Strong Plasmonic Enhancement of a Single PeridininâChlorophyll a

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Strong Plasmonic Enhancement of a Single Peridinin-Chlorophyll a-Protein Complex on DNA Origami-Based Optical Antennas Izabela Kaminska, Johann Bohlen, Sebastian Mackowski, Philip Tinnefeld, and Guillermo P. Acuna ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08233 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Figure 1. (a) Schematic illustrations of the OAs based on 100 nm Au (top) and 80 nm Ag (bottom) NPs selfassembled onto a pillar-shaped DNA origami (grey). A single natural light-harvesting complex PCP is positioned at the hotspot between the NPs, while a single dye Atto542 (green dot) is located in the base for co-localization. (b) Structure of a PCP monomer (peridinins (orange), chlorophylls (green) embedded in a protein scaffold (grey)).13 (c) Absorption (black) and emission (red) spectra of PCP complexes. 476x380mm (300 x 300 DPI)

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Figure 4. Correlation of fluorescence enhancement for excitation wavelengths of 532 and 640 nm, obtained for Au OAs (orange triangles) and Ag OAs (grey squares). 89x74mm (300 x 300 DPI)


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Figure 2. Intensity maps (10 × 10 µm) of PCP fluorescence obtained for Au OAs, Ag OAs, and DNA origami without NPs, plotted with the same intensity scale. Corresponding exemplary fluorescence transients measured for each of the three structures, obtained for the excitation wavelengths of 532 nm (green) and 640 nm (red), respectively. 236x167mm (300 x 300 DPI)


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Figure 3. Fluorescence enhancement distribution for 100 nm Au OAs (orange triangles), 80 nm Ag OAs (grey squares), and OAs without nanoparticles (black circles) as a function of fluorescence lifetime, for both excitation wavelengths: (a) 532 nm and (b) 640 nm. FE histograms for 100 nm Au dimers (c, d) and 80 nm Ag dimers (e, f) plotted logarithmically and fitted with log-normal distributions. 169x130mm (300 x 300 DPI)


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Strong Plasmonic Enhancement of a Single Peridinin-Chlorophyll a-Protein Complex on DNA Origami-Based Optical Antennas Izabela Kaminska†┴*, Johann Bohlen†‡, Sebastian Mackowski┴, Philip Tinnefeld†‡ and Guillermo P. Acuna†*

Affiliations: †

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. ┴

Institute of Physics, Faculty of Physics, Astronomy, and Informatics, Nicolaus Copernicus

University, Grudziadzka 5, 87-100 Torun, Poland. ‡

Department Chemie and Center for NanoScience (CeNS), Ludwig-Maximilians-Universitaet

Muenchen, Butenandtstr. 5-13 Haus E, 81377 Muenchen, Germany.

*Correspondence to: [email protected], [email protected]

KEYWORDS: plasmonics, fluorescence enhancement, DNA origami, optical antenna, photosynthetic complex, single-molecule detection.

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Abstract. In this contribution, we fabricate hybrid constructs based on a natural light-harvesting complex, peridinin-chlorophyll a-protein, coupled to dimer optical antennas self-assembled with the help of the DNA origami technique. This approach enables controlled positioning of individual complexes at the hotspot of the optical antennas based on large, colloidal gold and silver nanoparticles. Our approach allows us to selectively excite the different pigments present in the harvesting complex, reaching a fluorescence enhancement of 500-fold. This work expands the range of self-assembled functional hybrid constructs for harvesting sunlight and can be further developed for other pigment-proteins and proteins.

The irruption of the DNA origami technique1 more than a decade ago revolutionized the bottom-up fabrication of nanostructures2,3 with outstanding design flexibility and control, as well as ease of fabrication. Appealed by these characteristics, several groups have further developed the DNA origami technique and achieved a myriad of nanostructures4 with high level of reproducibility and detail. Furthermore, researchers quickly discovered the potential of this technique for nanophotonic applications, since DNA origami structures can act as molecular breadboards.5 Using this approach, single quantum emitters such as fluorophores,6 quantum dots,7 and even nanodiamonds can be positioned in the near-field of plasmonic metallic nanoparticles acting as optical antennas (OAs) with nanometric accuracy and precise stoichiometric control. This high degree of morphology control render the DNA origami technique ideal for the fabrication of hybrid light-harvesting structures and for probing their properties at the singlecomplex level, which can circumvent the challenges involved in the study and characterization of the fundamental photophysical and photochemical processes in bulk materials, where performance is often critically influenced by structural and chemical heterogeneity.8 In fact, previous efforts focused on self-assembling artificial light-harvesting complexes by combining fluorophores on a DNA origami structure in an attempt to mimic the architecture and performance of natural light-harvesting complexes.9,10 However, due to the complexity of natural photosynthetic systems, it is still challenging to artificially replicate their full functionality. 2 ACS Paragon Plus Environment

