本出版物中报告的研究得到了阿卜杜拉国王科技大学（KAUST）的支持。

注意： 使用前请务必查阅所有相关的材料安全数据表 （MSDS）。使用所有适当的安全措施和个人防护设备（安全眼镜、手套、实验室外套、全长裤、露趾鞋）。在生物通风橱中进行所有生物反应。
注：该方案旨在产生 2 x 10 10 个生物功能化的 NWs/mL，相当于 0.36 mg 铁/mL，涂有密度为 3 x10 4 抗体/NW 的抗 CD44 抗体。氧化铁铁（核壳）NW长2.5μm，直径为41.5nm。
1. 铁纳米线的释放
注：铁/氧化铁NW的制造过程在以前的出版物59中进行了详细解释。
<ol><li>在切割垫上，使用单刃刀片和小锤子将铝（Al）圆盘（图2A）切成小块（图2B），以装入2mL管中。使用镊子将小铝块转移到管中。</li>
	<li>用 1 mL 的 1 M 氢氧化钠 （NaOH） 填充含有小铝块的 2 mL 试管。确保所有铝片都用 NaOH 覆盖。</li>
	<li>将溶液在室温下在化学通风橱内工作30分钟。</li>
	<li>仅使用镊子取出Al块，并将释放的NW（黑色团簇， 图3A）保持在NaOH溶液中30分钟。不要改变NaOH溶液。</li>
	<li>通过将 2 mL 管放入磁性架中来收集 NW，并等待 2 分钟，然后除去旧的 1 M NaOH 溶液。用新鲜的 NaOH 溶液代替。</li>
	<li>将含有NW的2mL管超声处理30秒，并将其在化学通风橱内放置1小时。</li>
	<li>重复步骤 1.5-1.6 至少四次。</li>
	<li>通过将 2 mL 管放入磁性架中洗涤 NW 并等待 2 分钟。</li>
	<li>弃去NaOH溶液，用1mL无水乙醇代替。</li>
	<li>将管子超声处理 30 秒。</li>
	<li>将 2 mL 试管放入磁性架中并等待 2 分钟。</li>
	<li>弃去旧的无水乙醇溶液，用 1 mL 新鲜无水乙醇代替，超声处理 30 秒。</li>
	<li>重复步骤 1.11 和 1.12 至少四次。</li>
	<li>在室温下将 NW 保持在 1 mL 无水乙醇中，直到需要为止。</li>
	<li>使用电感耦合等离子体质谱（ICP-MS）测量铁的浓度，从而测量NWs的浓度。 注意：可以在此处暂停协议。但是，对于长期存储，除非需要，否则不要从 Al 模板中释放 NW。在不频繁超声处理的情况下，将释放的NW长时间沉淀在乙醇中会产生聚集体，需要更长的超声处理时间才能分离。</li>
</ol>2. 用APTES涂覆纳米线
注：在该方案中，100μL的APTES溶液（密度为0.946g / mL61）足以包被1.6×107m 2 / g的NW。如果纳米材料的比例或质量发生变化，则应相应地调整APTES体积。
<ol><li>使用 1 mL 移液管将步骤 1.14 中的 NW 转移到 5 mL 玻璃管中。</li>
	<li>要收集 2 mL 管中剩余的任何 NW，通过加入 1 mL 无水乙醇洗涤空的 2 mL 管两次，并使用 1 mL 移液管将其转移到 5 mL 玻璃管中。</li>
	<li>通过使用移液器，取 100 μL APTES 并将其直接添加到 NW 溶液中。</li>
	<li>涡旋 5 mL 玻璃管 10 秒。</li>
	<li>调整实验室蒸馏架夹具上的 5 mL 玻璃管。</li>
	<li>将5mL玻璃管的一半放入超声波浴的水中，超声处理1小时。</li>
	<li>从超声波浴中取出 5 mL 玻璃管，加入 400 μL 去离子 （DI） 水，然后加入 20 μL 1 M NaOH（碱性催化）。 注意： 首先加入去离子水很重要。</li>
	<li>如步骤2.5-2.6所述，调整5mL玻璃管，并超声再超声处理1小时。</li>
	<li>从超声波浴中取出 5 mL 玻璃管。</li>
	<li>在玻璃管旁边放置磁铁5分钟以收集NW。</li>
	<li>用 1 mL 新鲜无水乙醇代替上清液并超声处理 10 秒。</li>
	<li>重复步骤 2.10-2.11 四次。</li>
	<li>使用 1 mL 移液管将所有 NW 悬浮乙醇转移到新的 5 mL 玻璃管中。 注意：APTES涂层的NW可以储存在装有乙醇的玻璃管中，直到需要为止。该协议可以在此处暂停。</li>
