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Quantitative Evaluation of SERS Nanoparticles for Intracellular pH Sensing at a Single Particle Level Zhiqiang Zhang, Kazuki Bando, Kentaro Mochizuki, Atsushi Taguchi, Katsumasa Fujita, and Satoshi Kawata Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03276 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Analytical Chemistry

Quantitative Evaluation of SERS Nanoparticles for Intracellular pH Sensing at a Single Particle Level Zhiqiang Zhang,*,†,‡,∇ Kazuki Bando,†,§,∇ Kentaro Mochizuki,† Atsushi Taguchi,† Katsumasa Fujita,*,†,⊥,║ and Satoshi Kawata†, § † Department

of Applied Physics, Osaka University, Suita, Osaka 565-0871, Japan Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, 215163, Suzhou, China § Serendip Research, Osaka, Osaka 530-0001, Japan ⊥ Advanced Photonics and Biosensing Open Innovation Laboratory, AIST-Osaka Unversity, Suita, Osaka 565-0871, Japan ‡ CAS

║

Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka 565-0871, Japan

∇

These authors contributed equally to this work.

ABSTRACT: Intracellular pH is one of the key factors for understanding various biological processes in biological cells. Plasmonic gold and silver nanoparticles (NPs) have been extensively studied for surface-enhanced Raman scattering (SERS) applications for pH sensing as a local pH probe in a living cell. However, the SERS performance of NPs depends on material, size, and shape, which can be controlled by chemical synthesis. Here, we synthesized 18 types of gold and silver NPs with different morphologies such as sphere, rod, flower, star, core/shell, hollow, octahedra, core/satellites, and chainlike aggregates, and quantitatively compared their SERS performance for pH sensing. The SERS intensity from the most commonly utilized SERS probe molecule (para-mercaptobenzoic acid: p-MBA) for pH sensing was measured at the single nanoparticle level under the same measurement parameters such as low laser power (0.5 mW/µm2), short integration time (100 ms) at wavelengths of 405 nm, 488 nm, 532 nm, 584 nm, 676 nm, and 785 nm. In our measurement, the Ag chain, Ag core/satellites, Ag@Au core/satellites, and Au core/satellites nanoassemblies showed efficient pH sensing at the single particle level. By using p-MBA-conjugated Au@Ag core/satellites, we performed time-lapse pH measurements during apoptosis of HeLa cells. These experimental results confirmed that the pH measurement using p-MBA-conjugated Au@Ag core/satellites can be applied for long-term measurements of intracellular pH during cellular events.

KEYWORDS: SERS, nanoparticles, pH sensing, intracellular, apoptosis The pH value influences many biological processes such as proliferation and apoptosis, endocytosis, multidrug resistance, ion transport, and muscle contraction. It can greatly affect the structure and function of biomolecules such as DNA, protein, and enzyme. Typically, the intracellular pH in a normal mammalian cell ranges from 4.7 in lysosomes to 8.0 in mitochondria,1 whereas cancer cells have lower pH values.2,3 By monitoring the change in the pH, it not only helps to know the cell status and metabolic processes, but it can also help gain insights to cellular internalization pathways such as phagocytosis, micropinocytosis, receptor-mediated endocytosis, and non-specific endocytosis.4 The change in pH comes from protonation–deprotonation events inside the cell, which causes the large distribution of pH values. Therefore, a sensitive and fast detection of cellular pH microenvironment would facilitate better understanding of the dynamics of biochemical reactions such as the development of diseases and corresponding pathogenesis. Although the fluorescence technique and its relative pH probes have been well developed and used commonly to measure intracellular pH,5,6 it still cannot overcome several issues such as quenching, photobleaching, and autofluorescence background. Surface-enhanced Raman scattering

(SERS) has been extensively studied both theoretically and experimentally since its discovery on rough silver electrodes in 1970s.7-9 It can provide unique fingerprint information of biological and chemical samples with extremely high sensitivity from locally confined volume around the NPs. SERS provides ultrahigh sensitivity at the single molecular level and has several advantages over fluorescence methods, such as broader choice of excitation wavelengths, multiplex detection with high spectral resolution, negligible autofluorescence, and no quenching or photobleaching.10 Over the past few decades, booming developments in nanotechnology have accelerated the research for pursuing highly active SERS materials and substrates.11 Noble metal nanoparticles (NPs), such as gold and silver NPs, possess a unique optical property called localized surface plasmon resonance (LSPR), which plays a dominant role in SERS.12,13 The properties of LSPR on plasmonic NPs depend on their chemical composition, size, shape, and dielectric environment,14 which can be described by classical electrodynamic Mie theory.15-17 By chemical synthesis, a great diversity of shapes can be obtained, for example, sphere, shell/hollow, cube, rod, star, flower, plate, and polygons.18,19 Moreover, specific nanostructures consisting of multiple NPs

