DNA Origami-Based FÃ¶rster Resonance Energy-Transfer Nanoarrays

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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 23295−23302

DNA Origami-Based Förster Resonance Energy-Transfer Nanoarrays and Their Application as Ratiometric Sensors Youngeun Choi,†,‡,§ Lisa Kotthoff,†,‡ Lydia Olejko,† Ute Resch-Genger,‡,§ and Ilko Bald*,†,‡,§ †

Department of Chemistry, Physical Chemistry, University of Potsdam, 14476 Potsdam, Germany BAM Federal Institute for Materials Research and Testing, 12489 Berlin, Germany § School of Analytical Sciences Adlershof, Humboldt-Universität zu Berlin, 10099 Berlin, Germany ‡

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S Supporting Information *

ABSTRACT: DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye−dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Förster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 as a pH-inert FRET donor and FAM as a pHresponsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte-responsive dyes, demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in life sciences. KEYWORDS: DNA origami, nanoarray, FRET, ratiometric sensing, pH sensing

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INTRODUCTION

multichromophore systems, it is necessary to develop strategies to avoid fluorescence-diminishing dye−dye interactions. A powerful tool that allows for controlled arrangement of molecules with different functionalities is the DNA origami technology.6,7 As each oligonucleotide is addressable and can be easily modified chemically, a DNA origami structure can be folded to incorporate functionalities at predetermined positions. DNA origami structures have been meanwhile employed for the self-assembly of molecular and nanocrystal moieties8,9 including organic dye molecules,10−13 nanoparticles,14−16 quantum dots,17−19 and proteins.20−22 Because of their unique features, DNA origami nanostructures also present very interesting platforms for all applications utilizing FRET such as creating photonic wires,23,24 light-harvesting constructs,25−27 and sensing schemes.28 These FRET-based structures provide yet another crucial advantage for developing

Multiparametric fluorescence techniques utilizing molecular and nanoscale fluorescent reporters are widely used in the life and material sciences because of their ease of use, suitability for in vitro and in vivo imaging, sensitivity down to the singlemolecule level, and nanoscale resolution.1,2 A common strategy for signal enhancement is multichromophore systems. The efficiency of such systems is, however, largely controlled by dye−dye interactions. Depending on the orientation of the dye dipole moments, this can lead to the formation of H-type dimers and aggregates, which are commonly not or barely emissive and can act as an energy sink via Förster resonance energy transfer (FRET) between chemically identical, yet spectroscopically distinguishable fluorophores.3,4 FRET is a nonradiative energy-transfer process that occurs between an excited donor molecule and an acceptor molecule through dipole−dipole interactions. Its distance-dependent nature has prompted development of many analytical tools for sensing including biologically relevant molecules.5 However, for © 2018 American Chemical Society

Received: March 5, 2018 Accepted: June 19, 2018 Published: June 19, 2018 23295

DOI: 10.1021/acsami.8b03585 ACS Appl. Mater. Interfaces 2018, 10, 23295−23302

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ACS Applied Materials & Interfaces

