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Simultaneous Determination of Size and Position of Silver and Gold Nanoparticles in Onion Cells using Laser Ablation-ICP-MS Shuji Yamashita, Yukihisa Yoshikuni, Hideyuki Obayashi, Toshihiro Suzuki, Damon Green, and Takafumi Hirata Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05632 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019
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Analytical Chemistry
Simultaneous Determination of Size and Position of Silver and Gold Nanoparticles in Onion Cells using Laser Ablation-ICP-MS Shuji Yamashita,† Yukihisa Yoshikuni,† Hideyuki Obayashi,† Toshihiro Suzuki,† Damon Green,‡ and Takafumi Hirata*, † †Geochemical
Research Center, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. of Marketing & Technology, Teledyne Advanced Chemistry Systems, 14306 Industrial Road, Omaha, NE 68144, USA. ‡VP
ABSTRACT: Size distribution and mapping analyses of Ag and Au nanoparticles (NPs) have been made using an ICP-MS combined with laser ablation sampling technique (LA-ICP-MS). With the femtosecond laser (Ti:S laser) ablation system, the measured size distribution was modified toward smaller, whereas this disintegration could be reduced when the nanosecond laser (ArF Excimer laser) ablation was employed. This suggests that both the size and position of the NPs can be defined by the nanosecond LA-ICP-MS technique. More importantly, based on the peak-height analyses (PHA) of the measured signal intensity profiles, present form of the analytes, whether particulate or ionic form, could be defined. This is very important to investigate the transport of the NPs within biological samples. To demonstrate the unique feature of the technique, imaging analyses of Ag and Au NPs, together with distribution analysis of ionic form, were conducted on onion cells, prepared through a dosing experiments of the Ag and Au NPs. There were clear differences in both the increasing rates of the numbers of NPs and concentration range of the ionic form between the Ag and Au. The data obtained here demonstrate clearly that the LAICP-MS technique can become a major analytical tool to obtain both the size distribution and position of NPs from tissue samples.
Keyword: spICP-MS, nanoparticle, laser ablation, imaging spectrometry (spICP-MS) was developed for both the mapping and size distribution analysis of the NPs. With the conventional spICP-MS technique, the sizes,12 number concentrations,13,14 as well as the constituting element of the NPs, dispersed in the aqueous solution can be monitored.15 Typical analytical throughput of the analysis is restricted to about 150 particles per second, and this is mainly due to possible overlaps of the signal events from two or more NPs in the spICP-MS.16 Additionally, based on the signal intensity profile obtained with the spICP-MS, the present form of the analytes, whether particulate (NPs) or ionic form can be defined.17,18 Recently, Kálomista et al. reported that the signal intensity profile can reflect the shape of the NPs (e.g., spherical or rod shaped),19 and thus the spICP-MS technique provides various information such as size, shape, number concentration of the NPs. From these, the spICP-MS is now accepted as key technique for safety control of foods or drinks by FDA (Food and Drug Administration), JAS (Japanese Agricultural Standard), or EMA (European Medicines Agency).20 It is widely recognized that the distribution feature of the NPs can provide key information concerning both the transport and concentration mechanism of the NPs in solid materials. To derive the distribution map of the NPs (imaging analysis of NPs), the spICP-MS combined with laser ablation sampling technique would be the plausible choice (LA-ICP-
Nanoparticles (NPs) have been widely used in the various research fields, such as material, environmental, or biological sciences.1-3 Although many pioneering researches demonstrated the NPs could pass through the cell membranes,4-6 physicochemical interactions between the NPs and cell membrane have not been well investigated. To understand the biological impacts of the NPs, analytical techniques to obtain distribution (imaging) data of the NPs within solid materials, including biological tissues, are highly desired.7-9 For the distribution analyses of the NPs, instrumental techniques, such as secondary electron microscope (SEM), secondary ion mass spectrometer (SIMS),10 or surfaceenhanced Raman scattering (SERS)11 have been adopted. With these techniques, although distribution images with high-spatial resolution can be obtained, size of samples (analytical areas) for the imaging analysis is commonly restricted to ca. 100 × 100 µm scale, and thus, the imaging analysis for biological tissues, being >1 mm, is not a reality. Moreover, with the conventional techniques, size distribution analysis of the NPs cannot be achieved, and therefore, one may not monitor the time-dependent changes in the size of NPs through the biological activities or effects. To overcome this, new analytical technique based on a combination of laser ablation sampling technique and the single particle ICP-mass
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signal events originating from the NPs. In contrast, there is no difference in the response of signal detection between the conventional pulse counting and pulse counting with the attenuator device, therefore ion detection with shorter dwell times can also be made for ion currents >50 Mcps. This is very important for the detection of NPs being >50 nm. For the introduction of NPs dispersed in aqueous solutions, a combination of the conventional Micromist nebulizer and cyclonic spray chamber (Glass Expansion, Port Melbourne, Australia) was used. Introduction efficiency of NPs was about 5 % based on the comparison of sample uptake rate and the measured signal intensity data. For laser ablation sampling, two laser types were employed in this study; in-house CyberProbe UV1 femtosecond laser ablation system (wavelength of 260 nm; pulse duration of 210 fs) and Teledyne Cetac Analyte Excite Excimer laser ablation system (wavelength of 193 nm; pulse duration of 8 ns). Details of the instrumentation and operational settings are listed in Table 1.