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such as dimer, trimer, oligomer, chain, monolayer, and core/satellites can be fabricated by self-assembly.20,21 Among them, several plasmonic NPs have been extensively used in SERS for pH sensing.10,22-27 However, it is difficult to know which one is the best choice for fast pH sensing, because these SERS measurements were carried out by different researchers using different parameters such as SERS probe molecules, integration time, performance of the camera, excitation laser wavelength, and laser power. For example, the excitation wavelengths used are usually 532 nm, 633 nm, and 785 nm; the integration time differs from 10 s to 60 s; the laser power varies from a few mW to a hundred of mW; the NPs are isolated in solution or aggregated on the substrate. In this study, we synthesize 18 different types of gold and silver NPs such as sphere, rod, flower, star, cube, hollow, core/shell, self-assembled chainlike aggregates, and core/satellites nanostructures, and so on to quantitatively evaluate the NPs’ SERS efficiency for fast pH sensing. One of the most commonly utilized SERS probes molecule paramercaptobenzoic acid (p-MBA) is selected as a standard probe. NPs are immobilized on a glass substrate at the single particle level to avoid any effect of aggregation. Six excitation wavelengths from 405 nm to 785 nm are used to investigate the wavelength dependence of Raman scattering for each type of nanoparticle. Measurement parameters such as integration time, laser power, and objective lens are fixed for every test to guarantee the consistency of the Raman setup. A lineillumination mode is chosen to obtain adequate data for statistical analysis of Raman signal from different types of nanoparticle. The final SERS performance in the order of decreasing SERS efficiency is as follows: Ag chain, Ag core/satellites, Ag@Au core/satellites, Au core/satellites, and others. The Ag chain, Ag core/satellites, and Ag@Au core/satellites nanoassemblies showed fast pH sensing at the single particle level in SERS mapping. We also performed pH monitoring with functionalized NPs during apoptosis process of HeLa cells by time-lapse SERS imaging. The Ag@Au core/satellites NPs were injected to HeLa cells by electroporation before apoptosis was induced. The variation of intracellular pH was clearly observed during the progress of apoptosis with temporal and spatial information. EXPERIMENTAL SECTION Design of NPs. As shown in Figure 1, different types of metallic NPs for SERS evaluation are considered in this work. The materials are Au, Ag, and Au-Ag composite. The types of Au NPs are sphere,28 flower,29 star,30,31 rod,32 cube,33,34 prism,35,36 octahedron,37,38 and core/satellites.39 The types of Ag NPs are sphere,40 rod,41 decahedron,41 Au-coated decahedron,42 chain,43 Ag core/satellites,44 and Ag@Au core/satellites.44 The composite of NPs are Au@Ag coreshell,45 Au@Ag core/shell,46 and Au-Ag alloy hollow flower.47 The materials and the procedures to synthesize these NPs are shown in the Supporting information. Preparation of Isolated NPs Substrate for SERS. Each of the NPs were immobilized on glass substrates by electrostatic attraction force between oppositely charged NPs and the substrate. Except for positively charged NPs such as Au nanorod, Au octahedron, Au cube and Au triangle, all others are negatively charged NPs, and they can be immobilized on poly(diallyldimethylammonium) chloride (PDDA) modified substrate. For positively charged NPs, their surface charge was reversed by coating with a poly (sodium 4-styrenesulfonate)

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(PSS) layer. By adjusting the incubation time, the NPs density on the substrate can be controlled by monitoring with a darkfield microscope. As shown in Figure S1, the commercial glass bottom cell culture dish was cleaned by oxygen plasma for 3 min at 75 W. Then, 200 µL of 1 mg/mL poly-L-lysine (PLL) solution was dropped onto the glass and incubated for 20 min, followed by extensive washing with H2O. After drying with nitrogen flow, 50 µL of NPs solution was added on the glass for few tens of seconds, followed by rinsing with H2O. Then, the water in the cell culture was replaced with ethanol. Afterwards, 100 µL of 10 mM of p-MBA was added in the dish and incubated for overnight, followed by washing with ethanol. Finally, ethanol in the dish was replaced carefully with water. The density of NPs was confirmed by dark-field microscope and SEM imaging.

