Letter pubs.acs.org/NanoLett
DNA Origami Nanoantennas with over 5000-fold Fluorescence Enhancement and Single-Molecule Detection at 25 μM Anastasiya Puchkova, Carolin Vietz, Enrico Pibiri, Bettina Wünsch, María Sanz Paz, Guillermo P. Acuna,* and Philip Tinnefeld Institute for Physical & Theoretical Chemistry, and Braunschweig Integrated Centre of Systems Biology (BRICS), and Laboratory for Emerging Nanometrology (LENA), Braunschweig University of Technology, 38106 Braunschweig, Germany S Supporting Information *
ABSTRACT: Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration. Besides outperforming lithographic optical antennas, DNA origami nanoantennas are additionally capable of incorporating single emitters or biomolecular assays at the antenna hotspot. KEYWORDS: fluorescence enhancement, DNA origami, single-molecule detection, plasmonics, gold nanoparticles emission; a higher fluorescence signal is obtained due to an increase of the fluorophore’s quantum yield.23 In order to avoid the strong dependence of the fluorescence enhancement on the intrinsic fluorophore’s quantum yield, an alternative way to define a quantum yield independent fluorescence enhancement figure of merit was developed. Following this approach, the overall fluorescence enhancement is multiplied by the fluorophore’s intrinsic quantum yield.24 To date, the highest fluorescence enhancement, over 3 orders of magnitude, was obtained with dimer nanoantennas (DNs) using top-down lithography techniques.23,25 In both cases rather low quantum yield fluorophores were employed (under 10%) or a quenching agent was introduced, reaching a fluorescence enhancement figure of merit of approximately 90. Also with these structures, an observation volume in the subattoliter range was achieved leading to single-molecule occupancy at concentrations of 1 μM26 and 20 μM.25 Despite these impressive results, DNs are still not widely applied for biosensing. The reasons lie on the production difficulty, inherent to top-down lithography, but mostly on the challenge of positioning the biological assay of interest at the DN’s hotspot. Thus, these DNs are limited to FCS studies25,26 in which an elaborated model has to be applied to include a badly
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ollowing the pioneering works by Purcell1 and Drexhage2 plasmonic structures have been employed to control and manipulate molecular fluorescence. Depending on the plasmonic structure’s size, geometry, material, and relative position to the molecules, several effects were reported ranging from fluorescence quenching,3,4 enhancement,5−8 tailoring the dye’s emission directionality,9 photobleaching reduction,10,11 to light confinement below the diffraction limit.12−14 Although fluorescence quenching led to the development of optical rulers15 and light confinement enabled remarkable progress in the field of DNA sequencing,16 fluorescence enhancement holds promise for a realm of biosensing applications17 as well as for the detection of single molecules at biologically relevant concentrations.18,19 The interaction of a plasmonic structure with a fluorophore can lead to fluorescence enhancement based on the product of two factors. First is the enhancement of the absorption rate, which is proportional to the intensity of the electric field component parallel to the absorption transition dipole, at the fluorophore’s position. Second is the modification of the fluorophore’s quantum yield.5 Thus, for fluorophores with high quantum yields the strategy to obtain a significant fluorescence enhancement is to employ plasmonic structures that lead to a high electric field enhancement,20 typically gap-nanoantennas, although other geometries have been predicted and studied.21,22 Further enhancement can be obtained with fluorophores of low quantum yield. In this case, the interaction with the plasmonic structure may lead to a more efficient © XXXX American Chemical Society
Received: October 5, 2015 Revised: October 29, 2015
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Nano Letters defined interaction volume and the effect of the DN on photophysical parameters of the fluorophores.27 Bottom-up self-assembled antenna dimers based on colloidal nanoparticles (NPs) can also be produced, mostly using gold NPs functionalized with single stranded DNA oligomers with a thiol modification.28 In order to avoid aggregates, rather small particles were employed (typically under 40 nm),29,30 which together with the difficulty of aligning the dimer antenna to the incident field polarization lead to lower enhancement factors as compared to top-down lithographic DNs. Recently, selfassembled dimers were created by simply drying a colloidal gold NP solution.31 Beyond the production of dimer nanoantennas, the development of the DNA origami technique,32 enabled self-assembled templates where not only colloidal nanoparticles can be positioned with nanometer precision but also emitters or biomolecular assays can be placed in the hot-spot of the DN.33 Additionally, bigger particles can be employed and