Figure 1. (A) Folding process into DNA origami-based dye array. A triangular DNA origami is self-assembled first with extended staple strands to act as handles. The dye molecules are then positioned on the DNA origami by hybridization of the dye-labeled ssDNA with the overhanging ssDNA from the DNA origami. (B) FRET arrays and nomenclature used. Twelve positions from the DNA origami were chosen to act as dyelabeling sites, allowing to construct arrays of different sizes ranging from (2 × 1) to (3 × 4). The donor (orange) and acceptor (red) molecules were arranged in a checkerboard pattern while maintaining the ratio of donor to acceptor fixed at 1:1. (C,D) Normalized excitation (dotted line) and emission spectra (solid line) of donor and acceptor molecules with the spectral overlap of donor emission and acceptor excitation colored-in in gray, with (C) of FAM as a donor and Cy3 as an acceptor used for the systematic study of the nanoarray and (D) of coumarin 343 (C343) as a donor and FAM as an acceptor for its application as a pH ratiometric sensor. the desired pH. For more detailed description of folding scheme see the Supporting Information (Figure S-1). AFM Imaging. Atomic force microscopy (AFM) imaging was performed for each DNA origami sample to confirm their structure. The purified sample (2 μL) was adsorbed on freshly cleaved mica (Plano) with 28 μL of buffer (1× TAE with 15 mM MgCl2) and incubated for 30 s. After incubation, the sample was washed with ultrapure water and dried with compressed air. The imaging was performed under dry conditions (Flex AFM, Nanosurf) using cantilevers (Tap150 Al-G, Budget Sensors) with a resonance frequency of 150 kHz and a spring constant of 5 N m−1. An AFM image of a typical triangular DNA origami structure is shown in Figure S-2. Steady-State Fluorescence Spectroscopy. Steady-state spectroscopy measurements were performed with a spectrophotometer (FluoroMax P, HORIBA Jobin Yvon) with 3 mm quartz cuvettes (Hellma Analytics). Fluorescence emission was collected at a right angle to the excitation beam using an internal quantum correction system. For FRET nanoarray analysis using fluorescein (FAM) and cyanine 3 (Cy3), an excitation wavelength of 450 nm was chosen and for ratiometric pH sensing with C343 and FAM, an excitation wavelength of 400 nm. For collecting excitation spectra, the emission wavelengths were set to 600 and 550 nm. For all spectra, acquisition at an increment of 1 nm, integration time of 0.2 s, and bandpass of 5 nm were used. Time-Correlated Single-Photon Counting. The FRET efficiency is strongly distance-dependent according to eq 1

sensors as they can be designed as self-referenced dual wavelength probes. This eliminates analyte-independent signal distortions often encountered for single-color fluorescence intensity measurements.29−32 The broad applicability of such nanostructures requires not only the design of DNA origami nanoarrays with optimum brightness and spectroscopic performance but also with minimum cost. This can be achieved by a DNA template design, where dyes can be easily replaced to allow for simple adaptation to different tasks. Aiming to explore the potential of the DNA origami technology for light harvesting, signal amplification, and sensing, we assembled different donor and acceptor fluorophores on DNA origami structures and systematically assessed the influence of nanoarray size and dye pattern on their optical properties and FRET efficiency using steady-state and time-resolved fluorometry. The optimum design concept was then expanded to the design of ratiometric pH nanosensors utilizing pH-inert and pH-responsive dyes as FRET donors and acceptors, respectively.

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METHODS

DNA Origami Synthesis. The triangular DNA origami nanostructures were fabricated as previously described28 using M13mp18 (tilibit nanosystems) viral strand with 208 short oligonucleotides in TAE buffer (10× concentrated) containing 150 mM MgCl2 and ultrapure water (Merck Millipore). The solution was subsequently annealed from 80 °C and slowly cooled down to 8 °C in 2 h using a thermal cycler (PEQLAB, VWR). The solution was purified using centrifugal filters (100 kDa MWCO, Merck Millipore) by washing three times (6000 rpm, 7 min) with 1× TAE with 15 mM MgCl2 to remove excess staple strands. Subsequent hybridization was performed by mixing the DNA origami solution with dye-modified oligonucleotides (Metabion) in 1× TAE, 15 mM MgCl2 solution, heating up to 45 °C and cooling down to 25 °C over 1 h using the thermal cycler again. The final DNA origami solution was purified again using centrifugal filters six times. Final concentration of DNA origami in solution was set to approximately 5 nM for fluorescence measurements and checked using UV−vis absorption spectroscopy (NanoDrop 2000, Thermo Scientific). For pH ratiometric sensing, each pH buffer (1× TAE, 15 mM MgCl2) was prepared by adding appropriate amounts of HCl and NaOH, and the pH was measured using a pH meter (Orion 3 Star, Thermo Scientific). Buffers were exchanged at the second washing step of DNA origami folding to have

FRET efficiency =

R 06 6

R 0 + rDA 6

(1)

In eq 1, R0 is the Förster distance, at which the FRET efficiency is 50%, and rDA is the distance between donor and acceptor molecules. In order to avoid inaccuracies due to direct excitation of the acceptor, FRET efficiencies have been determined from the fluorescence lifetimes of the donor τ̅ FRET efficiency = 1 − DA τD

(2)

