Ultraphotostable Mesoporous Silica-Coated Gap-Enhanced Raman

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Ultraphotostable Mesoporous Silica-Coated Gap-Enhanced Raman Tags (GERTs) for High-Speed Bioimaging Yuqing Zhang,† Yuanyuan Qiu,‡ Li Lin,† Hongchen Gu,† Zeyu Xiao,*,‡,△ and Jian Ye*,†,§ †

State Key Laboratory of Oncogenes and Related Genes, School of Biomedical Engineering, ‡Department of Pharmacology, Institute of Medical Sciences & Translational Medicine Collaborative Innovation Center & Collaborative Innovation Center of Systems Biomedicineand, §Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, and △Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, P. R. China S Supporting Information *

ABSTRACT: Surface-enhanced Raman scattering (SERS) tags can be utilized as optical labeling nanoprobes similar to fluorescent dyes and quantum dots for bioimaging with additional advantages of fingerprint vibrational signals as unique optical codes and ultranarrow line widths for multiplexing. However, the development of the SERS imaging technique is much less well-established compared to the devlopment of fluorescence imaging mainly because of speed limitations. An effective strategy for improving the SERS imaging speed and simultaneously maintaining the photostability of SERS tags has not, to the best of our knowledge, been reported. In this work, mesoporous silica- (MS-) coated gap-enhanced Raman tags (GERTs) were designed with builtin Raman reporters for off-resonance near-infrared laser excitation and reduced photothermal effects, leading to ultraphotostability during laser irradiation. Additionally, they achieve large amplification of Raman signals by combining the chemical (CHEM) and electromagnetic (EM) enhancement effects due to the subnanometer core−shell junction, so SERS imaging can be performed in a dramatically reduced duration. With these unique structural and optical advantages, MS GERTs exhibit high storage, pH, serum, and photostabilities; strong Raman enhancements; and favorable biocompatibility. Therefore, MS GERTs achieve long-term cell imaging that can last for 30 min without being photobleached and also maintain decent imaging effects. Furthermore, MS GERTs enable continuous and stable imaging in living tissues for more than 30 min. With these advantages, MS GERTs might potentially have more biomedical applications. KEYWORDS: nanoprobe, bioimaging, plasmonics, surface-enhanced Raman scattering, photobleaching

1. INTRODUCTION

However, the development of the SERS imaging technique is much less well-established than the development of fluorescence imaging mainly because of its speed limitations. An effective strategy for improving the SERS imaging speed and simultaneously maintaining the photostability of SERS tags has not, to the best of our knowledge, been reported. Typical SERS tags include plasmonic nanospheres, nanorods, nanoshells, nanostars, nanoflowers, core−shell nanoparticles, and nanoaggregates.2 The ideal SERS tags for bioimaging would be desirable to enable high-speed Raman imaging and maintain ultraphotostability. Practically, imaging speed is an important factor to be considered. The imaging speed in current commercial Raman systems is mainly limited by the intrinsic inefficiency of the spontaneous Raman scattering process and the configuration in confocal Raman microscopies, where a single laser focus is scanned point by point on the sample by

Surface-enhanced Raman scattering (SERS) tags show ultrahigh sensitivities up to even the single-nanoparticle level, mainly due to strong Raman signals enhanced by near-field hot spots of plasmonic nanoparticles (NPs).1 As such, SERS tags can be utilized as optical labeling nanoprobes similar to fluorescent dyes and quantum dots for bioimaging.2−6 The fingerprint Raman vibrational signals from SERS tags serve as unique optical codes that differentiate themselves from the background signals coming from the surrounding substrate, solution, or biologic tissue. Additionally, the ultranarrow line widths of the Raman bands of SERS tags provide a much larger multiplexing capability than fluorescent tags. These crucial features of SERS tags enable them to be a potential technique for bioimaging applications such as live-cell imaging,7−11 tissue imaging,12,13 and in vivo imaging.14−19 Recently, SERS tags were found to exhibit great potential for the precise visualization of tumor margins, microscopic tumor invasion, and multifocal tumor spreading for surgery guidance.20−22 © 2017 American Chemical Society

Received: November 26, 2016 Accepted: January 11, 2017 Published: January 11, 2017 3995

DOI: 10.1021/acsami.6b15170 ACS Appl. Mater. Interfaces 2017, 9, 3995−4005

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Figure 1. (a) TEM image of MS GERTs. The scale bar is 50 nm, and the GERT inner gap is indicated by red arrows. A schematic diagram is shown in the inset. (b) Extinction spectra of GERTs and MS GERTs. The inset shows a photograph of aqueous MS GERTs. (c) Photostability measurement of time-resolved SERS spectra of solid MS GERTs on a silicon wafer during continuous irradiation for 30 min at a power density of 4.71 × 105 W/cm2. SERS spectra were acquired at 2-s intervals (785-nm laser, 10-ms exposure time/spectrum, and 100× objective lens). Spectra were background-subtracted and intensity-normalized to exposure time, laser power, and concentration of Raman reporters. (d) Three representative SERS spectra at selected irradiation times in panel c. (e) On-resonance excitation induces strong thermal effects and moderate EM enhancement on GERTs, whereas off-resonance excitation leads to weak thermal effects, moderate EM enhancement, but strong charge-transfer (CT) enhancement.

