Article Cite This: Anal. Chem. 2019, 91, 8383−8389
pubs.acs.org/ac
Cell-Penetrating Peptide Conjugated SERS Nanosensor for in Situ Intracellular pH Imaging of Single Living Cells during Cell Cycle Xiao-Shan Zheng,† Cheng Zong,† Xin Wang,‡ and Bin Ren*,† †
Downloaded via UNIV OF SOUTHERN INDIANA on July 23, 2019 at 10:53:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
MOE Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials (iChEM), College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ‡ School of Aerospace Engineering, Xiamen University, Xiamen 361005, China S Supporting Information *
ABSTRACT: Intracellular pH is an important modulator of cell functions, and its subtle change may dramatically affect the cellular activities and cause diseases. A reliable imaging of the intracellular pH is still a great challenge. We imaged the intracellular pH during the cell cycle at the single living cell level using newly designed cell-penetrating peptide conjugated pH nanosensors on a home-built in situ microscopic cell culture platform. The conjugated cell-penetrating peptide greatly enhanced the uptake of nanosensors without sacrificing the pH response. We observed a gradual alkalization from interphase to prophase and rapid acidification from prometaphase to telophase, reflecting variation and consumption of the species related to the energy storage during cell cycle. We realized SERS-based pH and fluorescence dual-mode imaging when the pH sensor was further modified with fluorescence dye. The integration of SERS imaging with in situ microscopic cell culture system offers great opportunity for revealing the intracellular pH-related biological and pathological processes.
I
addressed the reliability issue of SERS-based pH sensing by coating the pH nanosensors (Au nanospheres adsorbed with 4mercaptopyridine, 4-MPy) with bovine serum albumin (BSA) as a protective layer to form Au-(4-MPy)-BSA pH nanosensors (abbreviated as AMB) and successfully used them for the reliable pHi sensing of single living cells.21 However, the cellular internalization efficiency of AMB was still low and most of them were eventually trapped in lysosomes after a long incubation time, resulting in their uneven distribution in cells.22 Therefore, it is vitally important to find a strategy to enhance the cellular internalization efficiency and homogenize the distribution of nanosensors to faithfully reflect the pHi distribution. Various strategies, including ultrasound-mediated method, electroporation technique, and cell-penetrating-peptide (CPP) mediated method, have been developed to improve the cellular internalization efficiency of nanosensors.23−26 Among them, modification of nanosensors with cell-penetrating-peptide (CPP, such as Tat peptide) is receiving increasing interests. CPP can enable efficient and noninvasive delivery of nanosensors without toxicity and has been widely used in intracellular analysis, cell imaging, drug delivery, and etc.27−29
ntracellular pH (pHi) plays a critical role in the cell function and modulates various cellular activities including proliferation, signal transduction, and apoptosis. It is also an index of disease since subtle changes in pHi can result in major changes in metabolism and cause diseases. There is already some evidence demonstrating the link between pH and cancer progression including tumorigenesis and metastasis.1−3 Therefore, a technique with high sensitivity and spatiotemporal resolution is urgently needed for monitoring pHi. So far, several techniques including electrochemistry, NMR, fluorescence, and surface-enhanced Raman scattering (SERS) have been employed for the detection of pHi.4−8 Among the above various techniques, fluorescence and SERS show the unique advantages of capable of analysis at the single living cell level. SERS has been widely used for biological and biomedical applications as a noninvasive technique owing to its high sensitivity and good photostability, especially for biosensing owing to its capacity for smart design of SERS-based nanosensor with multifunction.9 SERS-based plasmonic nanoparticles labeled with functional molecules have been increasingly used for sensing the cellular microenvironment, including pH, gaseous content, and redox state.9−17 SERS-based pH sensing has been able to image the intracellular and extracellular pH distribution of single living cells.18−20 Yet, there are still some important issues to be addressed due to the complexity of cells. Previously, we © 2019 American Chemical Society