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A significant limitation of single light-harvesting structures is their dimension, which is typically smaller than the light penetration depth. As a result, light is not fully absorbed by the harvesting element. This shortcoming can be overcome by placing the light-harvesting complexes at the near-field of an OA capable of focusing the incident light at the harvesting pigment position, thus increasing the “apparent” absorption and emission cross sections of single light-harvesting complexes. Examples of this approach include the interaction between the natural light-harvesting complex 2 (LH2), active in the near infrared range, and gold nanorods tuned to exhibit longitudinal localized surface plasmons on an overlapping spectral range.11 The group of van Hulst demonstrated that an emission enhancement of 500× can be achieved when LH2 complexes are randomly deposited onto an array of gold nanorods of different dimensions, together with a 10× increment of the photostability, stronger than the one obtained with spherical gold nanoparticles.12 Another natural light-harvesting complex with interesting optical properties resulting from its pigment composition, a water-soluble peridinin-chlorophyll a-protein (PCP) complex present in the dinoflagellate Amphidinium carterae, has been coupled to silver island films (SIFs) to increase its performance. The plasmonic enhancement of single complexes resulted in an average and maximum emission intensity increase by the factor of 6 and 18, respectively.13 These works clearly demonstrated the potential of OAs to manipulate the optical properties of natural light-harvesting complexes and to boost their performance. However, further development of this plasmonic approach was hindered by the lack of techniques capable of deterministically positioning a single lightharvesting complex precisely at the hotspot of an optical antenna. In this contribution, we exploit the DNA origami technique to self-assemble an optical antenna dimer based on large, spherical metallic (gold and silver) colloidal nanoparticles (NPs) and to position a single PCP complex at the hotspot. This work extends the palette of species that can be incorporated to DNA origami structures, to natural light-harvesting complexes, and shows that the coupling to the OA dimer can enhance the emission of single harvesting structures by a factor of 500×. This approach represents a step towards the fabrication of self-assembled hybrid light-harvesting architectures. Results and discussion. The schematic illustrations of the self-assembled OAs used in our work, based on pillarshaped DNA origami structures and two metallic NPs, are presented in Figure 1a. At the top part of the construct, we have incorporated a biotin molecule, for subsequent binding of a PCP-streptavidin conjugate, and a single dye molecule Atto542 in the base for co-localization 3 ACS Paragon Plus Environment

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(green dot). The DNA origami structures with a single PCP complex were immobilized onto a neutravidin-modified glass coverslip and then incubated with metallic nanoparticles to create dimer OA constructs. Either two 100 nm Au NPs or 80 nm Ag NPs were bound at the top part, creating a hotspot gap of about 12 nm.14 More details about the NPs functionalization and OAs preparation can be found in the Materials and Methods, whereas details about the DNA origami structures are included in the Supporting Information (SI) and in references.14,15

Figure 1. (a) Schematic illustrations of the OAs based on 100 nm Au (top) and 80 nm Ag (bottom) NPs self-assembled onto a pillar-shaped DNA origami (grey). A single natural lightharvesting complex PCP is positioned at the hotspot between the NPs, while a single dye Atto542 (green dot) is located in the base for co-localization. (b) Structure of a PCP monomer (peridinins (orange), chlorophylls (green) embedded in a protein scaffold (grey)).13 (c) Absorption (black) and emission (red) spectra of PCP complexes.

We performed measurements for Au and Ag OAs with PCP complexes using a home-built confocal microscope with circularly polarized light (further details are included in Imaging and Analysis). In order to selectively probe the peridinin (Per) or chlorophyll a (Chl) molecules of every single PCP complex we used alternating excitation of 532 and 640 nm (more details in Materials and Methods). Figure 2 depicts typical PCP fluorescence intensity maps for Au and Ag OAs, together with constructs without NPs for comparison, plotted with the same intensity scale. The fluorescence intensity maps reveal that the presence of both Au and Ag OAs leads to a significant fluorescence enhancement of the PCP emission under both 532 and 640 nm excitations. In order to characterize the fluorescence enhancement induced by the OAs, fluorescence transients were recorded for each spot with alternating excitation. For further analysis, only transients showing co-localization between the PCP complex and Atto542, together with a single bleaching step of the latter, were considered. This approach ensures that only single DNA origami structures are included in the analysis. Exemplary pairs of fluorescence transients recorded for PCP complexes with Au and Ag OAs are included in Figure 2 (more examples together with a fluorescence transient measured for Atto542 can be found in Figure S2). Fluorescence transients of PCP complexes without OAs are also included in Figure 2 for internal referencing. Lines are color-coded: green is assigned to the excitation at the wavelength of 532 nm, whereas red - at 640 nm (in both cases detection is performed in the red channel, described in detail in Materials and Methods). For most cases, especially in 4 ACS Paragon Plus Environment