the dimer antenna can be aligned to the incident polarization. We have previously shown that the DNA origami technique can be used to build DN dimers with 100 nm colloidal gold NPs which exhibit a fluorescence enhancement of more than 2 orders of magnitude with a figure of merit of around 7520 and capable of detecting a single molecule in a 0.5 μM background34 for high quantum yield fluorophores (ATTO647N with a quantum yield of 0.65). In this contribution, we show that by a combination of factors including the improvement of the DNA origami structure, the reduction of the interparticle distance, alignment of the incident electric field polarization to the dimer orientation and introducing a quenching agent to reduce the fluorophore’s quantum yield, self-assembled DNs are substantially improved to compete with lithographically produced plasmonic structures and exhibit the additional advantage of placing objects in the plasmonic hotspot. Results and Discussion. Figure 1a shows a sketch of the DN that builds on a new DNA origami optimized for high yield and robust folding. Two 100 nm colloidal gold NPs forming a dimer are attached to the pillar-shaped DNA origami and a single ATTO647N fluorophore is incorporated at the center of the interparticle gap. The origami structure has a total height of 125 nm with the main shaft based on a 12-helix bundle. Over the last 29 nm, where the NPs are incorporated, the shaft is narrowed to a 6-helix bundle (see lower-left inset on Figure 1a). The NPs are functionalized with a thiolated T20 sequence and bound in the “zipper” configuration (see lower-right inset on Figure 1a and ref 35) to previously immobilized origami structures on a glass coverslip36 leading to an overall interparticle distance between 12 and 17 nm depending on the extent of steric effects on particle binding. Detailed information on the sample preparation can be found in the Supporting Information. A numerical simulation of the electric field intensity enhancement, in the vicinity of the 100 nm dimer structure, is included in Figure 1b. For a gap of 12 nm and an incident electric field polarization matching the dimer orientation at 640 nm, an electric field intensity enhancement factor of more than 500 is expected. As previously discussed, the metallic DN will also affect the fluorophore’s quantum yield depending on the fluorophore’s position, orientation, and quantum yield in the absence of the metallic nanostructure (hereafter termed “intrinsic” quantum yield). Figure 1c depicts a numeric simulation of the “effective” quantum yield37,38 at the dimer hotspot as a function of the intrinsic quantum yield for a dye oriented parallel to the dimer at an emission wavelength of
Figure 1. (a) Sketch of the DNA origami pillar (gray) employed to build the optical nanoantenna with two 100 nm Au nanoparticles together with a top-view (lower-left inset). The lower-right inset describes the “zipper” binding strategy to incorporate the Au nanoparticles to the origami structure. (b) Numerical simulation of the electric field intensity at the equatorial plane of the dimer structure with an interparticle distance of 12 nm at a wavelength of 640 nm and an incident electric field polarization parallel to the dimer orientation. (c) Numerical simulation of the fluorophore’s effective quantum yield at the nanoantenna hotspot as a function of the intrinsic quantum yield for a fluorophore at the hotspot aligned to the dimer orientation at a wavelength of 669 nm.
669 nm (peak emission of ATTO647N). The results show that the effective quantum yield at the hotspot lies between 0.775 and 0.825 depending on the gap distance considered for an intrinsic quantum yield higher than approximately 0.05. Therefore, for high intrinsic quantum yield fluorophores (close to 1) a reduction of the effective quantum yield occurs, whereas for low intrinsic quantum yield emitters the interaction with the dimer antenna leads to a significant (>10) enhancement. In order to take advantage of this enhancement, we employed a quenching agent, NiCl2 to reduce the intrinsic quantum yield of the ATTO647N fluorophore located at the antenna hot-spot.25,39 We focused on two concentrations of NiCl2 of 0.5 and 200 mM. In addition to quenching a high quantum yield fluorophore such as ATTO647N with NiCl2, we have also performed measurements with ATTO655 with an intrinsic quantum yield of approximately 0.3. We characterized the DNs performing fluorescence intensity and fluorescence lifetime measurements on a home-built B