Received: March 7, 2019 Accepted: May 29, 2019 Published: May 29, 2019 8383
DOI: 10.1021/acs.analchem.9b01191 Anal. Chem. 2019, 91, 8383−8389
Article
Analytical Chemistry
(see Figure S1) to obtain a high sensitivity for detection and an effective size for intracellular delivery. (2) AMB pH nanosensor: 100 μL of a 0.05 mM 4-MPy aqueous solution was added drop by drop to 10 mL of as-prepared Au colloids under vigorous stirring. Subsequently, 100 μL of a 2% BSA aqueous solution was added dropwise to the solution under vigorous stirring for 15 min. (3) Tat peptide conjugated AMB pH nanosensors: 100 μL of a 0.1 mg/mL aqueous solution of Tat peptide, Tat-TAMRA peptide, or Tat peptide without Cys terminal was added respectively to 10 mL of as-prepared AMB pH nanosensor under vigorous stirring to obtain three kinds of Tat peptide conjugated AMB pH nanosensors, namely AMBTat, AMB-Tat-TAMRA, or AMB-Tat (without Cys terminal). The prepared nanosensors should be kept overnight for complete conjugations before use. (4) Au-Tat-TAMRA nanoparticles: 100 μL of a 0.1 mg/mL aqueous solution of Tat-TAMRA peptide was added drop by drop to 10 mL of asprepared Au colloids under vigorous stirring for 15 min. Then, 1.0 mL aliquots of the above pH nanosensors or nanoparticles were added to Eppendorf tubes and centrifuged at 4000 rpm for 10 min. The supernatant was removed after centrifuging. Finally, the pH nanosensors or nanoparticles were mixed with 500 μL of PBS solutions or water and then added to the 96well plates for SERS measurements. Cell Culture and Sample Preparation for SERS Imaging. Human cervical cancer cells (CaSki cell line) were cultured in the high glucose DMEM (Hyclone) supplemented with 10% serum (Hyclone) and 1% penicillin/streptomycin (Hyclone). The cells were maintained at 37 °C in a humidified 5% CO2-containing incubator. To prepare the cell samples, the suspended cells were seeded onto a culture dish (Φ = 35 mm, NUNC) equipped with a cover glass in the center and cultured overnight for the attachment. Then a certain amount of pH nanosensors was added into the culture medium to interact with the cells for different time. After that, the culture medium was discarded and the cells were washed three times with a PBS buffer to remove the free nanoparticles. Finally, 2 mL of fresh culture medium was added into the culture dish again before SERS imaging. In Vitro Cytotoxicity. MTT assay was used to study the cytotoxicity of pH nanosensors on CaSki cells. First, CaSki cells were seeded at a density of 105 cells/well in a 96-well plate and incubated for 24 h. After removing the culture medium, 100 μL of fresh culture medium containing different doses of pH nanosensors were added into each well. The medium was discarded after 4 h incubation, and the wells were washed once with PBS buffer. Then 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 discarded and 100 μL DMSO was added to each well to dissolve the blue formazan crystal produced by proliferating cells. Cells treated with only culture medium served as a negative control group. The optical density (OD) at 490 nm of the sample was measured with a spectrophotometric microplate reader (BioTekELX800). All of the experiments were performed in quintuplicate and the relative cell viability (%) was indicated as a percentage relative to the untreated cells. Data Processing Procedure. The hyper-spectral data from SERS imaging were first optimized via baseline subtraction and noise reduction by using chemometric method before the pH analysis. Then the absolute intensity of peak 1093 cm−1 and the intensity ratios of peaks 1208 and 1274 cm−1 were calculated on the basis of the optimized hyper-
In particular, Tat peptide is an arginine-rich peptide with cationic property and can penetrate the negatively charged plasma membrane directly without the participation of endocytosis, which can accelerate the cellular internalization process.30 In this work, cysteine (Cys) terminated Tat peptide was conjugated to the BSA-coated pH nanosensors (abbreviated as AMB-Tat) to significantly enhance their cellular internalization efficiency. We used the pH nanosensors to in situ continuously monitor the pHi response and perform pHi imaging during the whole cell cycle on a home-built microscopic cell culture platform. On this platform the living cells were investigated under their normal and active state and could even proliferate during the long-term study. We observed every interesting periodic pHi variation during cell cycle which agrees well with the cell processes.