</ol>3. 纳米线的生物功能化
<ol><li>激活抗体 注：要获得大约 3 x 104 份抗体/NW，每 0.1 mg 铁使用 30 μL 抗体 （1 mg/mL）。<ol><li>在 2 mL 试管中，将 0.4 mg 3-3-二甲基氨丙基碳二亚胺 （EDC） 和 1.1 mg 磺基-N-羟基磺基琥珀酰亚胺 （Sulfo-NHS） 溶解在 1 mL 2-N-吗啉基乙磺酸水合物 （MES） （pH 4.7） 中。 注意：EDC/sulfo-NHS 混合物在使用前应新鲜并准备好。</li>
		<li>在新的 2 mL 管中，分别加入 30 μL 抗 CD44 抗体 （1 mg/mL）、960 μL 0.1 M 磷酸盐缓冲盐水（PBS，pH 7）和 10 μL EDC/磺基-NHS 混合物（在步骤 3.1.1 中制备）。</li>
		<li>在室温下将 2 mL 管置于 10 x g 的试管振荡器中 15 分钟。</li>
	</ol></li>
	<li>APTES包被纳米线的制备<ol><li>在步骤3.1.3的15分钟孵育期间，通过在APTES涂层的NWs管（来自步骤2.13）旁边放置磁铁来洗涤NWs2分钟以收集NWs。</li>
		<li>弃去乙醇，用 1 mL 的 0.1 M PBS（pH 7）代替，然后超声处理 10 秒。</li>
		<li>重复步骤3.2.1-3.2.2四次。</li>
		<li>如步骤3.2.1所述收集NW，并丢弃0.1M PBS。将NW保持在管中，无需任何溶液。</li>
	</ol></li>
	<li>抗体的附着<ol><li>将所有活化抗体溶液（在步骤3.1.3中制备）转移到NWs管（在3.2.4中制备）中，并超声处理10秒。</li>
		<li>将玻璃管置于旋转器中，在4°C下过夜。</li>
		<li>如步骤3.2.1所示使用磁铁收集NWs并弃去上清液。</li>
		<li>在4°C下加入1mL 2%牛血清白蛋白（BSA）溶液1小时以阻断反应。</li>
		<li>检查抗体功能化NW的抗原性，例如，使用免疫沉淀（IP）和蛋白质印迹（WB）测定。 注意：为了获得更好的结果，请在封闭步骤后立即在 IP、WB 或生物相容性测定中使用生物功能化的 NW。</li>
	</ol></li>
</ol>4. 生物相容性测定
注：为了研究NW的生物相容性，可以采用各种细胞活力测定和不同的细胞系。这里使用的NW的浓度是基于以前的出版物16。细胞接种应在NW生物功能化前一天进行。
注意：以下所有步骤都应在生物安全柜下完成。
<ol><li>在96孔板中，将悬浮在McCoy细胞培养基（100μL/孔）中的4×104 结肠癌细胞（HCT116细胞系）接种9个孔，并将其置于37°C和5%二氧化碳（CO2）的培养箱内过夜。</li>
	<li>如步骤3.2.1所示，使用磁铁收集NWS（来自步骤3.3.4），并用PBS洗涤NW，并用1mL 0.01M PBS（pH 7）替换旧溶液。重复此步骤三次。</li>
	<li>如步骤3.2.1所示，使用磁铁收集NWs，用加热的McCoy培养基洗涤NW（来自步骤3.2.1），并用1mL加热的McCoy培养基替换旧的PBS。重复此步骤三次。</li>
	<li>如步骤3.2.1所示，使用磁铁收集NW（来自步骤4.3），并用900μL加热的McCoy培养基替换1mL加热的McCoy培养基。NWs 浓度应为 0.02 mg NWs/mL。</li>
	<li>将96孔板（来自步骤4.1）从培养箱中取出到生物安全柜中。</li>
	<li>在生物安全柜下，从细胞中丢弃旧培养基，并用100μL悬浮的NWs（在步骤4.4中制备）代替。</li>
	<li>用手摇动板（在步骤4.6中制备），然后在37°C和5%CO2的培养箱内孵育24小时。</li>
	<li>第二天，从培养箱中取出96孔板（在步骤4.7中制备）。在生物安全柜下，使用多壁移液管向每个孔中加入11μL细胞活力试剂（材料表）。</li>
	<li>用板振荡器以10× g 的速度摇动板10秒。</li>
	<li>将板在培养箱中孵育1小时。</li>
	<li>从培养箱中取出96孔板（在步骤4.10中制备）。通过测量细胞活力试剂的吸光度（激发540nm，发射590nm）读取酶标仪（材料表）中的板。</li>
</ol>

