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Supporting Information - Nature
关注微信公众号SupportingInformation；Proximity-TriggeredSERSE；YunlongChen,LinDing,Wany；StateKeyLaboratoryofAnal；Engineering,NanjingUnive；DepartmentofPharmaceutic；London,LondonWC1N1AX,UK.；Experime
Supporting Information Proximity-Triggered SERS Effect for Raman Imaging of Protein-Specific Glycans on Cell Surface Yunlong Chen, Lin Ding, Wanyao Song, Min Yang, and Huangxian Ju* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P.R. China. Department of Pharmaceutical & Biological Chemistry, UCL School of Pharmacy, University College London, London WC1N 1AX, UK. Experimental Section Materials and Reagents. Chloroauric acid (HAuCl4?4H2O) was obtained from Shanghai Chemical Reagent Company (Shanghai, China). Trisodium citrate was obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Poly-L-lysine, sialic acid (Sia), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), poly(diallyldimethylammonium chloride) (PDDA), sodium butyrate (NaBu) and dibenzocyclooctyne-amine (DIBO) were purchased from Sigma-Aldrich Inc. (USA). Thiol PEG 1000 carboxyl (PEG) was obtained from Jenkem Technology Ltd. (Beijing, China). Tetraacetylated N-azidoacetyl-D-mannosamine (ManNAz), tetraacetylated N-azidoacetylgalactosamine (GalNAz), tetraacetylated N-azidoacetylglucosamine (GlcNAz), Alexa Fluor 647 DIBO alkyne and FITC conjugated EpCAM mouse anti-human mAb (clone VU-1D9) were obtained from Life Technologies (USA). Fluorescein labeled Sambucus nigra agglutinin (SNA) was purchased from Vector Laboratories (USA). MCF-7 and Ramos cells were from KeyGen Biotech. Co. Ltd. (Nanjing, China). Tris-EDTA buffer containing 10 mM TrisCHCl and 1 mM EDTA (TE, pH 8.0) was used as DNA stock solution. Phosphate buffer saline (PBS, pH 7.4) contained 136.7 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4, and 1.41 mM KH2PO4. To reduce the nonspecific binding of aptamer to cells, yeast tRNA (0.1 mg/mL, Sigma) was added to 5 mM MgCl2 contained PBS, which was used as the washing buffer. For temporary inhibition of sialidase, 500 μM Sia was added in the binding buffer (Sia-containing inhibition buffer). All other reagents were of analytical grade. All aqueous solutions were prepared using ultrapure water (? 18 MΩ, Milli-Q, Millipore). The epithelial cell adhesion molecule (EpCAM) aptamer (AP)S1 was purchased from Sangon Biological Engineering Technology & Co. Ltd. (Shanghai, China) with the sequence: 3'-HS-AAA AAA AAA AAA AAA GTC CGG TTG GGG GGT ACT GTT GCA CCC TGT CTG CGT TGG AGA CAT CAC. The 15 adenines at 3' terminal were the spacer sequence. A FITC-labeled EpCAM aptamer was used for fluorescence analysis. A random DNA sequence (RS) with sulfhydryl or FITC labeled at 3' terminal and the same length of the aptamer was used as the blank control. Apparatus. Mass spectra were obtained on a 4800 Proteomics Analyzer (Applied Biosystems, USA). The transmission electron microscopic (TEM) images were obtained on a JEM-2100 transmission electron microscope (JEOL Ltd., Japan). The UV-vis absorption spectra were recorded on a Nanodrop-2000C UV-vis spectrophotometer (Thermo, USA). Zeta potential analysis was performed on a Zetasizer (Nano-Z, Malvern, UK). Dynamic light scattering (DLS) was observed on a 90 Plus/BI-MAS equipment (Brook haven, USA). Flow cytometric analysis was performed on a Coulter FC-500 flow cytometer (Beckman-Coulter). The fluorescence cell imaging was performed on a TCS SP5 laser scanning confocal microscope (Leica, Germany). Raman spectra and images were gained on a Renishaw inVia confocal Raman microscope (Renishaw, UK) using 633 nm excitation. A 50-times telephoto objective was used for observation, spectral measurement and imaging. Preparation of Au Probes. Two sizes of gold nanoparticles (AuNPs) were prepared with different ratios of HAuCl4 to trisodium citrate.S2 The 10-nm AuNPs (Au10) were synthesized by adding 1.25 mL trisodium citrate (1%) to 50 mL boiling HAuCl4 solution (0.01%), while 40-nm AuNPs (Au40) were synthesized by adding 0.50 mL trisodium citrate (1%) to 50 mL boiling HAuCl4 solution (0.01%), which was used to prepare PDDA-coated Au40 (PDDA-Au40) by adding 250 μL PDDA (20%) and 250 μL NaOH (0.5 M) in the mixture. All the mixtures were stirred at 100 oC until the color became red or purple, and then cooled to room temperature. Prior to use, AuNPs were washed by centrifugation under 12000 rpm for Au10 or 6000 rpm for Au40 and resuspended in water. The concentrations of AuNPs were determined using UV-vis absorption spectrometry.S3 10 μL DTNB (10 mM in ethanol) and 10 μL PEG (1 mM) were added to 1 mL Au10 solution (10 nM) and stirred at room temperature overnight. After washed by centrifugation under 12000 rpm twice and resuspended to 1 mL in PBS buffer, the solution was mixed with 8 μL newly prepared EDC (50 mg/mL) and 10 μL DIBO (50 mM in DMSO), and allowed to react for 4 h. The obtained Au10-DTNB/PEG-DIBO (Au10 probe) was washed by centrifugation under 12000 rpm twice and resuspended to 1 mL in PBS buffer. 