Two-Photon Imaging of 3D Organization of Bimetallic AuAg

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Two-photon imaging of 3D organization of bimetallic AuAg nanoclusters in DNA matrix Katarzyna Brach, Magdalena Waszkielewicz, Joanna Olesiak-Banska, Marek Samoc, and Katarzyna Matczyszyn Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00873 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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Two-photon imaging of 3D organization of bimetallic AuAg nanoclusters in DNA matrix Katarzyna Brach, Magdalena Waszkielewicz, Joanna Olesiak-Banska*, Marek Samoc, Katarzyna Matczyszyn Advanced Materials Engineering and Modelling Group, Faculty of Chemistry, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland Corresponding Author: *E-mail: [email protected]

ABSTRACT: We report on two-photon excitation properties of small silver-doped gold nanoclusters (AuAgNCs) and on their three-dimensional arrangement in a hybrid system composed of DNA liquid crystals (LCs) and AuAgNCs. UV-Vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and multiphoton excitation spectroscopy were used to characterize the nanoparticles. We show that AuAgNCs exhibit two-photon excited luminescence (2PL) emission and second-harmonic generation (SHG) and that these properties remain the same in liquid crystalline matrix. The results are described in detail and discussed in the context of possible imaging application of AuAgNC and specific AuAgNCs organization induced by liquid crystalline ordering of DNA molecules.

KEYWORDS: two-photon luminescence, second-harmonic generation, DNA liquid crystals, silver-doped gold nanoclusters, dual nonlinear emission properties. 1 ACS Paragon Plus Environment

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Ultra-small nanoclusters are a relatively new class of metallic nanoparticles, which are especially intriguing because of their unique chemical and optical properties, such as strong photoluminescence, large Stokes shift, tunable emission in the visible spectrum, high photostability, good water-solubility, chirality and magnetism.1-8 Due to their size, structure and properties, nanoclusters bridge the gap between bigger nanoparticles and small molecules. Attractive luminescent properties of NCs overcome the limitations of standard fluorescent probes and quantum dots, which may exhibit high toxicity, poor photostability or photobleaching.9-13 Although there are many interesting reports about the application of gold nanoclusters as fluorescent probes in vitro and in vivo,14-22 most of them concern mainly one-photon excitation properties of nanoclusters. On the other hand, two-photon luminescence microscopy (2PLM) has many advantages over traditional one-photon optical microscopy techniques such as possibility of high-resolution deep imaging even in highly scattering environments like living tissues, localized red or infrared excitation in small focal volume and reduced phototoxicity and photobleaching. There are only few reports in which NCs have been successfully applied as fluorescent probes for in vitro two-photon imaging of human mesenchymal stem cells,23 cellular delivery in HeLa cells24-25 or SH-SY5Y human neuroblastoma cells.26 Gold nanoclusters have been shown to exhibit significant nonlinear optical properties with two-photon absorption cross-sections reaching several thousand GM at relevant wavelengths, which make them suitable also for photonic applications such as optical power limiting.5, 7, 27-28 Also second- and third-harmonic generation and multiphoton luminescence were observed,29 which are promising features for application in SHG microscopy and fluorescence microscopy. A majority of the investigations were conducted in solutions, however there were some reports concerning enhancement of optical properties in solid state30-31 or in DNA matrix.32 In addition,