MS). Laser ablation technique has been widely used for the elemental and isotopic analyses of trace-elements in the solid materials.21,22 For the quantitative analysis using the LA-ICPMS technique, data quality is severely controlled by the size distribution of the laser-induced aerosols. The introduction of large-sized particles, being >1 um, into the ICP can cause the signal spikes.25 Both the precision and accuracy of the measurements can be dramatically improved with the smaller size distribution of the laser-induced particles.23,24 To achieve this, shorter wavelength (i.e., ArF Excimer laser) and/or shorter pulse duration lasers (i.e., femtosecond laser) have been widely employed.23,24 This is well demonstrated for the metallic samples. With the ArF Excimer nanosecond laser, many signal spikes were found on the signal intensity profile of 63Cu from the metallic Cu.25 The signal spikes could be due to the release of large-sized particles produced through melting the metallic materials. The melting of the metallic materials could be reduced by the femtosecond lasers. With the femtosecond laser ablation (fs-LA) technique, both the higher sensitivity and smoothed signal intensity profile could be obtained even from the metallic materials.26 Because of higher energy transport efficiency of the fs-LA, the size of the laser-induced particles can be reduced, and this results in both the higher transmission and smoothed signal intensity profile. These data suggest that great care must be given for the choice of the laser types for the size distribution analysis of the NPs using the LA-ICP-MS technique. To evaluate the effect of laser types onto the size distribution of NPs, the signal intensities of individual events were carefully monitored, and the possible contribution of disintegration of the NPs through the laser ablation was carefully investigated in this study.
Table1. Instrumentation andsettings operational settings Table 1 Instrumentation and operational (1) ICP-MS instrument (Magnetic sector ICP-mass spectrometer) ICPMS Instrument AttoM Magnetic Sector ICP-MS Nu Instruments (Wrexham, UK) ICP forward power 1300 W Gas flow rates (Ar) Coolant gas 13.0 L min-1 Auxiliary Nebulizer Type of nebulizer Spray chamber Sample up take rate Sampler aperture Skimmer aperature Detection system Attenuator gain Detector Monitored isotopes Dwell time
Experimental Section Instrumentation. Since the typical signal duration time obtained from single NP would be 0.2 to 0.6 ms, data acquisition with shorter dwell time is highly desired to obtain quantitative and reproducible data from the transient signals. Hineman and Stephan demonstrated that the dwell time of shorter than 100 µs (0.1 ms) would be fast enough to obtain quantitative signal intensity data from the single NP.16 However, our preliminary results showed that the dwell time of 100 µs (i.e., sampling rate of 10,000 Hz) was not short enough to separate individual signal events induced by the introduction of small NPs of 50 Mcps could be monitored. One may consider that high-intensity signal can be monitored by an ion current monitoring system on the electron multipliers (e.g., analogue system). However, most of the analogue systems are not fast enough to monitor the transient signals obtained from single NP,28 and the system is therefore not capable of obtaining quantitative signal intensity profiles for individual
1.0 L min
-1 -1
0.94 L min Glass Expansion Micromist nebulizer (Coaxial Nebulizer) Water-cooled cyclonic spray chamber -1
0.2 mL min 1 mm 0.8 mm Pulse counting mode with attenuator device for the beam size >3 Mcps 1/100 ETP full size multiplier (IC) 107
Ag and 30 μs
197
Au
(2) Laser ablation system (UV femotosecond laser ablation system) a Instrument CyberProbe UV1 (Cyber Inc., Tokyo, Japan) Laser IFRIT Type-C Ti:S femtosecond laser Wavelength 260 nm Pulse duration 230 fs -2 Fluence 0.5 J cm Laser diameter 15 µm Repetition rate 30 Hz -1 Carrier gas He (0.78 L min ) -1
Makeup gas
Ar (1.1 L min ) mixed after the ablation cell using T-piece connector
Raster speed
30 µm s with Galvanometric optics
-1
(3) Laser ablation system (DUV Nanosecond laser ablation system) Instrument Analyte Excite (Teledyne Cetac, Omaha, USA) Laser ArF Excimer Laser Wavelength 193 nm Pulse duration 8 ns -2 Fluence 0.5 J cm Laser diameter 15 µm for size analysis and 3 µm for imaging analysis Repetition rate 30 Hz for size analysis and 5 Hz for imaging analysis -1 Carrier gas He (0.80 L min ) -1
Makeup gas
Ar (0.95 L min )
Raster speed
30 µm s for size analysis and 5 µm s for imaging analysis
a
-1
-1
Reference 26.
