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Simultaneous Rayleigh/Mie and Raman/Fluorescence Characterization of Molecularly Functionalized Colloids by Correlative Single-Particle Real-Time Imaging in Suspension Jörg Wissler, Martin Wehmeyer, Sandra Bäcker, Shirley K Knauer, and Sebastian Schlücker Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02528 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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Analytical Chemistry

Simultaneous Rayleigh/Mie and Raman/Fluorescence Characterization of Molecularly Functionalized Colloids by Correlative Single-Particle Real-Time Imaging in Suspension J. Wissler1, M. Wehmeyer1, S. Bäcker2, S.-K. Knauer2, S. Schlücker1* 1

Physical Chemistry I, Department of Chemistry, University of Duisburg-Essen and Center for Nanointegration Duisburg-Essen (CENIDE), Universitätsstrasse 5, Essen, 45141 Germany 2

Molecular Biology II, Department of Biology, University of Duisburg-Essen and Zentrum für Molekulare Biotechnologie (ZMB), Universitätsstrasse 5, Essen, 45141 Germany

Corresponding author: S. Schlücker, email: [email protected]

Abstract Many applications of nano- and microparticles require molecular functionalization. Assessing the heterogeneity of a colloidal sample in terms of its molecular functionalization is highly desirable, but not accessible by conventional ensemble experiments. Retrieving this information necessitates single-particle experiments which simultaneously detect both functionalized and non-functionalized particles via two separate imaging channels. In this contribution, we present an optical setup for performing correlative single-particle imaging using laser light-sheet illumination: the first detection channel records elastic light scattering (Rayleigh/Mie), while the second channel detects inelastic light scattering (Raman) or fluorescence. The instrument is tested with Raman reporter-functionalized SERS-active metal nanoparticles (core/satellite silver nanoparticles, dimers of gold nanoparticles) and fluorophore-functionalized colloids (fluorescent polymer microparticles, dye-labeled protein on gold nanoparticles).

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Introduction Nano- and microparticles play a central role in science and technology. Many applications of colloidal

particles

require

additional

processing

steps

which

involve

molecular

functionalization, for example, the introduction of functional coatings for encapsulation and subsequent conjugation chemistry. The visualization of colloids at the single-particle level is routinely performed by electron microscopy for determining parameters such as particle size and shape. However, this approach does not provide any chemical information on molecular functionalization. The chemical analysis of molecularly functionalized nano- and microparticles by optical spectroscopy is usually carried out at the ensemble level in cuvette experiments. A drawback of this approach is that only the mean value by ensemble averaging is obtained. Information on the functionalization state of individual particles is not accessible. Assessing the heterogeneity of a colloidal sample in terms of its molecular functionalization is highly desirable since for many applications it is intriguing to know whether really all particles have been molecularly functionalized or only a certain percentage. Retrieving this information necessitates single-particle experiments which simultaneously detect both functionalized and non-functionalized particles via two detection channels: one channel, which detects only the functionalized particles, and a second channel, which detects all particles, irrespective of their molecular functionalization. In this contribution, we present an optical setup for simultaneous two-channel experiments on single molecularly functionalized colloids in suspension in real-time. The development of this instrument was originally motivated by the analysis of gold particles (AuNP) functionalized with Raman reporter molecules (SERS nanotags/labels).1-6 It is generally accepted that surface-enhanced Raman scattering (SERS) dominantly arises from small clusters of AuNP (dimers, trimers, …), while the AuNP monomers exhibit a significantly lower enhancement.7,8 In most cases SERS colloids are crude mixtures of AuNP monomers and oligomers.