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the research about combination of LC and nanoparticles into one hybrid system has shown that liquid crystalline phase may be used as template for nanoparticles ordering, which may enhance and improve properties of both LC and nanoparticles.33-37 Here, we present an investigation concerning two-photon excitation properties of AuAgNCs in DNA LCs. First, we characterized the nanoparticles using common spectroscopic techniques and TEM combined with two-photon excited spectroscopy. Next, we prepared and investigated lyotropic DNA LCs doped with AuAgNCs using polarized light and fluorescence microscopy to analyze the distribution of nanoparticles within liquid crystalline matrix. We also exploited the advantages of 2PLM to further evaluate the properties of the prepared samples and asses the suitability of AuAgNCs as markers for multiphoton imaging as well as that of DNA LC as matrix for organization of nanoparticles. RESULTS AND DISCUSSION AuAgNCs characterization. The size and morphology of AuAgNCs were determined from TEM images (Figure 1A). The synthesis of AuAgNCs led to the formation of well dispersed spherical nanoparticles with the size of 1.78 ± 0.20 nm. Elemental analysis of AuAgNCs showed that Ag:Au mass ratio was equal to 1:75. The absorbance spectrum (Figure 1B) of AuAgNCs was similar to the ones reported before38 and showed strong absorption around 400 nm. Because of their small size induced discrete electronic structure and molecular-like properties, the optical properties of NCs are significantly different from those of plasmonic gold nanocrystals, in particular, the surface resonance plasmon band at 520 nm, characteristic for larger Au nanoparticles, is not observed in the case of NCs UV-Vis spectrum. This suggested also that NCs were the only product of the synthesis and no bigger particles were formed. The NCs were luminescent with a broad emission band at 600 nm (Figure 1C). The excitation spectrum shows 3 ACS Paragon Plus Environment

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the main peak at 405 nm. The luminescence quantum yield (QY) was measured by a comparative method using Rhodamine B as a reference and was equal to 13.0 %, markedly higher than the QY in the case of similarly sized gold NCs without silver doping.39 A

B

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Figure 1. (A) TEM image of AuAgNCs. (B) UV-Vis spectrum of AuAgNCs. (C) Excitation (dashed line, λem = 600 nm) and emission (solid line, λex = 400 nm) spectra of AuAgNCs. Two-photon excitation properties of AuAgNCs. 2PL signals from the solution of AuAgNCs were obtained in the range of excitation wavelengths from 800 nm to 1000 nm with the average power of incident light around 10 mW (Figure 2A). The 2PL intensity of AuAgNCs in the solution was found to decrease with the increase of the excitation wavelength, with the highest 2PL intensity for the excitation at 800 nm. We conducted the same experiments for a thin film of AuAgNCs obtained by depositing nanoparticles solution (CAuAgNC = 1 mg mL-1) onto a cover glass and complete solvent evaporation. Similarly to the solution of AuAgNCs, with the increasing excitation wavelength the decrease of 2PL was observed (Figure 2B). The maximum of the emission for the excitation at 800 nm was around 625 nm and with a decrease of excitation energy it was red shifted, up to 695 nm in the case of 1050 nm excitation. The red shift of luminescence maximum from AuAgNCs from solution and deposited on the glass substrate with the shift of incident laser wavelength suggest a different excitation and emission pathway. A similar behavior was 4 ACS Paragon Plus Environment

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observed e.g. for Ag nanoclusters.40 Weak SHG signal, increasing with the increase of excitation wavelength, was also recorded in the range of excitations from 850 nm to 1050 nm. We did not observe SHG with the excitation at 800 nm, which is probably due to the SHG generated in this case being strongly reabsorbed through single photon absorption (an inner-filter effect). A

B

Figure 2. 2PL emission spectra of (A) AuAgNCs aqueous solution (CAuAgNC = 1 mg mL-1) and (B) a thin film of AuAgNCs, obtained by depositing nanoparticles solution of CAuAgNC = 1 mg mL-1, at excitation wavelength from 800 nm to 1050 nm. DNA LCs doped with AuAgNCs. In a drying droplet of properly concentrated DNA solution the DNA molecules tend to self-assembly into a liquid crystalline hexagonal columnar phase near the border of the droplet.41 In the beginning of the drying process, DNA chains are homogeneously spread within the volume of the droplet, then, because of the capillary flow induced by contact line pinning and additional undulation caused by radial stress occurring at the end of drying, the DNA strands accumulate at the border of the droplet and liquid crystalline phase with characteristic regular zigzag pattern is formed.41 The orientation of DNA in this type of LC was already investigated in our group by means of 2PLM. The results indicated that DNA