■
EXPERIMENTAL SECTION Chemicals. Chloroauric acid (HAuCl4·4H2O), trisodium citrate dehydrate (C6H5N3O7·2H2O), 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. 4-Mercaptopyridine (4-MPy) and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Bovine serum albumin (BSA) was purchased from Bio Basic Inc. High purity (>98%) Tat peptide (KYGRRRQRRKKRGC), fluorescent labeling Tat peptide (Tat-TAMRA, 5TAMRAKYGRRRQRRKKRGC), and the Tat peptide without cysteine (Cys) terminal (KYGRRRQRRKKRG) were synthesized and provided by Chinese Peptide Company. All chemicals were used without further purification. Ultrapure water (Milli-Q, 18.2 MΩ) was used throughout all of the preparations. The PBS solutions of different pH values were prepared with Na2HPO4·12H2O, NaH2PO4·2H2O, and H3PO4 as needed and measured with a digital pH/ion meter. Instruments. The absorption spectra were collected with a UV−vis spectrometer (HITACHI, U-3900H). The morphology of the nanoparticles was characterized with a scanning electron microscopy (SEM) (HITACHI, S-4800). 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. 785 nm laser and 532 nm laser were used for all the measurements and imaging. For the detection of bulk solution samples: A 20× (NA 0.45) objective with a working distance of 4.5 mm and spot focused laser was used. The laser powers of 85 mW for 785 nm laser and 95 mW for 532 nm laser were used respectively and the acquisition time was 3 s for each spectrum. For the imaging of cell samples: The 50× (NA 0.45) objective with a long working distance and line focused laser was used. The laser power and acquisition time were 2.5 mW/pix and 2 s/shot for both lasers and each imaging. The total time for each imaging was approximately 3 min. Preparation of Different Nanoparticles, pH Nanosensors, and the Solution Samples for SERS Detection. (1) Au nanoparticles (AuNPs): 100 mL of a 0.01% 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 8384
DOI: 10.1021/acs.analchem.9b01191 Anal. Chem. 2019, 91, 8383−8389
Article
Analytical Chemistry spectral data. Finally, the intensities of 1093 cm−1 peak were used to construct the SERS image to show the distribution of pH nanosensors in the living cells while the intensity ratios were used to construct the corresponding pH distribution image. All the hyper-spectral data were automatically and objectively processed by a home-compiled software for data processing and for image construction.
shows a monotonic dependence of the intensity ratio with pH values, similar to our previously reported AMB nanosensors. These results indicate that the conjugation of Tat peptide has no advert effect on the pH response of nanosensor, which is key to design reliable pH nanosensors. The viability of CaSki cells incubated with AMB and AMB-Tat pH nanosensors for 4 h were compared via MTT assay (see Figure S3). It demonstrates that the conjugation of Tat has no adverse effect on cell viability. Generally, AMB-Tat has no obvious cytotoxicity especially at a low dose. These results indicate that the AMB-Tat can be reliably used for the pH analysis in living cells. As mentioned above, the positively charged arginine-rich chain of Tat peptides are in the exterior of the nanosensor after conjugation, which allows the direct interaction with the cell membrane to improve the intracellular delivery of nanosensors. To validate this assumption, the cellular internalization efficiencies of AMB and AMB-Tat were compared by SERS imaging with the strongest 1093 cm−1 peak of 4-MPy after incubation them with living CaSki cells for 2 h (Figure 1C). For AMB, only sparse nanosensors can be found and they distribute discretely inside the cells. In contrast, AMB-Tat nanosensors show a high loading density and uniform distribution throughout the whole cell following the cell contours (the corresponding microscopic image), which indicates a much higher cellular internalization efficiency of AMB-Tat compared with that of AMB and the effective conjugation of BSA with Tat peptides. In a control experiment using Tat peptide of the same sequence but without Cys terminal, we observed a much lower cellular internalization efficiency compared with Cys terminated AMB-Tat (see Figure S4 for SERS image) but a similar efficiency to that of AMB without Tat peptide in Figure 1C, which further shows the necessity of terminated Cys for a facile and successful bioconjugation of Tat peptide to the BSA coated nanosensor. Intracellular pH Imaging with AMB-Tat pH Nanosensor at the Single Living Cell Level. Benefiting from the conjugation of the Tat peptide, the cellular internalization process can be greatly shortened while the nanosensor loading can be markedly increased. Therefore, not only the potential adverse effect on cells due to the long-term incubation with nanomaterials can be avoided but also the quality of SERS pH imaging can be significantly improved with a higher spatial resolution. To determine the optimal incubation time and concentration of nanosensors, the cellular internalization of AMB-Tat