tumor margins for tumor surgery guidance.20 In these situations, the Raman imaging would require minimized photobleaching of the SERS tags, which typically comes from local photoheating effects and photochemical reactions (e.g., photo-oxidation and photoreduction). Normally, a larger laser power density and near-field enhancement inevitably lead to undesired amplified photobleaching effects in SERS tags.30−32 Therefore, from the perspective of tag stability, a lower laser power density and SERS tags with a smaller enhancement factor are preferred. Hence, in the typical system, simultaneously achieving high-speed imaging and great stability of SERS tags seems to be contradictory in SERS imaging. In this work, we have simultaneously achieved both highspeed imaging and great photostability with our special SERS

mechanically moving the stage to acquire a Raman spectrum at every pixel. A number of improvements have recently been performed to increase the imaging speed by utilizing a lineshaped focus18,23 and galvano mirrors.9,24,25 However, it still typically takes minutes to hours to acquire a Raman image over a large area with SERS tags.26,27 Therefore, in terms of imaging speed, a larger laser power density and SERS tags with a larger enhancement factor (namely, more and stronger near-field hot spots) are preferred to shorten the irradiation time at each pixel and, thereby, the total imaging time. Simultaneously, photostability of SERS tags upon continuous irradiation or in various media is crucial for biomedical applications, such as SERS-based quantitative analysis,7,28 time-lapse cellular imaging,9,23 in vivo pathway tracking of SERS tags,29 and repeated imaging of 3996


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were drop-cast on the silicon wafer and dried before testing. Au sphere−BDT samples were prepared as described in our previous work.33 Au sphere−R6G samples were prepared by mixing Au spheres and R6G aqueous solution together and then dropping the mixture onto the silicon wafer for drying. Notably, the Au spheres in the Au sphere−BDT NPs were synthesized using a seed-mediated process,33 whereas the Au spheres in the Au sphere−R6G NPs were synthesized by the sodium citrate reduction of HAuCl4 as developed by Frens.36 The four types of samples were all continuously irradiated for 30 min. Among them, Au sphere−BDT and Au sphere−R6G samples were irradiated with a 633-nm laser (2.16 × 105 W/cm2, 100× objective lens), whereas GERTs and MS GERTs were irradiated with a 785-nm laser (4.71 × 105 W/cm2, 100× objective lens). The MS GERTs were further continuously irradiated with a 785-nm laser for 30 min at higher power densities (1.18 × 106 and 2.36 × 106 W/cm2). All SERS spectra were acquired at 2-s intervals with a 10-ms exposure time/ spectrum The photostability of fluorescein isothiocyanate (FITC) fluorescence signals derived from Actin-Tracker Green stained cells was also determined (488-nm laser, 1.27 × 105 W/cm2, 60× objective lens, 1.0 NA) for 30 min. The fluorescence signal intensity was recorded at 2-s intervals. For the evaluation of the physiologic stability, UV−vis extinction spectra and SERS spectra of MS GERT solutions were collected over a period of 12 weeks. The concentration of the MS GERT solutions was 0.6 nM. Next, UV−vis extinction spectra and SERS spectra of MS GERT solutions of different pH values in the range of 3−12 were collected. The pH of the nanoparticle solutions was varied through the dropwise addition of HCl or NaOH solution, and the pH value was monitored with a pH meter (Mettler Toledo). Additionally, UV−vis extinction spectra and SERS spectra of MS GERT solutions in 10% fetal bovine serum and saline were collected over 96 h. 2.4. Cell Culture and Imaging. The human lung cancer cell line H1229 was obtained from American Type Culture Collection (ATCC), and all reagents for the cell culture were purchased from Gibco. The cells were cultured in RPMI 1640 Medium supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin, and 100 mg mL−1 streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. For cell Raman imaging purposes, the cells were allowed to adhere to the quartz-bottomed plates for at least 24 h and then incubated with MS GERTs at a final concentration of 0.02 nM for 6 h. Following the incubation, the cells were washed extensively with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at room temperature. Excess paraformaldehyde was removed with deionized water, and the samples were air-dried before SERS measurements. The Raman image of a whole cell could be obtained within 53 s with a 10ms exposure time per pixel (50 × 50 pixels, 100× objective lens), and the imaging time could be further shortened to 43 s (5-ms exposure time per pixel) and 35 s (1-ms exposure time per pixel). Furthermore, the cells were continuously irradiated with a 785-nm laser (4.71 × 105 W/cm2, 100× objective lens) for 30 min to evaluate the stability of cell Raman imaging, and 34 Raman images were obtained within 30 min. For fluorescence imaging, the cells were allowed to adhere to cover glasses for at least 24 h and fixed with 4% paraformaldehyde for 10 min at room temperature. Then, the cells were washed three times with PBS containing 0.1% Triton X-100 and blocked with PBS containing 4% BSA and 0.1% Triton X-100. The resultant cells were sequentially incubated with Actin-Tracker Green solutions for 20 min under darkness for labeling microfilaments. Following the incubation, the cells were washed three times with PBS containing 0.1% Triton X-100 and air-dried before measurements. Finally, cell fluorescence imaging was continuously performed with a 488-nm laser (2.44 × 104 W/cm2, 60× objective lens) for 30 min, and the fluorescence images were recorded every 1 min. 2.5. Cell Proliferation Assay. To evaluate the cytotoxicity of MS GERTs and CTAC-coated GERTs on cells, we investigated the effects of MS GERTs and CTAC-coated GERTs on H1299 cells at concentrations of 0.005, 0.01, 0.02, 0.05, and 0.1 nM. The H1299 cells were seeded in 96-well plates at a density of 3 × 103 cells per well and cultured in RPMI 1640 Medium at 37 °C in a humidified atmosphere containing 5% CO2. After 12 h, the culture medium was