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molecules within one columnar domain were parallel to each other and the domains with molecules oriented in different directions were separated by vertical wall defects.42 The polarized light microscopy observation of our sample confirmed the presence of LC with the characteristic zigzag pattern formed at the perimeter of the dried DNA droplet (Figure 3A). We observed multiple columnar domains separated by wall defects. The addition of AuAgNCs did not influence the LC phase formation as well the type of observed phase texture. Atomic force microscopy (AFM) measurements showed that the surface of the dried droplet edge was undulated (Figure 3B) and had a mountain-like shape with numerous peaks and valleys, which is consistent with literature reports.41-42 The maximum height of the undulated region (from a valley to a peak) was around 700 nm. The fluorescence microscopy observation of dried DNA droplets doped with NCs revealed that the nanoclusters were spread within the entire layer of the sample (Figure 3C). However, there were some particular regions of the sample, in which the intensity of the emission was significantly increased and characteristic emission pattern was observed. These regions corresponded to the peaks of the undulated LC phase. This kind of specific accumulation of AuAgNCs was correlated with the presence and ordering of DNA molecules into a lyotropic LC caused by capillary flow. In contrast, preparation of a thin film formed from a drying droplet of NCs solution with CAuAgNC = 1 mg mL-1 resulted in a formation of a “coffee ring” stain without a characteristic zigzag pattern and surface undulation.


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Figure 3. (A) Polarized light microscopy image of undulated columnar LC phase of DNA doped with AuAgNCs (CAuAgNC = 1 mg mL-1). (B) 2D AFM scan of the dried droplet edge surface. (C) Fluorescence microscopy image of undulated columnar LC phase of DNA doped with AuAgNCs taken in the same region of the sample as in Figure 3A (λexc = 460 – 495 nm, λem > 510 nm). One of the major difficulties in predicting the behavior of nanoparticles and the pattern of their distribution in liquid crystals is the complexity of prepared systems. There is a variety of variables which have to be taken into account, like the properties of mesogens and properties of nanoparticles such as their shape, size and coating. It was shown that even the same type of nanoparticles (size and shape), but with different coating can have different influence on the liquid crystal.43 Concentration of nanoparticles in liquid crystalline phase may be also one of the factors influencing the aggregation behavior of NCs. It was shown that depending on the 7 ACS Paragon Plus Environment

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concentration of nanoparticles different types of liquid crystalline textures and aggregation patterns can be observed, for example increasing the concentration of nanoparticles led to the increased aggregation of nanoparticles in liquid crystal.44-45 Comparing our current results to the ones obtained in the case of DNA LCs doped with gold nanorods (NRs),46 we could observe differential nanoparticles aggregation pattern. NRs were dispersed homogenously, whereas NCs also tended to accumulate in the defects. Taking into consideration the calculations of DNA base pair (bp) mole per nanoparticle mole, we obtained DNA bp:NC ratio ≈ 130:1 and DNA bp:NR ratio ≈ 160·104:1. However, if we look at the results in the category of weight/volume % concentration of nanoparticles doped into LCs, the difference between the nanoparticles concentration is much smaller (CAuAgNC = 1 mg mL-1 and CNR = 0.333 mg mL-1). We prepared DNA LC doped with AuAgNCs at the concentration of CAuAgNC = 0.25 mg mL-1, at which we could still observe defect correlated aggregation of nanoparticles, which was not present in the case of bigger NRs (Supplementary Information, Fig. S1). In order to exclude silver interaction with DNA, which could be responsible for specific defect correlated aggregation of NCs, we performed circular dichroism measurements (Supplementary Information, Fig. S2). There were no significant changes of DNA conformation in the presence of the clusters. Additionally, if silver had affinity to DNA, it would interact with all DNA molecules forming liquid crystal and in this case NCs would be spread homogenously in the volume of the sample. The orientation of DNA molecules and presence of walls separating columnar domains of different molecular orientation should not influence the interaction between silver and DNA, then it should not direct the aggregation of NCs. Furthermore, similar experiments conducted for DNA LCs doped with AuNCs have also shown specific aggregation of NCs (Supplementary Information, Fig. S3). We suggest that differences between NRs and