Figure 1. Types of NPs for SERS evaluation including Au and Ag with different morphologies and status of assembly.

Figure 2. Schematic diagram of the optical setup for slit-scanning Raman imaging, spectroscopic imaging and dark-field imaging or phase contrast imaging. Six different CW lasers (405 nm, 488 nm, 532 nm, 594 nm, 676 nm, 785 nm) were installed in the setup. FM, flip mirror; L, lens; GM, x-axis galvanometer mirror; DF, dark-field. The laser wavelength for the excitation was selected at each experiment and the laser beam propagates the common optical path.


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Line-Illumination Raman System with Dark-field Microscope. The Raman system used for SERS evaluation is described in the literature.48 The line-illumination Raman system can be more than 100 times faster than conventional point-illumination Raman microscopy, which benefits the evaluation of a large number of NPs. In our Raman system (Figure 2), an EM-CCD camera (iXon Ultra, Andor) was used because of its excellent performance such as faster readout rate and higher signal-to-noise ratio even for short exposure time. Six excitation lasers were used, ranging from 405 nm to 785 nm. For each excitation laser wavelength, the laser power was fixed at 0.5 mW/µm2, and the integration time was fixed at 100 ms for Raman signal collection. The line shaped laser was formed on the sample plane and scanned in a direction perpendicular to the laser line using a controlled galvanometer mirror. Raman scattering passed through two edge filters, which remove the scattering light below the excitation wavelength, and Raman signal was collected by the EM-CCD camera after passing through a spectrophotometer (MK-300, Bunkou-Keiki). An objective lens with NA 0.7 dry, 60× (S Plan Fluor ELWD 60×, Nikon) was used for the study of NPs’ SERS performance. For each sample, the SERS spectra of 100 NPs were measured and averaged. For time-lapse SERS imaging of HeLa cells, an objective lens with NA 1.27 WI, 60× (CFI Plan Apo IR 60×, Nikon) was used. The exposure time for each line was set to 100 ms. The sample was illuminated with intensity of 1.2 mW/µm2 and wavelength of 594 nm. Each SERS image was constructed from the measurements with 230 scanning lines and a frame interval of 5 minutes. The phase contrast image was recorded simultaneously by a cooled CCD (DS-Fi1c, Nikon) at every frame of the SERS images. pH Sensing of Immobilized NPs. Different buffer solutions with pH from 3.5 to 8.5 were dropped sequentially on the nanoparticle-decorated glass dish, followed by fast Raman scanning immediately within 30 seconds. The integration time was 100 ms for Ag chain, and 200 ms for core/satellite and rod NPs. The excitation laser wavelength was 594 nm. Induction of Apoptosis in HeLa Cells and Injection of the NPs. We used actinomycin D (act-D) to induce apoptosis in HeLa cells through inhibition of RNA synthesis.49 Figure S2 illustrates the schematic procedure of the experiment. HeLa cells were cultured on a glass bottom dish (P50G-1.5-14-F, Matek) with 3 µM act-D diluted with Dulbecco's modified Eagle's medium (DMEM) and placed in an incubator under 37 degree Celsius and 5 % CO2 for 2 hours. The medium was changed to that without act-D, and the cells were kept under the same condition for 10 hours. After that, the medium was removed and the dish was washed with PBS buffer twice and replaced with fresh DMEM medium containing p-MBA conjugated Ag@Au core/satellites. Electroporation was applied with an electroporator (NEPA21, NepaGene). Then we changed the DMEM to Hanks' Balanced Salt solution after washing the dish with PBS solution twice in order to remove excess amount of the satellite NPs. We immediately applied time-lapse SERS imaging with the slit-scanning Raman microscope (Figure 2). As control experiments, we demonstrated time-lapse SERS imaging of HeLa cells without act-D treatment using the same measurement conditions. Data Processing for Time-lapse SERS Imaging. We recognized the SERS hot-spots as places where the Ag@Au

