Smart Plasmonic Glucose Nanosensors as Generic Theranostic

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Smart Plasmonic Glucose Nanosensors as Generic Theranostic Agents for Rapid, Targeting-Free Cancer-Cell Screening and Killing Limei Chen, Haijuan Li, Haili He, Haoxi Wu, and Yongdong Jin Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2015 Downloaded from http://pubs.acs.org on June 1, 2015

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Analytical Chemistry

Smart Plasmonic Glucose Nanosensors as Generic Theranostic Agents for Rapid, Targeting-Free Cancer-Cell Screening and Killing Limei Chen, †, ‡ Haijuan Li, † Haili He, †, § Haoxi Wu, †,§ and Yongdong Jin *†

†State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Jilin 130022, P.R. China. ‡Department of Cell biology, Basic Medical College, Beihua University, Jilin 132013, China. §University of Chinese Academy of Sciences, Beijing 100049, P.R. China. *E-mail: [email protected]. Tel: +86-431-85262661. Fax: +86-431-85262661.

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ABSTRACT: Fast and accurate identification of cancer cells from health normal cells in a simple, generic way is very crucial for early cancer detection and treatment. Although functional nanoparticles, like fluorescent quantum dots and plasmonic AuNPs, have been successfully applied for cancer cell imaging and photothermal therapy, they suffer from the main drawback of needing time-consuming targeting preparation for specific cancer cell detection and selective ablation. The lack of a generic and effective method therefore limits their potential high-throughput cancer cell preliminary screening and theranostic applications. We report herein a generic in-vitro method for fast, targeting-free (avoiding time-consuming preparations of targeting moiety for specific cancer cells) visual screening and selective killing of cancer cells from normal cells, by using glucose-responsive/-sensitive glucose oxidase-modified Ag/Au nanoshells (Ag/Au-GOx NSs) as a smart plasmonic theranostic agent. The method is generic to some extent since it is based on the distinct LSPR responses (and colors) of the smart nanoprobe with cancer cells (typically have a higher glucose uptake level) and normal cells.

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Although prodigious efforts have been expended to find effective methods for cancer theranostics in recent years, cancer remains a major challenge to global public health. The difficulty of early detection and the lack of effective therapy and prevention lead to high mortality.1 In clinical detection of cancer, computed tomography (CT) and magnetic resonance imaging (MRI) are often used for detecting subtle and infiltrating focus, although they are sensitive but it is too expensive to perform them on all suspect patients, and also contrast agents used in CT and MRI are “always on”.2-4 Immunofluorescence assay is more useful for screening high-risk individuals, but it suffers the disadvantage of high individual variation, low positive predictive value, time-consuming procedure and the lack of a standardized method.4-6 Recently, detection of cancer biomarker plays an important role in the early diagnosis.7-10 In clinical medicine, a correct cancer diagnosis depends on pathological immunohistochemistry (IHC),11 in which multiple target-specific agents are introduced to determine specific disease markers, making it very timeconsuming and obtaining tumor tissue samples requires invasive procedures, which may increase the danger to the patient.12-14 Therefore, development of a sensitive, convenient and universal diagnostic method is a great challenge and will be very imperative to identify cancer, especially in early stage. Nano-materials and multifunctional nano-platforms with different shape and compositions have recently been used in cancer diagnosis, drug delivery and cancer therapy.7,15-20 Especially Au-based nanoparticles have attracted more attention because of their unique plasmonic optical and photothermal properties as well as their good biocompatibility.16,17,21-23 And bimetallic nanoparticles with a core–shell structure, due to more flexible tunability in local electric field and nanometer-scale structure than those of mono-component have proven to be better candidates for biosensing applications and ultrasensitive detections.25-29 Although we have very recently succeeded in using enzymatic Ag/Au-GOx nanoshells (NSs) for optical serum glucose sensing in vitro,28 the applicability of the probe for cell study is not obvious and still a challenge since the complexity of living cells. Given that cancer cells are known have higher glucose uptake rate than normal cells30,31 due to greater need for energy, such glucose-sensitive/responsive nanoprobes may find useful applications in cancer cell studies. In this study, we succeed in expanding its use for cell study and report a new approach for fast and sensitive in-vitro cancer cell imaging and discrimination by exploiting the Ag/Au-GOx NS as a smart nanoplasmonic theranostic agent. 3