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the absence of nanoparticles, single blinking and bleaching steps are measured, indicating the presence of a single PCP complex per DNA origami. In the presence of nanoparticles, multistep fluorescence transients are more common, which is likely related to the higher excitation power at the hotspot of the OAs. Multi-intensity levels for light harvesting complexes were already detected in early single-molecule studies.16

Figure 2. Intensity maps (10 × 10 µm) of PCP fluorescence obtained for Au OAs, Ag OAs, and DNA origami without NPs, plotted with the same intensity scale. Corresponding exemplary fluorescence transients measured for each of the three structures, obtained for the excitation wavelengths of 532 nm (green) and 640 nm (red), respectively.

The fluorescence intensity of each PCP complex is extracted from the first intensity level recorded on the fluorescence transients. In addition, for each PCP complex the fluorescence lifetime is determined (further details can be found in Imaging and Analysis). We calculated the fluorescence enhancement (FE) by normalizing the fluorescence intensity of the PCP complexes in the presence of the OAs to the average intensity of the complexes without NPs (1870 ± 225 and 364 ± 69 counts/sec for 532 nm and 640 nm excitation wavelengths, respectively). The obtained results are summarized in Figure 3. The populations of PCP without NPs (in black) are characterized by a fluorescence lifetime, which is in good agreement with the intrinsic value of approximately 3.75 ns.13 In contrast, the populations with OAs exhibit mostly a clear reduction in the fluorescence lifetime together with an enhancement of the fluorescence intensity, a signature of the interaction between the PCP complexes and OAs. OAs can affect the fluorescence properties of PCP complexes by increasing the electric field intensity at the complex’s position and thus enhancing the excitation rate and also by opening more pathways for the excited complex to decay. This typically leads to a reduction in the fluorescence lifetime and can either enhance or reduce the fluorescence quantum yield depending on the intrinsic quantum yield.17 For the excitation wavelength of 532 nm (Figure 3a) we observe similar FE values for both constructs, with a maximum of 145 and 120 for Au OAs and Ag OAs, respectively. These values are an order of magnitude larger than previous reports, where PCP complexes were simply deposited on SIFs.13 In the case of Chl excitation at 640 nm (Figure 3b), higher FE values are recorded for Au OAs, reaching values as high as 526-fold in contrast to 250-fold for Ag OAs. Such a dramatic increase of the optical response of an individual light-harvesting complex 5 ACS Paragon Plus Environment

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demonstrates the high – and yet unexplored - potential for improving performance of natural or artificial proteins using the DNA origami technology. Furthermore, the populations with OAs show a considerable dispersion (static heterogeneity) in both fluorescence intensity and lifetime as observed in previous works.18 Due to our fabrication process, samples can contain both dimer and monomer OAs together with a small number of DNA origami structures without NPs (represented by a few events in the Ag OAs sample, with a fluorescence lifetimes close to the intrinsic value). Dimer structures can yield both a stronger enhancement of the electric field intensity and quantum yield and therefore provide a higher fluorescence enhancement together with a more pronounced reduction in the fluorescence lifetime.18 This observation is supported by the strong correlation between fluorescence intensity and lifetime for the OAs populations in Figure 3. Additional effects that can introduce dispersion in the distributions are NPs size and shape, variation of the gap distance and orientation of the pillar structures on the glass slide.15,18–20 For the results obtained for each OA sample in Figures 3a and b, the corresponding FE histogram plot is included (Figures 3c-f). The distributions for both Au and Ag OAs exhibit a bimodal character in agreement with our observation that both monomer and dimer structures are sampled. In order to characterize each population, its contribution, and average FE value, we have fitted the distributions with double log-normal functions (dashed lines), as described previously15 (further details can be found in the SI). For the excitation wavelength of 532 nm, which corresponds to Per absorption, Au OAs (Figure 3c) yield an average FE of (11.0 ± 0.9) and (74.4 ± 4.2) for monomer and dimer structures, respectively. For Ag OAs (Figure 3e) slightly lower average enhancements were observed, equal to (9.7 ± 1.0) for monomer OAs and (59.1 ± 4.6) for dimer OAs. The results obtained for the 640 nm excitation, which populates directly excited states of Chl molecules in the PCP complex, exhibit comparable behavior. Monomer and dimer Au OAs (Figure 3d) yield average FE values of (54.7 ± 4.9) and (158.0 ± 5.9), respectively, whereas for Ag OAs (Figure 3f) these values are equal to (31.9 ± 5.6) and (117.1 ± 8.9). These results represent an improvement in the FE of approximately 25-fold compared to previous work13 and can be attributed mostly to the possibility to position single PCP complexes at the hotspot of OAs with nanometer precision and stoichiometric control, where they can experience the highest enhancement of the electric field intensity.13


