BSA-Coated Nanoparticles for Improved SERS-Based Intracellular pH

Nov 24, 2014 - Cheng ZongMengxi XuLi-Jia XuTing WeiXin MaXiao-Shan ZhengRen HuBin ... Jiaolai JiangSumeng ZouLingwei MaShaofei WangJunsheng ...
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BSA-Coated Nanoparticles for Improved SERS-Based Intracellular pH Sensing Xiao-Shan Zheng,† Pei Hu,‡ Yan Cui,† Cheng Zong,† Jia-Min Feng,† Xin Wang,∥ and Bin Ren*,†,‡,§ †

State Key Laboratory of Physical Chemistry of Solid Surfaces, ‡The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, and §Collaborative Innovation Center of Chemistry for Energy Materials, College of Chemistry and Chemical Engineering, and ∥School of Physics and Mechanical & Electrical Engineering, Xiamen University, Xiamen 361005, China S Supporting Information *

ABSTRACT: Local microenvironment pH sensing is one of the key parameters for the understanding of many biological processes. As a noninvasive and high sensitive technique, surface-enhanced Raman spectroscopy (SERS) has attracted considerable interest in the detection of the local pH of live cells. We herein develop a facile way to prepare Au-(4-MPy)-BSA (AMB) pH nanosensor. The 4-MPy (4-mercaptopyridine) was used as the pH sensing molecule. The modification of the nanoparticles with BSA not only provides a high sensitive response to pH changes ranging from pH 4.0 to 9.0 but also exhibits a high sensitivity and good biocompatibility, stability, and reliability in various solutions (including the solutions of high ionic strength or with complex composition such as the cell culture medium), both in the aggregation state or after longterm storage. The AMB pH nanosensor shows great advantages for reliable intracellular pH analysis and has been successfully used to monitor the pH distribution of live cells and can address the grand challenges in SERS-based pH sensing for practical biological applications.

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for multiplex detection; (4) noninvasive to biological samples; (5) negligible interference from autofluorescence of biological systems and Raman signal of water; and (6) free from quenching or photobleaching. These unique features make SERS an ideal technique for sensitive pH sensing. To realize SERS-based pH sensing, probe molecules with both sensitive pH response and strong Raman signals are adsorbed on the highly SERS-active nanoparticles. When the molecules are exposed to the environment of different pH values, the probe molecules experience protonation and deprotonation, leading to obvious changes in the relative intensity of some Raman marker peaks. The intensity ratio of the marker peaks of the protonated and deprotonated forms can be used for the sensitive pH sensing. Up to now, many molecules have been used as pH probe molecules, such as para-mercaptobenzonic acid (pMBA), 4-mercaptopyridine (4-MPy), para-aminothiophenol (pATP), 3-amino-5-mercapto-1,2,4-triazole (AMT), and 2-aminobenzenethiol (2-ABT). Among them, pMBA and 4-MPy are the most frequently used molecules because of their simple molecular structure, strong binding affinity with the metal nanoparticles, and sensitive pH response.10−17 When the pH sensor is internalized by the cells, it can produce different SERS signals in response to the cell

n biological systems such as cells, local pH of the cellular compartments is one of the most important regulating factors in the physiological activities of cells. Subtle changes in the pH can have dramatic effects on cells and organelle and cause diseases. Therefore, sensitive and reliable detection of the cellular pH microenvironment may facilitate the understanding of the changes during the occurrence and progression of diseases and corresponding pathogenesis at the molecular level. Optical techniques, which are noninvasive, free of ionizing radiation, and capable of multimodal imaging and real-time and quantitative detection, have become increasingly important in life sciences. Among the various optical techniques, fluorescence technique is well-established and has been the most commonly used to detect the cellular pH values.1 Nevertheless, it may suffer from quenching, photobleaching, and autofluorescence when used in biological systems. Raman spectroscopy has been widely used as a robust vibrational technique to provide molecular fingerprint information on various biomolecules, cells, and tissues, even under the in vivo conditions.2−5 When it is combined with plasmonic nanostructures, the Raman signal can be enhanced by several orders of magnitude, leading to the so-called surfaceenhanced Raman spectroscopy (SERS). After about 40 years’ development, SERS has been widely used in various interdisciplinary fields.6−9 It is especially advantageous when it is used for biomedical applications: (1) ultrahigh sensitivity up to single molecular level; (2) flexible excitation wavelengths from the visible to infrared region; (3) high spectral resolution © 2014 American Chemical Society

