纳米纤维阵列是在纳米相材料科学中心制造的，该中心是能源部科学用户设施办公室（提案ID：CNMS2019-103和CNMS2022-A-1182）。CNMS的支持是通过同行评审的提案系统授予的，并免费提供给打算发表其结果的成功申请人（http://www.cnms.ornl.gov/user/becoming_a_user.shtml）。我们感谢Kevin Lester和CNMS在纳米纤维阵列生产方面的帮助。我们感谢 John Caughmen 博士、Timothy McKnight 博士、Amber Webb 博士、Daryl Briggs 和 Travis Bee 对实验设计的重要讨论。我们感谢Adam Rondinone博士提供PECVD机器的原理图。我们感谢莱斯利·卡罗尔（Leslie Carol）的科学插图。这项工作由美国能源部生物成像科学计划、科学、生物与环境研究办公室、DE-SC0019104和美国农业部资助，电话：2021-67013-34835。JMM得到了美国农业部：国家粮食和农业研究所：农业和食品研究计划博士前奖学金2021-67034-35167的支持。

1. VACNFs生产（图2和图3）
<ol><li>用聚甲基丙烯酸甲酯（PMMA）495 A4旋转涂覆硅晶圆，在4000rpm下抗蚀，并在180°C下烘烤5分钟。 注意： 让晶圆冷却 10 秒，然后再进行下一步。</li>
	<li>旋转涂上第二层抗蚀剂（PMMA 950 A2）并在180°C下烘烤5分钟。</li>
	<li>使用电子束光刻技术在3 mm x 3 mm阵列中以指定的横向间距（间距：10μm或35μm）定义直径为300nm的催化剂点。</li>
	<li>在 30-40 mL 的 1：3 甲基异丁基酮：异丙醇 （IPA） 中显影光刻胶 1.5 分钟，然后用 IPA 冲洗并用 N2 干燥。 注意：使用明场显微镜（20倍物镜）检查点阵列。</li>
	<li>在硅蚀刻机的氧等离子体（渣渣）中暴露 6 秒，清洁残留光刻胶的晶圆。</li>
	<li>使用电子束蒸发首先沉积金属粘合层（Ti 或 Cr，10 nm），然后沉积第二层，即 Ni 催化剂（130 nm 或 150 nm）。在 Ti 或 Ni 金属沉积过程中，在 10 kV 时将电流保持在 0.2 A 以下，压力保持在 5 x 10-6 Torr 以下，并且沉积速率保持在 ~1 A/s 以进行视线沉积。</li>
	<li>使用顺序浴超声处理去除沉积在底层光刻胶层上的金属 （Ni），使 Ni 直接沉积在硅片上。这个过程称为升空。<ol><li>为此，准备3个装有丙酮的容器，并在丙酮中对晶圆进行超声处理1分钟;在室温（RT，20°C）下以35kHz的频率重复3次。用 IPA 冲洗并用 N2 晾干。 注意： 切勿让丙酮在晶圆上干燥。最后一次丙酮超声处理后，立即用IPA冲洗，然后用N2 气体干燥。如果在任何时候晶圆过早干燥（在IPA冲洗之前），则可能无法去除所有多余的金属和光刻胶，并且丙酮会留下残留物。</li>
	</ol></li>
	<li>使用扫描电子显微镜（SEM）检查催化剂形状的几何形状。要捕获 VACNF 芯片的 SEM 图像，请将载物台倾斜 30° 角并使用 1-3 kV 的电压，光束电流为 ~100 pA，工作距离为 ~5 mm。 注：催化剂应看起来像一个曲棍球（一个紧凑的圆柱体）（图4）。紧凑型圆柱体的高度应反映沉积在硅片上的镍的厚度。如果Ni点的轮廓很高，顶部看起来是凹陷的，类似于火山（图4），那么催化剂更有可能脱湿成多个Ni液滴，导致碳纳米纤维9（图4和图5）。这些问题可能是由于金属蒸发过程中的光刻胶熔化造成的，因为沉积时间长或剥离程序存在问题（图 4 和图 5）。</li>
	<li>将硅晶片切成四等份，并将其与乙炔/氨混合物一起放入直流等离子体增强化学气相沉积室（dc-PECVD）中。优化生长参数以控制纳米纤维的长度和锥度（尖端<200 nm）。<ol><li>使用 1-1.5 A 的电流和 440-560 V 的电压范围。在生长纤维时，使用氨流的预处理阶段，同时机器和基材加热到620°C。 确保在开始等离子体之前打开碳源（乙炔）10 秒。 注意：在需要优化PECVD机器运行参数的情况下，使用四分之一晶圆。为了生产长度大于 40 μm 且尖端直径小于 200 nm 的纤维，建议采用以下参数：Ni 催化剂厚度为 130 nm，使用电流为 1.75 A，生长时间为 70 min，乙炔：氨比为 45 标准立方厘米/分钟 （sccm）：100 sccm。Ni厚度为150nm，使用电流为1.5 A，生长时间为80 min，乙炔：氨比为48 sccm：100 sccm。无论催化剂厚度如何，都使用以下参数来生长 VACINF：生长温度为 620 °C，等离子体过程中的压力为 10 torr，喷头高度为 20 mm（图 6）。</li>
	</ol></li>