core/satellites NPs exist from the time series of SERS imaging datasets. A series of SERS intensity map at 1590 cm-1, which corresponds to the ring breathing mode of p-MBA, was made. The time series map shows the distribution of the satellite NPs in the cell. Thresholding and recognition of the center of each NP were done by Image J. The center coordinate of each NP was recorded, and the pH value at each coordinate was derived by obtaining the intensity ratio of the two vibrational modes corresponding to COO– and C=O mode, and the pH calibration curve taken in advance (Figure 7). This process was done by Matlab. RESULTS AND DISCUSSION Synthesized 18 Types of NPs. Although there have been a large variety of noble NPs with different shapes reported by different groups, we only selected 18 typical types of gold and silver NPs including sphere, rod, cube, octahedron, star, flower, triangle, decahedron, Au/Ag alloy hollow, core/shell, core/satellites, and random chainlike aggregate. Most of the NPs, as shown in Figure 3, were synthesized according to reported literatures by using modified protocols.28-47 For assemblies of NPs such as core/satellites, new protocols were developed to make samples that could be directly used for SERS measurement. For example, as shown in Figure S1, we used PLL as a linker to immobilize satellite NPs onto the larger core NPs, which benefits further modification of pMBA probes on assemblies. The typical SEM image and UVvis extinction spectrum for each of the 18 types of NPs are shown in Table S1.

Figure 3. Typical SEM images of 18 types of synthesized Au and Ag NPs. (1) Au sphere, (2) Au octahedron, (3) Au concave cube, (4) Au triangle, (5) Au@Ag core shell, (6) Au star, (7) Au flower, (8) Au rod, (9) Ag sphere, (10) Ag decahedron, (11) Ag rod, (12) Ag@Au core/shell, (13) Ag decahedron@Au core/shell, (14) Ag/Au alloy hollow flower,



chain of 1# spot (C) and averaged SERS spectrum (D) of all 14 spots. The objective lens was NA 0.7 dry, ×60; laser power was 0.5 mW/µm2; integration time was 100 ms. SERS Measurement of Various NPs. The thiol-based SERS probe molecule p-MBA has been extensively studied for pH sensing because of its unique SERS fingerprint spectrum on gold or silver NPs. The p-MBA has a strong affinity to gold or silver to form a uniform self-assembled monolayer with known packing density, and it can replace the capping agent on the surface of synthesized NPs such as PVP, citrate, and other surfactants, which exhibits a weak binding force with NPs. Its most significant Raman peak is around 1590 cm-1, which can be attributed to the aromatic ring breathing mode and commonly used as a reference peak for pH sensing, as shown in Figure S4. The intensity of this peak was used to quantitatively evaluate all NPs’ SERS performance in this study. 6

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(A) Ag Chain CCD Counts (x10^4)

CCD Counts (x10^4)

(15) Ag core/satellites, (16) Ag@Au core/satellites, (17) Au core/satellites, (18) Ag chain. All scale bars are 50 nm. Isolated NPs Substrate. In this work, the SERS performance of each nanoparticle type was quantitatively evaluated at a single particle level using an isolated nanoparticle substrate platform. The density of NPs on the substrate can be controlled by the density of nanoparticle solution or the incubation time. To confirm the distribution of NPs on the glass substrates after immobilization, the nanoparticle-decorated glass dishes were first checked by dark-field imaging, as shown in Table S1. For example, the dark-field image of the 72 nm Au nanospheres with an extinction peak around 540 nm clearly showed that the green color dots were separately distributed. The 75 nm Ag nanospheres, which has an extinction peak around 445 nm, appeared as isolated blue dots. The Au core/satellites, which has a second LSPR peak around 650 nm, appeared as separately distributed red dots. SEM imaging was used to further confirm the isolated NPs on the glass substrates. As shown in Figure S3, the NPs were distributed in isolation on the glass substrate.

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Figure 5. SERS intensity at 1590 cm-1 of representative NPs with different excitation wavelengths. (A) Ag chain, (B) Ag core/satellites, (C) Ag@Au core/satellites, (D) Au core/satellites. For each excitation wavelength, 300 spectra were averaged. The objective lens was 60x/0.7 NA; laser power was 0.5 mW/µm2; integration time was 100 ms. The error bar is standard deviation of 300 spectra.