Received: September 10, 2014 Accepted: November 16, 2014 Published: November 24, 2014 12250

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pH changes, ranging from pH 4.0 to 9.0. We systematically investigated the properties of AMB pH nanosensor by comparing with AM pH nanosensor, including the pH response, sensitivity, stability, and biocompatibility. The AMB pH nanosensor was successfully used to monitor the intracellular pH distribution of living cells. This study aims to emphasize the importance of reliability of SERS-based pH sensing and provide a facile way to prepare reliable SERS-based pH nanosensor.

environment with different pH values and probe the local pH changes in the microenvironment. Some efforts have been made to detect the local pH values of live cells by using SERS. Talley and co-workers used the pMBA-functionalized AgNPs as the pH sensor to probe the local pH information inside the cell for the first time.18 Kneipp and co-workers used the pMBA modified AuNPs as the pH nanosensor, which showed an effective pH response in the pH range of 2 to 8. By scanning over the entire live cell, they obtained the local pH distribution to reflect the cellular pH microenvironments.19 They further investigated the dynamic pH change in the endosome of live cells following the uptake of pH nanosensor, which gives insight into the maturation pathway of the endosome.20 This seminal work is of great significance for further applications of the SERS-based pH sensing in the study of the physiological and metabolic process in cells. Alternatively, Rector and coworkers designed a targeted pH nanosensor with 4-MPy adsorbed on the gold-core surface as the probe molecule and 2,4 ε-dinitrophenol-L-lysine (DNP) ligand attached on the silver-shell surface as the targeting molecules to the IgE receptor. They obtained the whole-cell pH distribution over time with a in vivo pH calibration curve and further studied the effect of temperature on the behavior of the pH nanosensor in cells as well as the pH response with the addition of dynamic external stimuli or the H+ flux inhibiting drugs.21−23 Aside from monitoring the cell process, efforts have also been made to improve the multimodality of the pH nanosensor. Moskovits and co-workers developed a multifunctional pH nanosensor for multimodal Raman and fluorescence imaging. The nanosensor was functionalized with a fluorescent coating and pH-sensitive molecule (4-MPy). The pH sensing part was detected with SERS to provide information on the local pH in cells, and the fluorescent part allows for tracking the attached fluorescent protein cargo, holding the potential in monitoring the drug delivery process.24 Recently, a silicon nanowire-based SERS endoscope was developed for more practical intracellular pH detection. It enables the detection of specific cellular compartments, such as the plasma or nucleus, and avoids the aggregation because of the immobilization of the nanoparticles.25 Although SERS-based pH sensing has found important application in live cell studies, there are still some challenges concerning the practical application for reliable pH sensing, especially in biological systems: (1) whether the selected peaks to monitor the pH response in bulk buffer solutions can be still reliable in the cellular systems; (2) whether the aggregation of nanosensor will affect the reliability of pH sensing because the tendency of aggregation will increase when nanoparticles are uptaken;26 (3) whether the nanoparticles with Raman probes or targeted molecule can remain intact, and will these molecules be replaced by the abundant biomolecules in the complex system of live cells, leading the important issue of stability and reliability. All the above questions are vital to reliable SERS-based cellular pH sensing and need to be investigated systematically. In this work, we developed a facile way to prepare the reliable SERS-based Au-(4-MPy-BSA) (AMB) pH nanosensor for intracellular pH analysis. The 4-MPy was chosen as the pH sensitive molecule adsorbed on the AuNPs to obtain the Au-(4MPy) (AM) pH nanosensor. The AM pH nanosensor was then functionalized with BSA molecules to form a biocompatible protective layer to obtain the reliable AMB pH nanosensor. The AMB pH nanosensor has a high sensitive response to local

