Direct Imaging of Single Plasmonic Metal Nanoparticles in Capillary

Direct Imaging of Single Plasmonic Metal Nanoparticles in Capillary with Laser Light-Sheet Scattering Imaging. Xuan Cao,. 1, 3. Jingjing Feng,. 2. Qi ...
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Direct Imaging of Single Plasmonic Metal Nanoparticles in Capillary with Laser Light-Sheet Scattering Imaging Xuan Cao, Jingjing Feng, Qi Pan, Bin Xiong, Yan He, and Edward S. Yeung Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03844 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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Analytical Chemistry

Direct Imaging of Single Plasmonic Metal Nanoparticles in Capillary with Laser Light-Sheet Scattering Imaging Xuan Cao, 1, 3 Jingjing Feng, 2 Qi Pan, 2 Bin Xiong, 1 Yan He, *, 1, 2 Edward S. Yeung 4 1

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha, 410082, P. R. China 2 Department of Chemistry, Tsinghua University, Beijing, 100084, P. R. China 3 Institute of Pharmacy and Pharmacology, University of South China, Hengyang Hunan province, 421001, P. R. China 4 Department of Chemistry, Iowa State University, Ames, IA 50011, U.S.A *Fax: +86-010-62780562

Email: [email protected]

ABSTRACT: Understanding the heterogeneous distribution of the physical and chemical properties of plasmonic metal nanoparticles is fundamentally important to their basic and applied research. Traditionally, they are obtained either indirectly via bulk spectroscopic measurements plus electron microscopic characterizations, or through single molecule/particle imaging of nanoparticles immobilized on planar substrates. In this study, by using light-sheet scattering microscopy with a supercontinuum white laser, highly sensitive imaging of individual MNPs flowing inside a capillary, driven by either pressure or electric field, was achieved for the first time. We demonstrate that single plasmonic nanoparticles with different size or chemical modification could be differentiated through their electrophoretic mobility in a few minutes. This technique could potentially be applied to high throughput characterization and evaluation of single metal nanoparticles as well as their dynamic interactions with various local environments. INTRODUCTION During the past two decades, noble metal nanoparticles (MNPs) that can give rise to localized surface plasmon resonance (LSPR) scattering and absorption have attracted considerable attentions due to their unique optical characteristics, and have found extensive applications in the fields of material science,1 chemical and biological sensing, 2,3 disease diagnosis and therapy,4,5 etc. So far many different methods have been developed to synthesize MNPs with various sizes, shapes and compositions, and modify their surfaces with various ligands and macromolecules.6 To understand and exploit their exceptional physical and chemical properties, 7 it is important to image and evaluate the obtained nanoparticles, as well as their interaction with the surrounding environment of interest, at the single particle level.8 This is because that unlike small organic molecules, most chemically prepared MNPs are inherently non-uniform physically, and such heterogeneities could have significant impact on their collective behaviors and performances.9 For better characterization of MNPs, researchers have explored a number of in-situ noninvasive plasmonic imaging techniques such as darkfield microscopy,10 differential interference contrast microscopy,11 orthogonal polarization microscopy,12 photothermal imaging13,14 and transient absorption imaging,15 in addition to electron and force microscopies. Most of these techniques, however, are only applicable to MNPs immobilized on or moving slowly adjacent to a planar glass substrate. To perform high speed single MNP imaging and evaluation, it is preferable to couple plasmonic imaging detection with high throughput micro-column flow analysis. Capillary electrophoresis (CE), as a major micro-column separation technique, has long been utilized for species identification in complex systems due to its high efficiency, high throughput and low sample loading.16 In CE, molecules or