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in good agreement with the product of the numerical simulations of Figure 1b,c. Using the intrinsic quantum yield of ATTO647N of 0.65, this value corresponds to a fluorescence enhancement figure of merit of 306, the highest reported to date. Analogous measurements were performed with different quencher (NiCl2) concentrations of 0.5 mM and 200 mM and also with a DNA origami pillar containing an ATTO655 fluorophore instead of the ATTO647N. The effect of the quencher on the fluorescence of ATTO647N was studied in two ways. On the one hand, we added the quencher in the single-molecule experiments of the dye attached to the DNA origami pillar but without gold nanoparticles. By comparing the brightness of single molecules with and without quencher, we found a fluorescence reduction to a factor of 0.58 for 0.5 mM and to a factor of 0.1 for 200 mM, respectively (see Figure S1 in the Supporting Information). On the other hand, we studied the quenching of ATTO647N labeled oligonucleotides by NiCl2 on the ensemble level in a spectrometer. In this case, the fluorescence is quenched to a factor of 0.13 for 0.5 mM and to a factor of 0.0034 for 200 mM NiCl2 (see Figure S2 in the Supporting Information). The difference in quenching efficiency is explained by the different local environments of the dye inside the DNA origami pillar on the surface or attached to an oligonucleotide diffusing in solution in combination with the interaction of the positively charged Ni2+ ions with negatively charged DNA.39 In addition, the quenching also leads to a biased selection of the brightest molecules when referencing is based on single-molecule measurements of fluorophores attached to DNA origamis immobilized on the surface. Figure 3a−c display histograms of the fluorescence intensities for the different quencher concentration with the same scale on the ordinate. Interestingly, the quencher does not noticeably alter the fluorescence intensity of ATTO647N when placed in the DN. Instead the intensity distributions appear similar, independent of quencher concentration. This is well in accordance with the simulations in Figure 1c, indicating that the quantum yield in the DN is rather independent of the intrinsic quantum yield over a broad range. Using the quenching of single fluorophores by Ni2+ in the immobilized DNA origami as a reference, we obtain a maximal fluorescence enhancement of 5468, which also constitutes the highest fluorescence enhancement reported to date for a DN. Comparing it to the quenching of the freely diffusing
confocal setup. For excitation, we employed an electro-optic modulator (EOM) to rotate the incident linearly polarized light (640 nm) at 20 Hz.40 Thus, we could match the incident electric field polarization with the DN orientation. Emitted fluorescence was spectrally separated using appropriate filters and detected using an avalanche photodiode and a module for time correlated single photon counting (TCSPC). Measurements were performed as follows. First, an image of a region of the sample was recorded and the position of the DNA origami structures was determined. Then, each structure was brought into the center of the confocal observation volume and fluorescence transients were recorded as exemplarily shown in Figure 2. Both reference (Figure 2, red, 10-fold more excitation
Figure 2. Single-molecule fluorescence transients for a DN (black line) and for a DNA origami structure without nanoparticles (red line) obtained using ten times more excitation intensity.
intensity) and transients of molecules in the DN dimer (black) exhibit blinking events and single-step photobleaching, typical features of a single molecule. Only transients with single-step photobleaching were considered to guarantee that only single molecules were studied. Molecules in the DN are, however, substantially brighter and their signal oscillates with the EOM rotation frequency of 20 Hz due to the symmetry and orientation of the DN.40 This oscillation is commonly not observed for the reference without DN (Figure 2, red) as fluorophores are attached to the DNA via a flexible linker and their dipole character, therefore, is averaged out at an integration time in the millisecond range. After smoothing, the maximum photon count of each DN is determined from the peaks of the transients. In the case of no quencher (Figure 3a), the maximum fluorescence enhancement factor obtained is 471
Figure 3. Photon count rate histogram for the dimer nanoantennas with no quencher (a) and with a NiCl2 concentration of 0.5 mM (b) and 200 mM (c). The axes on the right indicate the fluorescence enhancement depending on the reference employed. Immobilized reference refers to the quenching of the fluorophore bound to the DNA origami and immobilized on the surface. Diffusing reference refers to the quenching of labeled oligonucleotides in solution. C