In eq 2, τ̅DA is the donor’s fluorescence decay time when FRET occurs (amplitude weighted fluorescence decay time) and τD is the donor’s fluorescence decay time in the absence of the acceptor. Time-correlated single-photon counting measurements were performed using the FLS920 fluorescence lifetime spectrometer (Edinburgh Instruments; F900 software) equipped with a supercontinuum laser SC-400-PP (0.5−20 MHz, 400 nm < λ < 24 000 nm, pulse width ca. 30 ps, Fianium/NKT Photonics A/S). Fluorescence 23296


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Figure 2. (A) Normalized emission spectra for FRET nanoarrays on DNA origami triangles excited at 450 nm. The emission intensity at 515 nm (FAM) decreases and the intensity at 565 nm (Cy3) increases as the size of the nanoarray increases. The color of each spectrum correlates with the array size as shown on the upper panel. The black line is the emission spectrum of FAM in the absence of Cy3 for a (3 × 4) array. A sharp peak at 520 nm is visible when the arrays are smaller, which is the water Raman peak (marked with an asterisk), indicative of the overall low fluorescence intensity. (B) Fluorescence decay curves obtained for FAM by exciting at 490 nm and recording at 520 nm. With the size of the nanoarray increasing, the lifetime of FAM decreases. Black line is the lifetime decay of FAM in the absence of Cy3 for a (3 × 4) array. (C) FRET efficiencies E calculated based on amplitude averaged decay times, and emission intensity ratios plotted against the nanoarray. The FRET efficiency reaches a saturation point at (3 × 2) array (light blue), whereas the intensity ratio remains rising. All data points were determined in triplicate, from three independent measurements.

Figure 1B shows the five different nanoarrays that were assembled in a checkerboard pattern of donor and acceptor molecules using this method ranging from two dyes, (2 × 1) array, to the largest nanoarray having 12 dyes, (3 × 4) array. The ratio between donor and acceptor molecules was always maintained at 1:1. In these nanoarrays, the closest distance between two dyes on the same side of the DNA origami plane is 6 nm, with a DNA double helix between them and an interhelix gap of 1 nm for the triangular DNA origami design.7 The distance between neighboring donor and acceptor along the same DNA double helix (solid black line) is 7.7 nm. The distance between the dyes that are “diagonal” to each other is 9.7 nm (again, assuming an interhelix gap of 1 nm). As the closest two donor molecules on this checkerboard patterned nanoarray will be separated by 9.7 nm, fluorescence selfquenching is unlikely. For more details regarding the exact positions see the Supporting Information (Figure S-1). In order to determine the dye arrangements that lead to optimum FRET efficiency, we chose FAM as a donor and Cy3 as an acceptor. The Förster radius of this FRET pair is 6.7 nm.28 FRET Nanoarray Analysis. In the designed nanoarrays, a combination of effects leads to a change in the emission intensity ratio of the donor and acceptor molecules. Placing multiple donors around one acceptor leads to a light-harvesting effect funneling the excitation energy to the acceptor,25,26 thereby leading to an enhanced acceptor emission. Structures, where multiple acceptor molecules surround one donor molecule, increase the FRET efficiency because of the increase in the number of pathways that the donor can transfer its energy to the acceptor.1 As the assembled nanoarrays with the checkerboard pattern grow larger, the number of donor molecules surrounding an acceptor molecule increases up to 4 (and vice versa) within the spatial dimensions where FRET can occur. Figure 2 (upper panel) displays the different nanoarrays color coded according to the size of the array. This color code is subsequently used in all figures.

emission was collected at a right angle to the excitation beam using a multichannel plate (ELDY EM1-132/300, Europhoton). The excitation wavelength for FRET nanoarray analysis (FAM−Cy3) was set to 490 nm and the emission wavelength to 520 nm. For ratiometric pH sensing (C343−FAM), an excitation wavelength of 450 nm and an emission wavelength of 490 nm were employed. The obtained decay curves were fitted multiexponentially using the FAST software (Edinburgh Instruments) and the following equations (eq 3) n

I(t ) =

∑ Ai e−t / τi i=1

(3)

where τi is the decay time and Ai is the amplitude for each decay time component. The amplitude averaged decay time τ̅DA is given as n

τDA ̅ =

∑i = 1 Ai τi n

∑i = 1 Ai

(4)

The last decay component is fixed to the unquenched donor decay time. Fö rster distances for dye pairs used were calculated using PhotochemCAD software.