particles are differentiated under electroosmotic flow based on their electrophoretic mobility, which could contain the heterogeneity information for their evaluation. In particular, at the single molecule or single nanoparticle level, CE has been exploited to resolve conformational change and activities of individual enzyme molecules, 17,18probe small molecule-DNA interactions,19 and study the variations of single quantum dots, 20 etc. However, these single molecules or particles were almost all fluorescent and their efficient sensing all relied on fluorescence detection with advanced spectral filtering techniques. As far as we know, there are few reports on CE detection with plasmonic imaging. A major obstacle is the Rayleigh scattering background arising from the capillary walls. Unlike a smooth planar glass surface, the surface of the inner wall of a micron-sized capillary is usually optically uneven, often producing strong Rayleigh scattering interference when being illuminated with external light sources. But the Rayleigh scattering background and the LSPR target signal share the same detection wavelength, and cannot be eliminated by spectral filters, leading to severe interference to single particle sensing. As a result, only relatively large MNPs have been imaged inside capillaries or micro-channels.21,22 To bypass this problem, the intensity and the number of the nanoparticles have been detected off-column by using a sheath flow cuvette and a high-speed point detector, but at the cost of their in-column movement information.23 To minimize the Rayleigh scattering background for oncolumn detection, the prerequisite is to minimize the contact region between the incident light and the capillary tube. Recently, light-sheet microscopy or single plane illumination microscopy (SPIM) has gained much popularity due to its promising applications in 3D optical slicing of live biological samples such as embryos, cells and tissues.24,25 In a typical

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SPIM setup, the original output of a circular, single wavelength Gaussian beam from a TEM00 mode laser is shaped into a thin sheet of light through a series of optics, and illuminates the sample at 90 degree to the optical axis of the objective lens collecting the emission. When the thickness of the light sheet matches the depth of the focus of the lens, little sample outside the focal plane is illuminated theoretically, resulting in images with high resolution along the vertical direction, minimum out-of-focus background, and reduced Rayleigh scattering background.26 However, existing SPIM setups were all developed in the context of fluorescence microscopy and there has been no report on light-sheet LSPR imaging. For LSPR scattering imaging of MNPs with various sizes, shapes and compositions, a broadband white light source is generally required. But the conventional white light output from halogen tungsten lamp, mercury lamp or xenon lamp can hardly be collimated and compressed into a few-micron-thick light-sheet. In this work, we utilized a supercontinuum white laser as the light source, and developed a laser light-sheet microscopy technique for highly sensitive single MNP detection inside capillary. We demonstrated that individual gold nanoparticles flowing in a capillary could be readily visualized and differentiated. This novel technique can potentially be utilized to quantify the physical distributions of single MNPs in high speed and evaluate their dynamic interactions with different environments, and is expected to find wide applications in many areas of nanoparticle research.

EXPERIMENTAL SECTION Light-sheet imaging system. Figure 1 shows the schematic diagram of the laser light-sheet plasmonic imaging system. A NKT Photonics super-continuum white laser was used as the light source. The diameter of the output laser beam was about 0.6 mm. A wide-window band-pass filter restricted its wavelength range to the visible region. Two apochromatic lens with focal length f= 30 mm and 60 mm, respectively, were used to expand the diameter of laser beam to about 1.2 mm. The laser beam was shaped into a planar sheet by expanded it vertically with a pair of cylindrical lenses (f= 50 mm and 250 mm), followed by being compressed laterally with one cylindrical lens (f= 50 mm) and one illumination objective (f= 18 mm). The apochromatic lenses and the objective were all purchased from Thorlabs. The resulting light-sheet irradiated the sidewall of the square capillary at normal angle with a dimension of about 400 µm wide, 100 µm deep and 2.5 µm thick, forming a detection volume of ~0.02 nL. The scattering images were collected with a Nikon 20X Plan Apo objective and then acquired by an Olympus DP73 color CCD camera. All images are analyzed with the open source Image J software (National Institutes of Health). Single particle counting was performed using the Particle Analysis module. For determining their signal-to-noise ratio, the 24-bit RGB images were converted to 8-bit gray images, and the intensities of the particles and their surrounding background were measured separately. Capillary system. Fused silica square capillaries with outer and inner side length of 330 µm and 100 µm, respectively, were obtained from polymicro (Molex Inc., US). A 10-cmlong capillary was used in all of the experiments. A 1-cm window was cleared at the center of the capillary and washed twice with ethanol and water. Before experiment, the capillary was cleaned by the following sequence of solutions (1 ml

