F). This value matches well the antenna effect measured in solution, indicating that efficient energy transfer from a thousand of encapsulated dyes to a single acceptor at the NP surface results in the outstanding amplification. To the best of our knowledge, this is the largest antenna effect ever reported for optical biosensors, including those based on plasmonics.

To verify the number of acceptors in our nanoprobe, we analyzed emission traces of the individual particles and quantified the number of single-molecule bleaching steps, which is a signature of the single acceptor per particle. When excited through nanoantenna (at 550 nm), we estimated that 61±4% of NPs contained acceptors, among which 48±5% demonstrated single bleaching step, corresponding to a single acceptor per nanoprobe. Moreover, 67±7% of single step acceptor photobleaching events were accompanied by recovery of the donor emission (Figure 3G, Table S5). This opposite switching behavior of donor and acceptor emission is typically observed in single-molecule FRET measurements. [24] However, in our nanoprobe a single acceptor switches on/off emission of the entire NP containing ~1000 of dyes. Direct excitation of the acceptor in a total internal reflection (TIRF) mode with 640 nm laser (Figure 3H) showed that 52 ± 4% of acceptors underwent the single bleaching step, confirming that a large fraction of nanoprobes bear a single acceptor. The achieved >250-fold amplification of acceptor emission by the nanoantenna particles enabled single-molecule FRET detection (i.e. single-step bleaching of the acceptor and recovery of the donor) using simple epi-fluorescence microscope setup with a very low excitation irradiance comparable to ambient sunlight (36 mW cm -2 , Figure S15), which is >1000 times lower than that typically required for TIRF-based single-molecule detection. Then, we incubated the immobilized nanoprobes with increasing amounts of DNA target. Starting from 1 pM, individual particles switched their emission pseudo-color from red to green and the population of green species increased gradually (Figure S16). At the highest tested target concentration all nanoprobes switched their emission to green, showing the decrease in the FRET ratio parameter, A / (A + D) from 0.46 to 0.21 (Figure S17). The gradual switching indicates that the nanoprobe can be used for quantification of the nucleic acid targets. As our NPs bear only a single acceptor, the color switch of NPs corresponds to detection of the single nucleic acid molecule, so that we could practically calculate the number of target molecules hybridized with the nanoprobes. Therefore, we imaged a given area with immobilized nanoprobes and quantified the number of FRET-positive particles before and after incubation with DNA and RNA targets (Figure 4A). Without the target, no change in the number of FRET-positive nanoprobes was detected, whereas in case of DNA and RNA samples, 1433±87 and 768±24 particles, respectively, switched their color from red to green per 1600 µm 2 surface area (Figure 4 B-C). This "digital" detection of single-molecule targets correlated with corresponding drop in the average FRET ratio of NPs (Figure 4C, S18). Importantly, our nanoprobe enables detection of DNA/RNA targets with single-molecule sensitivity using basic epifluorescence microscope with excitation irradiance close to ambient sunlight (36 mW cm -2 ), something that has not been achieved to date. Here, the minimal detectable concentration of oligonucleotides is not limited by nanoprobe sensitivity but by the hybridization kinetics. Finally, we challenged our nanoprobes in another key application: to image in real time the single hybridization events between two labelled oligonucleotides. In the first experiment, immobilized donor nanoparticles bearing a single capture sequence were treated with TCS-ATTO665 during the imaging (Figure 5A). After addition of TCS-ATTO665, initially green NPs turned into red and then returned back to green (Figure 5C, Video S1). In the intensity traces, the single step jump of the acceptor emission was accompanied by the drop of the donor one, which corresponded to the single hybridization event. Then, the acceptor dye got bleached which resulted in exactly opposite single step changes: drop of the acceptor and jump of the donor. In the second experiment, we added shorter (8mer) TCS-ATTO665 (Figure 5B), which was expected to bind reversibly to PEMA-NPs bearing complementary capture sequence, similar to what is done in DNA-PAINT imaging methods. [25] Remarkably, we were able to follow continuous process of binding-unbinding of the oligonucleotideacceptor molecules to the donor NPs in our two-color images and the intensity traces, where donor and acceptor signals switched many times in the opposite way (Figure 5B, Video S2). The singlemolecule nature of these switching evens was confirmed by observed single steps in the intensity traces of the acceptor directly excited (at 640 nm) in TIRF microscopy (Figure S19). Thus, these two experiments show that, owing to >250amplification capability, our light-harvesting nanomaterial enables real-time detection of reversible and irreversible single oligonucleotide hybridization processes using a basic imaging setup with low excitation irradiance.

