方法文章

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

DOI：

10.3791/55030

⸱

2017年1月10日

 ,  ,  ,  ,  , 
1Department of Chemical and Biomolecular Engineering, University of California Berkeley, 2California Institute for Quantitative Biosciences (QB3), University of California Berkeley

本文内容

摘要

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我们提出了一种使用两亲性聚合物和 DNA 设计近红外荧光单壁碳纳米管 （SWNT） 电晕相的方案，以开发用于没有已知识别元件的分子靶标的传感器。

摘要

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半导体单壁碳纳米管 （SWNT） 是一类光学活性纳米材料，在近红外范围内发出荧光，与生物样品最透明的光学窗口相吻合。在这里，我们概述了将两亲性聚合物和多核酸吸附到 SWNT 表面的技术，以设计它们的电晕相并为小分子和蛋白质创建新型分子传感器。这些功能化的 SWNT 传感器既具有生物相容性又具有稳定性。聚合物通过 SWNT 和聚合物的直接超声处理或使用表面活性剂悬浮 SWNT，然后用聚合物透析，吸附到纳米管表面。这些传感器的荧光发射、稳定性和对目标分析物的响应使用吸光度和近红外荧光光谱法进行确认。此外，我们展示了传感器的表面固定在载玻片上，以使单分子荧光显微镜能够表征聚合物吸附和分析物结合动力学。

引言

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Single-walled carbon nanotubes (SWNTs) are atomically thin layers of carbon atoms rolled into long, thin cylinders that exhibit unique electronic and optical properties.1 Such properties include a band-gap producing near infrared (nIR) fluorescence emission via exciton recombination that is highly sensitive to its local environment. The nIR emission of SWNTs falls within the near infrared window in which the penetration depth of light is maximal for biological tissue.2,3 Additionally, SWNTs exhibit several unique features atypical in contrast to organic fluorophores: SWNT exhibit a large Stokes shift, do not photobleach, and do not blink.4

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方案

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Caution: Please consult all relevant material safety data sheets (SDS) before use. Nanomaterials may have additional hazards compared to their bulk material counterpart. Use all appropriate safety practices including engineering controls (fume hood, noise enclosure) and personal protective equipment (safety glasses, goggles, lab coat, full length pants, closed-toe shoes).

1. Preparation of Buffer, Surfactant, and Polymer Solutions

  1. Preparation of 100 mM NaCl solution
    1. Dissolve 584 mg of NaCl in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.

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结果

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SWNTs were suspended in aqueous solution using both surfactants and amphiphilic polymers by direct sonication and by dialysis exchange. Figure 1 shows SWNTs, grown using the iron carbonyl catalyzed method (HiPCO), suspended using SC, RITC-PEF20-RITC, and (GT)15-DNA. The optical density of a SWNTs with SDS (or polymer) increases dramatically after sonication and decreases upon removal of aggregates and contaminants through purification by centrifugation (

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讨论

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SWNTs are readily suspended in aqueous solution via direct sonication with SDS or ssDNA, as indicated by an increase in optical density provided by the colloidal dispersion of the resulting SWNT-polymer hybrid. SDS and ssDNA disperses and solubilizes bundles of SWNTs by adsorbing onto the SWNT surface through hydrophobic or pi-pi interactions. Additionally, other polymers, such as genomic DNA, amphiphilic polymers, conjugated polymers and lipids, can be adsorbed onto the surface of SWNTs by dialysis of samples suspended .......

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披露

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作者没有什么可透露的。

致谢

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这项工作得到了科学界面 （CASI） 的 Burroughs Wellcome Fund Career Award 的支持、Simons 基金会的资助以及大脑与行为研究基金会的青年研究员资助。

....

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材料

本文使用的材料清单
姓名公司目录编号评论
氯化钠Fisher ScientificS271-1
十二烷基硫酸钠Sigma AldrichL6026
胆酸钠水合Sigma AldrichC6445
tris 碱（Trizma 碱）Sigma Aldrich93362
盐酸Fisher ScientificA144-212
胺-PEG-胺，NH2-PEG-NH2Nanocs IncPG2-AM-5k
罗丹明 B 异硫氰酸酯Sigma Aldrich283924
荧光素异硫氰酸酯Sigma AldrichF7250
二氯甲烷Sigma Aldrich676853
二甲基甲酰胺Sigma AldrichD4551
N，N-二异丙基乙胺Sigma AldrichD125806
乙醚Sigma Aldrich673811
三（2-羧乙基）膦盐酸盐Sigma AldrichC4706 
5'-硫醇修饰的 DNA Integrated DNA Technologies
甲氧基聚乙二醇马来酰亚Sigma Aldrich63187
100 kDa 自旋过滤器Millipore
HiPCO 超纯化单壁碳纳米管IntegrisHiPco 超纯化
磷酸盐缓冲盐水Sigma AldrichP5493
防静电枪MiltyMilty Zerostat 3
离心机Eppendorf5415 D
超声仪Cole ParmerCV18
透析盒Thermo ScientificSlide-A-Lyzer G2 87722
BSA-生物素Thermo Scientific29130
中性亲和素蛋白Thermo Scientific31000
（3-氨基丙基）三乙氧基硅烷 （APTES）Sigma Aldrich440140
倒置显微镜蔡司Axio Observer.Z1
运动镜ThorLabsKM200-E03
潜望镜ThorLabsRS99
浸油蔡司Immersol 518f
100X 物镜蔡司平场复消色差物镜 100X 油， 1.4NA， PH3， 420791-9911-000
20X 物镜蔡司N-Achroplan 0.45 NA， 420953-9901-000 
盖板玻璃Healthrow ScientificHS159879H
盐酸多巴胺Sigma AldrichH8502 
红外 2D 阵列相机普林斯顿仪器NIRvana
红外 1D 传感器阵列普林斯顿仪器PyLoN 红外
nIR 光谱仪普林斯顿仪器SCT-320
平凸透镜ThorLabsLA1384
孔板（玻璃底）康宁4580
物 胺

参考文献

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  1. Dresselhaus, M. S., Dresselhaus, G., Avouris, P. Carbon Nanotubes. 80, Springer. Berlin Heidelberg: Berlin, Heidelberg. (2001).
  2. O'Connell, M. J., Bachilo, S. M., et al. Band gap fluorescence from individual single-walled carbon nanotubes. Science. 297 (5581), 593-596 (2002).
  3. Wang, F., Dukovic, G.....

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