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Figure 3. Fluorescence enhancement distribution for 100 nm Au OAs (orange triangles), 80 nm Ag OAs (grey squares), and OAs without nanoparticles (black circles) as a function of fluorescence lifetime, for both excitation wavelengths: (a) 532 nm and (b) 640 nm. FE histograms for 100 nm Au dimers (c, d) and 80 nm Ag dimers (e, f) plotted logarithmically and fitted with log-normal distributions.

One of the main advantages of our approach is that each individual PCP complex, coupled to either an Au or Ag OA, can be probed under green (Per) and red (Chl) excitation. Therefore, the correlation between FE532nm and FE640nm can be obtained for every single PCP complex positioned at the hotspot of the OAs. The results, included in Figure 4, are fitted with a linear function with a slope (intended as an average

) of 1.57 and 2.8 for Ag and Au OAs,

respectively. Higher FE for a direct chlorophyll excitation (640 nm) compared to the process mediated by the excitation energy transfer (EET) (532 nm) was previously shown in experimental13 and theoretical21 studies of single PCP complexes coupled to SIF. The higher average value of

for Au OAs as compared to the Ag OAs is in good agreement with

previous works, in which typically Au NPs lead to a strong fluorescence enhancement in the red and infrared spectral range, whereas Ag NPs are commonly applied for enhancements in the green spectral range.15 Nevertheless, we observed a surprisingly strong FE with the Au OAs in the green spectral range in contrast to measurements performed with the same type of OAs and a single fluorophore, Atto542.15

Figure 4. Correlation of fluorescence enhancement for excitation wavelengths of 532 and 640 nm, obtained for Au OAs (orange triangles) and Ag OAs (grey squares). Conclusions. In conclusion, we fabricate self-assembled hybrid structures where natural photosynthetic complexes are coupled to optical antennas. Precise positioning of the protein with respect to the hotspot is achieved by exploiting the DNA origami technique. We observe very large, exceeding 500-fold, plasmon-induced fluorescence enhancement of PCP, which is the highest value reported for this light-harvesting complex. We also present the first comparative studies of the direct and EET-mediated excitation for single PCP complexes placed in both Au and Ag OAs. The strongest fluorescence enhancement has been observed in the first case 7 ACS Paragon Plus Environment