Conclusion

In conclusion, we developed an organic nanomaterial that breaks the FRET barrier in existing luminescent nanoparticle systems. It is based on a light-harvesting nanoantenna, where ~1000 of strongly coupled rhodamine dyes with 52% fluorescence quantum yield are confined within a 20-nm polymeric particle. The obtained nanoparticle, being as bright as 87 Qdots-605 in single-particle microscopy (excitation at 530 nm), shows unprecedented 51% total FRET efficiency to a single acceptor hybridized at the DNAmodified particle surface, generating antenna effect (amplification factor) of ~250. To the best of our knowledge, none of reported fluorescent nanoparticles can combine such high brightness and FRET efficiency to a single acceptor at the surface. High loading of bright rhodamine dyes with their bulky counterions (50 wt%) and a specially designed polymer in this nanoantenna ensure short interfluorophore distances with minimal self-quenching that lead to excitation energy migration within 1000 encapsulated dyes and further FRET to a single accepter. As a result, overall energy transfer takes place beyond the Förster radius with minimal energy losses. Thus, our 20-nm particle is an efficient FRET donor, like a small dye molecule, but it is ~1000-fold brighter. On the other hand, based on screening of 10 different FRET acceptors, we found that dyes of carbopyronine family (ATTO665 and ATTO647N) are up to 35-fold more photostable in our system than a Cy5 derivative. Based on these findings, we constructed FRET-based nanoprobe for nucleic acids operating by strand displacement mechanism, where we combined our DNAfunctionalized nanoantenna particles (FRET donor) with a single hybridized ATTO665-based acceptor.

Single-molecule microscopy of the nanoprobes confirmed efficient FRET to a single acceptor, where one photobleaching step of the acceptor was accompanied by fluorescence intensity jump of entire particle containing ~1000 of dyes. The obtained nanoprobes enabled unprecedented FRET-based detection of single copies of short DNA and RNA as well as monitoring single oligonucleotide hybridization events by a simple epi-fluorescence microscope at low excitation irradiance (36 mW cm -2 ), close to ambient sunlight. For comparison, conventional single-molecule detection techniques would require in this case >1000-fold higher excitation irradiance, TIRF microscopy and minimized detection volumes to achieve good signal-to-noise ratio. [1a, 3] The developed nanomaterials will drastically simplify single-molecule detection and open a route to FRET assays for ultrasensitive detection of biomolecular markers, especially microRNA, mRNA and DNA, which are present in biological samples at very low concentrations. [26] Finally, owing to their compatibility with complex biological media, such as serum, our nanoprobes will enable detection of nucleic acid targets directly in biological fluids and, ultimately, application to target detection and imaging in cells. [27] RESEARCH ARTICLE

Light

Light-harvesting nanoprobes

Scheme 1 .

 1 Scheme 1. Design of light-harvesting nanoprobe. (A) Principle of lightharvesting nanoantenna based on polymeric nanoparticle loaded with cationic dye (R18) and its bulky counterion (F5-TPB). FRET from donor dyes (green) to the acceptor (red) is shown by magenta arrow, whereas excitation energy migration within donor dyes is shown by yellow arrows. (B).Synthesis of the nanoprobes with a single TCS-Acceptor and their response to the target.

Figure 1 .