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EXPERIMENTAL SECTION Chemicals and Materials. Silver nitrate (AgNO3), trisodium citrate, hydroxylamine hydrochloride (NH2OH•HCl), poly (L-histidine) (PLH) hydrochloride (Mw ≥ 5000), and glucose oxidase (GOx, from Aspergillus niger, Type II, ≥15 000 units/g) were purchased from SigmaAldrich. Gold chloride (HAuCl4•3H2O), and ascorbic acid were obtained from Beijing Chemical Works. All chemicals were used as received and without any further purification. Cell culture. HeLa, HepG2, A549, HL7702 and L929 cells were obtained from the American Type Culture Collection (ATCC, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) and contained 200 units mL-1 penicillin, 200 units mL-1 streptomycin (Invitrogen) at 37°C, 5% CO2 in a humidified atmosphere. The number of cells was counted to be ca. 5 × 106 cells mL-1. Synthesis of Ag/Au nanoshells and probe Ag/Au-GOx nanoshells. Ag/Au NSs were synthesized according to our previous method.28 Initially, colloidal Ag NPs (LSPR peak at ～ 430 nm) were synthesized with citrate as described previously.29 Subsequently, the prepared Ag sol (5 mL) was diluted with 5 mL water, and a freshly prepared reaction solution of 0.02 M NH2OH (0.1 mL) and 0.1% HAuCl4 (with varied volume from 75～300 µL) was added with magnetic stirring in a glass vial. The solution was then heated and refluxed for 7~8 minutes before cooling to room temperature. The probe Ag/Au-GOx NSs were prepared according to the previous protocol.28,29 Incubation of Ag/Au-GOx NS probes with cells. When the density of cells attained to 70 ～ 80%, the culture medium was removed and rinsed 3 times with PBS. Immediately the cells were incubated with 500 µL of the newly prepared Ag/Au-GOx NSs (0.74 × 1011 particles mL-1,32,33 in glucose-free PBS) for 20, 40 and 60 min at 37°C, 5% CO2 in a humidified atmosphere. In order to remove the extra Ag/Au-GOx NSs, the cells were rinsed 3 times with PBS after incubation with the probes. Dark-field scattering images of the samples were taken after different incubation time. Co-culture of malignant and benign cells. HepG2 and HL7702 cells were cultured separately under the same condition to the density 80% ～90%, digested by 0.25 % trypsin (in 10mM PBS, pH=7.4), counting cell numbers and then mixing the two kinds of cells in the ratio 1:1. The mixed cells were co-cultured in DMEM with 10% fetal bovine serum. Incubation with the Ag/Au 4