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EXPERIMENTAL SECTION Chemicals. 4-MPy and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Aldrich. Bovine serum albumin (BSA) was purchased from Bio Basic Inc. Choloroauric acid (HAuCl4·4H2O), trisodium citrate dihydrate (C6H5Na3O7·2H2O), potassium chloride (KCl), dibasic sodium phosphate (Na2HPO4·12H2O), sodium dihydrogen phosphate (NaH2PO4·2H2O), anhydrous phosphoric acid (H3PO4), and dimethyl sulfoxide (DMSO) were obtained from Sinopharm Chemical Reagent Co., Ltd. All the above chemicals were of regent grade and used as received without further purification. Ultrapure water (Milli-Q, 18.2 MΩ) was used throughout the experiments. The pH of the PBS solution was adjusted with Na2HPO4·12H2O, NaH2PO4·2H2O, and H3PO4 as needed and measured with a digital pH/ion meter. Preparation of AM and AMB pH Nanosensor and SERS Samples. The method for preparing the AM and AMB pH nanosensor is shown in Figure 1. First, Au colloidal

Figure 1. Schematic illustration of the procedure for the preparation of the Au-(4-MPy) (AM) and Au-(4-MPy)-BSA (AMB) pH nanosensor.

nanoparticles were synthesized according to a modified Frens’ method.27 In brief, 100 mL of 0.01% (w/v) HAuCl4 aqueous solution was refluxed to boiling under vigorous stirring. Then, 1 mL of 1% (w/v) sodium citrate aqueous solution was added quickly. The mixture was kept boiling for 30 min under stirring and cooled to room temperature, resulting in nanospheres with an average diameter of 40 nm (see Figure S1 of the Supporting Information). The Au nanoparticles (AuNPs) were subsequently functionalized with 4-MPy to obtain the AM pH nanosensor by adding 200 μL of 0.05 mM 4-MPy aqueous solution dropwise to 20 mL of as-prepared Au colloidal solutions under vigorous stirring. Then, 200 μL of 2% BSA aqueous solution was added dropwise to the obtained AM pH nanosensor solutions under vigorous stirring for 15 min to obtain the AMB pH nanosensor. For the SERS measurements, 1.0 mL aliquots of the pH nanosensor solution were added to Eppendorf tubes, and each tube was centrifuged at 4000 rpm for 10 min and the supernatant was then removed and discarded. After that, the pH nanosensor was mixed with 500 μL of PBS or other aqueous solutions and then added to the 96-well plates for SERS measurements. Characterization. The morphology of the AuNPs was characterized using scanning electron microscopy (SEM) 12251

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Figure 2. SERS spectra of AM and AMB pH nanosensor in PBS solutions of various pH values ranging from pH 4.0 to 9.0 in a step of 0.5 pH unit. (A) gives the raw spectra of AM and (B) is the normalized spectra of (A) with the strongest peak. (C) is the zoom-in spectra in the spectral range of 1150−1350 cm−1. (D−F) are similar to (A), (B), and (C) respectively, but for AMB. Each spectrum represents the average spectrum of 5 measurements.

containing pH sensitive nanoparticles with different doses (2− 50 μg/mL) were added into each well. Cells treated with culture medium only served as a negative control group. After 4 h coincubation, the medium was aspirated, and the wells were washed using PBS buffer. After that, 25 μL of MTT solution (5 mg/mL in PBS buffer) was added to the wells. After incubation for another 4 h, the solution was aspirated and 100 μL of DMSO was added to each well to dissolve the blue formazan crystal produced by proliferating cells. Cell viability was measured using a spectrophotometric microplate reader (BiotekELX800) at 490 nm. All experiments were performed in quintuplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated cells.

(Hitachi, S-4800). The absorption spectra were collected with a UV−vis spectrometer (HITACHI U-3900H). The average size of the AMB pH nanosensor dispersed in PBS solutions was measured using a dynamic light scattering instrument (Malvern, NanoZS). The SERS measurements were performed on the Nanophoton Raman-11 system equipped with an upright microscope (Nikon Eclipse 90i) and a 600 grooves/mm grating. A 785 nm laser line was used for all the measurements. For the SERS measurements of solution samples, a 20× (NA 0.45) microscope objective with a working distance of 4.5 mm and spot focused laser was used. The laser power and acquisition time were 88 mW and 3 s. For the SERS imaging of cell samples, the 50× (NA 0.45) microscope objective with a long working distance and line focused laser was used. The laser power and acquisition time were 3.0 mW/pix and 5 s/ shot. Cell Culture and Sample Preparation. Human cervical cancer cells (CaSki cell line) were cultured in DMEM (high glucose) (Hyclone) supplemented with 10% serum (Hyclone) and 1% penicillin/streptomycin (Hyclone). The cells were maintained at 37 °C in a humidified 5% CO2-containing atmosphere. For the SERS imaging, the suspended cells were seeded on a clean cover glass cultured for 24 h then the AMB pH nanosensor was added into the culture medium to interact with the cells for another 4 h. After that, the culture medium was discarded and the cells were washed three times with PBS before the SERS measurement. In Vitro Cytotoxicity. The biocompatibility and cytotoxicity of the AM and AMB pH sensitive nanoparticles was studied on CaSki cells by using the MTT assay. In detail, CaSki cells were seeded at a density of 1 × 105 cells per well in a polystyrene 96-well culture plate and incubated for 24 h. After the culture medium was removed, 100 μL of culture medium