作者没有什么可透露的。

与任何纳米材料制造和涂层方法一样，需要高质量的解决方案。释放（1 M NaOH）和官能化（MES）溶液可以重复使用多次。然而，在开始新工艺之前检查它们的pH值非常重要。在释放步骤中，用NaOH洗涤NW应至少进行四次。洗涤效果越好，NW的稳定性越好，它们的聚集度就越少。氧化层增强了NWs浸入乙醇或水中时的稳定性63。
用APTES和抗体包被NW后，NW的直径和长度受到影响。在这里，直径从 41.5 nm 增加到 70 nm，长度从 2.5 μm 减少到 1.6 μm，这是由于超声处理步骤破坏了 NW。因此，在生物功能化步骤之后表征NWs的形态至关重要。
抗体与NWs的结合依赖于胺基（在APTES上）和羧基（在抗体上）之间的共价相互作用。因此，确认APTES涂层的存在是一个重要的步骤，为此我们使用了EELS映射。涂层方法安全直接。它不需要高温或较长的孵育时间。此外，APTES涂层还可作为连接剂，使具有羧基的其他抗体或蛋白质实现共价连接。
在用抗体对NW进行生物功能化的情况下，生物功能化过程后抗体结合位点的抗原性可能会受到影响。IP 和 WB 方法可用于调查此问题。使用本协议中提到的生物功能化方法将允许抗体与对特定细胞受体具有高抗原性的NW结合。此外，用抗体对 NW 进行生物功能化增加了用目标受体 CD44 靶向细胞的能力。共聚焦显微镜证实了这一点。尽管未包被NWs的生物相容性很高（>95%），但向NWs添加APTES包被或抗体可提高其100%的生物相容性。
此外，包被和生物功能化方案是高效、经济和可重复的。它应该适用于任何其他铁-氧化铁纳米材料，因此应根据纳米材料的表面积和质量优化涂层和附着抗体的浓度。该协议可以在一般实验室的环境条件下安全地完成。生物功能化显著增强了纳米材料的生物相容性和靶向能力。总的来说，NW是纳米医学应用（包括多模态或组合治疗、细胞检测或引导以及生物传感）的非常有前途的材料。结合生物功能化，如本文所述，可以实现特异性细胞靶向，以提高精度和功效。