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NSs probe when we can distinguish them clearly from morphological characteristics after 48h, then checked under dark-field microscopy. Dark-Field imaging. Dark-field scattering images were obtained using an inverted Leica DMI6000B microscope (Germany) with a dark-field condenser (Leica 0.90 S1). In the dark-field equipment, the condenser delivers a narrow beam of white light from a tungsten lamp and collects only the scattered light from the samples. The dark-field images were captured by a Leica DFC450 C digital camera before and after probe nanoparticles incubation with cells or H2O2 respectively at different times as indicated in the text. Due to the enhanced absorption and scattering from gold nanoparticles (by a factor of 105), the scattered light from cells can be neglected. The Leica Application Suite software Leica DFC450 C from the camera manufacturer was used for image acquisition and has a feature for adjusting the white color balance for accurately capturing the color differences in samples. Cell Viability Study. For each MTT assay, 96 culture wells of different cells were prepared (~ 5000 cells in each of 96 wells). Six sets were treated with different cells, and one set served as the nontreated control. The complete assay was performed three times, and results were averaged. The concentration of Ag/Au NSs and Ag/Au-GOx NSs is ～0.74 × 1011 particles mL-1, 32,33 10 µL of NSs were added to each well and subsequently incubated with the cells for 24 h at 37 °C under 5% CO2. After 24 h incubation and exposure to the laser, 10 µL of MTT reagent was then added to each well and thoroughly mixed. Four hours later, 100 µL of Formazan solutions were added and oscillated 30 min with low-speed, the absorbance of the mixtures at 490 nm was measured using a multiwell plate reader. The cell viability was calculated as the ratio of the absorbance of the sample well to that of the control well and expressed as percentage viability, assigning the viability of nontreated cells as 100%. Photothermal ablation of cancer cells. All Cells were plated in 96 culture wells (~ 5000 cells in each of 96 wells). After 24 h (the density attained to 80%～90%), culture medium were removed by rinsing three times with PBS (10 mM with Na2HPO4•12H2O and KH2PO4). Fresh PBS was then added to the wells. Subsequently，the cells were incubated with freshly prepared probe Ag/Au-GOx NSs for a designated time as indicated in the text, then observed by Leica DMI6000B dark-field optical microscope with exposure time of 2.0 s. The photothermal efficiency of the NSs solutions were first investigated under various irradiation times (0, 0.5, 1, 1.5, 5



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2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10 min). The change of temperature in solution was determined by digital thermometer (Traceable 14-648-44 type). Then 500 µL of probe Ag/Au-GOx NSs with particle numbers of 0.74 × 1011 particles mL-1 (according to our previous study)32 were added to the culture dishes with a final volume of 1 mL in PBS at 37℃ for 20 min. After incubation, the free nanoparticles were removed and the remainder was rinsed with PBS buffer solution three times to remove unbound or non-specific bound Ag/Au-GOx NSs from the surface of the cells. The cancer cells were immersed in fresh PBS prior to irradiation for 10 min by the laser. The efficiency of photo-induced cancer cell thermal ablation was investigated with different times. Each well was exposed to the laser light with a focus area of 0.2 cm2 and then stained with trypan blue (final concentration 0.04%) for 5 min to test cell viability. Dead cells accumulated the dye and were stained blue, while living cells eliminated it and remained clear. After staining, the samples were imaged under 10× in bright field. Six replicates were done for each kind of cell type.

RESULTS AND DISCUSION Synthesis and properties of plasmonic glucose nanosensors. The Ag/Au-GOx NSs were prepared according to our previous method

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with some optimizations (mainly in Ag/Au ratio

and shell thickness) to obtain localized surface plasmon resonance（LSPR）red-shift of the nanoprobe as larger as possible (from visible to near-infrared region (NIR), for both enhanced sensitivity and theranostic applications) upon enzymatic reaction with cellular glucose. Scheme 1(ac) depict schematically the glucose-responsive enzymatic sensing strategy, the resulting LSPR shift, and cancer cell imaging and NIR photothermal therapy applications, respectively. The strategy is based on the selective dissolution of Ag from the starting Ag/Au NSs by enzymatically generated H2O2 in the presence of glucose and oxygen.28,29,34 During the enzymatic H2O2 etching process, the LSPR band of the nanoprobes red-shifted gradually with the particle’s morphology changing from complete NSs to porous ones. The magnitude of the spectral shift depends on the reaction time and concentration of glucose in the system. As cancer cells are known have higher glucose uptake rate than normal cells,30,31 cancer cells are usually believed to have more intracellular glucose content level than normal cells under certain conditions. When incubating such glucose-responsive/-sensitive NSs with living cells (normal or cancerous) at a certain condi6