was investigated at different incubation time and different nanosensor concentrations. As illustrated in Figure S5, the loading and distribution of nanosensors in the cells increase with both incubation time and concentration. The coincubation time of 4 h and concentration of 4 μg/mL were chosen as the optimal condition for the later living cell study after comprehensive consideration of both the cell viability and detection sensitivity. Figure 2 shows the pH imaging result of a single living CaSki cell after incubation with AMB-Tat for 4 h, together with the microscopic image and SERS intensity image of 1093 cm−1 peak of 4-MPy. The SERS intensity image indicates a high density of AMB-Tat inside the cell. It can be found that almost all the nanosensors distributed in the cytoplasm and no nanosensors distributed in the cell nucleus (the region showing no SERS intensity within the cell). It may be attributed to the nuclear envelope which surrounds the nucleus and blocks the entering of nanoparticles. The pH image was produced by
■
RESULTS AND DISCUSSION Preparation and Characterization of AMB-Tat pH Nanosensor. The scheme of AMB-Tat was illustrated in Figure 1A and the detailed synthetic procedure was given in
Figure 1. (A) Schematic illustration of the structure of AMB-Tat pH nanosensor (not drawn to scale). (B) pH response of AMB and AMBTat in PBS solutions shown with the intensity ratio of 1208 cm−1 to 1274 cm−1 peaks of 4-MPy as a function of pH values. The error bars indicate the standard deviations from 5 measurements. (C) Comparison of the cellular internalization efficiency of 10 μg/mL AMB and AMB-Tat being incubated with CaSki cells after 2 h, respectively. SERS intensity images were produced using the intensities of 1093 cm−1 peak.
the Experimental Section. The Tat peptide was designed with arginine-rich sequence (KYGRRRQRRKKRGC) and terminated with Cys. In this way, the Tat peptide can be conjugated to the BSA layer of AMB via the Cys residue by forming S−S bond and stay at the outmost layer of the nanosensor, as has been revealed in previous Gel electrophoretic analysis.31 Therefore, the exposed positively charged arginine-rich chain can directly interact with the negatively charged cell membrane to enhance the intracellular internalization efficiency of pH nanosensor. The UV−vis spectrum of AMB-Tat is almost the same as that of AMB (see Figure S1), indicating no aggregation occurred after the bioconjugation. The intensity ratios of the two peaks at 1208 and 1274 cm−1 from the SERS spectrum of 4-MPy of the AMB-Tat (see Figure S2 for full SERS spectra) were used for the pH sensing. For this purpose, we made the pH calibration curve using the intensity ratios of the two peaks detected in PBS solutions with different pH values containing AMB-Tat nanosensors. We measured in the physiologically important pH range from 4.0 to 9.0 in steps of 1.0 pH.3 The calibration curve (green) shown in Figure 1B 8385
DOI: 10.1021/acs.analchem.9b01191 Anal. Chem. 2019, 91, 8383−8389
Article
Analytical Chemistry
the pH calibration curve in Figure 1B. Through the pH distribution image, we can find a few point-like pH distributions ranging from pH 4.0 to 5.5, which may be ascribed to lysosomes.3 The dominant pH values are from pH 5.5 to 7.5, which is consistent with the cytoplasm pH. Some positions around the cell nucleus show pH values in the range of 7.5 to 9.0, indicating a perinuclear distribution of mitochondria (of higher pH) to supply energy for the metabolism in cell nucleus.3,4 This imaging quality has been greatly improved over reported results since most intracellular regions can produce signals and be analyzed. Such a homogeneous distribution of nanosensors is noteworthy because most of the previously reported ones cannot escape the ultimate fate of being trapped in lysosomes after long incubation process, which hinders investigation of the cellular activities in other subcellular organelles.23,32 In Situ Intracellular pH Imaging of Single Living Cells during Cell Cycle. We then used AMB-Tat to investigate some representative cellular processes of living cells, e.g. cell proliferation, which can provide insight into the pH dependent cell activities and pH related cell biology. For this purpose, We used the home-built in situ cell culture and microscopic detection system which is capable of controlling the optimal temperature and CO2 content for cells during detection (see pp S-6 and S-7 and Figure S6 in the Supporting Information for a detailed configuration and description) to perform pH
Figure 2. AMB-Tat for the intracellular pH imaging of a single living CaSki cell: Microscopic image was observed under the bright-field illumination; SERS intensity image was produced by using the intensities of 1093 cm−1 peak; pH distribution image was produced by correlating the intensity ratio of 1208 cm−1 to 1274 cm−1 peaks with pH values. The concentration of AMB-Tat was 4 μg/mL and the incubation time was 4 h. The pixel size in the pH distribution image was 0.8 μm × 0.8 μm.