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RESULTS AND DISCUSSION Design and Fabrication of FRET Nanoarray on DNA Origami. As shown in Figure 1A, DNA origami triangles were self-assembled including staple strands extended at their 5′-end by 21 nb either with (AAT)7 or with (CAA)7. These modified staple strands were used as handles to place dye molecules on the DNA origami platform at the allocated position. In the subsequent hybridization step, complementary DNA sequences [either (ATT)7 or (TTG)7, Table S-1] modified at the 5′end either with the donor molecule or with the acceptor molecule were added to the synthesized DNA origami. This allows the construction of a large number of different FRET nanoarrays without having to modify each staple strand with the dye molecule, making the folding process more flexible, efficient, and cost-effective. The labeling yield was expected to be no less than 80% according to that previously reported on hybridization efficiencies of single-stranded DNA (ssDNA) on DNA origami with slow dissociation rates.33−35 23297


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ACS Applied Materials & Interfaces The emission spectra of different nanoarrays in Figure 2A exhibit a high fluorescence intensity at 515 nm arising from the donor (FAM) and a second rise in intensity at 565 nm originating from the acceptor (Cy3). As the array grows larger from (2 × 1) to (3 × 4), in addition to the overall fluorescence intensity enhancement, the intensity ratio I(565 nm)/I(515 nm) increases from 0.9 ± 0.01 by 44% to 1.6 ± 0.03. The steady increase in the intensity ratio is plotted in Figure 2C. The enhancement in overall brightness of the nanoarray is also reflected by the relative decrease of the contribution of the water Raman peak (sharp peak at 520 nm, marked with an asterisk) to the measured signal as the array becomes larger. Subsequently, we performed time-resolved studies with these FRET arrays. The resulting decay curves are displayed in Figure 2B. The decay analysis was conducted using a multiexponential fit with four decay components with the last decay component fixed to unquenched FAM fluorescence decay time (τD = 4.5 ns). As follows from Figure 2B, the decay profiles show a rapid decrease in the donor fluorescence lifetime between the (2 × 1) array (dark blue, τ̅DA = 2.8 ± 0.2 ns) and (2 × 2) array (light blue, τ̅DA = 1.8 ± 0.02 ns). The difference in the donor fluorescence lifetime is not as significant for the larger arrays and reaches a minimum for the (3 × 4) array (magenta, τ̅DA = 1.5 ± 0.01 ns). The average lifetimes used here for the evaluation of our ensemble studies account for the multiple FRET pathways that exist within an array as well as the slight variations in lifetimes and lifetime components due to the labeling yields being below 100%. The absence and presence of a dye molecule can also influence the donor and acceptor decay kinetics with respect to amplitude and lifetime, respectively. The obtained average lifetimes were then used for the calculation of the distance-dependent FRET efficiency according to eq 2. The obtained FRET efficiency values, plotted in Figure 2C, rise from 0.52 ± 0.01 to 0.67 ± 0.001 for the (2 × 1) array and the (3 × 4) array. This equals to an increase by 22%. The FRET efficiency reaches a plateau for the (3 × 2) array, but the intensity ratio still increases. The discrepancy between the saturating FRET efficiency obtained from the lifetime measurements and the continuously increasing emission intensity ratio is attributed to the fact that light harvesting occurs, which enhances the emission intensity of the acceptor molecules by funneling excitation energy from its surrounding donor molecules, thereby steadily amplifying its fluorescence whilst not affecting the FRET efficiency.25,26 Analytical solutions and complex numerical solutions have been developed for FRET in two and three dimensions, and many efforts have been dedicated to verify such simulations experimentally.36−38 As the DNA origami structure provides exact location of each dye molecule, a simple method to estimate the FRET efficiencies of these nanoarrays with predetermined locations of donor and acceptor molecules is proposed and illustrated in Figure 3. For the FRET estimation, a circle with a radius of 10 nm (corresponding to a FRET efficiency of 10%) is drawn around each dye. Then, the ratio of the overlapping area of the circles (area within the dotted lines) over the total area is determined, that is, Aovlp/Atot. Although this approach does not provide a numerical solution for the FRET efficiencies of the nanoarrays, especially, because it cannot accurately depict the R−6 distance dependence of FRET from the donor to the acceptor molecules, it provides a quick insight into the changes of the FRET