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tion, the enzymatic NSs will react more with cancer cells and therefore the LSPR band of the reacted NSs attached on cancer cells surface will red-shift to NIR region for clearly visual identification by dark-field microscope since the scattering color of the plasmonic nanoprobes turns distinctly from yellow-green to orange or red (depending on the given LSPR sensitivity of a batch of nanoprobes used and the incubation/reaction time), while the nanoprobes with normal cells remain yellow-green colored (less reacted). Due to more spectral overlap of laser wavelength with the LSPR band of the reacted nanoprobes incubated with cancer cells, selective photothermal ablation of cancer cells is also achieved upon exposure cells to laser at 808 nm since the same amount of Ag/Au-GOx NSs incubated with cancer cells usually generate more heat than normal cells at a fixed laser frequency to destroy cells. The amount of HAuCl4 added during the preparation of precursor Ag/Au NSs (starting from Ag NPs)32 was found to be crucial for subsequent enzymatic H2O2 etching to obtain LSPR redshift of the nanoprobe as larger as possible and hence ensure their successful NIR cancer cell theranostic applications. The detailed optimized synthesis and characterizations of the precursor Ag/Au NSs can be found in the Supporting Information (Figure S1). Figure 1(a) shows representative LSPR responses of the typical GOx-free Ag/Au NS sample before and after reaction with different amount of hydrogen peroxide. The LSPR responses of the NS sample display a large band red-shift from ~ 495 nm to 795 nm, very sensitive to the concentration of hydrogen peroxide. We then use it to prepare the probe Ag/Au-GOx NS sample as described previously.28 As previously reported and shown in Figure 1b, due to high sensitivity of LSPR of the NSs (compared to solid AuNPs) in response to environmental changes, a pronounced ~ 50 nm redshift of the LSPR peak was observed after successive electrostatic attachment of poly (Lhistidine) and GOx onto the Ag/Au NS surfaces.28 The successful surface modifications were also confirmed by Zeta potential measurements.29 The as-prepared probe Ag/Au-GOx NS sample are enzymatic and very sensitive to glucose, with fast LSPR band red-shifts from ~ 540 nm to 946 nm within 5 min of glucose (conc. 0.1 mM) incubation. Figure 1 c&d show plasmonic darkfield scattering images of the well-dispersed probe nanoparticles before and after the enzymatic reaction with an excess amount of glucose, displaying a distinct color change from yellow-green to red – which forms the basis for subsequent cancer cell visual identification. The insets of Figure 1 c&d show their corresponding transmission electron microscopy (TEM) images, which clearly reveal the evolution of complete NSs (~ 60-70 nm in diameter) into porous NSs and more 7



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open framework structures after the enzymatic reaction. The Ag/Au bimetallic nature of the complex NSs were confirmed previously by TEM elemental mapping and EDX analysis.26 The preparation of the probe Ag/Au-GOx NS sample was quite reproducible from batch to batch and the probe suspension was stable at least one week when stored at 4 ℃. Dark field imaging identification of cancer cells. As we confirmed previously,29 GOx in the Ag/Au-GOx NSs system exhibits significantly improved enzymatic activities (than free GOx) toward higher temperatures and wider pH range.29 We therefore applied such smart and robust nanoplasmonic probes for in vitro visual discrimination of cancer cells from normal health cells. First, we choose the most common cancer cell - a malignant human cervical cancer cell line (HeLa) and a nonmalignant mouse embryonic fibroblast cell line (L929) as a control. Figure S2 shows the dark-field LSPR scattering images of the probe Ag/Au-GOx NSs attached on cell surfaces after incubation together for different period of time. Cells were incubated with 500 µL of Ag/Au-GOx NSs (～0.74 × 1011 particles mL-1, in PBS) for up to 1h. Dark-field scattering images of the samples were taken after 20, 40, and 60 min incubation time using a Leica DMI6000B microscope with dark-field condenser. As expected and obviously shown in Figure S2, although the coverage of the probe nanoparticles on cell surfaces are very similar, since cancer cells are known have higher glucose uptake rate (and hence higher intracellular glucose level under the test conditions) than normal cells, the Ag/Au-GOx NSs scatter mostly red light on the surface of HeLa cells but almost maintained green on that of L929 cells (due to relatively low glucose level and then imcomplete reaction) after 20 min incubation and enzymatic reaction. With the increasing of incubation and reaction time, the imaging difference between the tumor and non-tumor cells are diminished gradually due to reaching a reaction saturation of the nanoprobes in both systems, with the latter turns gradually red at 40 min and reach completely red after 60 min incubation and reaction. The results are repeated and quite reproducible. Thus, we choose reaction (incubation) time scale of ~ 15-20 min for fast and reliable visual discrimination of cancer cells from normal cells using the probe Ag/Au-GOx NSs. The universality of the approach was also preliminarily demonstrated by extending the experiment to other cancer cell lines (hepatocellular carcinoma HepG2 and human pulmonary carcinoma A549 cells) and normal human liver cell lines HL7702 cells. As shown in Figure 2, under the test conditions the tested three cancer cell lines are all orange/red-colored, 8