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RESULTS AND DISCUSSION Preparation and Characterization of AM and AMB pH Nanosensor. The AM and AMB pH nanosensor were prepared according to the procedure shown in Figure 1, and a detailed description is given in the Experimental Section. 4MPy was chosen as the reporter molecule because of the large Raman scattering cross section, simple molecular structure, strong binding with metal nanoparticles, sensitive pH response, and well-established spectral assignments. The key to the preparation of reliable pH nanosensors is the BSA coating over conventional SERS pH sensor as a protective layer. When nanoparticles are mixed with biological fluids such as serum or proteins, a protein corona will be formed on the surface of the nanoparticles, which will prevent the further interaction of the nanoparticles with other molecules.28 BSA is a commonly used coating protein molecule in the design of various SERS tags.29−32 It is believed that the disulfide bonds in the BSA corona can interact with the remaining Au sites on the 4-MPy modified AuNPs forming the strong Au−S bond. Therefore, a 12252

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Figure 3. pH response of (A) AM and (B) AMB pH nanosensor of different batches shown with the intensity ratio from 1208 to 1274 cm−1 as a function of pH values. The error bars indicate the standard deviations from 5 measurements. The fit line is given as a guide to the eyes.

response (see Figure S3 of the Supporting Information). Several typical pyridyl-related Raman peaks such as 1012, 1055, 1093, 1208, 1274, 1574, and 1607 cm−1 were obtained with high signal/noise ratio from the AM and AMB pH nanosensor. Among them, the intensity ratio of two pairs of bands at 1012 (ring breathing) and 1093 cm−1 (X-sensitive/C−S), 1574 (ring deformation with CC antisymmetric stretch) and 1607 cm−1 (ring deformation with CC symmetric stretch) are often chosen as the indicators in most SERS-based pH sensing by using 4-MPy because the intensity ratios of these peak pairs are found to change obviously at different pH values.21,34 As expected, the nanosensor shows high intensity even detected in the solution, benefited from the formation of aggregates. Comparing the SERS spectra of AM with AMB, we can find that AMB pH nanosensor has simpler spectra than AM pH nanosensor because the Raman peaks of AM pH nanosensor at 1093 and 1574 cm−1 appear to split. The main reason is that the AM pH nanosensor does not have any protective molecule, and the pyridyl group on the surface may interact with the anion in the solution.35 Therefore, when the AM pH nanosensor is redispersed in PBS solutions, the pyridyl group of 4-MPy may interact with the anion, which will result in the formation of aggregates and the peak splitting. Indeed, we found the AM pH nanosensor aggregated severely when redispersed in PBS solutions during the experiment (see Figure S5 of the Supporting Information). To verify this assumption, we obtained SERS spectra of AM and AMB pH nanosensor in H2O, 0.2 M KCl aqueous solution, and 0.2 M PBS solution (pH 7.0) (see Figure S4 of the Supporting Information). Here KCl was chosen as a control of different anion (Cl−). The results show that no peak splitting occurs in any solution for AMB pH nanosensor, while obvious peak splitting can be found in both KCl and PBS solutions for AM pH nanosensor. The results convincingly demonstrate that the SERS spectra of 4MPy would have interference by the anion for AM pH nanosensor without BSA coating. Furthermore, it can be imagined that the severe aggregation will induce a spectral change not purely influenced by the pH change but by the molecular interaction, which will result in a messy response. It is necessary to point out that the AMB pH nanosensor will also aggregate severely in PBS solutions due to the weakening of the repulsion between BSA molecules when the pH values (pH ≤ 5.0) are lower than the pI value of BSA (see Figures S2 and S5 of the Supporting Information). However, the pH value of the