Both the laser types and operating conditions can affect the disintegration of NPs. The lower fluence of the laser is important to minimize the contribution of laser-induced disintegration of the NPs. Benesova reported that the fluence of 0.1-1.0 J cm-2 would be appropriate value to ablate and also
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Analytical Chemistry μm, thickness about 40 μm) for the development of imaging technique of the NPs analysis. Moreover, onion cell tissues can be kept alive in water, in addition to being clear and easy to observe cell walls and nucleus. Prior to the imaging analysis, the single cell layer (approx. 1 × 1 cm) was exposed to either solutions containing Ag or Au NPs. Dosing experiments were conducted by floating the onion cell tissues on the 10 mL of solutions containing either Ag or Au NPs for 1-4 days at room temperature. The concentration of Ag and Au NPs was 108 particles mL-1. To minimize the contribution of the surface contamination of the NPs, for analytical procedure employed. two careful treatment was introduced in the imaging analysis. The first was the exposure procedure for the NPs. In this study, exposure of the NPs was made by floating the cell tissues on the solution containing the NPs (Fig. 1(a)). Hence, only one side of the tissue samples were contacted with the solution. After the exposure procedure, the tissue samples were carefully washed by the deionized water, and then the wet tissues were placed on the slide glass. Note that the sample side contacted with water was set to contact side to the slide glass (Fig. 1(b)). The sample surface, subsidized to the imaging analysis, were never contact with water containing NPs. However, one may consider that the several NPs should present on the bottom side of the cell tissues, where surface of the samples was contacted with the solution. To minimize the contribution of the NPs, present between the cell tissues and slide glass, soft ablation protocol was adopted in this study. Under the fluence and repetition rate of the laser ablation, the depth of the ablation pit was about 5-8 µm, which was significantly smaller than the typical thickness of the onion cell (e.g., 40 µm) (Fig. 1(c)). This suggests that the NPs presented in the bottom side of the cell tissues would not be ablated and introduced into the ICP, and thus, the remaining NPs between the cell tissue and slide glass were not monitored under the conditions employed here.