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Nano Letters ATTO647N oligonucleotides, we even observe a fluorescence enhancement of the immobilized fluorophore of up to 160 000. Overall, we present four different enhancement values: (i) one that is independent of intrinsic quantum yield (fluorescence enhancement figure of merit, up to 306), (ii) one that refers to the unquenched dye (up to 471), (iii) one that uses the quenched dye in the immobilized origami as a reference (up to 5468), and (iv) one that uses diffusing oligonucleotides in solution that are labeled with the same fluorescent dye (up to 160 000). The question arises which enhancement parameter is relevant in which situation. The fluorescence enhancement figure of merit (i) is the best parameter to describe the coupling between the nanophotonic structure and the emitter independent of its “intrinsic” quantum yield. The fluorescence enhancement of the unquenched emitter (ii) best describes the highest achievable brightness of a high quantum yield fluorophore and how the potential is maximized to sensitively detect a weak fluorescence signal amplified by a nanophotonic structure. This number could, for example, quantify the ability of detecting single molecules on low-tec point-of-need devices such as a modified smartphone.41 The latter two parameters ((iii) and (iv)) focus on the dynamic range of the fluorescence enhancement and describe the ability to detect a single molecule in a background of other labeled molecules. As such, they are a hallmark of the important ability to extend the dynamic concentration range of single-molecule measurements to the biologically relevant micromolar to millimolar regime16,19,42 including nanobiotechnological assays as, for example, single-molecule DNA sequencing.17,43 Realistically, the fluorescence enhancement for our DN might not quite reach the stated 160 000 (iv) because the intrinsic quantum yield would be so small that it would not completely recover in the DN. Still, if the nanoenvironments were identical, a substantially higher fluorescence enhancement than the one observed for the immobilized origami reference (iii, 5468) could be expected. For several applications, it might be desirable to directly employ low quantum yield fluorophores without the necessity to add an extra quencher. We demonstrate the feasibility of this approach by replacing the ATTO647N fluorophore by an ATTO655 fluorophore at the DNA origami pillar hotspot. ATTO655 has an intrinsic quantum yield of 0.3, comparable to the intrinsic quantum yield of ATTO647N bound to the DNA origami pillar hotspot with 0.5 mM NiCl2 (0.38, obtained as the product of ATTO647N intrinsic quantum yield of 0.65 in the absence of quencher times the quenching factor of 0.58). Figure S3 in Supporting Information shows the fluorescence enhancement obtained for ATTO655 together with the results for ATTO647N and 0.5 mM NiCl2 (previously included in Figure 3b) for comparison. Comparable enhancements values are obtained as expected, with the highest value for ATTO655 around 600 fold. Finally, we employ the DNs for single-molecule detection at high concentrations as this is important for breaking the singlemolecule concentration barrier.17 We record fluorescence scans and transients with circularly polarized incident light and we add a solution of oligonucleotides labeled with ATTO647N dyes to the chamber (see Figure 4a). We increase the concentration in steps to determine the highest concentration at which a single molecule can still be detected. In the absence of the quencher agent, the highest concentration reached was 5 μM. A single-molecule transient under these conditions is shown in Figure 4b (black line). When a solution of 0.5 mM
Figure 4. (a) Photograph of the chambered coverglass employed with a solution containing ATTO647N at a concentration of 25 μM and NiCl2 at a concentration of 0.5 mM (blue). (b) Single-molecule fluorescence transients at a background concentration of 5 μM (black) and 25 μM (red).
NiCl2 is added, single molecules are even detected at concentrations as high as 25 μM (Figure 4b, red line). The photograph in Figure 4a shows the chambered cover glass employed with a solution containing ATTO647N at a concentration of 25 μM and NiCl2 at a concentration of 0.5 mM. Despite the dark blue color of the solution, single molecules are detected. In principle, higher concentrations of NiCl2 should lead to single-molecule detection at even higher concentrations. However, we could not explore this regime because higher concentrations of NiCl2 led to nonspecific binding of fluorophores to the substrate creating a detrimental background. Conclusions. DNA nanotechnology has opened new vistas for placing molecules in self-assembled plasmonic nanoantennas. We introduce and combine four innovations to self-assembled nanoantennas including a further developed DNA origami template, reduced interparticle distances by zipper binding, alignment of the incident polarization to the dimer orientation and quenching of the fluorophore’s intrinsic quantum yield. With these advances, DNA origami based selfassembled DNs can outperform top-down lithographic nanoantennas in terms of fluorescence enhancement and singlemolecule detection at elevated concentrations. Finally, it is important to emphasize that our approach enables the immobilization of a single emitter (or a biomolecular assay) at the antenna hotspot so that all data were obtained from single-molecule transients. We obtained a fluorescence enhancement figure of merit of 306, this factor which is independent of the fluorophore employed, is the highest reported to date. We also employed a quenching agent to reduce the fluorophore’s intrinsic quantum yield. In agreement with our numerical simulations, the quenching agent only reduces the fluorescence intensity for origami structures without NPs since at the DN hotspot the interaction with the NPs counteracts the quenching induced by the NiCl2 leaving the overall fluorescence intensity essentially unaffected. Due to the different quenching efficiencies for fluorophores immobilized with the DNA origami and for fluorophores D