 1 Figure 1. Search for the best FRET acceptor. A) Chemical structure of dyes conjugated with TCS and used as FRET-acceptors. B) Normalized fluorescence spectra of PMMA-nanoprobes (PMMA-AspN3-1.6% polymer) with different acceptors and 30 wt% R18/F5-TPB dye loading. 20 mM phosphate buffer containing 30 mM NaCl and 12 mM MgCl2 was used. Excitation: 530 nm. C) Photobleaching kinetics of PMMA-nanoprobes formulated with different acceptors, immobilized on the glass surface and measured by microscopy. Excitation: 550 nm; power density: 0.41 W cm -2 .

Figure 2 .

 2 Figure 2. Development and evaluation of FRET nanoprobes. TEM images (scale bar 50 nm) and size distribution statistics for NPs of (A) PMMA-AspN3-5% and (B) PEMA-AspN3 polymers. C) Examples of single-particle intensity and (D) average intensity traces measured by microscopy for PMMA-AspN3-5% and PEMA-AspN3 NPs loaded with 50 wt% R18/F5-TPB. Excitation: 550 nm; power density: 0.41 W cm -2 . E) Absorbance and F) normalized fluorescence spectra of PEMAnanoprobes (50 wt% R18/F5-TPB dye) hybridized with 0 (NP-PEMA-T20) and 0.3, 1 and 2 TCS-ATTO665 acceptor. G) Fluorescence response of PEMA-nanoprobe (20 pM of TCS-ATTO665) to survivin DNA and RNA targets (200 pM, 3 h incubation at 20 °C). DNA targets containing 1, 2, and 3 mismatches were tested at the same conditions. The spectra in (F) and (G) were normalized at the donor emission. H) FRET ratio, A/(A + D), of PEMA-nanoprobes (10 pM of TCS-ATTO665) vs target DNA and RNA concentration (3 h incubation at 20 °C). A and D are the pick intensities of the acceptor (at 665 nm) and the donor (at 580 nm), respectively. Error bars are standard deviation (n = 3). 20 mM phosphate buffer containing 30 mM NaCl and 12 mM MgCl2 was systematically used.

Figure 4 .

 4 Figure 4. Detection of oligonucleotides with single-molecule sensitivity using PEMA-nanoprobes immobilized on a glass surface. A) Schematic presentation of

Figure 5 .

 5 Figure5. Imaging in real time single hybridization events using PEMA-SurC/T20 immobilized on a glass surface. Schemes of single-molecule irreversible (A) and reversible (B) hybridization of oligonucleotides TCS-ATTO665 of 12 nucleotides (12NA) and 8 nucleotides (8NA), respectively, with NP-PEMA-Surc/T20. Intensity traces of donor (black) and acceptor (red) of a single nanoprobe with addition of TCS-ATTO665 of 12NA (A) and 8NA (B) and corresponding two-color merged images of the same single particle. C) The same experiment as in (A): two-color merged images of a larger area at different times after addition of 50 nM of TCS-ATTO665. The excitation wavelength was 550 nm, 100x oil objective with power density 0.24 W cm -2 . Integration time was 400 ms. 50 wt% R18/F5-TPB dye loading was systematically used.

  highly emissive dyes capable to undergo >50% FRET to a single acceptor at the DNAfunctionalized particle surface. This nanomaterial enables unprecedented detection of single copies of DNA/RNA by a simple epi-fluorescence microscope with ultralow excitation power close to ambient sunlight. Nina Melnychuk, Sylvie Egloff, Anne Runser, Andreas Reisch and Andrey S. Klymchenko* Page No. -Page No.

Acknowledgements

This work was supported by the European Research Council ERC Consolidator grant BrightSens 648525. We thank Corinne Crucifix from the FRISBI platform for her help with the electron microscopy.