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distinctly different from the yellow-green color of the normal cells. For the sake of consistency of the experiments and results, the newly-prepared Ag/Au-GOx NS probes were immediately incubated with the cells after removed the culture medium and rinsed three times with PBS，and by fixing the incubation time (~ 20 min) we can visually identify all three tested cancer cell lines from two health normal cell lines in a facile and fast manner. The probe preparation and cell screening experiments are quite reproducible. We repeated more than 5 times starting from the nanoprobe preparation and all had similar results. More importantly, as shown in Figure 3, tumor cells can be easily visually identified from cocultured normal health cells (which simulates a clinical cell sample), making the nanoprobe and approach very promising for potential clinical use. We successfully identified tumor cells from a co-culture of malignant HepG2 and benign HL7702 cells (ratio 1:1), two cell lines from liver tissues with different morphological characteristics, which acts as a simulated clinical cell sample in this study. After ~ 20 min incubation with Ag/Au-GOx NSs the cells were immediately checked under dark field microscope. As shown in Figure 3, the HepG2 cancer cells can be easily identified from the co-cultured normal HL7702 cells due to distinct color difference. Figure S4 shows clearly the time evolution of the imaging process. There was only an inconspicuous color difference between co-cultured HepG2 and HL7702 cells when incubated with Ag/Au-GOx NSs for short 5 min; while 40 min incubation led to the diminishment of color difference again due to reaching a reaction saturation of the nanoplasmonic probes on the two cell types. However, the co-cultured malignant HepG2 and benign HL7702 cells can be well distinguished with incubation time of ~ 20 min under the test conditions. This is believed to be favorable and important for their potential clinical use as a smart agent for rapid and preliminary in-vitro cancer cell screening in patients and early diagnosis of cancer. To date, cancer has become one of the most serious global health threats. Therefore there is an urgent need to develop new detection techniques with greater sensitivity, specificity, rapidity and availability, especially with regard to clinical research in cancer pathogenesis, early diagnosis, and targeted therapy, prognosis and monitoring. Although currently computed tomography (CT), magnetic resonance imaging (MRI), immunofluorescence assay and pathological immunohistochemistry (IHC) methods have been used as powerful tools for clinical detection of cancer, they are either too expensive to perform them on all suspect patients or suffers the disadvantage of 9