Figure 4. Typical dark-field image of a Ag chain distributed on glass (A) and the SERS mapping image at the same position (B) excited by a 594 nm laser. The marked blue circles in (A) and (B) are fourteen randomly selected spots for SERS intensity analysis. The SERS spectrum from the single

The Ag chain obtained by self-assembly of 22 nm Ag nanospheres was chosen as a straightforward example to show the measurement process. As shown in Figure 4A-B, the red colored spots in the dark-field image corresponding with the Ag chains showed strong Raman signal in the SERS map obtained by line-illumination mode at the same positions, which indicates that the combination of dark-field imaging and fast line-illumination Raman imaging is highly efficient in measuring the NPs’ SERS signal in a large area. Also, the SERS spectrum from a single pixel can be easily extracted from the SERS mapping image, as shown in Figure 4C. After averaging all hot spots, a smoothed SERS spectrum can be obtained for quantitative comparison with SERS measurements of other nanoparticle types. To investigate the dependence of the nanoparticle’s SERS performance on different excitation wavelengths, six excitation laser wavelengths from 405 nm to 785 nm, which fully cover the resonance peaks of all NPs described in this study, were used to measure the SERS spectra for each type of


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nanoparticle. As shown in Figure 5A, the Ag chain showed weak SERS signal at 488 nm, and its SERS intensity reached the highest at 594 nm, and then the signal decreased significantly at 676 nm and further declined at 785 nm. For core/satellites nanoassemblies based on Ag nanospheres, the highest SERS intensity was at 532 nm (Figure 5B). For core/satellites nanoassemblies based on Au-coated Ag nanospheres (extinction peak at 560 nm), the optimal wavelength was the same at 594 nm (Fig. 5C). For Au core/satellites with a second extinction peak at 640 nm, the highest SERS intensity was at 594 nm (Figure 5D). We fixed the parameters for all measurements including the laser power for all excitation wavelengths (0.5 mW/µm2) and short integration time (100 ms) for Raman signal collection. It should be noted that we used much lower laser power and quite shorter exposure time than that of previously reported works. Unfortunately, all the single NPs showed extremely weak SERS signal under such measurement parameters, for example, the sphere, rod, star, flower, octahedron, cube, triangle, core/shell and hollow NPs.

Figure 6. Quantitative evaluation of the SERS performance of synthesized NPs by comparison of their SERS intensity at 1590 cm-1. The “Others” includes sphere, rod, star, flower, octahedron, cube, triangle, core/shell, and hollow NPs. The blue bars show total CCD counts while the orange bars show normalized CCD counts by each surface area of the NPs. Figure 6 and Table S2 showed the SERS performance of different types of NPs arranged in order of decreasing Raman intensity at 1590 cm-1 at the excitation wavelength that shows the strongest SERS signal for each nanoparticle. To compare the SERS performance of these 18 types of particles, the direct way is the SERS intensity obtained by CCD camera for each type of particle which can be marked as “cps/particle” as shown in Table S3. Because of the sizes of these18 types of particles are also different, the surface area or the number of SERS probes on the particle should be taken into consideration. Therefore, the other way to compare SERS enhancement is the normalization of SERS intensity by total number of SERS probes on the particle which can be marked as “cps/p-MBA”. It is clearly seen that the order of SERS performance of nanoparticles evaluated by “cps/particle” matches well with the order of that by the “cps/p-MBA”. From our experimental results, the SERS activities of NPs in the order from highest to lowest were as follows: Ag chain, Ag core/satellites, Ag@Au core/satellites, Au core/satellites, and others. The Raman intensity of the Ag chain showed about three times higher than that of Ag core/satellites and Ag@Au