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each) over a 0.5 h period (a) methanol-water (1: 1, v/v); (b) NaOH (0.1 M); (c) methanol-water (1: 1, v/v), and (d) phosphate buffer (1 mM, pH 7.0). After the capillary was thoroughly cleaned, it was glued at about 1 mm from one edge of a glass slide (10×76 mm). Another two pieces of supporting capillaries about 2-cm-long were glued near the other edge of the glass slide with a distance of 1 cm, which were aligned to be parallel with the main capillary and were used to eliminate the wedge interference. A coverglass was then put on top of the capillaries carefully, and the space in-between was filled with index matching fluid. The ends of the detection capillary were immersed in slot vials with 1 mM PB buffer. The sampling system consisted of a set of horizontally fixed slotted vials array as described in literature.27 The slotted vials for containing samples and reagents were produced from 0.2-mL microtubes (Porex, US) by fabricating 0.9-mm-wide, 1.0-mm-deep slots on the conical bottom of the tubes. The vials were horizontally fixed on the platform in an array, with the slot of each vial positioned horizontally to allow free passage of the sampling probe through all the vial slots sequentially by manually moving the platform along the direction of the array. The slotted sample vials containing reagent and carrier solutions were filled by 1 ml syringes. For counting of single particles, gravity-driven control flow was employed by hydrostatic pressure produced from height difference in liquid levels between the sample and the waste reservoir. The horizontally positioned waste reservoir was fixed at a level lower than that of the capillary, and was connected to the outlet of the capillary by a piece of tygon tube with inner diameter of 250 µm. Sample injection was performed while linearly moving the array of vials, allowing the inlet of the capillary to sweep into the slots, and residing in either the sample or buffer solution vials. The flow rates of the sample/buffer were adjusted to 20-100 nL/min by varying the level of the waste reservoir in relation to that of the reagents. For electrophoresis, the AuNR samples were diluted 50200 times from the stock solution and injected into the sample slotted-vial. The inlet and outlet of the capillary were immersed in the sample/buffer vial and the waste vial, respectively, and two pieces of platinum wires were inserted into the vials which were half-filled with the solutions. The sample introduction pressure was balanced by adjusting the height of the waste reservoir to make sure that the gravity flow did not exist. An electric field of 0-80 V/cm was then applied across the capillary by using a high-voltage power supply from Unimicro (Shanghai, China).

RESULTS AND DISCUSSION System design and optimization. Although laser-induced fluorescence detection for capillary electrophoresis has long been established over two decades ago, to the best of our knowledge, small plasmonic metal nanoparticles flowing through a capillary has not been imaged previously under laser illumination. The essential here is to perform LSPR scattering imaging in a highly scattering micro-column format. To address the technical issues, we took a systematic approach to design and optimize each component including the illumination, the capillary holder, and the detection light path, before integrated them together. Because a broadband illumination is required to image MNPs of various spectral signatures, we chose a supercontinuum(SC) white laser as the light source. The SC white light,

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Analytical Chemistry

Figure 1. Schematic diagram of supercontinuum laser light-sheet plasmonic imaging system for capillary electrophoresis detection. S: Shutter; SP: Short pass filter; AL1, AL2: Achromatic Lens; M: Reflection mirror; P1, P2: Polarizer; ACl1, ACl2, ACl3: Achromatic cylindrical lens; IL: illumination Lens; OL: Objective Lens; RL: relay lens system.