tags. We demonstrate the ultraphotostability of mesoporous silica- (MS-) coated gap-enhanced Raman tags (GERTs) (Figure 1a), allowing for high-speed Raman-based cell and tumor imaging. MS GERTs consist of plasmonic Au nanomatryoshka particles with embedded Raman reporters in the interior gap junction and with an external MS coating.33,34 Such nanostructures exhibit a variety of unique features. First, taking advantage of off-resonant laser excitation and the well protection from the solid metallic and MS shell, the photobleaching of the built-in Raman reporters can be minimized, and thus, MS GERTs exhibit ultraphotostability. Second, the subnanometer core−shell junction geometry in MS GERTs offers a large number of electromagnetic (EM)/ chemical (CHEM) hot spots, therefore achieving a total strong SERS enhancement, which allows for high-speed imaging. Additionally, the MS layer endows the GERTs with good biocompatibility. Because of the above features, the MS GERTs show remarkable photostability under a myriad of conditions, including continuous laser irradiation, in ethanol for 12 weeks, in solutions of different pH values, in saline solution, and 10% fetal bovine serum. Consequently, these superphotostable MS GERTs allow for fast and continuous in vitro imaging of cells and in vivo imaging of orthotopic tumors in living animals under continuous laser irradiation for 30 min at power densities of 4.71 × 105 and 2.21 × 105 W/cm2, respectively.

2. EXPERIMENTAL SECTION 2.1. Materials and Instrumentation. Tetraethyl orthosilicate (TEOS), sodium hydroxide (NaOH), and methanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Rhodamine 6G was obtained from Sigma-Aldrich. Actin-Tracker Green was received from Beyotime (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). All materials were used as received without any further purification. Nanopure water (18.2 MΩ) was used for all experiments. Transmission electron microscopy (TEM) images were collected on a JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan) operated at 200 kV. UV−vis spectra were obtained using a UV1900 UV−vis spectrophotometer (Aucybest, Shanghai, China). Fluorescence images were collected on a Nikon A1R MP microscope system (60× water immersion objective, 1.0 NA). An enzyme-linked immunosorbent assay microplate reader (Synergy 2, Bio-TEK) was used to measure the absorbance. 2.2. Synthesis of Mesoporous (MS) GERTs. GERTs (called nanomatryoshkas) were synthesized according to the procedure described in our previous work.33 Mesoporous silica coating was applied to the GERTs according to Gorelikov and Matsuura’s protocol with some modifications.35 The as-synthesized GERTs (40 mL) were washed three times by centrifuge for 5 min to remove excess reagents and then dispersed in 5 mL of aqueous cetyltrimethylammonium chloride (CTAC) solution (0.005 M). Then, 40 μL of 0.1 M NaOH solution was added with stirring. Following this step, 50 μL of 5% TEOS in methanol was added slowly to the solution under gentle stirring. This procedure was repeated three times at 30-min intervals, and then the mixture was reacted for 24 h at 30 °C. The resultant products were collected by centrifugation and washed with ethanol. Finally, template removal was performed by a highly efficient ionexchange method. The purified nanoparticles were dispersed in ethanol solution (25 mL) containing NH4NO3 (10 mg) and subjected to a sonic bath for 1 h. Then, the nanoparticles were washed by centrifuge for 5 min to remove excess reagents, and this procedure was repeated three times to obtain completely template-removed MS GERTs. 2.3. Stability Measurements of NPs. Raman measurements were carried out to evaluate the photostability characteristics of different NPs including GERTs, MS GERTs, Au sphere−1,4-benzenedithiol (BDT) NPs, and Au sphere−rhodamine 6G (R6G) NPs. All samples 3997


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ACS Applied Materials & Interfaces refreshed, and the NPs were added at final concentrations of 0.005, 0.01, 0.02, 0.05, and 0.1 nM for H1299 cells. Each concentration was used in at least five parallel tests. Then, the cells were cultured constantly for 3 days. A CCK-8 (Cell Counting Kit8, Dojindo) assay was used on the H1299 cells after 4 h, 1 day, 2 days, and 3 days according to the manufacturer’s instructions. The absorbance was measured spectrophotometrically using an enzymelinked immunosorbent assay microplate reader (Synergy 2, Bio-TEK). 2.6. Animal Imaging Experiment. All animal experiments were performed in compliance with Shanghai Jiao Tong University Animal Study Committee’s requirements for the care and use of laboratory animals in research. Six 20−22 g male severe combined immunodeficiency (SCID) mice were used for animal imaging studies. Human prostate cancer was selected as the research model because of its high incidence in men. The xenografted mouse models were generated by in situ injection of 106 PC3 cells per mouse (human prostate cancer cells in 200 μL of PBS) in the prostate region of male SCID mice. PC3 cells were obtained from ATCC and cultured under standard conditions. Raman spectroscopic measurements were performed when the tumor size reached 5 mm in diameter. For Raman imaging after intratumoral (i.t.) injection, the tumor-bearing mice were intratumorally injected with 20 μL samples containing 60 pM MS GERTs (in saline). Raman images of the tumor sites in the mice were obtained 4 h after i.t. injection with a 785-nm laser, 5× objective lens, and 2.21 × 105 W/cm2 power density. For Raman imaging after intravenous (i.v.) injection, the tumor-bearing mice were injected by tail vein with 200 μL samples containing 0.6 nM MS GERTs (in saline). Raman images of the tumor sites in the mice were obtained 20−24 h after i.v. injection with a 785-nm laser, 5× objective lens, and 2.21 × 105 W/cm2 power density. During the imaging, saline was added to the surface of the tumor tissues to prevent dehydration of the animals. Furthermore, a small part of the tumors of mice subjected to the i.t. injection of MS GERTs was continuously irradiated with a 785nm laser (5× objective lens, 2.21× 105 W/cm2 power density) for 30 min to evaluate the stability of tumor Raman imaging.