calculating the intensity ratios of the peaks at 1208 and 1274 cm−1 from the SERS spectra obtained at different positions, which were then converted into the pH values on the basis of
Figure 3. Real-time pH monitoring during the cell cycle of a single living CaSki cell by using the in situ cell culture and microscopic detection system. Microscopic images were observed under the bright-field illumination by focusing on the same position, SERS intensity images were produced by using the intensities of 1093 cm−1 peak, and pH distribution images were produced by correlating the peak intensity ratio of 1208 cm−1 to 1274 cm−1 with pH values. The concentration of AMB-Tat was 4 μg/mL and the pixel size in the pH distribution image was 0.8 μm × 0.8 μm. Orange, red, and yellow rectangle indicated the parent cell, the daughter cell #1, and the daughter cell #2, respectively. 8386
DOI: 10.1021/acs.analchem.9b01191 Anal. Chem. 2019, 91, 8383−8389
Article
Analytical Chemistry imaging under the normal state of living cells to allow in situ and long-term dynamic monitoring of cellular activities. Impressively, we observed an intact and clear mitosis process of a CaSki cell, such as line-up of chromosomes along metaphase plate during metaphase (t = 0), break of chromosomes at centromeres and movement of chromosomes to the opposite ends of the cell during anaphase (t = 6 min), and cytokinesis during telophase (t = 12 min) (shown in Figure S7). The results demonstrate that the in situ cell culture and microscopic detection system is competent for living cell study since the mitosis process is rarely observed under the conventional experimental conditions (sealed in PBS buffer). The long-term stability of cell culture and the SERS-based pH nanosensor enables the reliable and in situ monitoring of cellular processes of living cells. The pHi distribution and evolution during the whole cell cycle was monitored continuously on the in situ cell culture and microscopic detection platform. Benefited from the confocal microscopic system, we could focus the laser in the middle section of the cell (about 5 μm away from both the top and the bottom of cell in the Z-axis direction guided by the bright-field imaging) to ensure that we were monitoring the process always and only inside the cell. The pH imaging was started immediately after the addition of AMB-Tat pH nanosensors into the cell culture medium and continued for 6 h (Figures S8 and 3). From the SERS intensity image in Figure S8, we found that very few nanosensors were observed inside the cell in the first 30 min. However, after only 1 h incubation, the nanosensors already reached a high particle loading and distributed homogeneously over the whole cell following the cell morphology. Strikingly, from t = 2 h, this cell started to undergo mitosis and eventually split from a parent cell (t = 4 h) to two daughter cells (t = 5 h; Figure 3). The typical SERS spectra of nanosensors obtained after incubation with the cell for different time during the cell cycle were given in Figure S9. The pHi images of the cell during the mitosis process were shown in the right column of Figure 3. The seemingly random result shows a nice trend when we did statistical histogram analysis (Figure S10) and the pH distribution profiles for each time period were plotted in Figure 4A. The count of each curve in Figure 4A exhibits the number of nanosensors showing a specific pH value, which essentially reflects the pH distribution under this specific condition while the area of each curve indicates the total number of nanosensors being internalized. Therefore, we can analyze the result from two aspects: the number of nanosensors and pH distribution for the cell shown in Figure 3. As for the number of nanosensors (1) from interphase to prophase (t = 2−4 h), the number of nanosensors increased with the incubation time and cell growth; (2) from prometaphase to telophase and cytokinesis (t = 4−5 h), it is surprising to find that the number of nanosensors nearly remained constant although the cell had divided from one parent cell into two daughter cells, which may indicate that the internalization of nanosensors was inhibited at this stage; (3) during the resting phase G0 (t = 6 h), the number of nanosensors increased again, which means the cells started to uptake nutrition and energy for growth at this stage. It is noteworthy that SERS mapping of cells using the signal of the 4-MPy provides a quantitative way to estimate the number of intracellular nanosensors since the pixels showing SERS signals within the cell indicates the presence of nanosensors. We counted the numbers of pixels showing SERS signals in the
Figure 4. (A) Statistical profiles showing the pH distribution at different periods of cell cycle in Figure 3. (B) The counts of pixels showing SERS signals before (t = 4 h) and after (t = 5 h) cell division in the regions of the parent cell and the two daughter cells and (C) the corresponding pH distribution.