generated by nonlinear processes when a short-pulsed pump beam passing through a special photonic crystal, was the subject of intense studies in recent years.28,29 The commercial available SC laser could deliver a spectral range from ~380 nm to ~2,400 nm and produce ps SC pulses at sufficient high frequencies to function as quasi-CW sources. Similar to single wavelength lasers, the SC light is highly intense and collimated, but having a much shorter coherence length, making them suitable replacement of conventional incandescent lamps. The major concern in LSPR imaging in capillary electrophoresis is the Rayleigh scattering background from the glass capillary wall, which is over 100 m thick and is inherently optically heterogeneous. To reduce its contribution to the LSPR image, the incident laser beam was shaped into a thin light-sheet by using a combination of cylindrical lenses of different focal lengths. It struck through the middle of the square capillary, illuminated less than 3% (2.5 m /100 m) of the vertical cross section of its inner space, so that only the two sidewalls of the capillary could produce the Rayleigh scattering interference. The 2.5 m thickness of the illumination region matched the thickness of the focal plane of the 20X objective, ensuring that few out-of-focus particles could be observed. In this way, the signal-to-noise ratio (SNR) or contrast of the images of nanoparticles at the center of the capillary was significantly higher than that of striking the capillary with a large circular Gaussian beam. Notice that for efficient illumination and reduction of stray light, the capillary was immersed in the index-matching fluid and sandwiched between a glass coverslip and a glass slide. It was important to keep the capillary at about 1 mm away from the edge of the glass slide. If the distance was much less than 1 mm, the background level would be raised by the stray light from the immersion oil-air interface; if the distance was larger than 1.5 mm, that the being vertically focused light-sheet would hit the edges of the glass slide or the coverglass and be partially blocked. To align the position of the capillary, two criteria were examined: if a collimated laser beam struck exactly at the middle of capillary, its propagation direction would not be altered; if the capillary was placed symmetrically at the focal point of the lens, then the dispersion of beam after the focal plane should not deviate from that with the capillary removed. To keep a thin light-sheet while limiting the illumination area, the depth of the light-sheet was designed to be 100 m, the inner width of the square capillary, by choosing appropri-

ate focal lenses. In practice, due to the inevitable spatial variations and glass surface heterogeneities, the Rayleigh scattering background from the sidewalls could still be too high to obscure the signals from single metal nanoparticles near the capillary wall. In these situations, two detection modes can be of choice. One is the orthogonal polarization detection mode. In this mode, a pair of linear polarizers make the illumination light striking the capillary and the collected scattering light reaching the detector cross-polarized. Consequently, the Rayleigh scattering from the capillary sidewall is almost totally blocked and only anisotropic particles such as gold nanorods or aggregates of gold nanospheres could be observed due to their depolarization effect. That created an almost backgroundfree situation for single AuNR observation. The other is the slit-control mode. In this mode, there is at most a single polarizer in either the illumination or detection light path. A manually adjustable slit is put at the conjugate image plane of the detection objective, and can be adjusted to eliminate the sidewall Rayleigh scattering and to select the region-of-interest for observation. This mode can be applied to imaging both isotropic and anisotropic particles. Which detection mode to choose depends on the properties of the observed particles and the information desired. Signal-to-noise ratio. To demonstrate the performance of SC laser light-sheet plasmonic imaging, a capillary with some 35×70 nm large AuNRs immobilized on its inner wall surface was imaged with four different schemes (Figure 2): conventional darkfield microscopy (DFM) with oblique illumination, DFM with cross polarization detection, SC laser light-sheet imaging in the slit-control mode and SC light-sheet imaging in the orthogonal polarization detection mode. Traditionally, LSPR scattering imaging of ensemble as well as single MNPs are mostly performed using DFM equipped with an oblique circular illumination module, where the light source is usually a 100 W halogen tungsten lamp. But DFM is designed for examining samples attached to a planar glass surface. For micro-columns, the region of illumination is too large compared to the cross-section of the capillary, leading to strong Rayleigh scattering background. As shown in Figure 2A, the average background level was 34.5 and the noise value was 20.5 under the DFM scheme. The AuNRs adsorbed close to the middle of the tube had a signal-to-noise ratio (S/N) of only 5.4 and those near the capillary wall cannot be observed. Moreover, a 200 ms exposure time was required to reach such a poor S/N, because the light from the tungsten lamp is not well collimated and, to achieve oblique illumination, the major portion of the incoming light has to be blocked by the darkfield condenser. By applying orthogonal polarization detection to DFM (Figure 2B), the background interference was considerably reduced, but at a cost of further huge reduction of the illumination power and the corresponding signal level. Even though the exposure time of the color CCD camera increased from 200 ms to 2,000 ms, the particles on the capillary were still invisible. On the other hand, by using SC laser light-sheet illumination (SuperK Compact, 10 mW total power output from 500 nm to 700 nm) under the slit-control mode, the background level was only 1.1 and the noise value was just 0.89 (Figure 2C). For comparison, we intentionally had the slit only blocked the Rayleigh scattering from one side of the capillary wall, on which the AuNRs adsorbed could now be clearly observed. Benefit from the highly collimated and intense laser