100 μL AP (10 μM in TE buffer) and 1 μL PEG (1 mM) were added to 1 mL Au40 solution (1 nM) and stirred at room temperature overnight. Afterward, 100 μL NaCl (1 M) was added to the mixture stepwise within 12 h for stabilizing the obtained Au40-AP/PEG (Au40 probe), which was centrifuged (6000 rpm), washed with PBS twice, and finally resuspended in 1 mL binding buffer. For specificity demonstration, RS was used to replace AP for obtaining Au40-RS/PEG with the same procedure. To regulate the Sia level on cell surface EpCAM, sialidase was linked to the terminal carboxyl group of PEG through EDC-mediated carbodiimide chemistry by adding 10 μL sialidase (5 U/mL) and 8 μL EDC (50 mg/mL) to 1 mL Au40-AP/PEG solution. After reaction for 4 h, the obtained Au40-AP/PEG-sialidase was washed by centrifugation (6000 rpm) twice and resuspended to 1 mL Sia-containing inhibition buffer. Cell Culture. MCF-7 and Ramos cells were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (100 μg/mL), and streptomycin (100 μg/mL) at 37 oC in a humidified atmosphere containing 5% CO2, respectively. The cell numbers were determined using a Petroff-Hausser cell counter (USA).
Metabolic Labeling of Cell Surface Glycans. 50 mM metabolic labeling reagents, ManNAz (for labeling cell surface sialic acid), GalNAz (for labeling cell surface O-linked glycans) and GlcNAz (for labeling intracellular O-GlcNAc), were prepared in DMSO as the stock solutions, respectively. 200 μL MCF-7 cells were seeded in a confocal dish at the density of 1×105 cells/mL and incubated in medium containing one of the metabolic labeling reagents at 50 μM. The cells were then washed using PBS for 3 times before measurement. Ramos cells were incubated in medium containing the metabolic labeling reagent at 50 μM directly in the cell culture flasks and washed by centrifugation with PBS for 3 times before measurement. Flow Cytometric Analysis of Cell Surface EpCAM and Glycans. To confirm the existence of EpCAM on MCF-7 cell surface, an EpCAM negative cell line Ramos was used as control. MCF-7 and Ramos cells with concentration of 1×106 cells/mL were incubated with 20% FITC-conjugated EpCAM mouse anti-human mAb in PBS or 1 μΜ FITC-labeled aptamer in binding buffer respectively. After 30-min incubation at 37 oC, the cells were washed with PBS or washing buffer twice before flow cytometric test. For investigation of the dynamic change of cell surface EpCAM during NaBu treatment, MCF-7 cells were incubated with 1 mM NaBu for 1 to 7 days and subsequently analyzed with the same procedure. The Sia change on whole cell surface was analyzed by incubating NaBu-treated MCF-7 cells with 0.1 μM fluorescein labeled SNA in PBS containing 0.1 mM CaCl2 and 0.1 mM MnCl2 for 1 hour at 37 oC. The SNA could specifically recognize Sia group on cell surface. The cells were washed with PBS twice before flow cytometric detection. Fluorescence Imaging of Glycans and EpCAM on Cell Surface. 5 mM DMSO solution of Alexa Fluor 647 DIBO alkyne was prepared as a stock solution. To confirm the successful metabolic labeling, 25 μM Alexa Fluor 647 DIBO alkyne in PBS was added to the metabolized cells and incubated for 30 min at 37 oC. The cells were then washed using PBS for 3 times. 1 μΜ FITC-labeled aptamer or random DNA in binding buffer was added to the confocal dishes and incubated for 30 min at 37 oC. The cells were then washed with washing buffer for 3 times before confocal fluorescence imaging. Raman Imaging of Protein-Specific Glycans. The metabolically labeled MCF-7 cells seeded in confocal dishes were first incubated with 200 μL Au10 probe (10 nM) in PBS at 37 oC. After click reaction between azide and DIBO for 30 min, the cells were washed twice with PBS to remove excess Au10 probe. 