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Conventional cuvette SERS experiments do not permit to differentiate between their individual signal contributions. We were therefore interested in determining the percentage of SERS-active particles in suspension. Since elastic light scattering from AuNP is several orders of magnitude stronger than SERS, this signal channel serves as a reference for detecting all AuNP. The simultaneous detection of SERS from the same colloidal sample volume gives the corresponding information on both molecular functionalization (with or without Raman reporter molecules) and plasmonic enhancement (AuNP monomers versus AuNP oligomers). The correlative analysis of the two simultaneously recorded channels (elastic versus inelastic scattering) yields the percentage of the SERS-active AuNP. A sequential approach reported earlier, in which first Rayleigh and then Raman scattering was recorded, already provided an initial guess on the heterogeneity of colloidal samples with respect to its SERS activity. However, the two channels were not recorded simultaneously from the same particles.9 We therefore built an optical instrument for simultaneous real-time detection of both Rayleigh and Raman scattering from exactly the same particles at the same time in suspension, using laser light-sheet illumination10 for excitation in combination with a fast and highly sensitive array detector (EMCCD). The concept of simultaneous imaging molecularly functionalized single particles using Rayleigh/Mie and Raman scattering can easily be adapted to fluorophore-functionalized colloids. In this case, Rayleigh/Mie scattering is again employed for detecting all colloidal particles, while the corresponding fluorescence channel records only those particle which are functionalized with fluorophores. We tested our instrument for simultaneous single-particle imaging on Raman reporterfunctionalized as well as on fluorophore-functionalized colloids. Raman-functionalized colloids comprise highly plasmonically active silver superstructures (silver core/silver satellite nanoparticles)11

and

AuNP

dimers.12

Fluorophore-functionalized

colloids

comprise

commercially available fluorescent polymer microparticles and AuNP functionalized with an ACS Paragon Plus Environment


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Alexa dye-labeled protein. Results and Discussion Figure 1 shows a schematic of the experimental setup for simultaneous correlative singleparticle imaging using laser light sheet illumination. For technical details and specifications see Materials and Methods as well as the supporting information. Briefly, the 660 nm radiation from the laser is focused by a cylindrical lens on the sample containing the colloid.

Figure 1. Schematics of the optical setup for correlative single-particle real-time imaging with elastic light scattering (Rayleigh or Mie) and Raman/fluorescence detection. The molecularly functionalized colloid is illuminated using a laser light-sheet. The emitted light is collected by an objective with a high working distance in a 90° geometry. The elastically scattered light (upper channel) is reflect by a dichroic mirror (DC), attenuated by a neutral density filter (ND) and focused on one half of the EMCCD by a projection lens (L). The redshifted radiation (lower channel) passes through the dichroic mirror and a bandpass filter (BP) for blocking residual elastically scattered light and focused on the second half of the EMCCD by a projection lens. ACS Paragon Plus Environment

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The emitted radiation (elastic and inelastic scattering, fluorescence) is collected in a 90° geometry using a microscope objective (20x, NA0.75) with a long working distance of 1 mm. The elastically scattered light is reflected by a dichroic mirror (upper channel), attenuated by a neutral density filter and focused on the EM-CCD (first half of the chip). In contrast, longer wavelength radiation (Raman, fluorescence) is passing the dichroic mirror (lower channel). After blocking residual elastically scattered light by a bandpass filter the signal is focused on the EM-CCD (second half of the chip). The use of an EM-CCD provides two central advantages: high sensitivity combined with fast image acquisition in the milli-second range per image (video rate). Please note that we cannot discriminate between Raman and fluorescence contributions since the entire signal is integrated onto the chip (no spectral dispersion). Therefore, the corresponding sample should be characterized spectroscopically before. Overall, this approach allows us to simultaneously record two images from the same sample volume: either elastic and inelastic light scattering (Raman/SERS) or elastic light scattering and fluorescence. High optical collection efficiency, high detection sensitivity, and fast readout are necessary requirements for detecting single particles at video rates. We tested the performance of this setup for correlative single-particle imaging using two different colloidal samples: SERS-active colloids comprising Raman reporter-functionalized noble metal nanoparticles as well as fluorescently labeled polymer nanoparticles. Specifically, we were interested in characterizing the heterogeneity of the colloids at the single-particle level. Figure 2 shows the three different types of SERS nanoparticles employed in this study: i) ca. 52 nm gold nanoparticles (AuNP, Fig. 2 top) coated with 2-nitro-5-thiobenzoate as the Raman reporter; ii) silver superstructures (Fig. 2 middle) comprising a ca. 100 nm silver core with an ultrathin (few nm thick) silica shell. The silver satellite particles are attached to the Ag core and coated with 4-nitrothiophenol; iii) dimers of ca. 50 nm AuNP (Fig. 2 bottom) linked by



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1,4-benzenedithiol as the Raman reporter.