ms/spectrum using a 785-nm laser and a 100× objective lens. All spectra were background-subtracted, and their Raman intensities were normalized to the exposure time, laser power, and concentration of embedded Raman reporters. Figure 1c presents a representative time-resolved SERS trajectory, where the MS GERTs exhibit fairly stationary Raman signal intensity with two strong characteristic Raman bands of BDT at 1055 and 1555 cm−1 and one weak band at 1178 cm −1 , corresponding to the phenyl-ring breathing mode (CH inplane bending and CS stretching), the phenyl-ring stretching motion (8a vibrational mode), and the CH bending motion (9a vibrational mode), respectively.37,38 During SERS measurements, Raman blinking or fluctuation often occurs because of the effects of photoinduced heating39 and chemical reaction,40 configuration switching,41,42 and movement/diffusion of molecules.43,44 These effects can cause Raman-active molecules to burn into amorphous carbon, convert into other chemicals by oxidation, diffuse into and out of near-field hot spots, and transition between CHEM and EM enhancement state. Surprisingly, no such Raman blinking or fluctuation phenomena were observed during the photostability test of the MS GERTs for 30 min. Their stable SERS properties can be further demonstrated by the three representative SERS spectra with almost negligible change recorded before, during, and after irradiation shown in Figure 1d and by the lack of any obvious difference in the solid NPs on the silicon substrate from the bright-field images before and after continuous laser irradiation (Figure S1d). It is notable that such extraordinary SERS photostability and pronounced intensity of MS GERTs were realized with a large laser power density of 4.71 × 105 W/cm2 and a short exposure time of 10 ms/spectrum. In contrast, typical SERS measurements on structures or configurations with near-field hot spots such as nanosphere dimers and tipenhanced Raman scattering (TERS) usually utilize laser power densities that are 1−2 orders of magnitude lower and exposure times that are 2−3 orders of magnitude longer to maintain constant Raman spectra with sufficient signal-to-noise ratios.30,41,45,46 A number of structural advantages and optical features of MS GERTs might explain why they exhibit great photostability and pronounced intensity under such a high laser power density and short exposure time. First, the use of off-resonance laser excitation in the near-infrared (NIR) region (e.g., 785 nm) circumvents the plasmon resonance of GERTs in the visible region (e.g., 540 nm), which dramatically reduces the photoheating effects from the laser (Figure 1e). Second, the subnanometer core−shell junction geometry in GERTs offers many more EM/CHEM hot spots compared to conventional dimers and sphere-on-plane structures, where typically only one hot spot exists.47 Third, the electron transport across the molecular junction allows a total strong SERS enhancement with an intense CHEM [i.e., charge-transfer (CT)] enhancement and a moderate EM (i.e., near-field) enhancement.34 The strong CT enhancement occurs mainly because both ends of molecules covalently connect to the core and shell surface through Au−S bonds. The electrons can transfer from the highest occupied molecular orbital (HOMO) of the molecule to the unfilled states of Au, as well as from the occupied Au states to the lowest unoccupied molecular orbital (LUMO) of the molecule. We observed three effects that demonstrate the CT effect in MS GERTs: (1) red shift of the Raman bands,48,49 (2) broadening of the Raman bands,50 and (3) enhancement of Raman bands. Therefore, the photobleaching of the Raman

3. RESULTS AND DISCUSSION Figure 1a presents a TEM image of MS GERTs, which consist of a Au core−shell structure spaced by a uniform and nanometer-sized interior gap, with an MS layer coating on the surface (schematic diagram shown in the inset). The entire size of the MS GERTs was 82 ± 4 nm, and the MS layer had a homogeneous thickness of 12 ± 1.8 nm. The interior gap structure between the metallic core and shell (indicated by red arrows in Figure 1a) and the mesoporous morphology in the silica shell appeared to be quite clear in the MS GERTs. The gap size was relatively uniform primarily at ∼0.7 nm, which was determined by the thickness of the monolayer of embedded Raman-active 1,4-benzenedithiol (BDT) molecules.33,34 The asprepared GERTs without an MS coating exhibited a single pronounced resonance peak at 544 nm in water. Because of electron transport across the subnanometer molecular junction, those GERTs appeared homogeneous solid Au nanospheres without a gap in their optical extinction spectra.33 After application of the MS coating, the resonance peak showed a 5nm red shift due to the change in refractive index in the vicinity of the outer shell surface (Figure 1b). The inset in Figure 1b shows the typical ruby color of aqueous MS GERTs. To validate the extraordinary SERS photostability of MS GERTs, we chose the most challenging Raman measurement conditions, under which solid MS GERTs dried on a silicon wafer were continuously irradiated with a laser for 30 min at a power density of 4.71 × 105 W/cm2. According to our previous study,34 the SERS signal of MS GERTs at 532-nm excitation is much weaker than the signal at 785-nm excitation. Thus, SERS spectra were recorded every 2 s with an exposure time of 10 3998


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Figure 2. (Left) Schematic diagrams, (middle) photostability measurements of time-resolved SERS spectra of solid nanoparticles on a silicon wafer during continuous irradiation for 30 min, and (right) three representative SERS spectra at selected irradiation times: (a) Au sphere−R6G NPs, (b) Au sphere−BDT NPs, and (c) GERTs. SERS spectra were acquired at 2-s intervals (10-ms exposure time/spectrum and 100× objective lens). A 632-nm laser with a power density of 2.16 × 105 W/cm2 was used for Au sphere−R6G and Au sphere−BDT NPs, and a 785-nm laser with a power density of 4.71 × 105 W/cm2 was used for GERTs. Spectra were background-subtracted and intensity-normalized to exposure time, laser power, and concentration of Raman reporters.

reporters in the junction can be avoided to a large extent,30−32 and the MS GERTs still show great Raman signal enhancement. Next, the solid metallic cores and MS shells well protect the built-in Raman reporters to avoid possible desorption and minimize photoinduced chemical reactions by isolation from the environment. In addition, both ends of the BDT molecules covalently connect to the core and shell surface through Au−S bonds, accordingly minimizing any changes in molecular locations and orientations. All of these unique properties greatly enhance the photostability and sensitivity of the MS GERTs.