to minimize the disintegration of Au NPs.29 Hence, the fluence of 0.5 J cm-2 was used for both the ns- and fs-LA. Data reduction. With the data acquisition using the dwell time of 30 µs, the numbers of data produced per single second exceeds about 33,000, and therefore, an automated data reduction system is highly desired. In this study, we have developed in-house software for the detection of the signal events originating from the NPs, and also for the integration of signal intensity for individual signal events. Details of the software will be published elsewhere, and the only outline of the peak identification protocol will be described here. The signal intensity data obtained by the current spICP-MS system were stored as a csv format. Peak areas (integrated counts) were calculated by integrating the counts between starting and terminal channels. The starting channel was identified by the first data channel of continuous signal output (>1 counts), and the terminate channel was defined by the first channel of continuous nil signal output with more than 4 channels. As pointed out by Shaw and Donard, the major source of error in the size distribution analysis is the overlaps of the signal events from two or three NPs.30 They demonstrated that about 5 % of peaks can be overlapped when the number of events exceeded 100 under the assumption that the time duration of the signals of the single events was 575 µs. To minimize the contribution of overlaps of the signal events, all the peak shapes were always displayed on the software, and overlapped events were not used for further calculations. Calibration of size of NPs. With the spICP-MS system, the size of the NPs can be calibrated from the total signal intensity of individual signal events originating from the introduction of single NPs into the ICP. In this study, the calibration factor (ƒ) between the size of NPs and the signal intensity data were defined by signal intensity data obtained from commercially available 60 nm Ag and 60 nm Au NPs from Sigma Aldrich Inc. (St. Louis, USA). Hence, the calibration factors (ƒAg and ƒAu) were separately calculated from the mean signal intensity data obtained from >1000 events through the solution introduction technique. Size analysis of the NPs was carried out based on the assumption that the number of atoms contained in NPs is correlated with the ion counts found in the spICP-MS analysis as defined by 𝐷3 = 𝑓 × 𝑆 ... (1) where D is the diameter of the NP, S denotes the signal intensity. The calibration factor ƒ reflects both the ionization efficiency in the ICP and the transmission efficiency of ions from ICP ion source to the detector, and therefore, the ƒ must be defined to specific system setup and analysis conditions. In this study, the ƒ values were determined based on the measured signal intensity of constituent elements from NPs of known sizes (i.e., NPs for size calibration standard). Onion cells. To demonstrate analytical capability of the present LA-ICP-MS technique for both the size distribution and imaging analyses, onion cell tissues were used in this study. The onion was chosen because many pioneering researches have been conducted on onion cell tissues for research on the inside structure, mechanical properties and biochemical reactions of plant cell walls, as well as on the biological functions and element metabolism.31-36 Another important point to use the onion cell tissues is that the onion cell tissues is easy to sampling and is suitable size (about 100
Figure 1. Procedures for exposure experiments of Ag and Au NPs for onion cell tissues Results and Discussion Effect of laser ablation onto the size distribution of NPs. It is natural to consider that the size of the NPs can change through the process of laser ablation. As mentioned above, Benesova reported that the higher fluence being >1 J cm-2 can cause the disintegration of NPs,27 and the resulting size distribution of the NPs can be distorted by laser ablation. To test this, size distribution analysis was carried out using both the UV femtosecond and DUV nanosecond lasers. Aqueous solutions, containing 60 nm Au NPs of 107 mL-1, were dropped onto a high-purity Si-wafer. The NPs solutions were then dried at room temperature for 30 minutes, and the solution “cakes” were directly used for the size distribution analysis using the LA-ICP-MS. Prior to analysis by LA-ICP-MS, ƒAu was
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Analytical Chemistry
Figure 2(b) illustrates the resulting size distribution data for 60 nm Au NPs obtained by the laser ablation sampling technique. Both the UV femtosecond and DUV nanosecond lasers were used for the sampling. Calibration of the size of NPs was based on the ƒAu defined by the solution analysis on 60 nm Au NPs. The size distribution analysis revealed that the Au NPs with the original size (60 nm) were well preserved even after the laser ablation. However, scrutiny of the size distribution data shows that the size distribution by the UV femtosecond laser was modified toward smaller, suggestive of the contribution of laser-induced disintegration of the NPs (Fig. 2(b)). In fact, femtosecond laser can provide both higher adsorption and higher ablation efficiencies for metallic materials,26 and thus the NPs can be disintegrated to smaller sizes. The measured size distribution of the 60 nm Au NPs obtained by the DUV nanosecond laser was shown in Fig. 2(b). The most probable size of the Au NPs was 63 nm, showing good agreement with the data sheet (60 ± 12 nm). It should be noted that the width of the distribution peak at 60 nm (8 nm of FWHM) was significantly smaller than that obtained with UV femtosecond laser (24 nm of FWHM). Narrower distribution reflects the smaller contribution of both the laser-induced disintegration and agglomerations. The preservation of the original size (i.e., 60nm) found in the analysis using DUV nanosecond laser can be explained by lower ablation efficiency for the metallic materials, and thus the NPs on the Si-wafer were released with the small contribution of laser-induced disintegration or agglomerations. The data obtained here demonstrates clearly that DUV nanosecond laser is preferable for the size distribution analysis of the NPs using the LA-ICP-MS technique. In the case of Ag NPs, likely with the case of Au NPs, the measured size distribution was modified toward smaller when the UV femtosecond laser was used, whereas the disintegration could be reduced when the DUV nanoseconds laser ablation was adopted. Figure 2(b) also illustrates presence of the small size fraction (