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high individual variation, low positive predictive value, and time-consuming procedures. Furthermore, they are still not sensitive enough for early detection of cancers, especially on single cell level. Although Currently in clinics, 18F-fluorodeoxyglucose (18F-FDG), by exploiting a similar tactic (preferential uptake of 18F-FDG by tumors), has been widely used as the representative of radiopharmaceutical emitting positrons in PET-CT imaging for clinical detection of cancer,35 it’s radioactive and the imaging methodology lacks high sensitivity for early detection of cancers on cell level. Due to simplicity, greater sensitivity, and rapidity and availability, our smart plasmonic glucose nanosensor and the dark-field microscopy imaging technique would be promising for early detection of cancers, especially for high-throughput in-vitro cancer preliminary visual screening of cell samples for suspect patients. However, it worth to mention that, since it was reported that infectious (tuberculosis, sarcoidosis, pneumonia) and inflammatory processes, and other non-malignant processes (such as brown fat deposits, thyroid gland uptake, hyperplastic bone marrow, and gastrointestinal digestion) are commonly take up large amounts of glucose and may potentially lead to false positive results,3,36 further studies are needed before safe clinical use of the method. Furthermore, not all cancers exhibit high glucose uptake, such as mucinous carcinomas, prostate cancers, neuroendocrine tumors, and bronchioloalveolar cell lung carcinomas,36,37 the generality of the nanoprobe/approach (to what extent) also needs further assessment. And further work by taking advantage of plasmonic properties of the nanoprobe, for instance, by combined use of SERS detection, will make the proposed approach more powerful and reliable for rapid and sensitive, in-vitro screening and qualitative identification of cancer (on cell level). Selective NIR photothermal ablation of cancer cells. We also successfully applied such smart nonoprobes for selective photothermal ablation of cancer cells. Plasmonic nanoparticles and hybrids, such as silica core-Au NSs, Au nanorods and nanocages, had been successfully applied as efficient NIR photothermal absorbers for destroying cancer cells.38-41 In these studies, however, to achieve selective photothermal ablation of cancer cells (without affecting the surrounding nonmalignant cells), time-consuming specific nanoprobe LSPR band of the probe nanoparticles with cancer cells turns distinctly from visible to NIR region while that of thenanoparticles with normal cells remained in visible region, selective NIR photothermal ablation of cancer cells may achieved by using the generic probe nanoparticles without needing specific targeting moieties because the probe nanoparticles with cancer cells will generate more heat (at a fixed 10


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laser fluence and illuminating time) to destroy cells. For this study, a NIR laser at 808 nm was used, and the cell monolayer incubated with the probe nanoparticles solution for 20 min was submitted for the test. We tested first the NIR laser illumination induced temperature elevation profile of the probe nanoparticles in solution. Figure 4a shows the temperature elevation profiles of the precursor Ag/Au NSs, probe Ag/Au-GOx NSs before and after reaction with excess glucose, respectively, with the same particle dosage (~ 0.74 × 1011 particles), at a CW laser power of 1.37 W cm-2 for 10 min. The probe nanoparticles after reaction with glucose (curve c) showed significantly better photothermal efficiency than the other two, with solution temperature increasing to ~ 60 ℃ after 10 min laser exposure, almost 10 ℃ higher than that of the probe nanoparticles before the glucose reaction. The temperature continued to increase after 10 min laser exposure, while in probe Ag/Au-GOx NSs (before reaction with glucose) the temperature increased slowly and the GOxfree Ag/Au NSs nearly stop to increase in the last 2 min. The difference in temperature increment is therefore the basis for selective NIR photothermal ablation of cancer cells. After incubation with the probe Ag/Au-GOx NSs for 20 min, cells are exposed to CW NIR laser irradiation at various intensities of 70, 100, 150, 170, and 200 mW for 5 min each. The cells are then stained with trypan blue to test for their photothermal viability. As shown in Figure S3, from ~ 70 mW to 200 mW, the cancer cells appeared obviously ablated, especially at 170 mW, while the normal L929 cells were not affected seriously which started to be injured at 170 mW. Exposure to the NIR laser at and above 200 mW (1 W cm-2) caused photodestruction of both cells. So we choose the threshold laser intensity of 170 mW (0.85 W cm-2) and duration time of 5 min for photothermal cell ablation experiments. As shown in Figure 4b, all the tested cancer cells (HeLa, HepG2, and A549 cells) appeared obviously ablated, while the normal L929 cells were not affected seriously. To further assess the photothermal ablation effects on cancer cells, the standard methyl thiazolyl tetrazolium (MTT) assay was carried out to determine relative viabilities of cells after 24 h treatments with Ag/Au-GOx NSs and control Ag/Au NSs. In this assay, the absorbance of formazan (produced by the cleavage of MTT by dehydrogenases in living cells) is directly proportional to the number of live cells. As seen from Figure 4c, both Ag/Au NSs and Ag/Au-GOx NSs showed negligible cytotoxicity towards different cells, rendering them applicable for cell study. After 24 h treatments with Ag/Au NSs and Ag/Au-GOx NSs, the cells 11