core/satellites; the Ag core/satellites showed comparable intensity with Au-coated Ag core/satellites and showed higher Raman signal than Au core/satellites; the Ag rod showed a bit higher SERS signal than Au rod. For other NPs, their SERS intensities were too small to be measured in the conditions tested. It should be noted that the order of SERS NPs was summarized according to our specific experimental parameters such as lower laser power, shorter integration time for Raman signal collection, and one type of SERS probe molecule. Therefore, other types of NPs may show high SERS performance by changing the parameters. For example, the Au nanostar, which has intrinsic hot-spots, can provide high Raman enhancement by using other types of SERS probes or higher laser power or longer integration time. For example, Zhang et al.33 investigated the SERS activity of single Au nanostar with a 785 nm excitation wavelength by using a similar SERS probe 4-aminothiophenol (p-ATP), spotscanning mode with a laser power at 1.1 mW/µm2, and integration time of 20 seconds, which is 200 times longer than that of this work. Discussion of SERS Activity of NPs. To understand the SERS performance of different shaped NPs, two main factors should be considered: the surface area of nanoparticle or the number of SERS probe molecules, and the near-field electromagnetic (EM) field. According to the mechanism of plasmon resonance enhanced Raman scattering,13,50,51 the SERS enhancement factor is proportional to the strength of the EM field around the surface of the nanoparticle to the fourth power. Therefore, the near-field EM field is much more important for SERS performance of NPs. From the shape point of view, compared with other shaped NPs, the rodlike nanoparticle has much higher EM field enhancement at its two ends when the incident light is parallel to the longitudinal direction of the rod.52 The other way to get high near-field EM strength53-55 is to elaborately fabricate plasmonic hot-spots located at the gaps56-58 between several NPs through “top-down”59,60 or “bottom-up”56,61,62 methods. The number of hot-spots is another point to compare NPs’ SERS activity. For core/satellites nanoassemblies, the number of hot-spots depends on the number of satellite NPs on the core particle. However, the number of the satellites is limited because of the size of the core and satellite. To further increase the number of the hot-spots, self-assembly of small NPs into chainlike nanoaggregates is a simple route. In this work, the experimental results (Figure 6) showed that the particles with multiple hot-spots such as core/satellites and chainlike nanoassembly gave higher SERS activity while other particles including rodlike nanoparticles showed insignificant SERS intensity. To further understand the weak SERS enhancement of “Others”, we chose Ag rod and Au rod as a model. First, to confirm that the p-MBA molecules can successfully adsorb on the surface of nanoparticles, we first prepared the aggregated Ag and Au rod samples by a drying process and then incubated them into a 1mM of p-MBA solution with a same manner as before. As expected, the aggregated Ag rod and Au rod samples can easily show significant SERS “hot-spots”, as shown in Figure S4 and Figure S5. Therefore, the p-MBA molecules can adsorb on the surface of isolated Ag rod and Au rod nanoparticles in our experimental protocol. This result indicated that the SERS enhancement of single rod nanoparticle was too weak to detect. Additionally, we performed the dark-field imaging,


Analytical Chemistry SERS mapping imaging, and SEM imaging at the same position to confirm that these “hot-spots” did come from the aggregated nanoparticles but not from single particles. To further demonstrate the negligible SERS enhancement of single Ag rod and Au rod, we measured the Ag rod and Au rod samples with a high density which were prepared by incubation process. As shown in Figure S7 and Figure S8, the merged image of the dark-field, SERS, and SEM at the same position clearly shows that only aggregated Ag rod and Au rod can give SERS enhancement. Therefore, we concluded that our previous SERS results of Ag rod and Au rod samples were not from single rod particles but from the aggregated rod particles. For other shaped nanoparticles such as sphere, star, flower, core/shell, hollow, and octahedra particles, their SERS performance at the single particle level were same as rod nanoparticle’ case and they cannot meet the requirement of fast pH sensing at our measurement parameters (short integration time 100 ms, lower excitation power 0.5 mW/µm2). However, it has been well known that aggregated nanoparticles no matter what shapes they are, they can easily show much higher SERS enhancement than single particles. For a demonstration, we also prepared the Ag and Au sphere samples with higher density and lower density, as shown in Figure S9-S12. We can clearly see that only aggregated Ag and Au spheres can give SERS signals. Therefore, we can conclude that the single nanoparticle including sphere, rod, star, flower, core/shell, hollow, and octahedra are not suitable for single particle SERS sensing in our work, and only multiple “hot-spots” particles such as self-assembled chain and core/satellites particles which give higher SERS enhancements can be used for our purpose. (A) Dark-field Image of Ag Chain

−COOH

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core/satellites, and Au core/satellites were chosen for pH sensing test with short integration time because of their excellent SERS performance. Generally, pH sensing measurements based on SERS-active NPs are reported to be carried out in a concentrated solution with high laser power and/or long integration time.10,26,27,43,63 Here, we studied the pH sensing at the single particle level with lower excitation power (0.5 mW/µm2) and short integration time (100 ms or 200 ms). The SERS imaging scan started immediately after replacing different pH buffer solutions with pH from 3.55 to 8.55. The SERS spectra under different pH solutions were obtained from one fixed single chain (Figure 7A and Figure S13A). With increasing pH, the Raman peak at 1418 cm–1 increased while the other peak at 1698 cm–1 decreased, as shown in Figure 7B. The averaged SERS spectra also showed the same trend (Figure S13B). These two Raman peaks are attributed to the COO– and C=O stretching modes of p-MBA, respectively. Therefore, the intensity ratio of these two peaks is usually used to detect the change of pH environment around the NPs.64 In one SERS mapping image, all isolated Ag chains were accounted for by statistical analysis (Figure S13A). As shown in Figure 7C, the intensity ratio (I1418 cm–1/ I1698 cm–1) from Ag chain gradually increased with increasing pH.