Figure 2. Schematic structure (left column), TEM images (middle column) and SERS spectra (right column) of Raman reporter-functionalized noble metal nanoparticles for correlative single-particle Rayleigh and Raman imaging. Top: Gold nanoparticles (Ø ca. 52 nm, Ra = 2nitro-5-thiobenzoate; ensemble SERS spectrum), Middle: Ag/Ag superstructures (Ø ca. 150 nm, Ra = 4-nitrothiophenol; ensemble SERS spectrum), Bottom: Au-Au dimers (monomers Ø ca. 52 nm, Ra = thiophenol; single-particle SERS spectrum). We note that a fair and in-depth quantitative comparison of the different SERS intensities is not the scope of this study and is also far from being trivial since parameters such as the aggregation state of the colloid, its optical density, the laser excitation wavelength with respect to the plasmon peak of the colloid as well as the type of the Raman reporter molecule ACS Paragon Plus Environment

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need to be considered. Please note that the SERS spectrum in Fig. 2 bottom right is from a single AuNP dimer. In the following we therefore focus on the single-particle imaging experiments in colloidal suspension. We started with AuNP without Raman reporters as a negative control (Figure 3a). The single particles detectable in the Rayleigh channel at a frame rate of 10 images per sec are highlighted by red arrows. As expected, the corresponding Raman channel is dark which demonstrates the successful suppression of elastic scattering (cf. Fig. 1). We note that only in very rare cases we could observe a bright spot in the Raman channel, which we attribute to the presence of a very small fraction of AuNP clusters. Figure 3 (b) shows the results obtained for AuNP with Raman reporters. As expected, they can be detected at the single-particle level in the Rayleigh channel. However, despite their functionalization with Raman reporter

Figure 3. Correlative single-particle Rayleigh & Raman imaging (a) AuNP (Ø ca. 52 nm) without Raman reporter (negative control); (b) AuNP (Ø ca. 52 nm) with Raman reporter (2nitro-5-thiobenzoate). (a & b) In both cases Rayleigh scattering from all single AuNP but no Raman scattering can be detected (raw data; acquisition time: 100 ms, laser power: 100 mW). Scalebar: 20 µm.



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molecules, they are not detectable in the Raman channel at the single-particle level within the 100 msec acquisition time employed for every frame. This is in accordance with earlier SERS results on Raman reporter-functionalized AuNP monomers which were also not SERS-active at the single-particle level.7 Figure 4 shows the results for the Ag/Ag superstructures (Fig. 4a) and AuNP dimers (Fig. 4b). In the Rayleigh channel very bright spots from individual superstructures are observed, indicating their high scattering cross sections. Three of these Ag/Ag particles, which also show up in the Raman channel, are highlighted by red arrows (Fig. 4a right). Although some

Figure 4. Correlative single-particle Rayleigh & Raman imaging of Ag/Ag superstructures and Au-Au dimers (a) Ag/Ag superstructures (Ø ca. 200 nm, Ra = 4-nitrothiophenol) and (b) Au-Au dimers (monomers Ø ca. 52 nm, Ra = thiophenol, molecular bridge = 1,8octanedithiol). (a,b) In both cases intense Rayleigh scattering from all colloidal particles is observed. In the Raman channel not all Ag/Ag superstructures are detectable at the singleparticle level, while most of the Au-Au dimers are visible (raw data; acquisition time: 100 ms, laser power: 100 mW). The arrow number represents a recognized in-focus particle. Scalebar: 20 µm. ACS Paragon Plus Environment