	<li>使用SEM以30°倾斜评估所得纤维的几何形状，加速电位为1 kV。 注意：最佳纤维将是直的且不分支的。用电子束蒸发器无法控制Ni催化剂的晶体取向。结果，会有一些纤维由于催化剂脱湿而分支9.此外，晶体取向会导致VACNF生长到不同的高度，因此很难在所有VACNF上实现均匀的高度。</li>
	<li>以 1000 rpm 的速度将一层光刻胶 （SPR955） 旋涂在纤维上 45 秒。然后使用切割锯将 1/4 晶圆切成 3 mm x 3 mm 阵列。此时将光纤存放以备后用。 注意：如果计划将纤维转移到柔性基板上，请不要执行步骤 1.11。</li>
</ol>2. 将VACNF 转接到柔性基板上（图 7 和图 8）
<ol><li>合成纤维后，将SU-8 2015光刻胶以4000rpm的速度旋涂到晶圆或四分之一晶圆上45秒。</li>
	<li>将晶圆在95°C下软烘烤3分钟。</li>
	<li>使用接触对准器，用 3 mm 3 mm 阵列图案掩模曝光晶圆，该掩模与电子束光刻以 95 mJ/cm 2 的速度定义的图案对齐。 注意： 使用接近接触模式，曝光间隙比最高光纤大 20 μm。在此过程中的这一步中，纤维可能会被打翻。</li>
	<li>在95°C下进行曝光后烘烤6分钟。</li>
	<li>将晶圆在SU-8显影剂中显影15秒，用IPA冲洗，然后用N2干燥晶圆，从上到下移动。 注意： 显影晶圆时，请确保其完全浸没。</li>
	<li>在3000rpm下旋转涂覆带有薄光刻胶保护层（SPR 955 CM 0.7）的旋涂图案晶圆45秒，并在90°C下软烘烤30秒。</li>
	<li>通过将薄氧化硅层沉积在100°C下22个循环，将薄氧化硅层沉积到晶圆（2-3nm）上。</li>
	<li>使用切割锯将晶圆切割成 3 毫米 x 3 毫米的正方形。将切割锯对准晶圆上预先存在的图案。</li>
	<li>使用SEM以30°倾斜评估所得纤维的几何形状，加速电位为3 kV。</li>
	<li>如果存放芯片以供长期使用（>1 周），请在此处停止。将薯片存放在黑暗中。</li>
	<li>要将柔性基板与刚性基板分开，请将单个芯片置于丙酮中30分钟或直到SU-8开始卷曲。</li>
	<li>用IPA洗涤SU-8薄膜（附着在刚性基材上或从刚性基材上分离）5分钟，然后用水洗涤5分钟。运输时将薯片放入带有粘垫的商业盒子中。</li>
	<li>可选：将 SU-8 薄膜的无纤维面放在水溶性胶带上或薄硅橡胶上，上面有薄聚对苯二甲酸乙二醇酯 （PET）（12.5 μm 厚）背衬。<ol><li>使用镊子转移SU-8薄膜。按压 SU-8 正方形的边缘，使其粘在胶带/硅橡胶-PET 上;这是为了避免断裂光纤。此时，VACNF薄膜已准备好立即使用。</li>
		<li>要准备硅橡胶/PET 支架，请执行以下操作：使用两部分硅橡胶套件，将两部分（弹性体和交联剂）混合在一起。接下来，切出一个正方形的PET并将其粘在一个透明的塑料盘中。在PET顶部倒入一层非常薄的硅橡胶，并在80°C下固化1-2小时。</li>
	</ol></li>
</ol>3.在工厂使用纤维在刚性基材中使用溶液的方法（将一滴溶液放置在植物表面）（图1A）
<ol><li>使用前用丙酮（100%，5分钟），IPA（100%，5分钟）和ddH2O（5分钟）的增量洗涤去除光刻胶。</li>
	<li>将要刺穿的植物组织放在坚硬的表面上作为支撑。</li>
	<li>将 1 μL 染料或 DNA 液滴 （200 ng） 放在植物组织表面。</li>
	<li>将带有刚性基板的 VACNF 芯片放置在液滴顶部，纤维的方向使它们与液滴接触。 注意： 芯片的方向可以通过晶圆的“光泽度”来确定。芯片的闪亮面有纤维，不透明的一面没有。</li>
	<li>使用镊子的扁平面，敲击芯片。使用软头记号笔标记芯片接触的植物区域。交货后取出 VACNF 芯片。 注意： 敲击时施加的力的大小会因所使用的植物组织类型而异。建议在进行纤维穿刺之前练习攻丝。避免损伤植物组织。当人们可以在植物组织中看到VACNF芯片的轮廓时，损伤是显而易见的。</li>
	<li>对照重复步骤3.1-3.5（+染料/DNA，-纤维;-染料/DNA，+纤维;和-染料/DNA，-纤维）。-纤维是芯片的无纤维面。</li>
	<li>如果需要，将完整的植物或切除的植物器官储存在潮湿的室内，在长时间的条件下（16小时光照，8小时黑暗）。对于切除的器官，使用塑料培养皿和湿纸巾。</li>
</ol>4. 片上方法（将一滴溶液放在VACNF芯片上），刚性基板（图1B）
<ol><li>使用前用丙酮（100%，5分钟），IPA（100%，5分钟）和ddH2O（5分钟）的增量洗涤去除光刻胶。</li>
	<li>将 1 μL 染料或质粒 DNA （200 ng） 滴入具有刚性基板的 VACNF 芯片的纤维侧。确保将液滴放在芯片的中心并覆盖几根纤维。让液滴干燥15分钟。 注意： 芯片的方向可以通过晶圆的“光泽度”来确定。芯片的闪亮面有纤维，不透明的一面没有。</li>