(B) Single Ag Chain’s SERS Spectra −COO–

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Figure 7. Dark-field image of an isolated Ag chain on glass substrate (A). The particle marked by the white arrow in (A) is selected to show single particle SERS pH sensing. The SERS spectra of the marked singe Ag chain in different pH buffer solutions from pH 3.35 to pH 8.55 (B). The averaged ratio (n=14) of SERS intensity at the peak of –COO– and –COOH in different pH buffer solutions (C). The objective lens was 60x/0.7 NA; the laser power was 0.5 mW/µm2; the integration time was 100 ms. The error bar shows the standard deviation in the measurement of 14 spectra. Single Nanoparticle pH Sensing with Short Integration Time. The Ag chain, Ag core/satellites, Au-coated Ag

Figure 8. SERS spectra and pH sensing of Ag core/satellites with 532 nm excitation (A), Ag@Au core/satellites with 594 nm excitation (B), and Au core/satellites with 594 nm excitation (C). The objective lens was 60x/0.7 NA; laser power was 0.5 mW/µm2; integration time was 200 ms. The error bar shows the standard deviation of 20 spectra for the measurement of three different kinds of nanoparticles.

For core/satellites nanoassemblies of Ag, Ag@Au, and Au, their SERS spectra showed significant intensity change in two Raman stretching modes around 1400 cm–1 (COO–) and 1690 cm–1 (C=O) with increasing pH, as shown in Figure 8A-C. The COO– stretching peak changed to 1360 cm–1 for pH 3.35, and it redshifted with increasing pH. Also, the Raman shift of COO– stretching mode at pH 8.55 showed a blue shift compared to Ag chain and other reported values. Although the mechanism behind this finding is not yet fully understood, this interesting Raman shift of COO– stretching mode may provide a new method of pH sensing other than the ratio (ICOO–/ IC=O). It should also be noted that the ratio (ICOO–/ IC=O) by Ag@Au core/satellites showed more uniformity than that of the Ag core/satellite at higher pH values, which has smaller error bars.


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It should be noted that the error bars in Figure 7 and Figure 8A showed huge at high pH. One of the possibilities is that the repulsion between the negative charge of metal and p-MBA is high at higher pH. As a result, the intensity of the SERS signal is unstable and shows huge error at the neutral pH 7 or 8. Dynamic pH Imaging during Apoptosis Process. To demonstrate the capability of long-term intracellular pH sensing by SERS, we measured pH change during apoptosis of HeLa cells by using p-MBA conjugated Ag@Au core/satellites. We chose Ag@Au core/satellites because of their uniformity of enhancement and its chemical stability. After induction of apoptosis, the NPs were injected into the HeLa cells with the electroporation. Immediately after the introduction of NPs, simultaneous imaging with phase contrast and SERS were performed for 150 min. Raman imaging system in this work is a slit-scanning microscopy. Therefore, the resolution is same as the conventional microscopy in the direction of parallel to the slit and same as the confocal microscopy in the direction of perpendicular to the slit. The results of phase-contrast and SERS imaging are shown in Figure 9 A-B. The supporting information provides the movie of cell shrinkage by apoptosis. The SERS imaging was started about 2 hours before the cell shrinkage. The phase contrast images showed the morphological change along with the apoptosis process (Figure 9A). In Figure 9A, cell shrinkage and collapse were observed in the last 15 min of the measurement. Figure 9B shows the SERS intensity distribution of 1590 cm-1 assigned to a benzene ring breathing mode of p-MBA attached on the NPs. The p-MBA shows a strong peak at 1590 cm-1,65 therefore, the intensity distribution of the peak intensity represents the distribution of the NPs. The SERS images show that the NPs were evenly distributed in the cytoplasm except the nucleus. After 135 min, the cell began to shrink and the NPs were moved along with the shrinkage of the cell body. During the shrinkage, some of the NPs moved out of the focus region, and consequently, the hot spots disappeared from the focal plane.