parent cell before cell division (t = 4 h, orange rectangular region in Figure 3) and each daughter cell after cell division (t = 5 h, red and yellow rectangular regions in Figure 3) to minimize the signal variation due to the different aggregation states. As can be seen in Figure 4B and interestingly, the two daughter cells almost equally inherited the nanosensors (with 198 and 196 counts) from the parent cell during the cell division, which indicates a homogeneous distribution of intracellular nanosensors. We only observed a slight decrease between the sum of two daughter cells (394 counts) and that from parent cell (435 counts), which could be attributed to the further aggregation of nanosensors (overlap of the regions showing SERS signals) during the longer incubation time (from t = 4−5 h). As for the pH distribution, we found the following: (1) From interphase to prophase (t = 2−4 h), the pHi shows a trend of gradual alkalization (Figure 4A) and the regions around nucleus are more alkaline (Figure 3). Such an alkalization may be attributed to the increased mitochondria together with the activation and increased expression of H+ATPase since the cell should prepare energy for DNA duplication, protein synthesis, and cell growth at this stage.33 (2) From prometaphase to telophase and cytokinesis (t = 4−5 h), a dramatic and rapid acidification was observed (Figure 4A) and the regions around cell membrane become more acidic (Figure 3), which can be attributed to the initiation of glycolysis and increased glycolytic rate to generate metabolic acids since cellular energy is consumed during the division of the parent cell into two daughter cells.2,33 The statistical pH distribution trends of the parent cell and each daughter cell are given in Figure 4C. The two daughter cells exhibit almost the same pH distribution indicating their homogeneity. The 8387
DOI: 10.1021/acs.analchem.9b01191 Anal. Chem. 2019, 91, 8383−8389
Article
Analytical Chemistry
Figure 5. (A) Schematic illustration of the structure of AMB-Tat-TAMRA (not drawn to scale). SERS spectra of Tat-TAMRA aqueous solution (10−4 mg/mL), Au-Tat-TAMRA, AMB, AMB-Tat, and AMB-Tat-TAMRA nanoparticles dispersed in water by using laser of 785 (B) and 532 nm (C). (D) SERS and fluorescence dual-mode imaging of 4 μg/mL AMB and AMB-Tat-TAMRA incubated with CaSki cells for 4 h. The control sample is CaSki cells without any nanoparticles. The intensity of peak 1093 cm−1 of 4-MPy was used to reconstruct the SERS image and the peak area in the range of 1300 to 1700 cm−1 of TAMRA was used to reconstruct the fluorescence image.
AMB-Tat-TAMRA. In this way, the dual-mode (SERS and fluorescence) imaging integrated with the ability of pH sensing can be realized based on one single nanosensor. Figure 5D shows the dual-mode (SERS and fluorescence) images of different samples. In sharp contrast to the control experiments of cells without any nanoparticles and cells incubated with AMB in which only weak autofluorescence from the cells was observed, a high density of nanosensor with both high SERS signal and fluorescence signal from cells incubated with AMBTat-TAMRA was observed. Furthermore, the SERS image and fluorescence image colocalize well with each other, which clearly demonstrates the successful conjugation of TAMRAlabeled Tat peptide on the pH nanosensor. As TAMRA can be also labeled on drugs or antibodies, such nanosensor holds great potential for the multimodal imaging of drug release or targeting imaging of cancer cells together with the pH sensing ability.