Subsequently, we compared the photostability of the MS GERTs with those of three similarly sized SERS tags: Au sphere−R6G NPs, Au sphere−BDT NPs, and GERTs (Figure 2). These three nanostructures were all formulated using Au nanospheres with similar sizes of about 50 nm; however, the Raman reporter R6G was electrostatically absorbed on the surface of the Au nanospheres for the Au sphere−R6G NPs, the Raman reporter BDT was covalently conjugated with the Au surface for the Au sphere−BDT NPs, and the GERTs had the same structure as the MS GERTs but without an MS coating on the surface. Upon continuous laser irradiation (2.16 × 105 3999


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Figure 3. (a) Photostability comparison of Raman signals of SERS tags including Au sphere−R6G NPs, Au sphere−BDT NPs, GERTs, and MS GERTs: integrated peak areas at 1192 cm−1 for R6G molecules and at 1055 cm−1 for BDT molecules. Photostability measurements of FITC fluorescence signals were additionally performed (488-nm laser, 1.27 × 105 W/cm2, 60× objective lens) for comparison. (b,c) Photostability of MS GERTs with integrated peak areas at (b) 1055 and (c) 1555 cm−1 under continuous laser irradiation with different power densities for 30 min. (d) Three representative SERS spectra at selected irradiation times under 2.36 × 106 W/cm2 irradiation. (e−g) Stability measurements of SERS and extinction spectra for MS GERTs in (e) aqueous solutions of different pH values in the range of 3−12, (f) 10% fetal bovine serum, and (g) saline for different times.

W/cm2), the SERS intensity of the Au sphere−R6G NPs decreased significantly, about 2−3 orders of magnitude lower than that of MS GERTs, and the Raman characteristic bands of the R6G molecules (614, 766, 1192, 1306, 1361, 1509, and 1647 cm−1, corresponding to C−H, C−O−C, and C−C vibrations of the aromatic rings) exhibited shape modulation (Figure 2a),51 as confirmed by three representative SERS spectra recorded before, during, and after irradiation. To collect observable Raman signals within an exposure time of 10 ms, a high concentration (20 nM) of Au spheres had to be employed. During the irradiation, the stabilizer sodium citrate in Au sphere−R6G NPs could be damaged (Figure S1a), resulting in the aggregation of the Au nanospheres and the production of more EM hot spots. However, the increased EM effect boosts the photobleaching effect of R6G molecules, and the exposure of R6G molecules to the surrounding environment would also enhance the photochemical reaction under long-term irradiation, which, in turn, would further impair the SERS signal. Notably, the SERS intensity of the Au sphere−R6G NPs fluctuated during the irradiation period, probably because of the adsorption and/or desorption cycles of R6G molecules within the hot spots.52 Figure 2b shows the time evolution of the Raman spectra of Au sphere−BDT NPs during continuous irradiation (2.16 × 105 W/cm2). Compared to the Au sphere−R6G NPs, the Au sphere−BDT NPs exhibited a Raman intensity that was slightly higher but still 1−2 orders of magnitude lower than that of the MS GERTs. The Au sphere−BDT NPs presented less of a decrease and less fluctuation in SERS, and the characteristic Raman bands were still obvious after 30 min of irradiation. The stabilizer cetyltrimethylammonium chloride (CTAC) in the Au sphere−BDT NPs was easily photodamaged (Figure S1b) during the irradiation, but the BDT molecules exhibited a higher photostability than the R6G molecules, probably because the BDT molecules were covalently conjugated to the Au surface whereas the R6G molecules were electrostatically adsorbed on the Au surface. The Raman bands of the

BDT molecules in the Au sphere−BDT NPs still displayed about half the intensity after 30 min of irradiation, but sometimes, miscellaneous bands appeared during the irradiation (Figure S2). This can possibly be attributed to the generation of amorphous carbon during the photobleaching process.53,54 To highlight the essential role of a metallic Au shell, rather than an MS shell, in stabilizing the Raman reporters at the interior gap junction, we investigated the SERS photostability of GERTs without an MS coating. As shown in Figure 2c, the band intensity and shape of the Raman spectrum of the GERTs remained quite stable during the continuous irradiation even at twice the laser power density (4.71 × 105 W/cm2) compared to the Au sphere−BDT NPs, indicating that hiding the molecules in the nanogap can greatly improve the photostability of the molecules. The constant Raman spectra before, during, and after irradiation confirm their extraordinary photostability, even though the stabilizer CTAC was damaged during the irradiation (Figure S1c). Through further TEM examinations (data not shown), we found that the gap morphology did not change much after 10 min of continuous irradiation. However, TEM observations failed because of the photodamage of the carbon film on the Cu grid after longer irradiation times. In addition to stability, the GERTs exhibited strong signals at a much lower concentration (0.6 nM) than the signals in the hot spots of the Au sphere−R6G and Au sphere−BDT structures. Next, we quantitatively compared the photostabilities of these four SERS tags (Au sphere−R6G NPs, Au sphere−BDT NPs, GERTs, and MS GERTs) with fluorescein isothiocyanate (FITC), a type of conventional fluorescent dye, by plotting their photobleaching behaviors (see Figure 3a, dotted lines). The integrated peak areas at 1192 cm−1 for R6G molecules and at 1055 cm−1 for BDT molecules were chosen as representative for investigating their photobleaching behaviors from the timeresolved SERS spectra. These imaging tags exhibited different bleaching profiles. The fluorescence of FITC was quickly quenched in 5 min at an irradiation power density of 1.27 × 105 4000