Figure 9. Time-lapse SERS imaging of HeLa cells during apoptosis. (A) Phase contrast image of HeLa cells after drug

treatment (overnight). (B) SERS intensity map of 1590 cm-1 assigned to benzene ring breathing mode of p-MBA attached on the NPs. (C) local pH distribution map of the HeLa cell. Each pH value was calculated from the calibration curve taken in advance. The images show every 15 minutes/frame and the scale bar shows 10 µm (A-C). The white dotted area indicates a HeLa cell, which showed shrinkage during the measurement. (D) averaged pH from the white dotted HeLa cells over time every 5 minutes. The error bar shows standard deviation of the pH.

From the results of SERS imaging, we estimated pH values around the NPs as shown in Figure 9C. Every pH value was calculated by using the pH calibration curve taken in advance (Figure 7). The calculation result shows that the pH values at each position were different in the cells, which is presumably due to the difference in chemical environments in organelles or cytosol. In fact, the intracellular pH differs in each organelle.1 In Figure 9D, the pH averaged within the observed area exhibits a downward trend of pH with time (Figure 9D, Figure S14). On the other hand, the control experiment using HeLa cells without act-D did not show such a downward trend during the measurement (Figure S15). Due to the regulated proton gradient between the boundaries of each organelle, each organelle maintains a proper pH in normal conditions. However, in the apoptosis process, fragmentation of the organelles occurs in advance of cell shrinkage and it triggers acidification of the cell.66 In fact, such pH dynamics is related to the cascade and has been reported in literatures. One of the possibilities for the downward trend is that some acidic organelles, such lysosome, are fragmented.67 Since the pH in lysosome is around 4.7, while the pH in cytosol is around 7.2,1 the disruption of lysosomal membrane can induce a decrease in intracellular pH. Another possibility is the upregulation of V-ATPases, which deliver protons generated through hydrolysis reaction of ATP to lysosome during apoptosis.68 It is reported that low pH is necessary to induce cascade activation.69 These conditions can overlap and result in cytosolic acidification at an early state of the apoptosis process, which actually agrees with our experimental result showing the pH downward trend during the measurement. CONCLUSIONS Here, we synthesized eighteen types of gold and silver NPs such as sphere, rod, flower, star, cube, octahedra, hollow, core/shell, self-assembled chainlike aggregates, core/satellites nanostructures, and others. By combining dark-field imaging and fast line-illumination Raman imaging, the SERS performance of these NPs for pH sensing was quantitatively compared at the single particle level on glass substrate at six excitation wavelengths: 405 nm, 488 nm, 532 nm, 584 nm, 676 nm, and 785 nm. All SERS measurements were carried out under the same experimental parameters such as SERS probe (p-MBA), laser power (0.5 mW/µm2), integration time (100 ms), and objective lens (NA 0.7 dry, ×60). The order of SERS performance from highest to lowest at their optimal excitation laser wavelengths was as follows: Ag chain, Ag core/satellites, Ag@Au core/satellites, Au core/satellites, Ag rod, Au rod, Au rod, and others. The single Ag chain, Ag core/satellites, and Ag@Au core/satellites nanoassembly showed efficient SERS pH sensing. We also demonstrated the in vivo pH measurement with the synthesized NPs. Time-lapse SERS imaging was applied during apoptosis process using the p-MBA conjugated Ag@Au core/satellites. The experimental results indicate that the pH exhibited a decreasing trend with the progress of apoptosis, which agreed with previous



researches. Thus, this study shows that measurement of pH dynamics has great potential as a diagnostic tool such as identification of healthy/cancer cells or investigating a new drug with long term monitoring of cells. For further applications, not only the pH but also multi-functionalization of the NPs is necessary for observing a specific targeted event or organelle in cell events, which will provide a much more meaningful information by highly active SERS NPs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis procedures of 18 types nanoparticles; preparation and characterization of isolated nanoparticle substrate; Synchronized the dark-field imaging, SERS mapping imaging, and SEM imaging of single/aggregated nanoparticles on glass substrate at the same area; SERS mapping image of chainlike aggregate at different pH; preparation procedure for apoptosis triggering and injection of nanoparticles; Time lapse SERS imaging of HeLa cells during apoptosis and control experiment data (PDF) Movie of apoptosis (MP4)

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the NOF of Osaka University for technical support of SEM and UV-vis characterization. The authors acknowledge supports from JSPS KAKENHI (Grant 26000011), NSFC (Grant 51405483), and NSFJS (Grant BK20140377).

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