200 μL Au40 probe (1 nM) or Au40-RS/PEG (1 nM) in binding buffer was then added to the cells. After 30-min incubation at 37 oC, the cells were twice washed with washing buffer and then fixed with 4% paraformaldehyde solution for 15 min before Raman imaging. As control, the metabolically labeled Ramos cells in the cell culture flasks were treated with the same probe and incubation procedures. After washed by centrifugation, the treated cells were fixed in poly-L-lysine coated confocal dishes with 4% paraformaldehyde solution for 2 h. The Raman images of cells were obtained by the map image acquisition mode using static scan type at a center wavenumber of 1300 cm-1 with exposure time of 1 s, 2 times accumulation and 100% laser power. The imaging step was 1 μm ×1 μm. The strongest characteristic peak of DTNB was at 1330 cm-1, so the Raman images of cells were generated using signal to baseline map review mode from 1300 cm-1 to 1360 cm-1 by a WiRE 3.4 software, and the color scale of images were chosen as black to red corresponding to the background noise intensity and the maximum signal intensity respectively. The average Raman intensity of cells was obtained from statistics mean value by dividing the total red channel value with the total cell membrane length within the chosen area of the Raman image using Photoshop CS6 software. The Raman spectra of Au probes were taken by the spectral acquisition mode using static scan type at a center wavenumber of 1300 cm-1 with the same parameters as the Raman imaging. Size Characterization of AuNPs
Figure S1. TEM images of a) Au10 and b) Au40. Verification of PTSERS Effect
Figure S2. a) Zeta potential of PDDA-Au40. Inset: TEM image of PDDA-Au40. b) Raman spectra of (A) 1 nM PDDA-Au40 and (B) 10 nM Au10 probe in PBS, (C) mixture of 10 nM Au10 probe and 1 nM Au40 probe in PBS, (D) 1 nM PDDA-Au40 after incubation with 100 μM DTNB, (E) 10 nM Au10 added in (D), (F) 1 nM PDDA-Au40 after incubation with 10 nM Au10 probe. 三亿文库包含各类专业文献、幼儿教育、小学教育、专业论文、应用写作文书、各类资格考试、高等教育、外语学习资料、Supporting Information23等内容。　
　然而, 不允许在个别作者或部分著作权逐条记载稿件的 任何部分或 Supporting Information。 相关内容:在稿件的结尾部分必须包括一个简短的非句子格式并可以对放置在 ... 　当时我由于经验不足,把一个不属于这个范畴的 东西当成了 Supporting Information 传了过去,当然,审稿的时候没有 造成什么影响,但是编辑在回复里面却让我按照模版把 ... 　Supporting Information_药学_医药卫生_专业资料。1 Supporting Information Curcumin Analog WZ35 Induced Cell Death via ROS-dependent ER Stress and G ... 　7-Supporting Information_英语考试_外语学习_教育专区 暂无评价|0人阅读|0次下载|举报文档7-Supporting Information_英语考试_外语学习_教育专区。Supporting ... 　Supporting Information_英语学习_外语学习_教育专区。Supporting Information Supporting Information A mass spectrometry based method for differentiation of positional ... 　Supporting Information 3页 10财富值 Supporting Information 4页 免费 supporting information(1... 6页 1财富值 文献Supporting Informati... 26页 5财富值 ... 　?无论是“In press” 、“accepted”或“submitted”等的相关发表稿件副本需在发表或者其 他准备阶段中上传提交稿件 Supporting Information 以供审查。只有被 ... 　Take the first paragraph as an example, and show Ss what the supporting information is. Ask related sentence. Ss to to guess each the topic nd Think...Supporting Information Assistant 18Feb2014 Essay - 986 Words
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Supporting Information
Experience:
Re: 1.1 I currently work as a Floating Support Worker for Creative Support (previously as a Supported Housing Officer for Slough Borogh Council). My present role involves contacts with many local care and support agencies, also, with the voluntary sector. For example I regularly liaise with local GP surgeries, hospitals, Age Concern, Apetito, social services, occupation therapists and mental health professionals.
Re: 1.2 I have significant experience in providing one-to-one floating support to elderly and vulnerable clients living in the Council Housing properties and private properties in the Slough area. I have been working in this environment for 7 years and I am responsible at the moment for about 40 clients. My role involves assessing clients’ individual needs and providing housing related support in a non-judgemental and caring way on a daily basis. Every day I am exposed to personal problems of vulnerable people and their specific needs and wishes. After years of working in this challenging environment I gained substantial awareness of special needs of these people in our community.
Re: 1.3 I have to use my IT skills every day when entering the details of the visits to clients into an electronic database. This involves a significant amount of typing using a keyboard. I am also proficient in Microsoft Office. Moreover, I communicate remotely via Blackberry device with colleagues, appropriate agencies and I respond to urgent e-mails this way.
Knowledge:
Re: 2.1 I am fully trained and committed to equality and diversity policies. I apply them at my daily work.
Re: 2.2 I took part in many sessions regarding Berkshire-wide Safeguarding procedures on Safeguarding for Children and Adults. Therefore, I am very familiar with these matters and, again, I apply them daily at my current position. Also, I regularly attend Safeguarding Meetings with social services from Slough Council Borough were we discuss...
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