Figure 3. Estimating FRET efficiency of dye nanoarrays. (A) 2 × 2 array taken as an example of the prediction method. The red and orange dots represent the dye molecules, acceptor and donor, respectively. The distances between them are as they are on the DNA origami with 6.0 nm between the two closest dye molecules and 7.7 nm between dye molecules on the same DNA double helix. A gray circle is drawn with the dye molecule as a center point, with a radius of 10 nm where the FRET efficiency would be less than 10%. The total area for this array is the whole gray area, and the overlapping area is the darker gray area within the dotted line. (B) FRET efficiency calculated from donor decay lifetimes (data set connected with solid line) and Aovlp/Atot (data set connected with dash-dotted line).

efficiency as a function of the number of overall dye molecules that have a fixed position on a two-dimensional platform. This simple method can be easily adapted to larger arrays with multiple dye molecules and different donor−acceptor distances. In addition to the size of the nanoarray, the spatial arrangement of the donor and acceptor molecules significantly affects the FRET efficiency and the fluorescence emission intensity ratio. For smaller arrays, with three donor dye molecules and three acceptor dye molecules in total, the arrangement providing optimum light harvesting and maximum FRET efficiency is a checkerboard pattern of the donor and acceptor molecules [(3 × 4) array in Figure 2]. As the nanoarray size increases to six donor molecules and six acceptor molecules, the energy-transfer pathways become more complex and a checkerboard pattern might not show the best light-harvesting efficiency. This is underpinned by a saturating FRET efficiency, whereas the fluorescence intensity ratio still increases (Figure 2C). We therefore arranged the dye molecules in seven different ways, still maintaining the ratio of donor and acceptor molecules at 1:1, as depicted in Figure 4 for the (3 × 4) array. The emission intensity ratio determined from Figure 4A and the FRET efficiency determined from the amplitude weighted donor lifetimes in Figure 4B are summarized in Figure 4C. The least efficient nanoarray exhibits a behavior similar to that of the (2 × 1) array. One nanoarray, (3 × 4)_g, shows a higher emission intensity ratio than the checkerboard pattern, (3 × 4)_f, but the FRET efficiency was not significantly higher. However, if the array is not assembled in a checkerboard pattern, the donors are at closer distance and self-quenching can occur. Application as a Ratiometric pH Sensor. For the design of ratiometric sensors based on DNA origami nanostructures and FRET, we used the (3 × 4) array with a checkerboard pattern, for which no dye self-quenching is expected. C343 and FAM were chosen as a donor−acceptor pair, which is sensitive for the pH range of 5−8. The Förster radius of C343−FAM is 5.0 nm. Whereas C343 is pH-inert39 and can hence act as an internal reference signal for the sensor, FAM is pH-responsive 23298


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Figure 4. Arranging dye molecules on a (3 × 4) array in various patterns. Upper panel showing the seven different patterns of assembled donor and acceptor molecules. (A) Steady-state emission spectra recorded by exciting at 450 nm. The donor emission at 515 nm decreases, and the acceptor emission at 565 nm rises as the nanoarray pattern is assembled in a more efficient way. (B) Lifetime decay curves of donor recorded at 520 nm; excitation was at 490 nm, revealing a significant decrease in donor lifetime. (C) FRET efficiency obtained from averaged donor lifetimes and the emission intensity ratio of donor and acceptor plotted according to the nanoarray. The FRET efficiency seems to reach a maximum for the (3 × 4) _f nanoarray; the intensity ratio, however, increases still quite significantly between the (3 × 4)_f and (3 × 4)_g nanoarrays.

