Synergistic Combination of Unquenching and Plasmonic

Letter pubs.acs.org/NanoLett

Synergistic Combination of Unquenching and Plasmonic Fluorescence Enhancement in Fluorogenic Nucleic Acid Hybridization Probes Carolin Vietz,† Birka Lalkens,† Guillermo P. Acuna,† and Philip Tinnefeld*,‡,† †

Institute for Physical and Theoretical Chemistry, and Braunschweig Integrated Centre of Systems Biology (BRICS), and Laboratory for Emerging Nanometrology (LENA), Braunschweig University of Technology, Rebenring 56, 38106 Braunschweig, Germany ‡ Department of Chemistry, Ludwig-Maximilians-Universitaet Muenchen, Butenandtstr. 5-13, 81377 Muenchen, Germany S Supporting Information *

ABSTRACT: Fluorogenic nucleic acid hybridization probes are widely used for detecting and quantifying nucleic acids. The achieved sensitivity strongly depends on the contrast between a quenched closed form and an unquenched opened form with liberated fluorescence. So far, this contrast was improved by improving the quenching efficiency of the closed form. In this study, we modularly combine these probes with optical antennas used for plasmonic fluorescence enhancement and study the effect of the nanophotonic structure on the fluorescence of the quenched and the opened form. As quenched fluorescent dyes are usually enhanced more by fluorescence enhancement, a detrimental reduction of the contrast between closed and opened form was anticipated. In contrast, we could achieve a surprising increase of the contrast with full additivity of quenching of the dark form and fluorescence enhancement of the bright form. Using single-molecule experiments, we demonstrate that the additivity of the two mechanisms depends on the perfect quenching in the quenched form, and we delineate the rules for new nucleic acid probes for enhanced contrast and absolute brightness. Fluorogenic hybridization probes optimized not only for quenching but also for the brightness of the open form might find application in nucleic acid assays with PCR avoiding detection schemes. KEYWORDS: Fluorescence quenching, plasmonics, single-molecule experiments, nucleic acid hybridization pon interaction of a fluorophore with a fluorescence quencher, the electronic excitation energy is transferred between the two such that only very weak fluorescence is observed. This phenomenon is exploited for fluorogenic nucleic acid hybridization probes including strand displacement or competitive hybridization probes,1,2 molecular beacons,3−5 or TaqMan probes.6,7 To study the fluorophore−quencher interaction in these probes, we utilize a molecular beacon-like fluorescence quenching hairpin (FQH). It consists of a nucleic acid sequence labeled with a fluorescent dye on one end and with an organic quenching moiety on the other end. In the hairpin conformation, the fluorophore and the quencher are in close proximity, and thus fluorescence is suppressed. Upon hybridization to a complementary target sequence, the hairpin structure is opened. This hybridization reaction thereby displaces the fluorophore from the quencher and liberates the fluorescence. The fluorescence increase is hence an indicator of the presence of the target sequence. In the development of fluorogenic nucleic acid hybridization probes, the improvement of the contrast between the quenched and the unquenched form has been a focus.8 Most commonly, a combination of an organic fluorophore and an organic quencher molecule was applied. Alternatively, quenching by small metal nanoparticles was used to realize the dark state of the closed hairpin structure,

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resulting in highly sensitive and selective sensors.9−12 In all previous approaches, the contrast between opened and closed form was optimized by improving the quenching of the closed, dark form. On the other hand, the brightness of the unquenched form seemed to be an immutable constant as fluorescence dyes with close to 100% quantum yield have been available for a long time. Only recently, it has become possible to place fluorescent dyes in nanophotonic structures, so-called optical antennas, to increase their effective absorption cross section and to influence their radiative rate constants.13−18 This has enabled new ways of increasing the brightness of fluorescent molecules by several orders of magnitude.19 The antenna hotspots for enhanced fluorescence were, for example, created by larger gold or silver nanoparticles (>40 nm) that were optimally forming more complex structures such as a dimer with a controllable gap.16,20 DNA origamis have proven as superior scaffold for constructing optical antennas as they allow arranging nanoparticles and additionally offer handles to place other constituents such as fluorescent dyes in the nanophotonic hotspot.21−23 Received: September 7, 2017 Revised: September 27, 2017 Published: September 28, 2017 A