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Nano Letters diffusing in solution we have introduced two ways of estimating the fluorescence enhancement. The “immobilized” enhancement and the “diffusing oligonucleotides” enhancement, the latter being more appropriate for applications involving the detection of a single immobilized molecule in a highly concentrated solution containing other fluorescent molecules. The optical antennas in combination with a 200 mM concentration of NiCl2 lead to a fluorescence enhancement of 5468 and 160 000 for the “immobilized” reference and “diffusing oligonucleotides” reference, respectively. We have also demonstrated that these antennas can be employed for the detection of a single molecule at a concentration of 25 μM. The robust design and the further improvements indicate the potential of self-assembled nanoantennas for biosensing and emerging nanobiotechnological applications. Materials and Methods. Gold nanoparticles of 100 nm diameter were purchased from BBI Solutions and functionalized with 20T DNA-oligonucleotides containing a thiol modification on the 3′ end (Ella Biotech GmbH). Details of nanoparticles modification as well as DNA origami pillar design and its folding conditions can be found on the Supporting Information. Purified DNA origami pillars were immobilized on a biotin-labeled BSA (Sigma-Aldrich) surface by the linker molecule neutravidin (Sigma-Aldrich) and incubated later with diluted nanoparticle solutions for 48 h. Nanoparticles were diluted by 1× TE containing additionally 100 mM NaCl and 12 mM MgCl2 to reach an absorption of approximately 0:2−0:3 on the UV/vis Spectrophotometer (Nanodrop 2000, Thermo Scientific). Confocal single-molecule measurements were carried out in 1× PBS buffer containing 12 mM MgCl2 or 0.5 mM and 200 mM NiCl2. Confocal Measurement and Analysis. In single-molecule fluorescence experiments a custom built confocal microscope based on an Olympus IX-70 inverted microscope is used. A 78 MHz-pulsed laser beam at 640 nm (SuperK Extreme, NKT Photonics, Denmark) is used to excite the fluorophores. To adjust the laser intensity a variable neutral density filter is installed. A combination of an electro-optical modulator (EOM, LM 0202, Qioptiq), a linear polarizer and a quarter wave plate (AQWP05M-600, Thorlabs) is used to set the required polarization of the laser beams (linear, rotating linear). The collimated light beam is coupled into the oil-immersion objective (UPL FL 100 X/.6−1.30 Iris, Olympus) through a dual-band dichroic beam splitter (zt532/640rpc, CHROMA, U.S.A.) and is focused to the measurement chamber, which can be positioned accurately by a Piezo-Stage (E-501.00, Physik Instrumente GmbH&Co. KG, Germany). The emission of the fluorophores is collected by the same objective, focused on a 50 μm pinhole (Linos), and split spectrally at 640 nm by another dichroic beam splitter (640DCXR, Chroma, U.S.A.). One Single-Photon Avalanche Diode (SPCM, AQR 14, PerkinElmer, U.S.A.) accounts for the detection after appropriate spectral filtering (RazorEdge LP 647, Semrock, U.S.A.). The signal of the APD is registered by a TCSPC system (HydraHarp 400, PicoQuant, Germany) and evaluated using custom-made LabVIEW (National Instruments) software.
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Details on the preparation of the DNA origami samples and the functionalization of the gold nanoparticles. Figure S1 includes the results for the fluorescence quenching of single ATTO647N fluorophores bound to immobilized DNA origami at different NiCl2 concentrations, whereas Figure S2 depicts the results of the quenching of freely diffusing dye labeled oligonucleotides. Figure S3 shows the results for the fluorescence enhancement obtained for both an ATTO655 fluorophore placed at the antenna hotspot and an ATTO647N fluorophore together with 0.5 mM NiCl2. A list of all the staple strands and modifications is included in Table S1 and the temperature folding program for the DNA origami structure is included in Table S2. (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to Andreas Gietl, Birka Lalkens, and Phil Holzmeister for stimulating discussion and to Frank Demming from CST for assistance with the numerical simulations. This work was supported by a starting grant (SiMBA, EU 261162) of the European Research Council (ERC), and the Deutsche Forschungsgesellschaft (AC 279/2-1 and TI 329/9-1). C.V. acknowledges support from the Studienstiftung des Deutschen Volkes, and B.W from the Nanomet program GRK 1952/1 of the Deutsche Forschungsgesellschaft.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b04045. E
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