磁性纳米材料的一个独特特性是它们对磁场的响应能力1，可以有效地利用磁场以多种方式驱动它们，同时它们也可以通过磁共振成像 （MRI） 等进行监测。当施加高频交变磁场时，它们会产生热量，从而诱发体温过高，从而提供治疗选择1。另一种方法是光热处理，可以使用近红外 （NIR） 激光 2,3 实现。
在众多磁性纳米材料中，氧化铁在磁分离、热疗2,4、细胞引导5、药物递送6,7,8以及作为MRI造影剂9,10等生物应用中最受关注。这是由于它们的高生物相容性 11,12、大磁化强度 11,12、包被能力9、13、14、15、携带药物的能力 2,16、药物功能化的能力2,16 或/和靶向剂12,13,17，18，以及将光能转化为热的能力2.最近，MagForce开始使用氧化铁纳米颗粒进行热疗治疗的癌症患者临床试验19。
最近，磁性纳米线 （NW） 越来越多地用于生物医学应用 3,11,16,20,21,22。它们具有与磁性纳米珠相似的特性，但具有各向异性的形状和非常大的磁矩，这使得通过磁场23,24 进行非常有效的远程控制，包括低频驱动以诱导磁机械效应 25,26,27,28,29 .因此，NW已被用于不同的生物学应用，例如外泌体分离30细胞追踪21，癌症治疗3,11,16，药物递送16,31,32，以及作为MRI造影剂33。
具有特定细胞靶向能力的生物功能化磁性纳米材料在生物医学应用和精准医学方面具有巨大潜力34,35。为了附着这些靶向剂，需要对纳米材料进行表面改性。通常，它们需要一种提供官能团的涂层，这有利于处理剂的附着。在文献中，有大量的磁性纳米材料有机和无机涂层。根据可以固定在纳米材料上的官能团，这些涂层可分为四大类：基于羧酸基团的分子、聚合物、组氨酸和基于硅烷基团的分子。
基于羧酸基团的分子是表面改性方法之一。它利用高亲和力 在涂层上的负羧酸基团和磁性纳米材料上的正电荷之间36,37,38。羧酸与金属表面的结合过程可能涉及金属羧酸盐的产生或羧基与金属的粘附。然而，对于多段NWs，如铁/金或镍/金NWs，它们在生物应用方面具有极好的性能39,40，这种类型的涂层不容易应用。它需要同时使用两种不同的涂层：用于修饰金段的硫醇基团和用于磁段（铁或镍）的羧基38。基于羧基的分子的一些例子是血卟啉、庚二酸、棕榈酸和 3-[（2-氨基乙基）二硫代] 丙酸 （AEDP）38。使用聚合物对磁性纳米材料进行表面改性具有一些明显的优势。由于聚合物的大分子量，它增强了磁性纳米材料在溶液38中的稳定性。然而，它将显著增加纳米材料38的尺寸。聚乙烯吡咯烷酮 （PVP）、聚乙烯亚胺 （PEI）、精氨酸-甘氨酸-D 天冬氨酸 （RGD） 和聚乙二醇 （PEG） 是最常用的表面改性聚合物的一些例子。每个都有自己的功能并使用38。第三种表面改性方法是使用组氨酸涂层。组氨酸是一种具有组氨酸氨基酸侧链的蛋白质，对有限数量的磁性纳米材料（如镍38）具有高亲和力。它可用于蛋白质纯化过程38,41,42。组氨酸涂层也可以应用于多段NWs，例如镍/金NWs38。纳米材料表面的硅烷化是一个成熟的过程38,43,44。它基于一个硅原子通过三个单键与任何金属氧化物表面连接，同时该硅原子通过烷基链38,43,44与末端的官能团结合。这种涂层的优点是提供游离胺基团，并且它能够分别涂覆磁性和非磁性材料38,45，例如镍和金。因此，使用基于盐水基团的分子是生物功能化多链段NWs的实用途径。基于硅烷基团的分子的一些例子是（3-氨丙基）三乙氧基硅烷（APTES）和（3-氨丙基）三甲氧基硅烷（APTMS）38,45。
在涂层中添加靶向剂可以在病变细胞的诊断和治疗中发挥重要作用，同时最大限度地减少对健康组织的副作用46,47。在纳米材料表面添加靶向剂可增强细胞选择性结合和通过内吞受体的内化7。如果没有这些靶向配体，纳米材料会与细胞膜发生非特异性相互作用，与具有配体的纳米材料相比，细胞膜的结合速率较低48。靶向癌组织的挑战之一是它们与健康组织的特征相似性。因此，靶向的成功主要取决于确定用作生物靶标的适当配体49,50。各种靶向剂已被用于将纳米材料引导至癌细胞48,51（例如，CD44，因为它在癌细胞中的高表达与健康细胞相比 52,53,54,55）。
靶向药物可根据其成分及其复杂性分为三大类：基于适配体的靶向、基于配体的靶向和基于抗体的靶向。适配体是化学合成的短链 DNA 或 RNA-寡核苷酸，折叠成二维和三维结构，使其能够靶向特定抗原，最常见的是蛋白质56。基于配体的靶向包括肽和短氨基酸链57。基于抗体的靶向涉及使用整个抗体或抗体片段，例如单链可变片段或抗原结合片段51。使用这种方法的优点是具有两个与其特异性靶抗原具有高结合亲和力的结合位点，这使其具有极高的选择性58。结合位点类似于锁，抗原类似于钥匙58。
在这项工作中，使用的NW是通过电沉积到氧化铝膜上来制造的，这种方法在以前的出版物59中进行了详细描述。这里的重点是从膜中释放这些铁-氧化铁（核壳）NW，并用特异性抗体对它们进行生物功能化，以提供靶向能力。抗体不能直接与氧化铁-氧化铁NWs结合，需要连接子。用APTES包被NWs可提供游离胺基团，从而通过抗体上的羧基实现共价连接（图1）。APTES 涂层的优点是它能够同时适用于磁性21 和非磁性60 材料，例如铁/金或镍/金 NWs45。通常，该协议中解释的所有涂层和生物功能化步骤都可以与任何铁/氧化铁纳米材料一起使用。这里以铁/氧化铁NW为例。结果表明，抗体功能化的NWs对特异性细胞表面受体具有较高的抗原性，可用于不同的应用。示例包括细胞分离、药物递送、使用光热和/或磁机械处理的特定癌细胞治疗。