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NCs doping effects are caused mainly by different sizes and shapes of these nanoparticles. The rate at which liquid crystalline phase is formed in a drying droplet is high, thus bigger NRs are too heavy comparing to NCs to follow the capillary flow of DNA molecules from interior to the edge of a drying droplet. Since NCs are smaller it is easier for them to move and in order to minimize the total free energy they tend to aggregate in defects. Two-photon luminescence microscopy of DNA LCs doped with AuAgNCs. In order to evaluate the use of AuAgNCs as possible probes for biological imaging we investigated DNA LCs doped with the nanoclusters using 2PLM. 2PLM was already applied to explore DNA LCs doped with dyes42,47 or gold nanorods.46 The research provided useful information about the orientation of DNA molecules within the liquid crystalline matrix and the possible application of gold nanorods as imaging and theranostic probes. Figure 4A presents XY 2PL intensity scan of the undulated DNA liquid crystalline phase obtained for the phase region shown in Figure 4B. The results are in the agreement with onephoton fluorescence microscopy observations, where specific aggregation of nanoparticles correlated to the position of the peaks formed within the undulated phase. The distance between neighbouring peaks was twice the distance between vertical wall defects, and it varied from 8 to 25 µm. Consequently, the same values were obtained for the distance between linearly organized AuAgNCs. The experimental conditions were optimized in order to obtain good signal to noise ratio and to avoid the photobleaching and photodamage of the sample. The 2PL intensity scans could be performed with the average power of incident light up to 1.5 mW. With the advantage of the intrinsic 3D resolution of 2PLM, we performed several scans in the same region, but with different Z positions of the undulated phase formed at the perimeter of a droplet (Figure 4C). It was found that AuAgNCs not only tended to aggregate on the top of the 9 ACS Paragon Plus Environment

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peaks of the undulated LC phase, but also accumulated within the whole volume of the phase forming a vertical wall defect. It means that in this type of LCs, self-assembly of NCs is templated by phase defects. DNA LCs offer a possibility for 3D self-assembly of nanoparticles on liquid crystalline template. A

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C

Figure 4. (A) 2PL intensity raster scan of undulated phase (CAuAgNC = 1 mg mL-1) with (B) the corresponding optical image (size of the image 80 x 80 µm), λexc = 800 nm. (C) 2PL intensity raster scans performed at several Z positions of undulated phase at the perimeter of dried droplet of DNA doped with AuAgNCs (CAuAgNC = 1 mg mL-1), λexc = 800 nm. We performed additional 2PLM measurements, which proved the multimodal imaging abilities of AuAgNCs. A slight modification of the experimental setup allowed us to collect the signals originating from SHG emission. Figure 5 presents XY intensity raster scans obtained from the same region of the sample, in which we detected signals from both 2PL and SHG at the same time (Figure 5A) and SHG signals, which were separated from 2PL (Figure 5B). The contribution of 2PL in the two-photon excited emission of AuAgNCs was in this case significantly greater than that of SHG. However, it was still possible to detect SHG signals that were consistent with the aggregation pattern of nanoclusters. The results suggest great potential of AuAgNCs in nonlinear optical imaging of biological samples.