	<li>处理叶子或其他切除器官时，将它们放在坚硬的表面上。使用完整的植物时，在应用VACNF的器官下方放置一个坚硬的表面。</li>
	<li>在15分钟的干燥步骤之后，放置VACNF芯片，使纤维侧与植物组织接触。用镊子的后端敲击芯片。 注意： 敲击时施加的力的大小会因所使用的植物组织类型而异。建议练习敲击芯片。</li>
	<li>重复工厂方法的步骤 3.6-3.7。</li>
</ol>5. 使用片上方法将 SU-8 薄膜中的 VACNF 应用于植物组织（图 1C）
<ol><li>将 1 μL 染料或 DNA 液滴 （200 ng） 放在 SU-8 薄膜的纤维侧，并使其干燥 10 分钟。确保将液滴放在芯片的中心。 注意： 根据所使用的基材，干燥时间会有所不同。</li>
	<li>使用一把锋利的镊子，将VACNF薄膜放在植物表面。 注意：SU-8 胶片在空气中放置的时间越长，胶片就越脆。为限制丢失 SU-8 胶片的风险，请确保所有工厂/样品和设备都靠近 SU-8 胶片。</li>
	<li>轻轻地将一个小化妆器卷在 VACNF 薄膜上。用软头记号笔标记放置柔性基材的区域。使用胶带从植物表面去除柔性基质。 注意： 滚动化妆涂抹器时施加的力的大小会因使用的植物组织而异。在进行生物分子或染料递送之前练习应用 VACNF 薄膜。当人们可以看到植物组织中VACNF薄膜的轮廓时，对植物组织的可见损伤是显而易见的。</li>
	<li>对照重复步骤5.1-5.3，并按照工厂方法的步骤3.7所述储存植物。</li>
</ol>6. 所有输送方式的显微镜和图像分析
<ol><li>使用共聚焦显微镜对样品进行成像，使用适合输送的荧光探针/报告基因的发射和激发波长。 注意：瞬时转化所需的时间因植物种类和传递的标记物而异。例如，在拟南芥中48小时后检测到荧光标记物的表达，而在生菜叶中则在96小时后检测到荧光标记物的表达7。</li>
	<li>成像时，尽量聚焦在纤维脱落的区域。纤维将具有不同的方向。输送的成功与否并不取决于断裂纤维的外观。 注意：由于 PECVD18 中纤维形成导致氮化硅层的形成，纤维将具有最常见的激发/发射设置。</li>
	<li>每个样本至少捕获 5 张图像。产生的信号会有所不同。</li>
	<li>使用 ImageJ19 测量 20 μm x 20 μm 共聚焦图像区域7 中的荧光值作为总荧光（积分密度）。</li>
</ol>

使用碳纳米纤维阵列将生物分子和染料高效递送到植物中

<ol>
	<li>Canto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+expression+systems+in+plants:+potentialities+and+constraints.">Transient expression systems in plants: potentialities and constraints.</a> Advances in Experimental Medicine and Biology. 896, 287-301 (2016).</li><li>Gou, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+protoplast+transient+expression+system+for+gene+functional+studies+in+strawberry+(Fragaria+vesca).">Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca).</a> Plant Cell, Tissue, and Organ Culture. 141, 41-53 (2020).</li><li>Baltes, N. J., Gil-Humanes, J., Voytas, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+and+agriculture:+Opportunities+and+challenges.">Genome engineering and agriculture: Opportunities and challenges.</a> Progress in Molecular Biology and Translational Science. 149, 1-26 (2017).</li><li>Ren, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+protoplast+isolation+and+transient+expression+system+for+functional+characterization+of+flowering+related+genes+in+Cymbidium+orchids.">Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in Cymbidium orchids.