stable serum protein corona can be formed on AuNPs, which plays important roles to ensure the reliability of the AMB nanosensor. On one hand, the BSA corona can provide an effective protection layer due to steric effect to prevent the aggregation of AuNPs, when they are in direct contact with the interference species or cellular components.33 On the other hand, the repulsion between BSA molecules can further prevent the aggregation of the AMB pH nanosensors. In addition, the cytotoxicity of nanosensor can be reduced since BSA is a common protein molecule. A broader UV−vis spectrum of AMB pH nanosensor (see Figure S1 of the Supporting Information) indicates that a slight aggregation occurred after the subsequential addition of 4-MPy and BSA. That is, the AMB pH nanosensor consists of both monodispersed nanospheres (dominantly) and some gold nanoaggregates formed by 2−3 gold spheres as shown in Figure 1 and Figure S2 of the Supporting Information. The small amount of nanoaggregates dominates the SERS signal because of the dramatically enhanced electromagnetic field in between the nanoparticles due to the coupling effect. The coupling between nanoparticles leads to a red shift and broadening of the localized surface plasmon resonance band. In the present experiment, we chose a 785 nm wavelength to obtain strong SERS signal and avoid interference of the autofluorescence of live cells. Furthermore, the final size of these nanoaggregates is within the range that can still be easily internalized into the live cells. Therefore, we believe it is the nanoaggregates that provide the high SERS sensitivity, and the BSA coating can prevent the gold nanospheres from further aggregations. We will demonstrate the roles of BSA coating in the following experiments. First, the BSA coating prevents further aggregation of the pH nanosensor. Then, with the protection of BSA coating, the reporter molecule can survive under the complex conditions, such as high ionic strength solution or even the heterogeneous biological system, which ensures the sensitivity and stability of pH nanosensor. Finally, the biocompatibility can be improved and the BSA-coated pH nanosensor can be successfully used for reliable intracellular pH sensing. Characterization of pH Response in Bulk Solution. SERS spectra of AM and AMB pH nanosensor in PBS solutions of various pH values from pH 4.0 to 9.0 in steps of 0.5 pH are shown in Figure 2. SERS spectra of Au and Au-BSA nanoparticles did not show obvious signal that may interfere with the SERS of 4-MPy no matter in the intensity or the pH 12253

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Figure 4. (A) The SERS spectra and (B) the corresponding pH calibration curve of AMB pH nanosensor before and after storage for 7 days. The black lines and error bars represent the average spectrum and the standard deviations from 5 measurements, respectively. The fit line is given as a guide to the eyes.

cell culture medium is about 7.0−7.5. Therefore, before uptaken by the cell, the BSA-coated nanoparticles will not form large aggregates. Furthermore, the SERS spectral feature and the pH response still remain unchanged owing to the BSA coating which can effectively prevent the interaction of reporter molecules with the anion. The latter feature is especially important for the application of pH nanosensor under complex conditions. As aforementioned, the two pairs of peaks of 1574 cm−1/ 1607 cm−1 or 1012 cm−1/1093 cm−1 were often selected to monitor the pH response.21,34 However, in this study we prefer to use the peaks of 1208 cm−1 (C−H in-plane bending) and 1274 cm−1 (C−N stretching) for the following reasons. (1) The intensity ratio of peaks 1208 and 1274 cm−1 (hereinafter referred to as the intensity ratio) changes with the pH values monotonically (see Figures 2F and 3B). (2) Their peak intensities are comparable, which can minimize the error when calculating the intensity ratio. (3) They can still be detected with explicable intensity ratio when the AMB pH nanosensor is introduced into the live cells (see Figure S6 of the Supporting Information). Figure 3 shows the dependence of the intensity ratio on the pH for AM and AMB pH nanosensor of different batches. It can be seen that the AMB pH nanosensor has excellent reproducibility in a wide pH range from 4.0 to 9.0 with small deviations even for the samples of different batches. The intensity ratio increases with the increasing pH of the PBS solutions, which can be attributed to the increased number of the deprotonated pyridyl. However, for the AM pH nanosensor, no regular response was found for samples of different batches (see Figures 2C and 3A). This is easy to understand because the AM pH sensor severely aggregates, and the 4-MPy molecules adsorbed on one gold sphere may interact with the gold surface of other nanoparticles. Such kind of interaction will lead to a messy response contributed by the single-end- and double-end-bonded 4-MPy. The double-end-bonded species can be effectively avoided after the BSA coating, which reflects the importance of BSA modification. The slight variation in the calibration curve of AMB pH nanosensor for different batches may be a result of slight difference in the aggregation state of AuNPs and the detection condition. Therefore, in the present study, we made the pH calibration curves for each batch. We