Figure 5. FRET nanoarray for pH sensing. The emission spectra were recorded at four different pH values for the (2 × 1) array (A), 2 × (2 × 1) array (B), 6 × (2 × 1) array (C), and (3 × 4) array (D) always exciting at 400 nm. The first narrow peak marked by an asterisk is the water Raman peak which decreases relatively as the number of dye molecules increases, as shown in Figure 2. The emission intensity of the donor at 487 nm decreases as the pH is increased, as the emission of FAM is restored at 520 nm allowing for FRET to occur. The emission profiles differ significantly between the nanoarrays, with an increase in the number of dye molecules accounting for a more pronounced pH-induced change in emission. The pH responsivity is further enhanced by arranging the dye molecules into a checkerboard pattern. (E) pH dependence of the emission intensity ratio for the nanoarrays studied. All data points were obtained from three measurements. The pH responsivity clearly depends on the number of dye molecules per nanoarray. The pH sensitivity between pH 6 and pH 8 is further enhanced for the (3 × 4) array relative to that of the 6 × (2 × 1) array. (F) Nanoarrays on triangular DNA origami for pH sensing, with green circles representing C343 and orange circles FAM.

are referred to as 2 × (2 × 1), and DNA origami with six FRET pairs, which contain the same number of dyes as the (3 × 4) array, as 6 × (2 × 1). As the distance between each FRET pair is at least 18 nm, no interaction between the different FRET pairs is to be expected. The emission spectra (Figure 5A−D) reveal only a single emission peak at 490 nm at pH 5 because the emission of FAM is quenched. With rising pH, this peak turns into a weak shoulder and a second more pronounced band peaking at 520 nm appears, which arises from the acceptor FAM. The sharp peak at 464 nm is the water Raman peak which decreases in normalized intensity as the array becomes larger, as also shown in Figure 2. The emission

and indicates the proton concentration.3 The independence of C343 to pH and the strong dependence of FAM to pH were confirmed beforehand and are shown in the Supporting Information (Figures S-3 and S-4). The normalized excitation and emission spectra of the respective dyes are shown in Figure 1D. Excitation of C343 at 400 nm followed by energy transfer to FAM results in emission at 520 nm. The nanoarrays constructed from the FRET pair as well as the resulting emission spectra are shown in Figure 5. We compare the performance of simple FRET pairs (2 × 1) and their multiples, in which the FRET pairs are not interacting with each other, to (3 × 4) arrays. DNA origami structures with two FRET pairs 23299


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Figure 6. FRET nanoarray for pH sensing. The fluorescence decay curves of C343 were recorded at 490 nm (excited at 450 nm) at four different pH values for the (2 × 1) array (A), 2 × (2 × 1) array (B), 6 × (2 × 1) array (C), and (3 × 4) array (D). The fluorescence lifetime of the donor decreases as the pH rises, with FAM restoring its fluorescence emission and allowing FRET process to take place. The FRET efficiency does not change much between the 2 × (2 × 1) array and 6 × (2 × 1) array, indicating that the FRET pair do not interact. (E) pH-dependent FRET efficiencies obtained from the donor fluorescence lifetimes. The pH sensitivity of the (3 × 4) array is again at an optimum among the four nanoarrays studied. (F) Nanoarrays on triangular DNA origami assembled for pH sensing with green circles representing C343 and orange circles representing FAM.

structure of the DNA origamis at each pH was confirmed via AFM imaging showing their shape withstood the different pH environments (Figure S-6).

spectrum changes its profile with changing pH more rapidly for the (3 × 4) array than for the (2 × 1) array. This indicates the improved sensitivity for pH sensing. The intensity ratios between donor (487 nm) and acceptor (520 nm) are plotted against the pH for the differently sized nanoarrays (Figure 5E). The exponential fits of the pH dependence of the FRET efficiencies for the (2 × 1), 2 × (2 × 1), and 6 × (2 × 1) arrays reveal an increasing pH sensitivity of the nanoarrays with increasing number of dye molecules. The pH sensitivity is further improved by locating the dye molecules in a checkerboard pattern such as in the (3 × 4) array, boosting the emission intensity ratio to the highest value at pH 8. Subsequently, we performed time-resolved studies with the nanoarrays at different pH values (Figure 6). The decay profiles show a rapid decrease in the donor fluorescence lifetime with rising pH, as the fluorescence emission of FAM is restored thus enabling the FRET process to take place more efficiently. The resulting decay curves were analyzed using a triple-exponential fit for pH 5 and a biexponential fit for pH 6− 8, with the last decay component being set to the fluorescence decay time of unquenched C343 (τD = 5.65−5.71 ns). Although there was no trend in the C343 lifetimes when changing the pH and C343 should not be deprotonated at pH 5,39,40 we observed a small, yet not negligible contribution from an emissive species with a very small lifetime in the ps range. This required an extra decay component for multiexponential fit analysis. We tentatively attribute this to a stronger interaction between the C343 molecules and the DNA at low pH. FRET efficiencies were obtained from the amplitude weighted average lifetimes of the donor (τ̅DA) using eq 2. The results including an exponential fit of the data points are summarized in Figure 6E. Again, the (3 × 4) array shows the best sensitivity to pH changes (further quantifications are shown in Figure S-5) with sensitivities comparable to smallmolecule pH ratiometric sensors.41 The integrity of the