</a> International Journal of Molecular Sciences. 21 (7), 2264 (2020).</li><li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanovehicles+for+plant+modifications+towards+pest-and+disease-resistance+traits.">Nanovehicles for plant modifications towards pest-and disease-resistance traits.</a> Trends in Plant Science. 25 (2), 198-212 (2020).</li><li>Davern, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+nanofiber+arrays:+a+novel+tool+for+microdelivery+of+biomolecules+to+plants.">Carbon nanofiber arrays: a novel tool for microdelivery of biomolecules to plants.</a> PLoS One. 11 (4), e0153621 (2016).</li><li>Morgan, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+broadly+applicable+method+for+transient+transformation+of+plants+using+vertically+aligned+carbon+nanofiber+arrays.">An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays.</a> Frontiers in Plant Science. 13, 1051340 (2022).</li><li>Melechko, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertically+aligned+carbon+nanofibers+and+related+structures:+Controlled+synthesis+and+directed+assembly.">Vertically aligned carbon nanofibers and related structures: Controlled synthesis and directed assembly.</a> Journal of Physics D: Applied Physics. 97, 041301 (2005).</li><li>Melechko, A. V., Desikan, R., McKnight, T. E., Klein, K. L., Rack, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+vertically+aligned+carbon+nanofibres+for+interfacing+with+live+systems.">Synthesis of vertically aligned carbon nanofibres for interfacing with live systems.</a> Journal of Physics D: Applied Physics. 42 (19), 193001 (2009).</li><li>Nelson-Fitzpatrick, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Novel Materials for the Design of Cantilever Transducers [dissertation]. , (2011).</li><li>Fletcher, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+flexible+arrays+of+vertically+aligned+carbon+nanofiber+electrodes+to+temperature-sensitive+substrates.">Transfer of flexible arrays of vertically aligned carbon nanofiber electrodes to temperature-sensitive substrates.