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Figure 4. Raman and fluorescence images for H1299 cells stained with MS GERTs and fluorescence probes. (a) Representative superstable Raman images obtained from a single cell: bright-field image (left) and Raman images at different irradiation times (t = 0, 10, 20, and 30 min). Each Raman image was obtained within 53 s with a power density of 4.71 × 105 W/cm2 and a 10-ms exposure time per pixel. (b) Bright-field image (left) and corresponding cell fluorescence images at different irradiation times (t = 0, 2, 4, and 6 min; 2.44 × 104 W/cm2 power density). (c) Representative Raman images obtained from a single cell: bright-field image (left) and Raman images acquired with different exposure times (t = 10, 5, and 1 ms) per pixel. The laser power density employed was 4.71 × 105 W/cm2. The scale bars are 5 μm in panels a and c and 20 μm in panel b.

W/cm2, and then the bleaching time constant (τ) was obtained by fitting the decay curves to the equation I = Ae(−t/τ) (solid lines).31 The time constant obtained for FITC was only 55.8 s, suggesting that FITC experienced severe photobleaching property. The signals of the Au sphere−R6G NPs decayed seriously in the beginning, preserving only ∼20% of the original Raman intensity after 5 min of irradiation and even becoming negligible after ∼10 min of irradiation. Comparatively, the Au sphere−BDT NPs also decayed seriously in the first few minutes, but they seemed to be slightly more photostable than the Au sphere−R6G NPs at the same power density, retaining ∼30% of the original Raman intensity after 30 min of laser irradiation. The time constants for the Au sphere−R6G and Au sphere−BDT NPs were dfound to be 147 and 1019 s, respectively, which means that they were more stable than FITC even at a larger power density (2.16 × 105 W/cm2). Moreover, these results also reveal that the covalent connection between the BDT molecules and the Au surface was more stable than the electrostatic adsorption between the R6G molecules and the Au surface. In striking contrast, the Raman intensities of the GERTs and MS GERTs decreased only slightly even under more than twice of the laser power density compared with the Au sphere−R6G and Au sphere−BDT samples, preserving over 80% of their initial intensities after 30 min of laser irradiation. Notably, the Raman intensity of the GERTs exibited a slight increase at the beginning of irradiation, and this might have been caused by the increase in the NP density within the laser spot due to the photodamage and movement of the CTAC stabilizer under the continuous irradiation (Figure S1c).52 The time constants of the GERTs and MS GERTs were found to be 9680 and 13443 s, respectively, much greater than those of the other tags. A comparison of the time constants of the GERTs and MS GERTs also demonstrates that the MS shell played a certain role in improving the photostability of the NPs, as has been reported in previous works.55,56 The time evolutions of the Raman spectra in terms of the integrated peak areas at the representative 1509 cm−1 peak for R6G molecules and 1555 cm−1 for BDT molecules are shown in Figure S3, exhibiting

overall photobleaching trends similar to those in Figure 3a for all SERS tags. We further increased the laser power density to investigate the photostability of the MS GERTs. The time evolutions of the Raman spectra of the MS GERTs under three different laser power densities (4.71 × 105, 1.18 × 106, and 2.36 × 106 W/ cm2) were recorded, and the integrated peak areas of the corresponding Raman peaks at 1055 and 1555 cm−1 are plotted as a function of the irradiation time (dotted lines) in Figure 3b,c. The bleaching time constants were obtained by fitting the decay curves with the same equation as described above (solid lines). The obtained time constants are 13443, 7291, and 3878 s for the peaks at 1055 cm−1 and 55059, 8812, and 5372 s for the peaks at 1555 cm−1 at the three different power densities of 4.71 × 105, 1.18 × 106, and 2.36 × 106 W/cm2, respectively. One can see that the bleaching time constant of the BDT molecules in the MS GERTs decreased with increasing laser power density, but it was still fairly large at the highest power density, which means the MS GERTs exhibited excellent photostability even at intensely high power density. We also noticed that the time constant of the 1555 cm−1 peak was larger than that of the 1055 cm−1 peak at a given power density. We presume that this is possibly due to the fact that the closed-loop structure of the phenyl ring (corresponding to the peak at 1555 cm−1) is more stable than the single C−S bond (corresponding to the peak at 1055 cm−1) under continuous irradiation. Figure 3d shows three typical SERS spectra of MS GERTs at the highest power density of 2.36 × 106 W/cm2 before, during, and after 30 min of continuous irradiation. The characteristic Raman bands of the BDT molecules are still quite obvious after 30 min of continuous irradiation with more than 50% of the initial intensity, thus demonstrating their great potential for long-term SERS-based bioimaging. For bioimaging, it is desirable that SERS tags maintain their photostability under storage conditions and in various physiologic environments (e.g., at different pH values and in serum) without inducing any cytotoxicity. GERTs synthesized with a coating of CTAC molecules exhibit severe toxicity (see Figure S4).51,52 Moreover, CTAC-coated NPs easily aggregate 4001


Research Article


Figure 5. Imaging of orthotopic prostate tumors in mice model. (a) (i) Bright-field image, (ii) corresponding Raman image (2.21 × 105 W/cm2 power density), and (iii) overlay image of prostate tumor and bladder in human prostate-tumor-bearing mice after i.t. injection with MS GERT solution (20 μL of 60 pM). Prostate tumors were developed near the bladder. (b) (i) Bright-field image, (ii) corresponding Raman image (2.21 × 105 W/cm2 power density), and (iii) overlay image of prostate tumor and bladder in human prostate-tumor-bearing mice after i.v. injection with MS GERT solution (200 μL of 0.6 nM). Prostate tumors were developed near the bladder. (c) Representative superstable prostate tumor Raman images obtained from the microregion in panel a with different irradiation times (t = 0, 10, 20, and 30 min). Each Raman image was obtained within 2 min using a 5× objective lens. All scale bars are 1000 μm.