DOI: 10.1021/acs.nanolett.7b03844 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. Experimental design. (a) DNA origami pillar equipped with an 80 nm silver nanoparticle and an FQH. Generally, the dye quencher pair (ATTO647N, red sphere and BBQ650, black sphere) is positioned to expect quenching (left inset, closed) until binding of a target sequence opens the FQH and fluorescence is released (right inset, opened). (b−d) Confocal scan images of single-molecule experiments. Green spots represent the fluorescence of ATTO542 indicating the locations of DNA origami pillars. Red spots represent the fluorescence of ATTO647N corresponding to opened FQH. A colocalization of both events is depicted in yellow. Scan images of the closed FQH, after target addition with opened FQH and after photobleaching are shown in b−d, respectively. See Figure S3 for scan images of all systems used.

fluorescence (see Figure 1a insets). The target ssDNA is complementary to both the loop and one-half of the stem. Upon opening the FQH, fluorescence is restored. As we anticipated substantial heterogeneity for a complex system consisting of DNA origami pillar, FQH, and a silver nanoparticle, we carefully designed a single-molecule assay that provides maximum information on the single-construct level. We first immobilized the DNA origami pillar equipped with 8 biotins on a bovine serum albumin (BSA)/biotin/ neutravidin coated glass coverslip. The DNA origami pillar also carried the FQH in the closed, quenched form. Subsequently, silver nanoparticles were added that could bind to capturing strands protruding from both sides of the DNA origami pillar during incubation overnight. Probably for steric reasons, in the presence of the hairpin only one nanoparticle bound per DNA origami pillar (see Figure S1). The DNA origami pillars are additionally equipped with one ATTO542 dye to identify their position on the coverslip. First we carried out confocal single-molecule imaging on a custom-built setup for pulsed interleaved excitation29 using alternating 532 (for ATTO542) and 640 nm (for ATTO647N) laser excitation [see Figure 1b−d and Figure S3 for false-color images of green excitation/green emission (green) and red excitation/red detection (red)]. From these scans, we identified the positions of the DNA origami pillars due to the ATTO542 fluorescence in the green spectral channel. Additionally, we extracted the intensity of single ATTO647N molecules upon

In this work, we placed a FQH in the hotspot of an optical antenna and studied the effect of the nanophotonic structure on the fluorescence of the quenched and the unquenched state. Previous experiments indicated that quenched fluorophores are enhanced more than high quantum yield fluorophores.14,24−27 Hence, a reduction of the contrast between the closed and opened conformation might be anticipated. In contrast, we show that a careful design of the quenching mechanism in the FQH enables a contrast increase between the closed and opened conformation in combination with an absolute intensity enhancement of the opened conformation. This synergy of hairpin quenching with fluorescence enhancement of the bright form opens unexpected potentials for new generations of fluorogenic nucleic acid hybridization probes with increased brightness. For studying the influence of the proximity of a plasmonic nanostructure on the behavior of a fluorescence quenching hairpin (FQH), an optical antenna consisting of an 80 nm silver nanoparticle (NP) attached to a DNA origami pillar20,28 was modified for incorporating an FQH (Figure 1, SI).20,28 Similar to a molecular beacon,3 the FQH consists of a hairpin-like structure with 15 nucleotides forming the stem and 15 nucleotides forming the loop. This design allows experiments on the single-molecule level avoiding short opening events at room temperature. In the closed configuration, quencher (BBQ650) and fluorophore (ATTO647N) are in close proximity suppressing B

DOI: 10.1021/acs.nanolett.7b03844 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 2. Relative fluorescence intensity and total fluorescence enhancement for FRET quenching. Design of the FRET FQH (green) without (a) and with (b) a target (blue). The target opens the FQH and releases the fluorescence. (c) Relative intensities of the closed FQH with (blue) and without 80 nm nanoparticle (purple). The intensity is relative to a free single fluorophore (Atto647N). (d) Total enhancement observed for the FRET FQH with (blue) and without nanoparticle (purple). Results of 136 molecules are shown; error bars show standard errors.

Figure 3. Relative fluorescence intensity and total fluorescence enhancement for contact quenching. Design of contact-quenched FQH (green) without (a) and with (b) a target (blue). The target opens the FQH and releases fluorescence. (c) Relative intensities of the closed FQH with (blue) and without 80 nm nanoparticle (purple). The intensity is relative to a free single fluorophore (Atto647N). The inset shows a magnified view around 0.0 fluorescence intensity. Negative values are reported after background correction probably due to noise. (d) Total enhancement observed for the contact-quenched FQH with nanoparticle (blue) and without nanoparticle (purple). Results of 217 molecules are shown; error bars show standard errors.