</a> Advanced Materials. 18 (13), 1689-1694 (2006).</li><li>Keller, S., Blagoi, G., Lillemose, M., Haefliger, D., Boisen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+thin+SU-8+films.">Processing of thin SU-8 films.</a> Journal of Micromechanics and Microengineering. 18 (12), 125020 (2008).</li><li>Wouters, K., Puers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusing+and+swelling+in+SU-8:+insight+in+material+properties+and+processing.">Diffusing and swelling in SU-8: insight in material properties and processing.</a> Journal of Micromechanics and Microengineering. 20 (9), 095013 (2010).</li><li>Jamal, M., Zarafshar, A. M., Gracias, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentially+photo-crosslinked+polymers+enable+self-assembling+microfluidics.">Differentially photo-crosslinked polymers enable self-assembling microfluidics.</a> Nature Communications. 2, 527 (2011).</li><li>Williams, R., Goodman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wetting+of+thin+layers+of+SiO2+by+water.">Wetting of thin layers of SiO2 by water.</a> Applied Physics Letters. 25 (10), 531-532 (1974).</li><li>Kundu, A., Nogueira Campos, M. G., Santra, S., Rajaraman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+vascular+delivery+of+agrochemicals+with+micromilled+microneedles+(&#181;MMNs).">Precision vascular delivery of agrochemicals with micromilled microneedles (&#181;MMNs).</a> Scientific Reports. 9, 14008 (2019).</li><li>Acanda, Y., Welker, S., Orbovi&#263;, V., Levy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+agroinfiltration+method+for+transient+gene+expression+in+Citrus.">A simple and efficient agroinfiltration method for transient gene expression in Citrus.</a> Plant Cell Reports. 40 (7), 1171-1179 (2021).</li><li>Pearce, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+properties+of+SiNx+coatings+as+stable+fluorescent+markers+on+vertically+aligned+carbon+nanofibers.">Synthesis and properties of SiNx coatings as stable fluorescent markers on vertically aligned carbon nanofibers.</a> AIMS Materials Science. 1 (2), 87-102 (2014).