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were exposed to NIR laser with the intensity 0.85 W cm-2 for 10 min. MTT assay was carried out after 24 h. The cell viabilities of the three cancer cells after 10 min photothermal ablation are ~ 35% for HepG2, 5% for A549 and 1% for HeLa cells, respectively, while that of the normal L929 cells is nearly 85% (Figure 4c). The cell viability for the three different cancer cells showed also a big difference, which may originate from their different NS uptake dosage. This has been further confirmed experimentally. As shown in Figure S5, with the incubation time increasing the probe NSs not only attached on the surface of the cell membrane, but also enter gradually into the cytoplasm; and the uptake rate of probe NSs varied with cell types, with more probe NSs entered into cytoplasm of HeLa cells (than A549 cells, Figure S5c cf. Figure S5d) after incubating with Ag/Au-GOx NSs probes, respectively, for 20 mins at the same condition. The control cells without treatment of the nanoprobes were irradiated at the same NIR laser power under the same illumination conditions and all survived. In order to exclude the possible influence of the moderate level of enzymatically produced H2O2 on cell death, a control experiment by incubating the A549 cells with high concentration of H2O2 (ranging from ~ 1 mM to 16.7 mM) was also performed. As shown in Figure S6, the result confirmed that the cell viability was not obviously influenced by even higher concentrations of H2O2 in our experiments. These results suggest that Ag/Au-GOx NSs can be used as an efficient and generic agent for selective NIR photothermal ablation of cancer cells, without needing time-consuming probe design for specific cancer cell targeting. The threshold energy to kill the cancer cells in our work is found to be 0.85 W cm-2, which is lower than that needed in the case of the Ag/Au hollow NSs and Au nanorods.42,43 This makes our Ag/Au-GOx NSs very promising for potential practical theranostic applications.

CONCLUSIONS In conclusion, we applied the glucose-responsive/sensitive Ag/Au-GOx NSs for the first time as a smart nanoplasmonic probe for fast, and targeting-free visual discrimination and screening of cancer cells from normal cells by using dark-field scattering imaging. Selective ablation of cancer cells, without needing time-consuming probe design for specific cancer cell targeting, also achieved upon exposure cells to a laser at 808 nm, since cancer cells incubated with the particles require less the laser energy to destroy photothermally due to more spectral overlap of the 12


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probe LSPR band with laser wavelength. Due to the simplicity, effectiveness, and universality, the developed method may be useful in the early detection of tumor and therapeutic applications. Other possibilities for using the smart Ag/Au-GOx NSs for biomedical applications may include tracking of glucose metabolism inside the cells, glucose- or H2O2-responsive gene/drug delivery, and so on. It can have an important impact on biomedicine.

ACKNOWLEGMENT This work has been financially supported by the National Natural Science Foundation of China (Grant No. 21475125, 21175125) and the Hundred Talents Program of the Chinese Academy of Sciences.