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A

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Figure 5. Comparison between (A) 2PL combined with SHG intensity raster scan and (B) SHG intensity raster scan (λexc = 920 nm) of nanocluster doped undulated DNA phase (CAuAgNC = 1 mg mL-1). CONCLUSION In summary, we synthesized monodispersed thiol-stabilized silver-doped gold nanoclusters and showed their two-photon excitation properties. Then we introduced the NCs into DNA matrix, where the nanoparticles emitted strong 2PL and weak SHG signal. In the presence of liquid crystalline DNA the optical properties of nanoclusters remained unchanged, which makes them attractive probes for imaging of biological samples. Additionally, we observed that undulated liquid crystalline phase of DNA formed at the perimeter of a drying droplet induced the 3D organization of nanoclusters in the wall defects. It is an easy method for nanoclusters ordering, which may find the possible application in photonic devices construction. Further investigation concerning the properties and distribution of NCs within different types of liquid crystalline phases is required to fully understand the phenomena of liquid crystalline induced NCs aggregation and these studies are currently performed in our laboratory. METHODS 12 ACS Paragon Plus Environment

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Chemicals. Deoxyribonucleic acid sodium salt from salmon testes, HAuCl4•3H2O (99.9%), glutathione (≥98.0%) and AgNO3 (99+%) were purchased from Sigma Aldrich. All chemicals were used as received, except when mentioned specifically. Milli-Q water of resistivity 18.2 MΩ•cm was used to prepare all solutions. AuAgNCs synthesis. AuAgNCs were prepared according to a slightly modified protocol of Le Guevel.38 Aqueous gold salt solution was mixed with silver nitrate at an Ag:Au ratio equal to 1:47. Glutathione was used as a reduction agent at a molar ratio of 1:1 to gold. The sample was incubated for 48 h at 65 °C. The purification steps consisted of centrifugation at 3000 rpm for 5 min and double precipitation with acetone. DNA liquid crystal preparation. The DNA liquid crystal preparation consisted of deposition of 10 µL of AuAgNCs doped DNA solution (CDNA = 10 mg mL-1 and CAuAgNC = 1 mg mL-1) on the top of a thin cover slip and drying under 55 °C until total solvent evaporation. The samples were stored at room temperature. Instrumentation. The morphology of AuAgNCs was examined using a FEI Tecnai G2 20 XTWIN transmission electron microscope. The UV-Vis absorption spectra were recorded in 10 mm quartz cuvettes at room temperature using a JASCO V-670 spectrophotometer. The fluorescence

spectra

were

measured

at

room

temperature

using

a

FluoroMax-4

spectrofluorometer. The ICP analysis was performed with ARL 3410 ICP (Fisons Instruments). The samples were observed under polarized light and using a conventional widefield epifluorescence Olympus BX60 optical microscope. The surface of dried droplets was investigated by AFM in tapping mode (Dimensional V scanning probe microscope, Veeco). Two-photon excited luminescence measurements were performed with a custom-built two-photon microscope in which luminescence was excited in the range of 800 nm to 1050 nm with a mode-locked


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Ti:sapphire laser (Chameleon, Coherent Inc.). The laser beam was focused tightly by a high numerical aperture Nikon Plan Apo oil immersion objective (100×/1.4 NA) or by a Nikon LU Plan Fluor objective (100×/0.9 NA). The sample was mounted on an XYZ piezoelectric scanning stage (TRITOR 102) and two-photon excited emission was collected in epi-fluorescence mode through the same microscope objective. The emitted signal was separated from the incident light on a dichroic mirror and detected by avalanche photodiodes operating in the photon counting regime. SHG intensity raster scan was recorded in the same experimental setup with additional Semrock 460/14 nm BrightLine® single-band bandpass filter placed before the avalanche photodiodes. The two-photon excited emission spectra were recorded using a Shamrock 303i Spectrograph from Andor. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGMENT This

work

was

supported

by

the

National

Science

Centre

under

grant

DEC-

2013/09/B/ST5/03417, DEC-2013/10/A/ST4/00114 and by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Technology. SUPPORTING INFORMATION AVAILABLE Polarized light and fluorescence microscopy images (Figures S1 and S3), circular dichroism measurements (Figure S2) This information is available free of charge via the Internet at http://pubs.acs.org/. 14 ACS Paragon Plus Environment

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