believe in the future, this slight difference may be overcome after the standardization of the synthesis and detection procedures. Stability, Reliability, and Cytotoxicity of AMB pH Nanosensor. As aforementioned, the stability and reliability of pH nanosensor are vital to reliable pH sensing, especially in biological systems. Herein we performed systematic comparison of the stability and reliability of the AMB pH nanosensor with that of the AM pH nanosensor. From the viewpoint of practical application, the shelf stability of a pH sensor is an important property. Figure 4 shows the SERS spectra obtained from the freshly prepared AMB pH nanosensor and from that after storage for 7 days. It reveals that no significant change can be observed in both the peak position and the intensity (see Figure 4A). We demonstrate that the BSA protected AMB pH nanosensor remains SERS-active for at least 1 week. This considerable stability can be attributed to the stabilization of the BSA coating to the nanoaggregates. Figure 4B shows that the pH responses are a little different before and after storage for 7 days. The slight variation in the calibration curve before and after storage may also be a result of slight difference in the aggregation state of AuNPs, and the detection conditions are the same for the samples of different batches. However, such a minor change in the pH response is already quite good for practical application. All these results indicate the AMB pH sensor has a long-term stability. On the other hand, the AM pH nanosensor without BSA coating was found to aggregate too severely to be used for pH detection after storage for only 1 day. Although the AMB pH nanosensor exhibits good stability by BSA coating, it is unavoidable that the aggregates may form when they are used under the complex conditions, such as in solutions of high ionic strength or in the complex biological system. Therefore, it is essential to verify that such an aggregation will not change the pH response to ensure the reliability of the pH nanosensor. We first used a small amount (10 μL, ca. 6 mol/L) of saturated NaCl aqueous solution to intentionally induce the further aggregation of AMB pH nanosensor due to the weakening of the repulsion between the BSA molecules. By extending the interaction time of the pH nanosensor with NaCl, we could obtain different aggregation 12254

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Figure 5. SERS response of AMB pH nanosensors in PBS solutions of pH 5.5 (A, B, and C) and pH 7.0 (D, E, and F) with different aggregation states adjusted by addition of the saturated NaCl aqueous solution. (A and D) show the full spectra, and (B and E) are the expanded spectra for the region of 1150−1350 cm−1 after normalization with the peak at 1208 cm−1. The peak intensity ratios from 1208 to 1274 cm−1 are shown as a histogram for the solution of (C) pH 5.5 and (F) pH 7.0. The “NaCl+PBS-#2” sample has a more serious state of aggregation than the “NaCl+PBS#1” sample. A higher aggregation state is achieved by extending the interaction time of pH nanosensor with NaCl.

shown in Figure 6. The results show that the signal intensities decrease slowly with the increase of the incubation time. However, the signal intensity can still maintain about 70% even after incubation for 48 h compared with that of 0 h. It reveals that the reporter molecules are not obviously influenced or replaced by the complex cellular components, which ensures the reliability of application of pH nanosensor in cell systems.

states. Then the aggregated solutions were dispersed into the PBS solution (500 μL, 0.2 mol/L). Such an attempt is to change only the state of aggregation but maintaining the pH values. We have measured the pH values of the mixture by a pH meter and an approximately 0.07 pH decrease has been observed after the addition of NaCl. Therefore, the influence of ionic strength on the pH can be neglected. The SERS spectra of AMB pH sensor with different states of aggregations dispersed in PBS solutions of different pH (5.5 and 7.0) are shown in Figure 5. The PBS sample serves as a control without NaCl, and the “NaCl+PBS-#2” sample has a more severe aggregation than the “NaCl+PBS-#1” sample. The results reveal that no significant change could be observed in both the peak position and the intensity. More importantly, the intensity ratios of different samples (see Figure 5, panels C and F) show only small deviations and are in line with previous results shown in Figure 3B. Thus, the further aggregation will not affect the reliability of the pH response, which is highly important for applications under complex conditions such as biological systems. For the application in biorelated systems, the stability and the cytotoxicity of pH nanosensor in biological systems, for example culturing medium, should be considered. The stability is mainly concerned with the competitive replacement of the reporter molecule by the biomolecules in the biological culture medium or in live cells. To address this issue, we incubated AMB pH nanosensor with the cell culture medium containing various biomolecules at 37 °C to mimic the condition of interaction between pH nanosensor and cell. SERS spectra were recorded at different incubation times, and the results are

