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.

注意:使用前请咨询所有相关的材料安全数据表（SDS）。相比，他们的散料对口纳米材料可能有额外的危害。利用一切适当的安全实践，包括工程控制（通风橱，隔音罩）和个人防护设备（安全眼镜，护目镜，实验室外套，全长裤，封闭趾鞋）。 1.缓冲液，表面活性剂和聚合物溶液的制备<ol><li> 的100mM NaCl的溶液制备 <ol><li>溶解584毫克的NaCl在80毫升去离子水。加去离子水使总体积为100毫升。 </li></ol></li><li> 3％十二烷基硫酸钠的制备（SDS）溶液 <ol><li>溶解3克的SDS在80毫升去离子水。加去离子水使总体积为100毫升。 </li></ol></li><li> 2％的胆酸钠的制备（SC）溶液 <ol><li>溶解2克草皮鎓胆水合物在80毫升去离子水。加去离子水使总体积为100毫升。 </li></ol></li><li> 成像缓冲器的制备（1×的Tris:20毫摩尔Tris，100mM的NaCl）中 <ol><li>溶解22.23克Tris碱并使用磁力搅拌棒和板在500毫升去离子水58.44克氯化钠。 </li><li>小心加入浓HCl直到达到pH为8.1。 </li><li>加入去离子水，达到1升的最终体积</li></ol></li><li> RITC-PEG-RITC聚合物的合成 <ol><li>二氯甲烷和二甲基甲酰胺（DMF）的混合物:在1毫升1:1的溶解胺双官能聚乙二醇（PEG）（5 100kDa或20kDa的，0.1摩尔/升）和若丹明异硫氰酸酯（RITC，0.2摩尔/升）。 </li><li>添加0.2摩尔/升的N，N-二异丙基乙胺（DIEA）。 </li><li> 3小时后，沉淀用10倍体积的乙醚，随后通过真空过滤的。 </li><li>重新溶解在DMF和重复乙醚沉淀弗洛通过真空过滤结婚。 注意:其他异硫氰酸酯改性分子（ 如，异硫氰酸荧光素，FITC）可以连接到PEG或其它胺改性的聚合物使用类似的方法。 </li></ol></li><li> PEG化-DNA的制备（PEG-DNA）的 <ol><li>结合100三（2-羧乙基）膦盐酸盐（TCEP）溶液（0.5M，pH 7.0）中以44.9微升微升5&#39;-巯基 - 修饰的DNA（1毫克/10μL在0.1M NaCl）中，并加入到4.855毫升去离子水。 </li><li>搅拌1小时。 </li><li>溶解500毫克甲氧基聚乙二醇马来酰亚胺的5ml磷酸盐缓冲盐水。 </li><li>结合DNA和PEG溶液（10毫升总），搅拌24小时。 </li></ol></li></ol> 2.单壁碳纳米管（单壁碳纳米管）悬浮液的制备<ol><li> 单壁碳纳米管的清洗以除去催化剂和杂质。 <ol><li>添加200-300毫克没有洗过的单壁碳纳米管的成塑料离心管中CONTA进不去45毫升去离子水。 </li><li>涡旋2分钟，超声处理用水浴超声5分钟的溶液中。需要注意的是超声器设置仪器之间的不同，所以检查的SWNT的解决方案增加了光学密度（ 即变黑），以确保单壁碳纳米管分散。 </li><li>离心20分钟将溶液在16100 xg离心并丢弃上清液。 </li><li>加入约45毫升的新鲜去离子水。 </li><li>重复步骤2.1.2-2.1.4高达8倍。 </li><li>删除尽可能多的水尽量小心，不要去打扰丸状的单壁碳纳米管，并允许单壁碳纳米管颗粒在空气中干燥。 </li></ol></li><li> 单壁碳纳米管的核酸悬浮液 <ol><li>溶解在0.1M NaCl的核酸（NA）为100毫克/毫升的浓度。 </li><li>使用抗静电枪从一次性锅铲，微量离心管和单壁碳纳米管的库存除去静电。在通风橱中，加入Na溶液的20微升至0 980微升.1 M氯化钠，然后加入1毫克的单壁碳纳米管。 </li><li>使用超声发生器3毫米直径的尖端，超声处理10分钟，在40％的幅度，在冰浴中的溶液中。 </li><li>两次离心DNA的单壁碳纳米管溶液中90分钟，16100×g的。