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Crafts, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+in+plants.">Translocation in plants.</a> Plant Physiology. 13 (4), 791 (1938).</li><li>Martens, H. J., Hansen, M., Schulz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caged+probes:+a+novel+tool+in+studying+symplasmic+transport+in+plant+tissues.">Caged probes: a novel tool in studying symplasmic transport in plant tissues.</a> Protoplasma. 223, 63-66 (2004).</li><li>Liu, J., Essner, J., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+supercapacitor+based+on+coaxially+coated+manganese+oxide+on+vertically+aligned+carbon+nanofiber+arrays.">Hybrid supercapacitor based on coaxially coated manganese oxide on vertically aligned carbon nanofiber arrays.</a> Chemistry of Materials. 22 (17), 5022-5030 (2010).</li><li>Saleem, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+and+cost-effective+growth+of+vertically+aligned+carbon+nanofibers+using+spin-coated+polymer-stabilized+palladium+nanocatalysts.">Low temperature and cost-effective growth of vertically aligned carbon nanofibers using spin-coated polymer-stabilized palladium nanocatalysts.</a> Science and Technology of Advanced Materials. 16, 015007 (2015).</li><li>Merkulov, V. I., Lowndes, D. H., Wei, Y. Y., Eres, G., Voelkl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+growth+of+individual+and+multiple+vertically+aligned+carbon+nanofibers.">Patterned growth of individual and multiple vertically aligned carbon nanofibers.</a> Applied Physics Letters. 76 (24), 3555-3557 (2000).</li><li>Retterer, S. T., Melechko, A., Hensley, D. K., Simpson, M. L., Doktycz, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+control+of+catalyst+nanoparticles+for+the+synthesis+of+high+density+carbon+nanofiber+arrays.">Positional control of catalyst nanoparticles for the synthesis of high density carbon nanofiber arrays.</a> Carbon. 46 (11), 1378-1383 (2008).</li><li>Rooks, M. J., Wind, S., McEuen, P., Prober, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+30-nm-scale+structures+for+electron+transport+studies+using+a+polymethylmethacrylate+bilayer+resist.">Fabrication of 30-nm-scale structures for electron transport studies using a polymethylmethacrylate bilayer resist.