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CONCLUSIONS

We have created various FRET nanoarrays of different sizes and patterns of donor and acceptor molecules at a ratio of 1:1 using DNA origami structures. Steady-state and time-resolved fluorescence studies revealed that the emission intensity ratio of donor and acceptor molecules as well as the FRET efficiency is optimized by arranging the dye molecules in a checkerboard pattern as well as increasing the size to a (3 × 4) array. In this array, the dyes also exhibit minimum self-quenching. The DNA nanoarrays used here are very versatile and enable to simply replace and choose the dyes according to the envisaged application. On the basis of their improved FRET efficiency as well as increase in overall emission intensity, the (3 × 4) nanoarrays were used in a proof-of-concept study as a ratiometric pH sensor using the pH-inert donor C343 and the pH-responsive acceptor FAM. The drastic changes of the FAM emission intensity in the pH range of 6 to 8 clearly demonstrate the potential of this approach for pH sensing. In summary, this ensemble study provides the basis for developing bright and sensitive ratiometric sensors based on DNA origami structures, where the stoichiometry and the precise location of the dye molecules are predefined. We demonstrate that simply the arrangement of dye pairs into well-defined arrays improves the performance of the sensor in terms of brightness and sensitivity. The DNA nanoarrays can be easily extended to the detection of other analytes enabling a broad range of applications. As an example, the DNA nanoarray sensor platform can be equipped with fluorophores responsive to biologically relevant metal ions. This can be utilized, for example, for the design of intracellular probes, as cellular uptake of DNA origami nanostructures has been 23300


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ACS Applied Materials & Interfaces previously demonstrated.42−44 Moreover, single particle or single DNA origami studies and their comparison to ensemble measurements are planned for the future. This could be used, for example, to address the dye-labeling yield of the DNA origami structures.