<table>
	<tbody>
		<tr>
			<td>2 mL tube (snap-cap Microcentrifuge)</td>
			<td>Eppendorf, Fisherscientific</td>
			<td>05-402-7</td>
			<td></td>
		</tr>
		<tr>
			<td>2-N-Morpholino EthaneSulfonic acid hydrate 99% (MES)</td>
			<td>Thermscientific</td>
			<td>AC172590250</td>
			<td>Concentration 0.1 M and pH 4.7</td>
		</tr>
		<tr>
			<td>3-3-Dimethyl-aminopropyl Carbodiimide (EDC)</td>
			<td>Thermofisher</td>
			<td>PG82079</td>
			<td></td>
		</tr>
		<tr>
			<td>3-AminoPropyl-Tri-Ethoxy-Silane (APTES)</td>
			<td>Sigma Aldrich</td>
			<td>919302</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL glass tube</td>
			<td>Fisherscientific</td>
			<td>03-339-22C</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate ( flat bottom)</td>
			<td>Sigma Aldrich</td>
			<td>CLS3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD44 antibody</td>
			<td>BD Biosciences</td>
			<td>550990</td>
			<td>Clone 515, concentration 1 mg/mL</td>
		</tr>
		<tr>
			<td>APTES ((3-Aminopropyl)triethoxysilane), 99%</td>
			<td>Sigma Aldrich</td>
			<td>919-30-2</td>
			<td>Concentration 99%</td>
		</tr>
		<tr>
			<td>BCA assay (Pierce BCA Protein Assay Kit)</td>
			<td>Thermofisher</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin solution (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>9048-46-8</td>
			<td>Concentration 35%</td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Thermofisher</td>
			<td>50116047</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell viability reagent</td>
			<td>AlamarBlue,Thermofisher</td>
			<td>DAL1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Colon cancer cells - HCT116 cell line</td>
			<td>ATCC</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardwood Hammer</td>
			<td></td>
			<td></td>
			<td>Any hammer tool can be used, there is no specific brand.</td>
		</tr>
		<tr>
			<td>Inductively coupled plasma Mass Spectrometer (ICP-MS)</td>
			<td>Perkin Elmer</td>
			<td>ELAN 9000 ICP-MS</td>
			<td>The used software is &#34;Elan instrument control session&#34;</td>
		</tr>
		<tr>
			<td>Laboratory Retort Stand with Clamp</td>
			<td>RVFM</td>
			<td>13-0140</td>
			<td>This is used to handle the 5 mL glass tube in the sonicator bath.</td>
		</tr>
		<tr>
			<td>Magnetic rack (DynaMag-2 Magnet)</td>
			<td>Thermofisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#8217;s 5A Medium 1x</td>
			<td>Gibco</td>
			<td>16600082</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader (Bio-Rad xMark Absorbance Spectrophotometer)</td>
			<td>Bio-Rad</td>
			<td>1681150</td>
			<td>Microplate Manager 6 software (#168-9520)</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 10x</td>
			<td>Gibco</td>
			<td>14200067</td>
			<td>Concentration 0.1 M (No calcuim, no magnesium)</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1x</td>
			<td>Gibco</td>
			<td>14190136</td>
			<td>Concentration 0.01 M (No calcuim, no magnesium)</td>
		</tr>
		<tr>
			<td>Plate shaker (Microplate Genie)</td>
			<td>Scientific Industries (Genie)</td>
			<td>SI-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Edge Razor blades</td>
			<td>Polysciences</td>
			<td>08410-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodum hydrixide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>1310-73-2</td>
			<td>Concentration 1 M, pH 13</td>
		</tr>
		<tr>
			<td>Sulfo-N-HydroxySulfosuccinimide (sulfo-NHS)</td>
			<td>Thermofisher</td>
			<td>106627-54-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>ATCC</td>
			<td>30-2101</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>VWR</td>
			<td>10136-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube shaker (Eppendorf Thermomixer R Mixer, 2.0 mL)</td>
			<td>Eppendorf, Fisherscientific</td>
			<td>05-400-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath (2510)</td>
			<td>Branson</td>
			<td>2489502</td>
			<td></td>
		</tr>
	</tbody>
</table>