SUPPORTING INFORMATION AVAILABLE Optimized synthesis of Ag/Au NSs, time optimization for cell imaging and optimization of NIR laser power for photothermal ablation of cancer cells and the dark-field images of cocultured HepG2 and HL7702 cells for different times and the dark-field images of HeLa cells after incubation with Ag/Au-GOx NSs for different times and the light-field images of A549 cells after incubation with different amount of H2O2. This material is available free of charge via the internet at http://pubs.acs.org.

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Scheme 1. Schematic of smart glucose nanoprobe preparation, LSPR response and in-vitro cancer cell theranostic applications. (a) Schematic of enzymatic Ag/Au-Gox nanoprobes with complete Ag/Au NS (yellow-green) before and after the enzymatic H2O2 etching to partially dissolve Ag and form porous NS (red). (b) During the reaction process, the LSPR band of the resultant plasmonic nanoprobes red-shifts gradually from visible to NIR region. The magnitude of the LSPR band shift depends on the reaction time and concentration of glucose in the system. Due to a distinct response difference in LSPR band shift of cancer cells (red) and normal cells (yellow-green) that arisen from their different glucose uptake level, the plasmonic properties of the nanoprobes can be exploited for (c) in-vitro dark-field microscopy-based visual identification and NIR photothermal therapy of cancer cells, respectively.

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Figure 1. Optical properties and dark-field scattering imaging of the plasmonic nanoparticles. a) LSPR responses of the typical precursor (enzyme-free) Ag/Au NSs sample before (Ⅰ0 )and after (Ⅰ1 andⅠ2) reaction with different amount of hydrogen peroxide ( 0.3 M). Ⅰ0: 0 µL; Ⅰ1: 15 µL; Ⅰ2: 30 µL. b) LSPR responses of the probe Ag/Au-GOx NSs sample after GOx immobilization and reaction with 15 µL of glucose solution （0.1 M） for different times: 2-5 min. c-d) Darkfield LSPR scattering images and corresponding TEM images of the probe Ag/Au-GOx NSs sample before (c) and after (d) reaction with an excess amount of glucose.

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Figure 2. Visual identification and screening of cancer cells from normal cells. Bright field microscopic images (the 1st row), and dark field scattering images (the 2nd and 3rd rows) of two non-tumor cell lines (L929, HL7702) and three tumor cell lines (HeLa, HepG2, and A549) after incubation with control Ag/Au NSs and probe Ag/Au-GOx NSs under same conditions for ~ 1520 min. At the test conditions the tumor cell lines with the probe nanoparticles turn orange/red, distinctly different from that of normal cells (yellow-green), as the enzymatic reaction of the probes proceed more rapidly and completely with higher intracellular glucose level of the tumor cells.

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Figure 3. Visual identification of cancer cells from co-cultured normal cells. (A-C) Bright-field images of the independent culture of HepG2, HL7702 cells with different shape, and their coculture, respectively. (D-F) Dark-field images of HepG2, HL7702, and co-cultured HepG2 + HL7702 cells, respectively, after incubation with Ag/Au-GOx NSs for 20 min.

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Figure 4. Selective NIR photothermal ablation of cancer cells. a) The temperature elevation profiles of the precursor Ag/Au NSs suspension (curve a), the probe Ag/Au-GOx NSs suspension before (curve b) and after (curve c) etching by H2O2, under irradiation with a CW laser power of 1.37 W cm−2 for 10 min. All nano-materials have the same particle dosage (～ 0.37 × 1011 particles). b) Optical microscope images of control L929 cells and cancer cells (HeLa, HepG2, and A549 cells) incubated with or without Ag/Au-GOx NSs after irradiation with a CW laser (with wavelength of 808 nm) at 170 mW (0.85 W cm-2) for 5 min and subsequent staining with trypan blue. c) MTT cell viability assay of L929, HeLa, HepG2 and A549 cells after incubation with Ag/Au NSs and Ag/Au-GOx NSs and laser exposure.

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Smart Plasmonic Glucose Nanosensors as Generic Theranostic

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