工程分子识别与单壁碳纳米管仿生聚合物
Summary
We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.
Abstract
Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.
Introduction
单壁碳纳米管（SWNT）的原子的碳原子薄层轧成表现出独特的电子和光学性质长而细的圆柱体。 1这些性质包括一个带隙产生近红外（NIR）荧光经由激子的重组也就是它的本地环境高度敏感发射。 SWNT的NIR发射落在近红外窗口，在其中的光的穿透深度为最大的生物组织内。 2,3此外，单壁碳纳米管表现出相对于有机荧光非典型几个独特的功能：SWNT呈现大的斯托克斯位移，不要光漂白，不闪烁。 4最近，利用这些特性已导致新颖分子传感器的与应用到生物学各种各样的发展。 5,6-未修改然而，单壁碳纳米管是不溶于水，并且获得各个单壁碳纳米管的悬浮液可能是一个挑战。 7,8 BundliNg和在溶液中的单壁碳纳米管的聚集可以混淆他们的带隙的荧光，使2它们不适合用于传感应用。
在水溶液中分散个别碳纳米管要求修改其表面以防止疏水驱动聚集。 9虽然共价修饰可以呈现的单壁碳纳米管的水溶性，10以及赋予特异性结合化学，在单壁碳纳米管的晶格缺陷部位减少或减轻它们的荧光发射。通过疏水和π-π堆积相互作用吸附到纳米管表面13 –相反，单壁碳纳米管的官能化可以通过使用表面活性剂，脂质，聚合物和DNA 9,11来完成。周围表面官能化的单壁碳纳米管的产生的化学环境被称为其电晕相。扰动电晕相位可对激子的纳米管表面上行驶的很大的影响，引起调制到SWNT氟escence排放。它是电晕相和可利用通过将特异性结合模式到单壁碳纳米管的大的表面积，以开发新的分子传感器的SWNT荧光之间的这种敏感的关系。扰动在结合分析物的单壁碳纳米管的电晕相位可以导致在局部电介质环境的变化，电荷转移，或引入晶格缺陷，所有这些都可以调节SWNT的荧光发射以作为信号转导机制。 14这种方法是在新型荧光传感器的发展用于检测许多不同类型的分子中的包括DNA，15,16葡萄糖17和小分子如ATP，18活性氧19和一氧化氮。 20,21然而，这些方法被限制的，因为它们依赖于对靶分析物的已知结合形态的存在。
近日，一个更通用的应用程序蟑螂对设计荧光传感器用单壁碳纳米管与两亲杂，磷脂和多核酸非共价地官能化的发展。这些分子吸附到碳纳米管表面，以产生单个的单壁碳纳米管22的高度稳定的悬浮液– 25具有独特的电晕阶段可以特异性结合蛋白26,27或小分子包括神经递质多巴胺。 28 – 30工程电晕相位分散的单壁碳纳米管和特异性结合目标分析物被称为电晕相分子识别（CoPhMoRe）。 28小尺寸，低毒性，高稳定性和CoPhMoRe单壁碳纳米管传感器unbleaching NIR荧光使他们为延长时间分辨测量体内检测的最佳候选。 6最近的工作表明它们在植物组织应用活性氮和氧的光学检测。 31为CoPhMoRe SWNT传感器特别激动人心的应用是用于神经递质如在体内 ，多巴胺标签免费检测的电位在其他技术，如电化学感测或免疫组织化学，从缺乏空间分辨率，时间分辨率和特异性的痛苦。
设计和发现CoPhMoRe SWNT传感器迄今被分散库的大小和化学多样性限制，限制找到一个传感器为特定的目标的可能性。到目前为止，研究人员已经只是触及可结合，共同块，生物，可作为功能活跃分散剂SWNT仿生传感器的聚合物的表面。在这里，我们提出了两个单壁碳纳米管的分散和表征他们的高通量筛选和单一的单壁碳纳米管传感器分析荧光不同的方法。具体来说，我们概述了与多核苷酸oligom涂层单壁碳纳米管的方法器使用直接超声处理以及如何通过透析通过表面活性剂交换官能与两亲聚合物的SWNT。我们使用若丹明异硫氰酸酯（RITC-PEG-RITC）官能化（GT）15 -DNA和聚乙二醇作为例子。我们证明了使用的（GT）15-DNA单壁碳纳米管作为用于检测多巴胺CoPhMoRe传感器。最后，我们概述用于执行单分子传感器测量，其可用于表征或单分子检测程序。
Protocol
Representative Results
Discussion
单壁碳纳米管可以容易地悬浮在通过直接超声水溶液用SDS或ssDNA的，由所得到的单壁碳纳米管的聚合物混合的胶态分散体提供的增加光密度所指示的。 SDS和单链DNA分散并通过疏水性或π-π相互作用吸附在SWNT表面溶解的单壁碳纳米管束。此外，其他的聚合物，例如基因组DNA，两亲聚合物，共轭聚合物和脂质，可以通过使用SC或SDS悬浮样品的透析吸附到单壁碳纳米管的表面上。亲水性聚合物，如PEG，?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Simons Foundation grant, and a Brain and Behavior Research foundation young investigator grant.