biofunctionalization of magnetic nanomaterials

磁性纳米材料在不同的生物医学应用中受到广泛关注。用特定的靶向剂对这些纳米材料进行生物功能化是提高其诊断和治疗疗效，同时最大限度地减少副作用的关键方面。与非磁性纳米材料相比，磁性纳米材料的优势在于它们能够以非接触方式和远距离响应磁场。这允许引导或积累它们，同时也可以监控它们。最近，具有独特功能的磁性纳米线（NWs）被开发用于生物医学应用。这些NW的大磁矩可以通过磁场更有效地远程控制它们的运动。这已在癌症治疗、药物递送、细胞示踪、干细胞分化或磁共振成像中取得了巨大成功。此外，通过模板辅助电化学沉积进行NW制造提供了一种通用的方法，可以严格控制NW特性。特别是铁纳米管和铁-铁-氧化铁（核壳）纳米管由于其高磁化强度和低毒性而适用于生物医学应用。
在这项工作中，我们提供了一种使用针对在大量癌细胞中过表达的特定细胞表面标志物的特异性抗体对铁/氧化铁 NW 进行生物功能化的方法。由于该方法利用了氧化铁表面的特性，因此也适用于超顺磁性氧化铁纳米颗粒。NW 首先涂有 3-氨丙基-三乙氧基-硅烷 （APTES） 作为连接子，抗体共价连接到该连接子上。APTES包被和抗体的生物功能化通过电子能量损失谱（EELS）和zeta电位测量得到证实。此外，使用免疫沉淀和蛋白质印迹法测试NWs上抗体的抗原性。通过共聚焦显微镜和细胞活力测定研究了生物功能化NWs的特异性靶向及其生物相容性。