如果使用的PEG-DNA，纯化出多余的和未反应的DNA和PEG使用100kDa的自旋滤波器。加入足够的PBS，以填补自旋过滤器和旋转9300 XG为1.5分钟，重复此洗涤步骤3次。 </li><li>收集和保存上清液，小心不要去打扰含碳纳米管束和聚集沉淀。丢弃在根据适当的纳米材料的机构危险废物过程中沉淀。 </li><li>测量在632纳米用紫外/可见分光光度计，以确定使用ε= 0.036升/厘米·毫克按照朗伯 - 比尔定律和适当的稀释因子悬浮SWNT的近似浓度的溶液的吸光度。 </li></ol></li><li> 两亲聚合物悬浮单壁纳米碳管<ol><li>从一次性锅铲，微型离心管和单壁碳纳米管股票去除静电。在通风橱中，加入5毫克的SWNT至5ml的2％悬浮剂溶液（可选地，SDS溶液可以使用）。 </li><li>使用超声发生器6毫米直径的尖端，超声处理1小时，在40％的幅度，在冰浴中的溶液中。 </li><li>使用超速离心机和珍藏上清15万XG 4小时离心机样品。 </li><li>溶解两亲性聚合物（ 例如，RITC-PEG-RITC）的SC-SWNT溶液的1重量％。 </li><li>透析用3.5 kDa的透析膜的高分子的SC-SWNT溶液对1升的去离子水或缓冲液5天。改变出的水或小时2和小时4.较大透析膜可用于后缓冲，只要它允许移除所选择的表面活性剂，但单壁碳纳米管和两亲聚合物二者的保留。 </li><li>用紫外/可见分光光度计，以确定测量在632 nm处的溶液的吸光度用ε= 0.036 L /厘米*毫克按照朗伯 - 比尔定律及相应的稀释倍数暂停单壁碳纳米管的大致浓度。 </li></ol></li></ol> 3.表面固定化单壁碳纳米管传感器的制备<ol><li> BSA的生物素和的NeutrAvidin储备溶液的制备 <ol><li>溶解10毫克冻干的BSA - 生物素在1mL去离子水中，制成10毫克/毫升储液，并储存于4℃。 </li><li>在2mL去离子水中溶解10毫克的NeutrAvidin蛋白（NAV，去糖基化抗生物素蛋白的蛋白质），以使一个5毫克/毫升贮备液。商店等分于-20℃。解冻等分试样可以在4℃保存数天。 </li></ol></li><li> 准备BSA生物素涂显微镜载玻片 <ol><li>清洁的显微镜载玻片和0.17毫米遮玻璃（或以适合的显微镜物镜）用去离子水，随后用甲醇，丙酮和去离子水的最后冲洗。 </li><li&gt;通过在干净的显微镜载玻片放置双面胶带的几件相距约5mm创建信道。通过采取长玻璃盖玻片并按下它到双面胶带的顶部密封的通道。可以肯定的是集中在显微镜载玻片使盖玻片和幻灯片的边缘不冲水。 </li><li>加入100微升BSA - 生物素储液至900 1×μL的Tris缓冲液以1毫克/毫升的最终浓度。 </li><li>由溶液吹打到一个端部和使用组织芯吸走溶液在另一流的BSA的生物素溶液进入通道的50微升。孵育5分钟，随后由3-5刷新用0.1M NaCl的50微升。 </li><li>稀释的960微升1×Tris缓冲液5毫克/毫升的NAV库存40微升至0.2毫克/毫升的最终浓度。 </li><li>由溶液吹打到一个端部和使用组织芯吸走溶液在另一流的NAV溶液进入通道的50微升。孵育2-5分钟接着3-5刷新用0.1M NaCl的50微升。 </li><li>在成像缓冲器稀悬浮SWNT的储备溶液以1-10毫克/升的浓度和流量将溶液的50微升到通道并孵育5分钟。 </li><li>轻轻洗去使用成像缓冲器的50微升多余的单壁碳纳米管。 </li></ol></li><li> APTES硅烷化显微镜载玻片的制备 注:APTES硅烷化载玻片允许的方式固定带负电荷的DNA包裹的单壁碳纳米管的玻璃基板的表面上。 <ol><li>制备在乙醇（3-氨基丙基）三乙氧基硅烷（APTES）的10％溶液。 </li><li>通过使用3.2.2所述的步骤做渠道，流入100 1X的微升Tris缓冲液。 </li><li>冲洗与APTES溶液的信道和孵育5分钟。用1X Tris缓冲液清洗。 </li><li>稀释在成像缓冲器悬浮DNA的单壁碳纳米管的储备溶液以1-10毫克/土地流的浓度的溶液进入通道和incub 50微升吃了5分钟。 </li><li>轻轻洗去使用成像缓冲器的50微升多余的单壁碳纳米管。 </li></ol></li></ol> 4.荧光光谱法和单壁碳纳米管传感器的显微镜<ol><li> 近红外荧光显微镜 <ol><li>执行使用激光激发并用的InGaAs传感器阵列用于成像配备倒置显微镜的表面固定化的单壁碳纳米管的成像。 </li><li>引导激光束进入上使用可调节的可调安装座反射镜和一对设置到端口高度后安装虹膜倒置显微镜的背照明端口。确保梁是水平和直线通过确认放置后续镜之间，并在进入照明端口之前当其穿过两个虹膜。如果有必要，使用潜望镜装配调整光束的高度。删除会减弱光束照明端口上的任何短通滤波器的热量。 </li><li>将适当的过滤器立方体进入显微镜反映激发光到目标，并收集发射光。这通常包括用激发波长高于截止，（ 例如 ，750 LP）与发射长通滤波器（ 例如，850的LP），以撞击所述传感器进一步减少散射的激发光的分色长通滤波器。此外，发射滤波器可以被选择为选择性对特定手性的单壁碳纳米管的排放。 </li><li>通过插入合适的对准立方体用含有1mM针孔两个偏置，磨砂盘更换物镜进行微调整的光束对准。对准束两者的上下对准盘片的中心。 </li><li>附加使用适当的适配器和0.5X透镜在必要时，以容纳传感器尺寸的2D的InGaAs传感器阵列到显微镜的侧成像端口。 </li><li>使用100X油浸物镜（1.4 NA），适用无荧光浸油，并将immobilized SWNT样品到显微镜阶段。 </li><li>提高物镜直到油触点护罩玻璃的底部（＃1.5，170微米厚）。使如果必要的调整，以客观衣领成像条件， 例如，温度，玻璃厚度等 </li><li>慢慢提升与激励源的目标和监视的InGaAs相机的荧光信号。荧光强度应逐渐增加，因为焦平面接近表面和固定传感器成为焦点。 </li></ol></li><li> 近红外荧光光谱仪 可以使用相同的显微镜装置进行，但通过将所收集的光出显微镜主体并进入光谱仪和InGaAs线阵探测器荧光光谱:注。 <ol><li>使用运动坐骑和镜子额定NIR，直接朝着光谱仪的入射狭缝的光路。聚焦光线降低到POI核苷酸上的入口用的聚焦透镜狭缝（ 例如 ，平凸中，f = 150毫米）。这种对准是由衰减激光功率为&lt;1毫瓦，并用1"镜更换显微镜物镜并使用50/50光束分离滤波器立方体完成的，激发光随后将通过出口离开显微镜主体和可用来调节反射镜和透镜将光束聚焦到入口狭缝。 </li><li>更换显微镜物镜和长通滤波器后，放置一个孔板上舞台，并记录使用分光计和的InGaAs阵列的焦点对准样品的光谱。 </li></ol></li><li> 测量的（GT）15 DNA的单壁碳纳米管对多巴胺可逆反应 <ol><li>安装传感器被覆通道到显微镜台上，用100X油物镜和的InGaAs相机带入焦点。加在磷酸盐缓冲盐水（PBS）100μM的多巴胺溶液50μL到流动通道和记录在荧光强度的变化。 </li><li>用磷酸盐缓冲盐水洗出多巴胺溶液，并观察在各个单壁碳纳米管传感器的荧光的变化。 </li></ol></li><li> 孔板筛查单壁碳纳米管传感器的响应分析 注:可使用使用机动阶段控制的透明孔板进行不同的分析物的筛选。一种理想的孔板是透明的可见光和红外和具有黑色侧壁以最小化孔之间的串扰。 <ol><li>悬浮SWNT传感器（ 例如 ，5毫克/升浓度）到每个孔，足以覆盖阱均匀，典型地&gt; 100微升的用于96孔板和384孔板的底部&gt; 30微升的移液管等体积。 </li><li>移液器分析（ 例如 ，2μL，100μM最终体积）从库中筛选到孔板。准备一式三份各分析考虑到潜在的富裕以及变异，fluctuat激发强度或温度的离子。 </li><li>提高目标（ 例如 ，20X achroplan，0.45 NA），同时监测利用分光计和砷化铟镓阵列发射光谱。物镜的最佳位置将投入焦平面大致在孔中的样品体积，这应该对应于在测得的强度最大的中间。 </li><li>对于每个样品孔，录制1-10敞口收集光谱。 </li><li>比较的发光光谱为每孔含有无需额外的分析物只是单壁碳纳米管来量化荧光响应的控制。 </li></ol></li></ol>