Andria Andriuzzi

Géraldine Michel

Claudiu V Dimofte

How Brand

How Brand Conversations on Social Media Prompt Jealousy in Brand Relationships

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

H1: Brands are perceived as more human when they use FFAs addressed to consumers. H2: The use of FFAs addressed to other consumers produces higher brand humanization perceptions for consumers of low than for consumers of high brand attachment. H3: Consumers display more jealousy after observing brand FFAs addressed to others. H4: Brand positioning moderates the focal effects, such that the links between the use of FFAs and (a) brand humanization and (b) jealousy is stronger for brands positioned on warmth relative to those positioned on competence. Study 1 (N = 188 adults from an online panel) addresses H1 and H2 in a 2 (brand FFA: absent vs. present) x 2 (brand attachment: low vs. high) between-subjects design. Participants imagined browsing their Facebook newsfeed and noticing a particular brand-consumer conversation. Brand FFAs were manipulated by including (or not) appreciative expressions into the brand's posts. Participants indicated the extent to which they anthropomorphized the brand on six 7-point semantic differential scales (adapted from [START_REF] Hudson | The Influence of Social Media Interactions on Consumer-Brand Relationships: A Three-Country Study of Brand Perceptions and Marketing Behaviors[END_REF]α = .89). A two-way ANOVA on perceptions of anthropomorphism with the two experimental factors as predictors uncovered a significant effect of brand attachment (F(1, 182) = 26.57, p < .001) and a significant interaction between the two factors (F(1, 182) = 7.19, p < .01). Consumers not particularly attached to the brand perceived the brand as more human when employing FFAs (vs. when not), but this effect was absent for consumers strongly attached to the brand.

Study 2 (N = 397 undergraduates) assesses the extent to which consumers would perceive the brand as more humanlike and express more positive brand perceptions and less jealousy when they (vs. another consumer and vs. control) would be the target of a brand's flattery (H3). The design was thus a three-cell (FFA target: self vs. other vs. control) betweensubjects comparison. Participants were asked to provide a specific brand that they had a relationship with, which was subsequently employed by the software in the study's cover story. The subsequent procedure and measure were similar to Study 1. A one-way ANOVA on the brand anthropomorphism item revealed a significant main effect of FFA: F(2, 389) = 5.65, p < .01. Respondents perceived that a brand's use of FFAs, whether aimed at the self (M = 5.05) or another consumer (M = 5.20) appeared as more humanlike than the same brand not employing FFAs (M = 4.60). Furthermore, participants observing an FFA aimed at the self reported less jealousy (M = 5.19) than those observing the brand aiming the FFA at another consumer (M = 4.57) or those noting a brand conversation that did not include an FFA (M = 4.53; F(2, 389) = 17.75, p < .001).

Study 3 (N = 126 undergraduates) adds a brand positioning variable such that more jealousy was expected when the brand was positioned on warmth rather than competence (H4). The design was thus a 3 (FFA target: self vs. other vs. control) x 2 (brand positioning: on warmth vs. competence) between-subjects. The cover story and measures replicated Study 2. Participants assessed brand positioning on a 6-point semantic differential item anchored at 1 = definitely warmth-based and 6 = definitely competence-based. A two-way ANOVA with the two experimental factors as predictors and the brand feelings item as the dependent measure revealed a significant main effect of FFA: F(2, 120) = 4.16, p < .02. Participants observing an FFA aimed at the self reported less jealousy (M = 5.43) than those observing the brand aiming the FFA at another consumer (M = 4.93) or those noting a brand conversation that did not include an FFA (M = 4.81). However, this effect did not vary significantly by brand positioning, potentially due to the study's low power.

While much research addresses the effects of social media in general, little is known about how brand flattery impact consumer response. This research identifies the relationships between brand flattery, brand humanization, and brand jealousy, thus contributing to literature on brand-consumer relationships.

SUMMARIZING TABLE

other vs. control

There is jealousy when the brand aims its FFA at another consumer. Study 3: H3, H4 3 (FFA target: self vs. other vs. control) x 2 (brand positioning: on warmth vs. on competence)

There is jealousy when the brand aims its FFA at another consumer.

No clear effect of brand positioning.