640 nm excitation in the red spectral channel. In the next step, we incubated the coverslip with 1 nM solution of the target DNA overnight. Coverslips were glued on the microscope to minimize overnight drift. Next, we scanned the same region of the surface again and analyzed the fluorescence of the same ATTO647N molecules. After binding of the target DNA, many hairpins opened, and unquenched fluorescence was released (see Figure 1c). In a third step, we photobleached the ATTO647N molecule and scanned the surface a third time (Figure 1d). This allowed extracting intensity values for quenched and unquenched forms as well as for the background for each single DNA origami pillar FQH construct. To evaluate

the influence of the plasmonic structure on the FQH assay, this set of experiments was carried out for the DNA origami pillar nanoparticle FQH constructs as well as for a control without nanoparticles. In a first realization, we designed an FQH using FRET from the dye ATTO647N to the dark quencher BBQ650 (Figure 2a). The quencher was attached to the 3′ end of the hairpin while ATTO647N was labeled internally on the other side of the hairpin at a distance of 10 base pairs (closed conformation).8 For this dye-quencher pair we expected 83% quenching efficiency for Förster energy transfer at a calculated distance of 5.0 nm.30,31 Opening of the FQH by the target C

DOI: 10.1021/acs.nanolett.7b03844 Nano Lett. XXXX, XXX, XXX−XXX

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and 3.6-fold plasmonic fluorescence enhancement of the unquenched form. To study why the remaining fluorescence of 1.7% of that of a single ATTO647N is not enhanced in the optical antenna, we carried out another set of control experiments. To this end, the closed contact-quenched FQH on the DNA origami pillar was measured at 70-fold higher excitation intensity and compared to a control sample without FQH. For many transients recorded, photobleaching steps were visualized indicating the presence of a weakly emitting and bleachable fluorescence impurity (see Figure S5). This impurity was found for both the FQH sample and the control sample and yielded similar intensities supporting the idea that the contact-quenched FQH is indeed completely dark. The remaining detected fluorescence of ∼1.7% is ascribed to bleachable impurities. The results are summarized in Figure 4 where the fluorescence intensities relative to an unperturbed dye molecule

sequence yields the opened form in which the dye is displaced from the quencher (Figure 2b). In the absence of nanoparticles, we found a most probable quenching efficiency of ∼85% close to the expected value (see pink bars in Figure 2c). The distribution is broadened toward higher values most likely due to structural heterogeneity or photophysical effects. The presence of the silver nanoparticle strikingly reduces the quenching and further broadens the intensity distribution (blue bars in Figure 2c). An average quenching of only 17% is observed. In the absence of nanoparticles and after opening, the hairpins (see scan in Figure 1c) emit fully unquenched fluorescence as determined from fluorescence lifetime measurements (exemplarily in Figure S4) that yielded unperturbed values. For this configuration, an increase of 2.9 ± 0.4 fold was recorded, whereas in the presence of nanoparticles slightly higher values (3.7 ± 0.3 fold) were obtained (see Figure 2d). As the dye is already brightened in the closed conformation in the optical antenna, the contrast from quenched to unquenched form is similar. In this case the plasmonic effect is not additive with the hairpin quenching, but it is almost completely compensated by the brightness increase of the quenched form. As a net effect, the optical antenna similarly increases the brightness of the quenched and unquenched forms, and the positive effect is limited to overall moderately brighter molecules. An influence of the plasmonic nanoparticles on the FRET rate between dye and quencher likely explains why the quenched form is not enhanced even more than the unquenched form.32−34 To circumvent these limitations and obtain a higher total enhancement or contrast, we studied an alternative quenching mechanism. We reasoned that quenching and enhancement might be additive if the enhancement of the quenched form could be prevented by completely suppressing fluorescence in the quenched form. To this end, we redesigned the dye− quencher interaction to promote the formation of ground state complexes that exhibit a negligible radiative rate.35 The redesigned hairpin promotes direct contact between the dye ATTO647N and the quencher BBQ650 (see sketch in Figure 3a,b). Our assignment of quenching mechanisms is corroborated by the absorption spectra of the components involved (see Figure S2). The closed FRET FQH shows absorption spectra which represent the sum of the individual constituents. In contrast, the contact-quenched FQH exhibits a blue-shifted shoulder at 605 nm indicative for ground state complex formation with H-type geometry.35 Upon opening of the contact-quenched FQH the absorption spectrum also resembles the sum of the individual constituents. For the contact-quenched FQH, we carried out the same series of experiments on the same molecules as described for the FRET FQH above. Without nanoparticles, the hairpin is strongly quenched with a remaining fluorescence intensity of 1.7 ± 0.6% relative to the unquenched hairpin (purple bars in Figure 3c). Interestingly, bound to the optical antenna, the brightness of the quenched form is identical with an average fluorescence intensity of also 1.7 ± 0.2% of that of a single unquenched ATTO647N dye (blue bars in Figure 3c). Opening the hairpin recovers the fluorescence completely for the hairpin without antenna and 59 ± 21 times brighter hairpins are observed (Figure 3d). For the optical antenna, the unquenching yields brighter dye molecules that are even 217 ± 31-fold brighter than the quenched hairpins (Figure 3d). This implies that we observe full additivity of a 59-fold unquenching