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(13) Olejko, L.; Cywinski, P. J.; Bald, I. Ion-Selective Formation of a Guanine Quadruplex on DNA Origami Structures. Angew. Chem., Int. Ed. 2015, 54, 673−677. (14) Heck, C.; Prinz, J.; Dathe, A.; Merk, V.; Stranik, O.; Fritzsche, W.; Kneipp, J.; Bald, I. Gold Nanolenses Self-Assembled by DNA Origami. ACS Photonics 2017, 4, 1123−1130. (15) Prinz, J.; Heck, C.; Ellerik, L.; Merk, V.; Bald, I. DNA Origami Based Au-Ag-Core-Shell Nanoparticle Dimers with Single-Molecule SERS Sensitivity. Nanoscale 2016, 8, 5612−5620. (16) Acuna, G. P.; Moller, F. M.; Holzmeister, P.; Beater, S.; Lalkens, B.; Tinnefeld, P. Fluorescence Enhancement at Docking Sites of DNA-Directed Self-Assembled Nanoantennas. Science 2012, 338, 506−510. (17) Du, K.; Ko, S. H.; Gallatin, G. M.; Yoon, H. P.; Alexander Liddle, J.; Berglund, A. J. Quantum Dot-DNA Origami Binding: A Single Particle, 3D, Real-Time Tracking Study. Chem. Commun. 2013, 49, 907−909. (18) Bui, H.; Onodera, C.; Kidwell, C.; Tan, Y.; Graugnard, E.; Kuang, W.; Lee, J.; Knowlton, W. B.; Yurke, B.; Hughes, W. L. Programmable Periodicity of Quantum Dot Arrays with DNA Origami Nanotubes. Nano Lett. 2010, 10, 3367−3372. (19) Deng, Z.; Samanta, A.; Nangreave, J.; Yan, H.; Liu, Y. Robust DNA-Functionalized Core/Shell Quantum Dots with Fluorescent Emission Spanning from UV-Vis to Near-IR and Compatible with DNA-Directed Self-Assembly. J. Am. Chem. Soc. 2012, 134, 17424− 17427. (20) Kuzuya, A.; Kimura, M.; Numajiri, K.; Koshi, N.; Ohnishi, T.; Okada, F.; Komiyama, M. Precisely Programmed and Robust 2D Streptavidin Nanoarrays by Using Periodical Nanometer-Scale Wells Embedded in DNA Origami Assembly. ChemBioChem 2009, 10, 1811−1815. (21) Shen, W.; Zhong, H.; Neff, D.; Norton, M. L. NTA Directed Protein Nanopatterning on DNA Origami Nanoconstructs. J. Am. Chem. Soc. 2009, 131, 6660−6661. (22) Vogel, S.; Rackwitz, J.; Schürman, R.; Prinz, J.; Milosavljević, A. R.; Réfrégiers, M.; Giuliani, A.; Bald, I. Using DNA Origami Nanostructures To Determine Absolute Cross Sections for UV Photon-Induced DNA Strand Breakage. J. Phys. Chem. Lett. 2015, 6, 4589−4593. (23) Nicoli, F.; Barth, A.; Bae, W.; Neukirchinger, F.; Crevenna, A. H.; Lamb, D. C.; Liedl, T. Directional Photonic Wire Mediated by Homo-Förster Resonance Energy Transfer on a DNA Origami Platform. ACS Nano 2017, 11, 11264−11272. (24) Stein, I. H.; Steinhauer, C.; Tinnefeld, P. Single-Molecule FourColor FRET Visualizes Energy-Transfer Paths on DNA Origami. J. Am. Chem. Soc. 2011, 133, 4193−4195. (25) Hemmig, E. A.; Creatore, C.; Wünsch, B.; Hecker, L.; Mair, P.; Parker, M. A.; Emmott, S.; Tinnefeld, P.; Keyser, U. F.; Chin, A. W. Programming Light-Harvesting Efficiency Using DNA Origami. Nano Lett. 2016, 16, 2369−2374. (26) Olejko, L.; Bald, I. FRET Efficiency and Antenna Effect in Multi-Color DNA Origami-Based Light Harvesting Systems. RSC Adv. 2017, 7, 23924−23934. (27) Albinsson, B.; Hannestad, J. K.; Börjesson, K. Functionalized DNA Nanostructures for Light Harvesting and Charge Separation. Coord. Chem. Rev. 2012, 256, 2399−2413. (28) Olejko, L.; Cywiński, P. J.; Bald, I. An Ion-Controlled FourColor Fluorescent Telomeric Switch on DNA Origami Structures. Nanoscale 2016, 8, 10339−10347. (29) Christ, S.; Schäferling, M. Chemical Sensing and Imaging Based on Photon Upconverting Nano- and Microcrystals: A Review. Methods Appl. Fluoresc. 2015, 3, 034004. (30) Meier, R. J.; Simbürger, J. M. B.; Soukka, T.; Schäferling, M. A FRET based pH probe with a broad working range applicable to referenced ratiometric dual wavelength and luminescence lifetime read out. Chem. Commun. 2015, 51, 6145−6148. (31) Zhang, X.; Xiao, Y.; Qian, X. A Ratiometric Fluorescent Probe Based on FRET for Imaging Hg2+Ions in Living Cells. Angew. Chem., Int. Ed. 2008, 47, 8025−8029.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b03585.

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AFM images, DNA sequences, and additional spectral data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ute Resch-Genger: 0000-0002-0944-1115 Ilko Bald: 0000-0002-6683-5065 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Deutsche Forschungsgemeinschaft (DFG, BA 4026/5-2), by the University of Potsdam, the Federal Institute for Materials Research and Testing (BAM), and the DFG project GSC 1013 (SALSA).

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DNA Origami-Based FÃ¶rster Resonance Energy-Transfer Nanoarrays

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