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Analytical Chemistry illumination, the S/N was over 200 with just a 10 ms exposure time of the CCD camera. Under the orthogonal polarization mode (Figure 2D), the background as well as the noise level could become even lower. The S/N reached ~250 with a 20 ms exposure time of the CCD camera. That was at least 40 times of sensitivity increase compared to the DFM scheme. We note that under this mode, the apparent LSPR scattering intensity of the immobilized anisotropic AuNRs would be dependent on their orientation, but that would not be a problem for AuNRs rotating in the free solution.

Capable of “seeing” small MNPs one-by-one, it is now possible to count them quantitatively. Figure 3A shows the single particle counting results of different concentrations of 40 nm AuNP solution (obtained via sequential dilution of the original solution) flowing through the capillary. For each sample, a continuous 100 frames recorded at a frequency of 10 Hz were analyzed. It can be seen that a good linear relationship (R2 = 0.9977) between the average counts in one frame and the AuNP concentration was obtained. According to the uncertainty of the measurements associated with the current setup, we estimated that reliable counting could be achieved for AuNP concentration down to about 0.2 pM. 50

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Figure 2. Scattering images and their signal-to-noise comparisons of single AuNRs absorbed on a capillary obtained using different schemes: (A) traditional DFM with oblique illumination, (B) traditional DFM with cross polarization detection, (C) SC laser lightsheet imaging in the slit-control mode and (D) SC light-sheet imaging in the orthogonal polarization detection mode. Middle columns: single AuNR plasmonic scattering images on top of the Rayleigh scattering background from the capillary wall. Middleto-right and right-most columns: enlargement of the single AuNR images adsorbed near the center of the capillary and the corresponding line-graphs. Left-most columns: the line-graphs of the Rayleigh scattering images across the wall. The white arrow shows the single AuNR near the capillary wall and the enlarged images were placed in left-top corner of the middle column image.

Single particle imaging, counting and differentiation on-the-flow. With the capability to detect gold nanoparticles absorbed inside the capillary with high S/N in a short exposure time, we can now image, count and differentiate particles in the flow. The AuNRs in Figure 2 had an average dimension of 35×70 nm. Since the LSPR scattering coefficient is proportional to the 6th power of the nanoparticle radius, this lightsheet imaging apparatus was estimated to be capable of seeing single ~20 nm Au nanoparticles with S/N of ~3. To confirm that, we prepared small AuNRs with average size of 17×40 nm, whose scattering cross-section was close to a 22 nm spherical Au nanosphere according to the Mie theory. As shown in Movie 1, the small AuNRs flowing inside the capillary were clearly visualized with average S/N of 7.2 under the orthogonal polarization mode by using the compact SC laser and a CCD exposure time of 100 ms. By using a high power SC laser (SuperK Extreme, >100 mW in the visible region), even smaller particles such as 13 nm AuNPs could be imaged individually on-the-flow (Movie 2).