dramatic acidification from parent cell to daughter cells can be still observed, which is in accordance with the result in Figure 4A. (3) During the resting phase G0 (t = 6 h), the pH values recover to between pH 5.5 and 7.5, which is almost the same as that at the initial stage (t = 2 h), indicating the end of this cell cycle. Thereafter, each daughter cell becomes an individual cell and is preparing for its new cell cycle. All of the above results demonstrate that it is important to carry out single cell experiment under in situ cell culture environment. In this way, we are capable of real-time and long-term monitoring the dynamic cellular activities and revealing more exciting information on living cells under their normal states that is not achievable in previous works. SERS-Fluorescence Dual-Mode Imaging with DyeLabeled Tat Conjugated pH Nanosensor. If we use the Tat peptide labeled with a fluorescent dye (such as TAMRA, see Figure 5A) while preparing the Tat-conjugated pH nanosensor (abbreviated as AMB-Tat-TAMRA), we may have a bonus: the pH nanosensors now can further produce the fluorescence signal of TAMRA in addition to the pH sensitive SERS signal of 4-MPy. TAMRA has an absorption at 542 nm. Therefore, the AMB-Tat-TAMRA can be excited by 532 nm laser to produce strong fluorescence signal and can be excited by 785 nm laser (which is far from 542 nm) to produce strong SERS signal free of fluorescence background. As compared in Figure 5B,C, only SERS signal of 4-MPy was obtained by 785 nm excitation while dominant fluorescence signal of TAMRA was obtained by 532 nm excitation from
■
CONCLUSIONS In summary, we have rationally modified the BSA-protected SERS-based pH nanosensor with Tat peptide conjugated pH nanosensor (AMB-Tat) to dramatically improve its cellular internalization efficiency without sacrificing the pH response. This excellent sensor enabled the investigation of the pHi evolution of single living cells during the whole cell cycle. We for the first time observed the pHi evolution showing gradual alkalization, rapid acidification and recovery to equilibrium pH during the cell cycle, which can be related to the events and 8388
DOI: 10.1021/acs.analchem.9b01191 Anal. Chem. 2019, 91, 8383−8389
Article
Analytical Chemistry
(12) Krafft, C.; Schmitt, M.; Schie, I. W.; Cialla-May, D.; Matthäus, C.; Bocklitz, T.; Popp, J. Angew. Chem., Int. Ed. 2017, 56, 4392−4430. (13) Kneipp, J. ACS Nano 2017, 11, 1136−1141. (14) Cao, Y.; Li, D.-W.; Zhao, L.-J.; Liu, X.-Y.; Cao, X.-M.; Long, Y.T. Anal. Chem. 2015, 87, 9696−9701. (15) Cui, J.; Hu, K.; Sun, J.-J.; Qu, L.-L.; Li, D.-W. Biosens. Bioelectron. 2016, 85, 324−330. (16) El-Said, W. A.; Kim, T.-H.; Chung, Y.-H.; Choi, J.-W. Biomaterials 2015, 40, 80−87. (17) Kneipp, J.; Kneipp, H.; Wittig, B.; Kneipp, K. Nano Lett. 2007, 7, 2819−2823. (18) Jaworska, A.; Jamieson, L. E.; Malek, K.; Campbell, C. J.; Choo, J.; Chlopicki, S.; Baranska, M. Analyst 2015, 140, 2321−2329. (19) Luo, R.; Li, Y.; Zhou, Q.; Zheng, J.; Ma, D.; Tang, P.; Yang, S.; Qing, Z.; Yang, R. Analyst 2016, 141, 3224−3227. (20) Xu, M.; Ma, X.; Wei, T.; Lu, Z.-X.; Ren, B. Anal. Chem. 2018, 90, 13922−13928. (21) Zheng, X.-S.; Hu, P.; Cui, Y.; Zong, C.; Feng, J.