under acid, alkaline, electrolyte, and cell medium conditions.57 In contrast, the MS GERTs exhibit much improved biocompatibility. A CCK-8 assay was applied to investigate the proliferation and cell viability of H1299 cells cultured with different concentrations of MS GERTs for 4, 24, 48, and 72 h (Figure S4). The results showed that the MS GERTs are noncytotoxic to cells within the concentration range of 0−0.1 nM. Thus, the MS layer improved not only the photostability but also the biocompability. We then explored the photostability of MS GERTs in aqueous samples of different pH values and incubated them with 10% fetal bovine serum and saline for 96 h. The Raman intensity at 1055 cm−1 (black lines) and the peak position of the plasmon resonance (red lines) were found to be quite stable during the incubation time (Figure 3e-3g). Moreover, the negligible broadening of the extinction spectra indicates that no obvious aggregation occurred (Figures S5 and S6). Finally, we explored the photostability of the MS GERTs in ethanol, which serves as the long-term storage medium for protecting the MS shell from hydrolysis and contamination and as a conducive medium for bioapplications. The shapes of the Raman spectra and extinction spectra of the MS GERTs remained almost unchanged during 12 weeks of storage in ethanol without any protection (Figures S5d and S6d). Subsequently, we investigated the capability of the photostable MS GERTs for cell-based high-speed imaging. Figure 4a shows SERS images of H1299 cells stained with the MS GERTs. The SERS-based images of the cells were almost identical during 30 min of continuous irradiation, which is in good accordance with the above discussion of the excellent photostability of MS GERTs. In contrast, the signals from FITC fluorescent-labeled cells almost disappeared within 6 min of irradiation as a result of severe photobleaching of FITC (Figure 4b), even though the laser power had been greatly reduced. In our experiments, a SERS image of a single cell labeled with MS GERTs could be acquired within 53 s (2500 spectra acquired from an area of 27 μm × 28 μm) with an exposure time of 10 ms per pixel. The bright-field image and

Raman image of the cell also coincided well (Figure S7). More importantly, we could further shorten the imaging time on cells with a high-resolution image (50 pixels × 50 pixels). In Figure 4c, the single-cell Raman image was acquired within 44 s at an exposure time of 5 ms per pixel; the exposure time could even be shortened to 1 ms per pixel, so that a high-resolution singlecell image was obtained within 35 s. Notably, the reason that good imaging results can be obtained within such a short time is that the MS GERTs provide sufficiently strong signals. The SERS images in this work were achieved by scanning the cells point by point by moving the mechanical stage. In such a mode, stage movement occupies most of the time spent for acquiring images. If some improvements, such as utilization of galvano mirrors,9 could be implemented, a single-cell high-resolution SERS image could potentially be realized in less than 3 s, which is highly favorable for the real-time observation of cells. To demonstrate the potential of MS GERTs for in vivo applications, we performed Raman imaging of the MS GERTs using orthotopic prostate cancer mice models. Figure 5 shows representative Raman images of prostate tumors in living mice. As an initial test, we performed intratumoral (i.t.) injection of the MS GERTs (20 μL, 60 pM) into the prostate tumors, waited for 4 h to allow for sufficient diffusion of the NPs inside the tumors, and subsequently performed the Raman imaging. A SERS image was acquired over a large area (7040 μm × 4864 μm) covering the bladder and the prostate tumor (Figure 5a). Considering that repeated imaging of tumors might be needed for in vivo biomedical applications, we selected a microregion (black dotted rectangle in Figure 5a, over an area of 2304 μm × 1536 μm) of the prostate tumor to perform repeated imaging (Figure 5c). During the imaging, we added saline to the surface of the tumor tissues to prevent dehydration of the animals. A total of 18 high-resolution Raman images of tumors were obtained within 30 min using an exposure time of 1.86 s per pixel, and the signals of these repeated images were found to be quite stable, indicating the superior photostability of the MS GERTs in the tumors. Furthermore, we performed intravenous (i.v.) injection of the MS GERTs (200 μL, 0.6 nM) and 4002


Research Article


81471779, 81571763, and 81622026), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. TP2014028), the Science and Technology Commission of Shanghai Municipality (No. 13ZR1422100), and Shanghai Jiao Tong University (No. YG2016MS51) for their financial support.

obtained Raman images of the prostate tumors 24 h thereafter. Because of the enhanced permeability and retention (EPR) effect of nanoparticles, the MS GERTs were able to passively accumulate inside the tumor regions and yield signals. As expected, pronounced Raman signals from the prostate tumor but not the normal surrounding tissues were acquired (Figure 5b). As such, the MS GERTs were confirmed to be capable of maintaining ultraphotostability in the blood circulation system and in acidic tumor tissues, indicating the great potential of this type of SERS tag for in vivo applications.