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The authors have nothing to disclose.

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，?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-1</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium dodecyl sulfate</td>
			<td>Sigma Aldrich</td>
			<td>L6026</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium cholate hydrate</td>
			<td>Sigma Aldrich</td>
			<td>C6445</td>
			<td></td>
		</tr>
		<tr>
			<td>tris base (Trizma base)</td>
			<td>Sigma Aldrich</td>
			<td>93362</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine-PEG-amine,NH2-PEG-NH2</td>
			<td>Nanocs Inc</td>
			<td>PG2-AM-5k</td>
			<td></td>
		</tr>
		<tr>
			<td>rhodamine B isothiocyanate</td>
			<td>Sigma Aldrich</td>
			<td>283924</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein isothiocyanate</td>
			<td>Sigma Aldrich</td>
			<td>F7250</td>
			<td></td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>676853</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-diisopropylethylamine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D125806</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>673811</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride&#160;</td>
			<td>Sigma Aldrich</td>
			<td>C4706&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#8217;-thiol-modified DNA&#160;</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methoxypolyethylene glycol maleimide</td>
			<td>Sigma Aldrich</td>
			<td>63187</td>
			<td></td>
		</tr>
		<tr>
			<td>100k Da spin filters</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HiPCO Super purified single walled carbon nanotubes</td>
			<td>Integris</td>
			<td>HiPco SuperPurified</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr>
			<td>anti static gun</td>
			<td>Milty</td>
			<td>Milty Zerostat 3</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5415 D</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra sonicator</td>
			<td>Cole Parmer</td>
			<td>CV18</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis cassettes</td>
			<td>Thermo scientific</td>
			<td>Slide-A-Lyzer G2 87722</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-biotin</td>
			<td>Thermo scientific</td>
			<td>29130</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidin protein</td>
			<td>Thermo scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane (APTES)</td>
			<td>Sigma Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer.Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>kinematic mirrors</td>
			<td>ThorLabs</td>
			<td>KM200-E03</td>
			<td></td>
		</tr>
		<tr>
			<td>periscope</td>
			<td>ThorLabs</td>
			<td>RS99</td>
			<td></td>
		</tr>
		<tr>
			<td>immersion oil</td>
			<td>Zeiss</td>
			<td>Immersol 518f</td>
			<td></td>
		</tr>
		<tr>
			<td>100X objective</td>
			<td>Zeiss</td>
			<td>Plan-apochromat 100X oil, 1.4NA, PH3, 420791-9911-000</td>
			<td></td>
		</tr>
		<tr>
			<td>20X objective</td>
			<td>Zeiss</td>
			<td>N-Achroplan 0.45 NA, 420953-9901-000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cover glass</td>
			<td>Healthrow Scientific</td>
			<td>HS159879H</td>
			<td></td>
		</tr>
		<tr>
			<td>dopamine hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>H8502&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>infrared 2d array camera</td>
			<td>Princeton Instruments</td>
			<td>NIRvana</td>
			<td></td>
		</tr>
		<tr>
			<td>infrared 1d sensor array</td>
			<td>Princeton Instruments</td>
			<td>PyLoN IR</td>
			<td></td>
		</tr>
		<tr>
			<td>nIR spectrograph</td>
			<td>Princeton Instruments</td>
			<td>SCT-320</td>
			<td></td>
		</tr>
		<tr>
			<td>planoconvex lens</td>
			<td>ThorLabs</td>
			<td>LA1384</td>
			<td></td>
		</tr>
		<tr>
			<td>wellplates (glass bottom)</td>
			<td>Corning</td>
			<td>4580</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering molecular recognition with bio-mimetic polymers on single walled carbon nanotubes

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.

单壁碳纳米管悬浮在同时使用表面活性剂和通过直接超声处理和通过透析交换两亲聚合物水溶液。 图1显示了单壁碳纳米管，采用羰基铁催化法（的Hipco）增长，使用SC，RITC-PEF20-RITC暂停，（GT）15 -DNA。用SDS（或聚合物）一个单壁碳纳米管的光密度在超声处理后显着增加，并在除去聚集物和污染物通过纯化通过离心（ 图1）减小。在632吸光?...

Watch this Scientific Journal Video about Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes at JoVE.com

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

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.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

engineering-molecular-recognition-with-bio-mimetic-polymers-on-single

Research

JoVE Journal

Bioengineering

8.1K 次观看.  University of California Berkeley. 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.

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

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

科研

生物工程

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

同时合成单壁碳纳米管和石墨在电弧等离子体磁增强

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

通过创建一个的合成Curli的操纵子工程粘附的细菌

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

凝血因子VIII的脂质纳米管螺旋组织

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...