重要的是将铝（Al）圆盘（图2A）切成小块（图2B）以适合所使用的管子。向Al块中加入1M NaOH后，应立即开始反应，这可以通过产生气泡来观察。如果在1分钟内没有发生反应，或者反应非常快，溶液完全浑浊，有白云，请立即除去旧的NaOH溶液并用新溶液代替。检查NaOH溶液的pH值。它应该是pH>12。当NW在前30分钟内用NaOH从Al碎片中释放出来时，NW（黑色团簇， 图3A）将漂浮在NaOH溶液中。在释放NW的最后一步中，解决方案应该是均匀的。不应存在NW集群（图3B）。如果用磁铁收集NW3分钟，则溶液应为澄清，NW沉淀应为黑色（图3C）。如果颗粒是灰色的，请丢弃试管并重新开始新的铝样品。
在释放过程中，NWs获得了厚度约为5 nm的天然氧化铁层，这与之前的报道相似11,16,20。该氧化层对NWs的生物相容性16、官能化16,18和磁性20具有重要贡献。然而，将NW保留在模板中（即，不释放它们）直到需要时才能防止它们对它们产生任何环境影响。释放过程后NWs的平均直径和长度分别为2.5 μm和41.5 nm。单个NW的质量可以如表1所示计算，并通过电感耦合等离子体质谱（ICP-MS）确认。在这里，每个氧化铝盘含有约0.3毫克的铁。
为了减少释放后NW的聚集并实现进一步的功能化，NW涂有APTES。该涂层提供游离胺基团，并具有涂覆磁性和非磁性材料的能力39,45，使其适用于涂覆多段 NW，例如铁/金 NW。在纳米线涂层过程中，重要的是要关注两件事。首先，根据 NW 的表面积和质量计算 APTES 分子62 所需的体积。这些计算结果见表2。其次，保持纳米线连续运动，以防止其团聚和 APTES 涂层阻挡 NW 表面的某些部分。例如，在该协议中，纳米线分别在APTES包被和抗体功能化过程中在超声浴和旋转器下孵育。每个APTES分子包含一个硅原子和一个末端官能胺基团21。因此，电子能量损失谱（EELS）图可用于通过在NW表面上显示硅原子（粉红色）来确认APTES涂层的存在（图4A）。相比之下，未涂层的NW显示树皮蓝色的铁原子（图4B）和浅蓝色的铁/氧混合原子（图4B）。非涂层NW的相应EELS映射（图4C，4D）显示中心（图4C）的铁与氧强度高于表面（图4D），表明铁 - 铁氧化物（核壳）结构。EELS映射（图4C，4D）确定NWs上的氧化铁层比Fe2O3多3，这与先前的出版物20相似。
可以使用 IP 和 WB 测定来确认附着抗体的抗原性，其中可以在阳性样品上观察到条带，但在阴性对照上则不可见（图 5）。请注意，要观察透明条带，请勿使用少于 0.1 mg NWs/10 x 106 个细胞。BCA测定（材料表）结合 表3 中的一些计算结果，用于量化NW上的抗体数量。
此外，使用zeta电位测量来阐明表面功能化。APTES上的末端胺基团降低了未包被NWs的负电荷， 如图6所示。与APTES包被的NW相比，抗体功能化的NWs也改变了电荷（图6）。所有 zeta 电位测量均在 pH 值为 7 时进行。
抗体功能化NW的特异性细胞靶向可以使用共聚焦显微镜确认（图7）。在开始任何应用之前，都应测试任何新纳米材料的生物相容性。因此，使用了细胞活力测定，并证实了非包被、APTES 包被和抗体包被的 NW 即使在高浓度下也具有生物相容性（图 8）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61360/61360fig01.jpg"> 图 1：示意图表示纳米线的涂层和生物功能化方法。 （A） 用 APTES 涂覆铁 NW。（B） 使用 EDC + Sulfo-NHS 激活抗体，在末端 （C） 具有抗体功能化纳米线。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61360/61360fig02.jpg"> 图 2：铁沉积铝盘。 （A） 切割前的铝盘。（B） 红线表示切割圆盘的位置。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61360/61360fig03.jpg"> 图 3：纳米线释放步骤。 （A）纳米线簇漂浮在NaOH溶液中。该图像是在加入NaOH和去除Al膜后10分钟拍摄的。（B）悬浮在乙醇中的纳米线。这张照片是在最后一个释放步骤中拍摄的，紧接在超声处理步骤之后。（C）磁铁收集的黑色纳米线颗粒。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61360/61360fig04.jpg"> 图 4：电子能量损失谱 （EELS） 图。 （A） APTES涂层纳米线（APTES-NW）。蓝色和粉红色分别代表铁原子和硅原子。（B）无涂层纳米线（NW）。树皮蓝色和浅蓝色分别代表铁和铁/氧混合物的映射。（C）核心和（D）非涂层NW外壳的相应EELS映射。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61360/61360fig05.jpg"> 图 5：抗体偶联纳米线的功能化和活性的确认。 通过免疫沉淀（IP）和蛋白质印迹（WB）证实了CD44-NWs的抗原性。+Ve：代表阳性对照（全细胞裂解物）。-Ve：代表阴性对照（仅未涂覆的NW）。– CD44细胞：代表不表达CD44抗原的细胞;+ CD44细胞：代表表达CD44抗原的结肠癌细胞。这是 n = 3 个独立实验的代表性印迹。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61360/61360fig06.jpg"> 图 6：NW、APTES-NW 和抗体的 Zeta 电位值。 条形表示平均值±标准误差。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61360/61360fig07.jpg"> 图 7：共聚焦显微图像显示了特定的靶向。 CD44-NWs附着（A 和 C），而Isotype-NWs（阴性对照）未附着（B 和 D）。红色、蓝色和绿色分别代表 CD44-NW 的细胞膜、细胞核和簇。A和B分别是C和D的明场图像。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61360/61360fig08.jpg"> 图 8：与不同配方的纳米线一起孵育的结肠癌细胞 （HCT116） 的细胞活力研究。 孵育 24 小时后用 NW 处理细胞，然后与 NW 孵育 24 小时。所有实验均在37°C的培养箱内进行。 条形表示平均值±标准误差。 <a href="https://www.jove.com/files/ftp_upload/61360/61360fig08large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>参数</td>
			<td>价值</td>
			<td>单位</td>
			<td rowspan="6"></td>
			<td rowspan="6"></td>
		</tr><tr><td>Fe NW 长度 （h）</td>
			<td>2.6</td>
			<td>微米</td>
		</tr><tr><td>半径（直径/2） （r）</td>
			<td>0.01679231</td>
			<td>微米</td>