-M.; Wang, X.; Ren, B. Anal. Chem. 2014, 86, 12250−12257. (22) Kneipp, J.; Kneipp, H.; Wittig, B.; Kneipp, K. J. Phys. Chem. C 2010, 114, 7421−7426. (23) Behzadi, S.; Serpooshan, V.; Tao, W.; Hamaly, M. A.; Alkawareek, M. Y.; Dreaden, E. C.; Brown, D.; Alkilany, A. M.; Farokhzad, O. C.; Mahmoudi, M. Chem. Soc. Rev. 2017, 46, 4218− 4244. (24) Shangyuan, F.; Zhihua, L.; Guannan, C.; Duo, L.; Shaohua, H.; Zufang, H.; Yongzeng, L.; Juqiang, L.; Rong, C.; Haishan, Z. Nanotechnology 2015, 26, No. 065101. (25) Bhardwaj, V.; Srinivasan, S.; McGoron, A. J. Analyst 2015, 140, 3929−3934. (26) Lin, J.; Chen, R.; Feng, S.; Li, Y.; Huang, Z.; Xie, S.; Yu, Y.; Cheng, M.; Zeng, H. Biosens. Bioelectron. 2009, 25, 388−394. (27) Fales, A. M.; Yuan, H.; Vo-Dinh, T. Mol. Pharmaceutics 2013, 10, 2291−2298. (28) Hu, F.; Zhang, Y.; Chen, G.; Li, C.; Wang, Q. Small 2015, 11, 985−993. (29) Hossain, M. K.; Cho, H.-Y.; Kim, K.-J.; Choi, J.-W. Biosens. Bioelectron. 2015, 71, 300−305. (30) Schwarze, S. R.; Dowdy, S. F. Trends Pharmacol. Sci. 2000, 21, 45−48. (31) Ignatovich, I. A.; Dizhe, E. B.; Pavlotskaya, A. V.; Akifiev, B. N.; Burov, S. V.; Orlov, S. V.; Perevozchikov, A. P. J. Biol. Chem. 2003, 278, 42625−42636. (32) Büchner, T.; Drescher, D.; Traub, H.; Schrade, P.; Bachmann, S.; Jakubowski, N.; Kneipp, J. Anal. Bioanal. Chem. 2014, 406, 7003− 7014. (33) Orij, R.; Urbanus, M. L.; Vizeacoumar, F. J.; Giaever, G.; Boone, C.; Nislow, C.; Brul, S.; Smits, G. J. Genome Biol. 2012, 13, R80.
species involved in energy storage and consumption during cell cycle. The platform offered by this study for investigation of the pH related cellular processes under the normal cell culture condition may shed new light on the deep understanding of diseases. We ambitiously predict that the future trend in the single living cell study should ensure the normal cell viability and activity during detection in order to reveal the true nature of life, so that we can do in a “prospective” rather than “retrospective” way.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01191.
■
Various figures showing the characterizations of pH nanosensor including UV−vis spectra, SEM image, pH dependent SERS spectra, in vitro cytotoxicity, and cellular internalization efficiency; description and configuration of the in situ cell culture and microscopic detection system; imaging, SERS spectra, and statistical analysis of the real-time pH sensing during cell cycle (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Xiao-Shan Zheng: 0000-0003-2567-5365 Bin Ren: 0000-0002-9821-5864 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We acknowledge the financial support from MOST (2013CB933703) and NSFC (21633005, 21621091, and 21503171).
■
REFERENCES
(1) Dai, Y.; Xu, C.; Sun, X.; Chen, X. Chem. Soc. Rev. 2017, 46, 3830−3852. (2) Webb, B. A.; Chimenti, M.; Jacobson, M. P.; Barber, D. L. Nat. Rev. Cancer 2011, 11, 671−677. (3) Casey, J. R.; Grinstein, S.; Orlowski, J. Nat. Rev. Mol. Cell Biol. 2010, 11, 50−61. (4) Roos, A.; Boron, W. F. Physiol. Rev. 1981, 61, 296−434. (5) Ning, L.; Li, X.; Yang, D.; Miao, P.; Ye, Z.; Li, G. Anal. Chem. 2014, 86, 8042−8047. (6) Gillies, R. J.; Ugurbil, K.; den Hollander, J. A.; Shulman, R. G. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 2125−2129. (7) Han, J.; Burgess, K. Chem. Rev. 2010, 110, 2709−2728. (8) Talley, C. E.; Jusinski, L.; Hollars, C. W.; Lane, S. M.; Huser, T. Anal. Chem. 2004, 76, 7064−7068. (9) Cialla-May, D.; Zheng, X. S.; Weber, K.; Popp, J. Chem. Soc. Rev. 2017, 46, 3945−3961. (10) Schlücker, S. Angew. Chem., Int. Ed. 2014, 53, 4756−4795. (11) Zheng, X.; Zong, C.; Xu, M.; Wang, X.; Ren, B. Small 2015, 11, 3395−3406. 8389
DOI: 10.1021/acs.analchem.9b01191 Anal. Chem. 2019, 91, 8383−8389