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(excitation at 640 nm), for both gold (526×) and silver (250×) dimers, which is in qualitative agreement with previous reports. Although several pathways of future optimization of this structure can be envisioned, the results described in this work substantially expand the spectrum of DNA origami functional constructs for light-harvesting, and can be potentially developed for other photosynthetic proteins to fabricate solar energy conversion nano-devices. Materials and Methods. Peridinin-chlorophyll a-protein (PCP) is a water-soluble pigment-protein complex present in dinoflagellate Amphidinium carterae with optical properties which result from its pigment composition. Each monomer contains eight peridinin (Per) and two chlorophyll a (Chl) molecules embedded in a protein scaffold.22 The high Per to Chl ratio determines strong and broad absorption in the visible spectral range, with Per absorbing in the range from 400 to 550 nm, and Chl from 350 to 450 nm (Soret band) and from 600 to 670 nm (Qx/Qy band) (Figure 1c). PCP can be probed either in a direct excitation process via Chl or in the excitation energy transfer (EET) mediated process via Per which subsequently transfer excitation energy to Chl, with a yield close to 100% (Figure S1).23 The emission of PCP at 673 nm originates from Qy transition of Chl molecules. To realize the idea of probing selectively Per or Chl in every single PCP complex we have used an alternating excitation of 532 nm and 640 nm (more details can be found in Imaging and Analysis). At the same time, at a wavelength of 532 nm we have probed a fluorophore Atto542 located in the base of the construct. Measurements of co-localization, emission of Atto542 in a green channel and PCP in a red channel, enabled the identification of PCP complexes bound to DNA origami structures. Silver and gold NPs, 80 and 100 nm in diameter, respectively, were purchased from BBI Solutions and functionalized with 25T DNA-oligonucleotides containing a thiol modification at the 3’ end (Ella Biotech GmbH) like describe elsewhere.14,19 Briefly, 2 mL of nanoparticles solution was mixed with 20 mL 10%-Tween20, 20 mL of a mixture of 4:5 potassiumphosphate buffers and 12 mL of 100 µM DNA oligonucleotides 25T containing a thiol modification at the 3’ end. The mixture was stirred at 40 °C during a stepwise salting process with 1×PBS buffer containing 3.3 M NaCl, to the final concentration of 750 mM NaCl. In order to remove free oligonucleotides, the solution of nanoparticles was spinned down and the pellet was dissolved in 1×PBS buffer containing 10 mM NaCl, 2.11 mM KH2PO4, 2.89 mM K2HPO4, 0.01 % Tween20 and 1 mM EDTA. The purification procedure was repeated five more times. 8 ACS Paragon Plus Environment

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DNA origami pillars were prepared like described elsewhere.14,19 The details on DNA origami design, DNA sequences and folding program can be found in the Supplementary Information. The DNA origami pillars were incubated with the solution of PCP complexes for 2 h. This was followed by the incubation with biotin-modified DNA-oligonucleotides complementary to the oligonucleotides protruding from the base of the DNA origami pillars for 5 h. Such prepared structures were immobilized on the glass surface of a Lab-Tek chamber (Thermo Fisher Scientific) coated with BSA-biotin/neutravidin (Sigma-Aldrich). Afterwards, immobilized DNA origami pillars were incubated for 48 h at 4 °C with nanoparticles solution diluted to the absorption of 0.1-0.15 (Nanodrop 2000, Thermo Scientific) with 1×TE containing 12 mM MgCl2 and 100 mM NaCl. Finally, the sample was washed with 1×TE containing 12 mM MgCl2 to remove unbound NPs and single-molecule fluorescence measurements were performed. Imaging and Analysis. Single-molecule fluorescence measurements were performed on a custom built confocal microscope based on an Olympus IX-70 inverted microscope. 80 MHz-pulsed laser beams (532 nm LDH-P-FA-530B and 639 nm LDH-D-C-640, both Picoquant) are alternated by an acousto-optical tunable filter (AOTFnc-VIS, AA optoelectronic) and used to excite PCP complexes and a dye, Atto542. A combination of a linear polarizer (LPVISE100-A, Thorlabs) and a quarter wave plate (AQWP05M-600, Thorlabs) is used to set the circularly polarized light. After passing a dual band dichroic beam splitter (z532.633, AHF), the light beam is focused by an oil-immersion objective (UPLSAPO 100XO, NA 1.40, Olympus) on the measurement chamber, which can be positioned accurately by a piezo-stage. The emission of the fluorophores is collected by the same objective, focused on a 50 µm pinhole (Linos), and split spectrally by another dichroic beam splitter (640DCXR, AHF) between the green (Brightline HC582/75, AHF; RazorEdge LP 532, Semrock) and red (Bandpass ET 700/75m, AHF; RazorEdge LP 647, Semrock) detection channels. Fluorescence is detected by APDs (tSPAD 100, Picoquant) and the signals registered by a TCSPC system (SPC-830, Becker&Hickl) and evaluated using custom-made LabVIEW (National Instruments) software.

ASSOCIATED CONTENT Supporting Information.


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The following files are available free of charge. Details on raw fluorescence enhancement data and detailed information on DNA origami design (PDF).

Corresponding Author *E-mail: [email protected], [email protected] Present Addresses Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We acknowledge funding by the Deutsche Forschungsgesellschaft (AC 279/2-1 and TI 329/91). IK is grateful for the support by the Mobility Plus grant 1269/MOB/IV/2015/0 from the Polish Ministry of Science and Higher Education (MNiSW). PT acknowleges the excellence cluster

NIM

(Nanosystems

Initiative

Munich).

SM

acknowledges

support

by

2016/21/B/ST3/02276 project from the National Science Center of Poland.

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