</a> Journal of Vacuum Science &amp; Technology B: Microelectronics Processing and Phenomena. 5 (1), 318-321 (1987).</li><li>Lee, G., Sohn, D. K., Seok, S. H., Ko, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+hole+density+variation+in+the+PECVD+reactor+showerhead+on+the+deposition+of+amorphous+carbon+layer.">The effect of hole density variation in the PECVD reactor showerhead on the deposition of amorphous carbon layer.</a> Vacuum. 163, 37-44 (2019).</li><li>Fowlkes, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+catalyst+particle+crystallographic+orientation+in+vertically+aligned+carbon+nanofiber+synthesis.">Control of catalyst particle crystallographic orientation in vertically aligned carbon nanofiber synthesis.</a> Carbon. 44 (8), 1503-1510 (2006).</li></ol>

作者没有什么可透露的。

在本文中，我们提出了构建垂直排列的碳纳米纤维阵列的方法，将纤维转移到柔性基质上，并将纤维在刚性或柔性基质中应用于植物，用于将生物分子或染料递送给植物。我们描述了两种通用方法，即片上方法和工厂上方法，用于沉积引入的材料，并展示了在刚性基板上的纤维以及使用VACNF薄膜的片上方法的成功结果。这些纤维的应用在实践和理论上比传统的植物转化方法（粒子轰击、通过PEG或电穿孔的原生质体转化）更简单，可用于对农杆菌介导的转化不敏感的植物。然而，只有少数细胞被转化。
垂直排列的碳纳米纤维是在橡树岭国家实验室纳米相材料科学中心通过其用户程序生产的。用户可以申请使用该设施生产VACNF。或者，VACNF芯片可以在洁净室中使用带有碳源的直流等离子体增强化学气相沉积机生产22,23。使用所描述的方法，有几个步骤对于纤维的生产、纤维转移和 VACNF 芯片/薄膜的应用至关重要。要使纤维应用起作用，纤维必须是直的，并且在尖端具有 <200 nm 的锥形直径才能成功输送到植物细胞中 6,7（图 3）。为了制造特定尺寸和间距的碳纳米纤维，可以改变多种参数，包括网点大小、横向间距和沉积的催化剂量。为了选择用于碳纳米纤维生产的最佳网点尺寸，从各种网点尺寸生长纤维（如图5所示）。我们发现直径为 300 nm 的光纤效果最好，因此选择了这种网点尺寸（图 5）。在找到正确的参数后，我们希望使用具有 >50% 光纤且具有理想几何形状（直且尖端直径 <200 nm）的芯片。为了检查纤维的几何形状，我们使用扫描电子显微镜对VACNF芯片/薄膜样品的随机视场进行成像。
此外，纤维必须具有一定的最小长度才能在植物细胞内实现输送。生产不同长度纤维的重要性在于，可以使用更长的纤维来穿透更深的组织层。较长的纤维（长度>40 μm）对于柔性薄膜至关重要，因为纤维转印的工作原理是将纤维从其基部折断，并且需要在纤维顶部分层SU-8。用于该协议的SU-8层的工作厚度为20-35μm。在各种植物（弯曲或扁平）的表皮内完成输送所需的最小高度为 10-15 μm 6,7。因此，长度为 >40 μm 的纤维是 VACNF 薄膜所必需的。在生产碳纳米纤维时，需要考虑几个不同的参数：催化剂材料、催化剂几何形状、催化剂材料的厚度以及PECVD腔内的条件（气体比、压力、温度、电流、喷淋头高度和生长时间）8,9,24,25。为了生产Morgan等7和Davern等6使用的长度超过25 μm的碳纳米纤维，我们增加了Ni催化剂的用量，改变了乙炔氨的比例，并增加了电流和生长时间。此外，我们更加关注催化剂材料的几何形状。为了产生高大的直纤维，沉积的催化剂需要具有曲棍球形状，而不是类似于火山的形状（图4）。火山结构是由升空后光刻胶的残余物产生的。为了防止火山的形成，在电子束光刻过程中使用双层PMMA来形成底切26。底切有助于沉积金属催化剂的剥离（图2）。催化剂的厚层对于高VACNFs的生长很重要。Merkulova等人24研究了VACNFs的形态。VACNF的垂直排列是由于Ni催化剂尖端型生长和垂直于衬底的直流电位排列（图6）。喷淋头描述了PECVD反应器的几何形状（图6），并作为电场电位的来源27。