Materials
| sodium chloride | Fisher Scientific | S271-1 | |
| sodium dodecyl sulfate | Sigma Aldrich | L6026 | |
| sodium cholate hydrate | Sigma Aldrich | C6445 | |
| tris base (Trizma base) | Sigma Aldrich | 93362 | |
| hydrochloric acid | Fisher Scientific | A144-212 | |
| Amine-PEG-amine,NH2-PEG-NH2 | Nanocs Inc | PG2-AM-5k | |
| rhodamine B isothiocyanate | Sigma Aldrich | 283924 | |
| fluorescein isothiocyanate | Sigma Aldrich | F7250 | |
| dichloromethane | Sigma Aldrich | 676853 | |
| dimethylformamide | Sigma Aldrich | D4551 | |
| N,N-diisopropylethylamine | Sigma Aldrich | D125806 | |
| diethyl ether | Sigma Aldrich | 673811 | |
| Tris(2-carboxyethyl)phosphine hydrochloride | Sigma Aldrich | C4706 | |
| 5’-thiol-modified DNA | Integrated DNA Technologies | ||
| methoxypolyethylene glycol maleimide | Sigma Aldrich | 63187 | |
| 100k Da spin filters | Millipore | ||
| HiPCO Super purified single walled carbon nanotubes | Integris | HiPco SuperPurified | |
| phosphate buffered saline | Sigma Aldrich | P5493 | |
| anti static gun | Milty | Milty Zerostat 3 | |
| centrifuge | Eppendorf | 5415 D | |
| ultra sonicator | Cole Parmer | CV18 | |
| dialysis cassettes | Thermo scientific | Slide-A-Lyzer G2 87722 | |
| BSA-biotin | Thermo scientific | 29130 | |
| Neutravidin protein | Thermo scientific | 31000 | |
| (3-Aminopropyl)triethoxysilane (APTES) | Sigma Aldrich | 440140 | |
| inverted microscope | Zeiss | Axio Observer.Z1 | |
| kinematic mirrors | ThorLabs | KM200-E03 | |
| periscope | ThorLabs | RS99 | |
| immersion oil | Zeiss | Immersol 518f | |
| 100X objective | Zeiss | Plan-apochromat 100X oil, 1.4NA, PH3, 420791-9911-000 | |
| 20X objective | Zeiss | N-Achroplan 0.45 NA, 420953-9901-000 | |
| cover glass | Healthrow Scientific | HS159879H | |
| dopamine hydrochloride | Sigma Aldrich | H8502 | |
| infrared 2d array camera | Princeton Instruments | NIRvana | |
| infrared 1d sensor array | Princeton Instruments | PyLoN IR | |
| nIR spectrograph | Princeton Instruments | SCT-320 | |
| planoconvex lens | ThorLabs | LA1384 | |
| wellplates (glass bottom) | Corning | 4580 |
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