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Figure 3. Single MNP counting and differentiation on-the-flow. (A) Single particle counting results for different concentrations of 40 nm AuNPs under pressure driven flow. (B) Calculated electrophoretic velocity of single 35×70 nm AuNRs as a function of the applied voltage under the electric field driven flow. (C) Distribution of the electrophoretic mobility of three different AuNRs of LSPR maximum at 708 nm, 648 nm and 588 nm, respectively.

The particles move with the same rate in gravity driven flow. In capillary electrophoresis, however, the electrophoretic mobility of the MNPs would differ based on their size, shape and surface charge, which allows them to be separated accordingly. For individual moving particles or molecules being imaged in CE, measurement of their flowing rate simply transfers to measuring the distance of their directional move during the exposure time of one frame of the CCD camera. Figure 3B shows that there is a good linear correlation between the average flowing rate of 35×70 nm AuNRs and the high voltage applied across the capillary. As a proof of concept experiment, we prepared 3 batch of AuNRs with LSPR maxima centered at of 708 nm, 648 nm and 588 nm via oxidative etching, respectively, and have them flowing through the capillary sequentially at the same electric field strength of 40 V/cm. Figure 3C shows the distribution of the electrophoretic mobility of the 3 AuNRs. The broad distribution of each indicates that the physical properties of the AuNRs were inherently heterogeneous, but it was clear that their average values differ significantly. In another demonstration, the CTAB coated 35×70 nm AuNRs were modified with more positively charged MUTAB molecules and neutral PEG molecules, respectively and were subject to CE separation at electric field strength of 72 V/cm

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Analytical Chemistry

(Movie 3 and Movie 4). It can be seen that the 2 AuNRs with different surface coatings move in obviously different rates. The MUTAB modified AuNRs moved obviously faster than the PEG modified ones. Taken together, these experimental results demonstrate that this method could potentially be utilized for evaluation of various types of plasmonic NPs.

CONCLUSION With supercontinuum laser illumination, we have developed light sheet plasmonic scattering microscopy for in situ real time observation of plasmonic metal nanoparticles flowing through capillaries. Rayleigh scattering background interference arising from the glass capillary walls were successfully reduced and high S/N imaging of MNPs inside capillaries were achieved with either a narrow slit or orthogonal polarization detection. The fast movement of small MNPs with scattering cross-section equivalent to a ~20 nm AuNP or less was successfully captured. Single MNP counting as well as differentiation of single MNPs of different physical characteristics were demonstrated under either gravity or electric field driven flow conditions. Given the fast growth of applications of plasmonic metal nanoparticles in various fields, we expect this highly sensitive and high throughput plasmonic imaging technique to be potentially useful in characterization and evaluation of MNPs as well as their dynamic interactions with surrounding environments.

ASSOCIATED CONTENT Supporting Information Supporting information is available free of charge via the Internet at http://pubs.acs.org. Detailed synthesis procedure of nanoparticles (PDF) Movie 1. Visualization of 17×40 nm AuNRs movement in capillary under gravity flow applying orthogonal polarization detection model. (AVI ) Movie 2. Visualization of 13 nm AuNPs movement in 50% glycerol solution under gravity flow applying slit control detection model. (AVI ) Movie 3. Visualization of 35×70 nm MUTAB modified AuNRs movement in capillary under electric field driven flow. (AVI ) Movie 4. Visualization of PEG modified 35×70 nm AuNRs movement in capillary under the same applied voltage (AVI)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +86-010-6278 -0562. Fax: +86-010-6277-1149

Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China with grant numbers of 91027037, 21127009, 21425519 and 21221003, Natural Science Foundation of Hunan Province 13JJ1015, Hunan University 985 fund and Tsinghua University Startup fund. We thank Dr. Jianzhang Pan and Dr. Qun

Fang from Zhejiang University for the assistance in the slottedvials and high speed CE based sampling system.

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