Figure 6. SERS spectra of AMB pH nanosensor obtained after incubated with cell culture medium for different time. The black lines are the average spectra, and red error bars represent the standard deviations from 5 measurements. 12255

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The main purpose of the pH nanosensor is for the detection of cellular pH. Therefore, it is important to test the cytotoxicity. For this purpose, we used the classical MTT assay to investigate the cytotoxicity of AM and AMB pH nanosensor on CaSki cells. The comparison of CaSki cell viability following incubation with different dose of AM and AMB pH nanosensor for 4 h are shown in Figure 7. No obvious cytotoxicity was

Figure 7. A comparison of the viability of CaSki cells after incubation with different doses of AM and AMB pH nanosensors for 4 h. Figure 8. Use of AMB pH nanosensor in live cell pH imaging: (A) Bright-field microscopic image, (B) SERS intensity image using the 1208 cm−1 peak, and (C) pH image of a CaSki cell after incubation with AMB pH nanosensor for 4 h.

observed for AMB pH nanosensor. In contrast, the cell viability decreases gradually with the increasing dose of AM pH nanosensor. The result indicates that the biocompatibility of pH nanosensor has been improved by BSA coating. From the above results, we can conclude that the AMB pH nanosensor has the advantages of high sensitivity, good stability, and biocompatibility, and thus holds great potential for reliable intracellular pH detection. Intracellular pH Detection with AMB pH Nanosensor. Figure 8A shows the bright-field microscopic image of one CaSki cell after 4 h incubation with the AMB pH nanosensor. The presence of AMB pH nanosensor inside the cell can be verified by SERS imaging over the whole cell. Benefiting from the high sensitivity, we were able to perform fast SERS imaging in only about 5 min, using the line-imaging mode of the Nanophoton instrument. The SERS image was represented using the intensity of peak 1208 cm−1 of 4-MPy and shown in Figure 8B. The result indicates a discrete distribution of the nanosensor throughout the whole cell and many of the nanosensors were taken up via the process of endocytosis to form aggregates in the late endosomes.26 The typical SERS spectra of AMB pH nanosensor obtained from the cell are shown in Figure S6 of the Supporting Information. The pH distribution was presented in the false color (see Figure 8C), by using the intensity ratio of the peaks of 1208 and 1274 cm−1, which is converted into the pH scale following the pH calibration curve. A pH distribution ranging from pH 5.0 to 9.0 was observed, and most of the pH values fall between 5.0 and 6.8 by the statistical analysis over the image. This result indicates that reliable microenvironment pH image can be successfully monitored using the AMB pH nanosensor.

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CONCLUSIONS In summary, to address the challenges in reliable SERS-based pH sensing, we developed a facile way to prepare the robust AMB pH nanosensor. Owing to the presence of the BSA coating as the protective layer, the pH nanosensor has a highly sensitive response to pH changes ranging from pH 4.0 to 9.0. It also shows good biocompatibility, stability, and reliability under various complex conditions. It has been successfully used to detect the intercellular pH distribution. The ease of preparation, outstanding stability, and biocompatibility endows the AMB pH nanosensor with a great potential for reliable pH analysis of cellular systems, the long-term dynamic monitoring of cellular processes (such as the differentiation of stem cells), and the comparative analysis of pH distributions of the control system (normal and cancer cells). In addition, the AMB pH nanosensor can be further optimized by replacing the AuNPs by other nanoparticles with higher SERS activity such as Au@ AgNPs to obtain higher sensitivity or functionalized with specific biorecognition molecules for targeting purpose. Such a targeted pH sensing will be even more meaningful for biological systems, which is now underway in our lab.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 12256

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge support from MOST (Grants 2013CB933703 and 2011YQ03012406), NSFC (Grants 21021120456, 21321062, and 21227004), and MOE (Grant IRT13036).

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dx.doi.org/10.1021/ac503404u | Anal. Chem. 2014, 86, 12250−12257