Figure 4. Contrast of the FQH with FRET quenching and with contact quenching. Fluorescence enhancement for all cases is investigated as average with standard errors. The red line denotes the signal of a single, unquenched dye not affected by nanoparticles. The enhancement can be split into a part caused by unquenching (below red line) and by plasmonic enhancement (above red line).

are displayed for the different samples studied. For the FRET quenching, plasmonic fluorescence enhancement is achieved, but the contrast does not increase as the intensity of the quenched form increases similarly. For the contact-quenched FQH, the quenching is more pronounced and is not reduced by the plasmonic nanostructure. In combination with an absolute intensity enhancement of the opened FQH, the contrast is increased revealing the full additivity of quenching and enhancement. Fluorogenic nucleic acid hybridization probes have a broad range of applications and were optimized for high contrast between quenched and unquenched forms. Hitherto, contrast improvement implied optimized quenching of the dark form. Here, we demonstrate that fluorogenic nucleic acid hybridization probes can further be improved by additionally increasing the brightness of the opened form with optical antennas. While previous studies indicated that the combination with antenna fluorescence enhancement might have detrimental effects on the quenching of the dark form, we devised a strategy to synergistically combine quenching with plasmonic enhancement in a contact-quenched hairpin. We first showed that FRET quenching is indeed reduced by the D

DOI: 10.1021/acs.nanolett.7b03844 Nano Lett. XXXX, XXX, XXX−XXX

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presence of the optical antenna. In contrast, contact-induced quenching can quantitatively quench a fluorescent dye to 0% quantum efficiency, and the full additivity of quenching and enhancement is obtained. Our study opens the way for a modular design of bioassays, in which fluorescence assays are placed in well-controlled plasmonic nanoreactors using DNA nanotechnology. The modularity will allow separate optimization of biomolecular assays (like faster binding kinetics) and plasmonic enhancement systems (like stronger enhancement by adapting the antenna geometry) for optimized overall performance. In addition, the advantage of an absolute brightness increase also enables facilitated detection. We envision that a single target molecule generates such strong and specific signal that on the one hand the usage of less sensitive detection devices is possible and on the other hand even low concentrations are detectable without the need of further molecular amplification, opening up new possibilities in the field of molecular diagnostics.

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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.7b03844. Experimental methods, fluorescence lifetime and intensity distributions, absorption spectra, further experimental details, analysis details, details on the design of the DNA origami structure, list of staples, folding program, AFM image of DNA origami structure (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +49 89 2180 77549. ORCID

Carolin Vietz: 0000-0002-8417-4168 Guillermo P. Acuna: 0000-0001-8066-2677 Notes

The authors declare the following competing financial interest(s): The authors have filed a provisional patent application, EP17159353.6, on the described method of combining nucleic acid hybridization probes with plasmonic enhancement.

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ACKNOWLEDGMENTS We thank Kristina Hübner for AFM images and Tim Schröder, Mario Raab, Ija Jusuk, and Sarah Ochmann for stimulating discussions. This work was funded by the BMBF (Point-ofCare-Diagnostik mit Einzelmolekül-Nachweis POCEMON, 13N14336) and the Deutsche Forschungsgesellschaft (AC 279/2-1 and TI 329/9-1). C.V. is grateful to the Studienstiftung des deutschen Volkes for a scholarship. We acknowledge funding of the state ministry for research of lower saxony in the frame of the “Quantum- and Nanometrology” (QUANOMET) strategic research area. Quanomet is part of the LUH-TUBS research alliance.

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REFERENCES

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DOI: 10.1021/acs.nanolett.7b03844 Nano Lett. XXXX, XXX, XXX−XXX