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of the other Ag/Ag superstructures appear as bright spots in the Rayleigh channel, e.g. the particle in the top left corner (Fig. 4a left), they are not detectable in the corresponding Raman image (Fig. 4a right). We assume that all satellites in the Ag/Ag superstructures are coated with Raman reporter molecules (see Fig. 2) since during the synthesis an excess of Raman reporter molecules upon incubation of the colloid was employed. We therefore conclude that not all Ag/Ag superstructures are SERS-active enough to be detectable at the single-particle level within 100 msec. For the singe-particle analysis of the AuNP dimers we changed to glycerol as a highly viscous suspension medium. We expected that this slows down the diffusion and would eventually enable us to also temporally resolve the rotation of the dimer around its axis. The Rayleigh channel in Fig. 4b shows numerous single dimers of AuNP. In addition to the brightest particles directly from the focal plane of the light-sheet (ca. 22-24 particles in the Rayleigh channel of Fig. 4b) also particles from out-of-focus regions are observable via their “corona” (cf. light sheet in Fig. 1: vertical illumination profile with the Gaussian-shaped laser beam). The red arrows indicate those particles which are detectable in both the Rayleigh and the Raman channel (ca. 15 particles). Since the percentage of dimers also detectable in the Raman channel is higher than in the case of the superstructures, we conclude that the AuNP dimers are highly SERS-active and additionally relatively uniform with respect to their SERS intensity. Further correlative Rayleigh/Raman images of dimers together with quantitative information on the percentage of SERS-active particles including a comparison of their SERS intensities (histograms) as well as the corresponding colocalization comparisons are contained in the supporting information. Figure 5 shows the results for correlative Rayleigh/fluorescence single-particle imaging experiments using fluorophore-labeled micro- (Fig. 5a) and nanoparticles (Fig. 5b), respectively. According to vendor specifications, the beads in Fig. 5 (a) are 2 and 3 µm in size. The 3 µm size particles are stained with multiple fluorescent dyes, whose emission



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spectra are compatible with many commonly used fluorochromes for flow cytometry. They can be excited with light between 400 nm and 650 nm to yield emission in all fluorescence channels for calibration of the laser settings. The fluorescence spectra of the bead suspension (ensemble level, cuvette experiments) excited at 405, 488, 532, 635 and 660 nm, respectively, are shown in the supporting information. The fluorescence spectra excited in the blue and green exhibit the strongest emission. From these ensemble experiments (average fluorescence) we cannot infer whether the signal arises from beads with multiple fluorophores per bead or from single-color-encoded beads. This information can only be obtained from correlative Rayleigh/fluorescence microscopic experiments at the single-particle level. Figure 5(a) shows a correlative Rayleigh/fluorescence image of the fluorescent beads obtained with 660 nm laser excitation. The Rayleigh channel (signal attenuated by an OD 4.5 filter) shows four beads, while only two of them are visible in the corresponding fluorescence channel (raw data). Since the two other particles from the Rayleigh channel are not observed in the fluorescence channel, we conclude that they do not contain red fluorophores. The same phenomenon was also observed from additional Rayleigh/fluorescence images (supporting information). Overall, we conclude that not all beads contain all fluorophores, but seem to be mixtures of particles with a different fluorophore composition. Also in the case of the 52 nm AuNP - Survivin protein- Alexa 660 conjugates (Fig. 5b) not all particles are detectable in both the Rayleigh and the fluorescence channel. Again, we conclude that not all AuNP are functionalized homogeneously by the fluorescently labeled protein (Survivin-Alexa 660).


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Figure 5. Correlative single-particle Rayleigh/Mie and fluorescence imaging of multi-color fluorescent beads and Alexa660-Survivin-AuNP. (a) Multi-color fluorescent micron-sized beads; (b) AuNP (Ø ca. 52 nm) coated with Alexa660 dye-labeled Survivin protein. (a,b) In both cases not all colloidal particles are visible in the corresponding fluorescence channel (acquisition time: 100 ms, laser power: 10 mW (a) and 100 mW (b)). The arrows highlight particles from the focal plane. Scalebar: 20 µm.