		</tr><tr><td>Fe 密度 （D）</td>
			<td>7.87</td>
			<td>克/厘米3</td>
		</tr><tr><td>1 Fe NW 体积 （V）= π r^2h</td>
			<td>2.30E-15型</td>
			<td>厘米3</td>
		</tr><tr><td>1 Fe NW 质量 = V x D</td>
			<td>1.80E-08型</td>
			<td>微克</td>
		</tr><tr><td>1 Fe NW 表面积 = 2π r^2 + 2πrh</td>
			<td>型号：2.76E-01</td>
			<td>微米2</td>
			<td>2.8E+05</td>
			<td>纳米2</td>
		</tr></tbody></table>表 1：铁 NW 质量的计算。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>参数</td>
			<td>价值</td>
			<td>单位</td>
		</tr><tr><td>APTES 密度/100 μL*</td>
			<td>9.50E-01型</td>
			<td>g</td>
		</tr><tr><td>APTES的分子密度（MW）</td>
			<td>2.21E+02</td>
			<td>克/摩尔</td>
		</tr><tr><td>APTES 摩尔数 = 质量/MW</td>
			<td>4.29E-03型</td>
			<td>摩尔</td>
		</tr><tr><td>APTES 分子数/100 μL**</td>
			<td>2.57E+21</td>
			<td>分子</td>
		</tr><tr><td>APTES 尺寸 ***</td>
			<td>5.00E-01</td>
			<td>纳米</td>
		</tr><tr><td>APTES 表面积</td>
			<td>2.00E-01型</td>
			<td>纳米2</td>
		</tr><tr><td>西北表面积****</td>
			<td>2.76E+05</td>
			<td>纳米2</td>
		</tr><tr><td>0.3mg NW 中的 NW 数量 ****</td>
			<td>1.70E+10</td>
			<td>NW公司</td>
		</tr><tr><td>在 NW 周围创建一层所需的 APTES 分子数量</td>
			<td>1.38E+06</td>
			<td>APTES分子</td>
		</tr><tr><td>0.3 mg NW 所需的 APTES 分子数</td>
			<td>2.35E+16</td>
			<td>APTES分子</td>
		</tr><tr><td colspan="3">*参考编号 61</td>
		</tr><tr><td colspan="3">** 分子数 = 摩尔数*阿伏伽德罗数 （6E+23）</td>
		</tr><tr><td colspan="3">参考编号 62</td>
		</tr><tr><td colspan="3">从表 1</td>
		</tr></tbody></table>表2：计算NWs包被所需的APTES分子数。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> </td>
			<td>Y型IgG抗体</td>
			<td>西北</td>
		</tr><tr><td>一种抗体的质量数</td>
			<td>2.3E-13微克</td>
			<td>1.8E-08微克*</td>
		</tr><tr><td>一种抗体的表面积</td>
			<td>23 纳米2</td>
			<td>2.6E+05 纳米2</td>
		</tr><tr><td>基于BCA测定</td>
			<td colspan="2">每 1 毫克 0.3 毫克</td>
		</tr><tr><td>每mg NW中0.3mg抗体中抗体分子和NW的数量</td>
			<td colspan="2">~1E+15 抗体 ** 每 ~5.6E+10 NWs</td>
		</tr><tr><td>每个NW的抗体分子数</td>
			<td colspan="2">每 1 NW ~2E+04 抗体***</td>
		</tr><tr><td colspan="3">*根据表1计算</td>
		</tr><tr><td colspan="3">**0.3 mg 的抗体数量是通过将我们从 BSA 测定中获得的抗体数量 （0.3 mg） 除以 Y 形 IgG 抗体的平均分子量 （180 kDa = 3E-16 mg） 来计算的。</td>
		</tr><tr><td colspan="3">根据一个 Y 形 IgG 抗体分子的表面积 （~23 nm2） 和一个 NW 分子的表面积 （~2.6E+05 nm2），大约 1E+04 个抗体足以在 NW 上形成单层。在我们的案例中，抗体的密度是预期量的两倍。这可能与 APTES 涂层有关，该涂层提供了更多的臂，允许抗体的高附着。这种情况确保了 NW 被抗体完全覆盖，并且无论 NW 的方向如何，抗体与细胞表面抗原结合的机会都会很高。但是，如果抗体密度低于预期数量（1E+04抗体分子），则细胞与NW之间的结合几率会降低。</td>
		</tr></tbody></table>表3：纳米线上抗体数量的计算。

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Biofunctionalization of Magnetic Nanomaterials

在这项工作中，我们提供了一种用抗体对磁性纳米材料进行生物功能化的方案，用于特定的细胞靶向。例如，我们利用铁纳米线来靶向癌细胞。

磁性纳米材料的生物功能化

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

biofunctionalization-of-magnetic-nanomaterials

Research

JoVE Journal

Bioengineering

2.5K 次观看.  King Abdullah University of Science and Technology. 在这项工作中，我们提供了一种用抗体对磁性纳米材料进行生物功能化的方案，用于特定的细胞靶向。例如，我们利用铁纳米线来靶向癌细胞。

视频: Biofunctionalization of Magnetic Nanomaterials

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

磁性镊子的扭曲和扭矩的测量

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

磁共振显微镜下切除活体脑组织的代谢支持

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Methods Article

磁性纳米材料的生物功能化