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(1) Nie, S.; Emory, S. R. Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering. Science 1997, 275, 1102−1106. (2) Wang, Y.; Yan, B.; Chen, L. SERS Tags: Novel Optical Nanoprobes for Bioanalysis. Chem. Rev. 2013, 113, 1391−1428. (3) Pei, H.; Zuo, X.; Zhu, D.; Huang, Q.; Fan, C. Functional DNA Nanostructures for Theranostic Applications. Acc. Chem. Res. 2014, 47, 550−559. (4) Pei, H.; Li, F.; Wan, Y.; Wei, M.; Liu, H.; Su, Y.; Chen, N.; Huang, Q.; Fan, C. Designed Diblock Oligonucleotide for the Synthesis of Spatially Isolated and Highly Hybridizable Functionalization of DNA−Gold Nanoparticle Nanoconjugates. J. Am. Chem. Soc. 2012, 134, 11876−11879. (5) Xu, H.; Li, Q.; Wang, L.; He, Y.; Shi, J.; Tang, B.; Fan, C. Nanoscale Optical Probes for Cellular Imaging. Chem. Soc. Rev. 2014, 43, 2650−2661. (6) Pei, H.; Liang, L.; Yao, G.; Li, J.; Huang, Q.; Fan, C. Reconfigurable Three-Dimensional DNA Nanostructures for the Construction of Intracellular Logic Sensors. Angew. Chem., Int. Ed. 2012, 51, 9020−9024. (7) Sha, M. Y.; Xu, H.; Natan, M. J.; Cromer, R. Surface-Enhanced Raman Scattering Tags for Rapid and Homogeneous Detection of Circulating Tumor Cells in the Presence of Human Whole Blood. J. Am. Chem. Soc. 2008, 130, 17214−17215. (8) Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K. In Vivo Molecular Probing of Cellular Compartments with Gold Nanoparticles and Nanoaggregates. Nano Lett. 2006, 6, 2225−2231. (9) Kang, J. W.; So, P. T.; Dasari, R. R.; Lim, D. High Resolution Live Cell Raman Imaging Using Subcellular Organelle-Targeting SERSSensitive Gold Nanoparticles with Highly Narrow Intra-Nanogap. Nano Lett. 2015, 15, 1766−1772. (10) Lu, G.; De Keersmaecker, H.; Su, L.; Kenens, B.; Rocha, S.; Fron, E.; Chen, C.; Van Dorpe, P.; Mizuno, H.; Hofkens, J.; Hutchison, J. A.; Uji-i, H. Live-Cell SERS Endoscopy Using Plasmonic Nanowire Waveguides. Adv. Mater. 2014, 26, 5124−5128. (11) Huefner, A.; Kuan, W.; Müller, K. H.; Skepper, J. N.; Barker, R. A.; Mahajan, S. Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with Surface-Enhanced Raman Spectroscopy and Chemometrics. ACS Nano 2016, 10, 307−316. (12) Schlücker, S.; Küstner, B.; Punge, A.; Bonfig, R.; Marx, A.; Ströbel, P. Immuno-Raman Microspectroscopy: In Situ Detection of Antigens in Tissue Specimens by Surface-Enhanced Raman Scattering. J. Raman Spectrosc. 2006, 37, 719−721. (13) Lutz, B. R.; Dentinger, C. E.; Nguyen, L. N.; Sun, L.; Zhang, J.; Allen, A. N.; Chan, S.; Knudsen, B. S. Spectral Analysis of Multiplex Raman Probe Signatures. ACS Nano 2008, 2, 2306−2314. (14) Qian, X.; Peng, X.; Ansari, D. O.; YinGoen, Q.; Chen, G. Z.; Shin, D. M.; Yang, L.; Young, A. N.; Wang, M. D.; Nie, S. In Vivo Tumor Targeting and Spectroscopic Detection with Surface-Enhanced Raman Nanoparticle Tags. Nat. Biotechnol. 2007, 26, 83−90. (15) Zavaleta, C.; De La Zerda, A.; Liu, Z.; Keren, S.; Cheng, Z.; Schipper, M.; Chen, X.; Dai, H.; Gambhir, S. Noninvasive Raman Spectroscopy in Living Mice for Evaluation of Tumor Targeting with Carbon Nanotubes. Nano Lett. 2008, 8, 2800−2805. (16) Keren, S.; Zavaleta, C.; Cheng, Z.; de La Zerda, A.; Gheysens, O.; Gambhir, S. Noninvasive Molecular Imaging of Small Living Subjects Using Raman Spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 5844−5849. (17) Zavaleta, C. L.; Smith, B. R.; Walton, I.; Doering, W.; Davis, G.; Shojaei, B.; Natan, M. J.; Gambhir, S. S. Multiplexed Imaging of

4. CONCLUSIONS In conclusion, MS GERTs were designed with built-in Raman reporters for off-resonance NIR laser excitation. As a result, the reporters were well protected by the solid metallic shell, mesoporous silica coating, and reduced photothermal effect during laser irradiation. Significantly, they exhibited a large amplification of Raman signals by combining the CHEM and EM enhancement effects, so that SERS imaging could be achieved with reduced duration and laser power density. We further demonstrated that, with these unique structural and optical advantages, MS GERTs exhibit great photostability for sensitive and fast SERS-based cell and tumor imaging. More specifically, the MS GERTs exhibit high storage, pH, serum, and photostabilities; strong Raman signals; and favorable biocompatibility. Thus, they are particularly suitable for highspeed, long-term, and real-time bioimaging under the continuous laser irradiation. As promising SERS tags, they might also hold great potential for other biomedical applications.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b15170. Bright-field images of samples before and after 30 min of continuous laser irradiation, Raman spectra of Au sphere−BDT nanoparticles during 30 min of continuous laser irradiation, photostability comparison of Raman signals of four types of SERS tags, cell viability analysis of MS GERTs and GERTs stabilized by CTAC, detailed extinction spectra and Raman spectra of MS GERTs in Figure 3, and bright-field image and overlays of brightfield and Raman images in Figure 4a (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jian Ye: 0000-0002-8101-8362 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.

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ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation of China (Nos. 21375087, 21511130019, 4003


Research Article

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Ultraphotostable Mesoporous Silica-Coated Gap-Enhanced Raman

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