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Conclusions This proof-of-concept study demonstrates the feasibility of simultaneously performing correlative single-particle imaging of molecularly functionalized nano- and microparticles with either Rayleigh/Raman or Rayleigh/fluorescence detection. This approach provides two images recorded simultaneously from the same sample volume and their direct comparison gives information on the percentage of particles showing up in the Raman or fluorescence channel, respectively. A systematic analysis of many sets of images recorded at video rate in combination with a suitable software could then resolve the underlying distribution function for an entire ensemble of colloidal particles. This paves the way to quantitatively assess the functionalization yield of molecular modification steps in nano- and micoparticle synthesis at the single-particle level, i.e. information which is otherwise difficult or most likely impossible to be determined at the ensemble level.


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Material and methods Correlative single-particle imaging: Light scattering and/or fluorescence was excited by the 660 nm radiation from a diode-pumped solid state laser (Laser Quantum, model PSU Type SMD12, maximum power 1 W). The setup used an inverted microscope configuration for collection of the scattered light (cf. Fig. 1). For the SERS imaging of single nanoparticles, the setup was used in light-sheet illumination due to the better signal-to-noise ratio. The emitted light was collected in 90° geometry by a 20x microscope objective (NA 0.75, WD 1 mm, Nikon) and directed towards the opto-splitter optics (Gemini W, Hamamatsu) containing dichroic filters (FF-665-Di02-25x36, Semrock). The elastically scattered light is reflected off the dichroic filter and then attenuated by a neutral density filter. The inelastically scattered light was directed towards a long pass razor edge filter (LP02-664RU-25, ODabs > 6 at 664 nm, Semrock) for blocking residual Rayleigh scattering which passed the dichroic filter. Both channels were then spatially combined and projected by the opto-splitter optics onto an EMCCD camera (ImageX2, Hamamatsu) for simultaneously detecting elastic and inelastic scattering from the same nanoparticles within the sample volume. Electron microscopy: Transmission electron microscopy (TEM) was performed using a Zeiss EM 910 instrument with an acceleration voltage of 120 kV. Scanning electron microscopy (SEM) images were acquired with a JSM-JOEL 7500F field-emission scanning electron microscope at 15 kV acceleration voltage using SE2 detection. Nanoparticle suspensions were prepared on Quantifoil Cu-400 mesh TEM grids with carbon support. 1-2 µL of the aqueous sample was placed on the TEM grid and was dried on air for 2 h before exposing it to vacuum environment of the electron microscopes. Image acquisition and processing: Image recording was performed with Hamamatsu HoKaWo 2.9.10. Image processing was performed with Fiji / ImageJ v1.51.13,14 Materials: Amicon spin filter columns of different MWCO (Merck Millipore, Ultra-4 type) ACS Paragon Plus Environment


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were used for separation and purification of molecularly functionalized gold nanoparticles. Mechanical components were obtained from Thorlabs and Edmund optics. Optical components were obtained from Thorlabs, Edmund optics, Qoptics and Hamamatsu. An EMCCD camera (ImageX2) for detection was obtained from Hamamatsu. Dichroic and band pass filters were obtained from a AHF (Tübingen, Germany) and Semrock. Chemicals: The following chemicals were purchased from Sigma-Aldrich Germany and used without further purification unless stated otherwise: 3-mercapto-n-propyl-trimethoxysilane , 4-nitrothiophenol, thiophenol, 1,8-ocatanedithiol. The hydrophilically stabilized Raman reporter 5,5’-dithiobis-2-nitrobenzoic acid-monoethylene glycol, DTNB-MEG, was prepared as described in Jehn et al.15 Aqueous solutions were prepared using purified deionized water (18.2 MΩ cm, MilliQ Millipore). Fluorescent microbeads with a nominal size of 2 to 3 µm for FACS calibration (MACS calibration beads, #130-093-607) were purchased from Miltenyi. Amine-reactive Alexa 660 succinimidyl ester (#A20007, NHS ester) was purchased from ThermoFisher. Synthesis of Alexa 660-labeled His6-tagged Survivin: His6-tagged Survivin was expressed and purified according to published protocols.16 Alexa 660-labeled Survivin was synthesized and purified using a standard protocol for protein labeling provided by the vendor. Fluorophore-labeled His6-tagged Survivin was conjugated to Ni-NTA-functionalized 52 nm gold nanoparticles.16 Synthesis of Raman reporter-functionalized gold nanoparticles: Gold nanoparticles with a mean diameter of ca. 52 nm were synthesized according to published protocols using the seeded growth method.17,18 The colloidal particles were incubated with the corresponding Raman reporter (1 mM in ethanol). Synthesis

of

Raman

reporter-functionalized

silver

core/silver

satellite

(Ag/Ag

superstructures): The Ag/Ag superstructures were synthesized according to a published ACS Paragon Plus Environment

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protocol.19 We adopted the protocol for obtaining Ag/Ag superstructures with a diameter of ca. 200 nm. The colloidal particles were incubated with the corresponding Raman reporter (1 mM in ethanol). Synthesis of molecularly bridged Raman reporter-functionalized gold nanoparticle dimers: Gold nanoparticle dimers were obtained by a solid-phase supported approach.12 1,8octanedithiol was employed as a molecular linker for controlling the gap size. The colloidal particles were incubated with the corresponding Raman reporter (1 mM in ethanol).

Acknowledgements We gratefully acknowledge support from the German Research Foundation (DFG) within the Collaborative Research Center SFB 1093 “Supramolecular Chemistry of Proteins” (project A09 “Raman spectroscopic monitoring of protein recognition by supramolecular ligands”).



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References (1) Schlücker, S. ChemPhysChem 2009, 10, 1344-1354. (2) Graham, D. Angew Chem Int Ed 2010, 49, 9325-9327. (3) Wang, Y. L.; Schlücker, S. Analyst 2013, 138, 2224-2238. (4) Wang, Y. Q.; Yan, B.; Chen, L. X. Chem Rev 2013, 113, 1391-1428. (5) Schlücker, S. Angew Chem Int Ed 2014, 53, 4756-4795. (6) Lane, L. A.; Qian, X. M.; Nie, S. M. Chem Rev 2015, 115, 10489-10529. (7) Zhang, Y. Y.; Walkenfort, B.; Yoon, J. H.; Schlücker, S.; Xie, W. Phys Chem Chem Phys 2015, 17, 21120-21126. (8) Cassar, R. N.; Graham, D.; Larmour, L.; Wark, A. W.; Faulds, K. Vib Spectrosc 2014, 71, 41-46. (9) Wark, A. W.; Stokes, R. J.; Darby, S. B.; Smith, W. E.; Graham, D. J. Phys. Chem. C 2010, 114, 18115-18120. (10) Fuchs, E.; Jaffe, J. S.; Long, R. A.; Azam, F. Opt Express 2002, 10, 145-154. (11) Gellner, M.; Steinigeweg, D.; Ichilmann, S.; Salehi, M.; Schütz, M.; Kömpe, K.; Haase, M.; Schlücker, S. Small 2011, 7, 3445-3451. (12) Yoon, J. H.; Lim, J.; Yoon, S. ACS Nano 2012, 6, 7199-7208. (13) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. Nat Meth 2012, 9, 671-675. (14) Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J.-Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Nat Meth 2012, 9, 676-682. (15) Jehn, C.; Küstner, B.; Adam, P.; Marx, A.; Ströbel, P.; Schmuck, C.; Schlücker, S. Phys Chem Chem Phys 2009, 11, 7499-7504. (16) Wissler, J.; Bäcker, S.; Feis, A.; Knauer, S. K.; Schlücker, S. Small 2017, 13, 1700802. (17) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss Faraday Soc 1951, 55-75. (18) Bastus, N. G.; Comenge, J.; Puntes, V. Langmuir 2011, 27, 11098-11105. (19) Xie, W.; Schlücker, S. Nat Commun 2015, 6, 7570.


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