为了用电子束光刻技术定义催化剂点阵列，我们应用了电子束光刻胶（聚甲基丙烯酸甲酯），然后使用电子束在光刻胶上以特定形状和特定位置在光刻胶上打小孔。将所需直径的孔放置在具有定义间距（间距）的规则网格上，并在将基板加载到机器之前将指定所需图案的文件加载到电子束光刻工具中。除了纤维高度外，纤维转移成功的另一个关键参数是在丙酮浴中花费的时间。VACNF薄膜需要在丙酮浴中放置足够长的时间，以使其边缘开始卷曲;如果它们在丙酮浴中放置的时间太短，它们就更难从芯片上取下，并且可能会破裂。芯片越老，它们在丙酮浴中停留的时间就越长。在丙酮浴之后，将薄膜/芯片置于异丙醇和水中以除去接触丙酮并去除纤维上的保护性光刻胶。
为了进行旋涂，将晶圆或晶圆片放置在旋涂机的真空吸盘上，并使用旋涂机的测试功能验证晶圆的中心位置。将光刻胶的小水坑（直径~2.5 cm）施加到晶圆的中心并纺丝（3000 rpm，持续45秒） 图8中包括纺涂前后的纤维图像，显示了纤维几何形状（高度、方向和间距）的保存情况。纤维的存在会导致光刻胶在纤维底部涌出，并导致层比预期的更厚。VACNF生长后的旋涂已被其他组探索11,18。
该工艺中另一个至关重要的步骤是确保对VACNF芯片/薄膜施加适量的力。输送机制依赖于纤维通过镊子在刚性基质 6,7 上敲击的冲击力或在柔性基质上使用迷你化妆涂抹器滚动在细胞壁上进行小穿刺。纤维可能会也可能不会断裂并保持嵌入植物细胞6,7 而不会影响结果，但结合检查染料吸收和组织损伤的练习对于获得正确的压力是必要的。此外，在使用 VACNF 芯片/薄膜递送 DNA 后选择适当的成像时间点也很重要，因为可检测表达的时间因植物物种和递送载体类型而异7（图 16）。
尽管这种方法广泛适用于植物，但它有一些局限性。例如，在VACNF薄膜中添加一层薄薄的氧化硅并不总是导致薄膜完全亲水，因为在SU-8的顶部添加了光刻胶保护层。如果这个问题成为现实，则可以将更厚的氧化硅层应用于VACNF。为了测试薄膜是疏水的还是亲水的，可以将它们放入水中。如果薄膜下沉，它们是亲水的，如果它们漂浮，它们是疏水的。此外，生产的纤维批次之间可能存在差异。在直流-PECVD机器中生长光纤时，有几个参数可以改变;该协议中描述的是两种不同量的Ni催化剂的一组参数。此外，Ni催化剂的晶体取向无法控制28 ，一些支化将不可避免地导致纤维的产生。
虽然我们在本文中展示了使用刚性和柔性底物将荧光素染料和 DNA 递送至植物细胞，但该方法应广泛适用于其他生物分子和基因修饰方法，例如，用于苹果或其他水果等植物系统的 RNAi 沉默，在这些系统中，产生稳定的转基因系需要数年时间。此外，这些纤维还可用于递送遗传编辑材料或用于植物的稳定转化。

植物转化（瞬时和稳定）尚未在所有植物组织和物种中广泛实现。植物的瞬时转化是质粒中编码的基因暂时导入植物但不能稳定地掺入基因组的过程。使用颗粒轰击、农杆菌、电穿孔或聚乙二醇处理原生质体的传统方法速度慢或可能很麻烦。此外，它们并不适用于所有植物物种1、2、3、4。使用纳米材料进行 DNA 递送是一个新兴领域，仍处于起步阶段5.纳米材料，特别是碳纳米纤维，也已成功用于将蛋白质、葡聚糖和染料输送到植物叶子，而不会引起伤口反应6。这项工作的目标是提供一种详细的协议，用于使用一种纳米材料，即碳纳米纤维，将生物分子或染料输送到植物。在这里，我们专注于DNA作为首选的生物分子，它允许各种植物器官中的细胞瞬时转化。
此前，Morgan等人7 展示了使用附着在刚性硅衬底上的碳纳米纤维来瞬时转化生菜， 本氏猪笼草 和杨树的叶子，以及拟南芥的叶子和根。尽管在各种器官上都取得了成功的转化，但纤维更难应用于具有弯曲表面的植物组织，例如根或果实。我们推断，纳米纤维的柔性背衬可能会通过更好地符合器官的形状来提高其输送效率。
在此，我们详细介绍了用于制造和设计垂直排列的碳纳米纤维、将 VACNF 转移到柔性基质以及将 VACNF 应用于植物的刚性和柔性基质以递送生物分子和染料的方法。采用直流催化等离子体增强化学气相沉积（dc C-PECVD）和Ni催化剂制备了碳纳米纤维。如Melechko等人所述，使用电子束光刻、金属蒸发和剥离过程的组合来控制Ni催化剂点的位置、直径和高度8,9。使用双层电子束光刻胶，可以在基底上沉积较厚的镍催化剂，以产生更长的纤维10。纤维从刚性基板到柔性基板的转移基于对Fletcher等人11中描述的方法的修改，目前的方法放弃了使用无定形碳层或牺牲光刻胶层。SU-8 通过纤维转移的升空是通过利用 SU-812,13,14 烘烤不足和曝光不足产生的固有拉伸应力来实现的。SU-8 是一种复杂的聚合物，具有天然疏水性，这使得其难以用于促进 DNA 递送。为了抵消SU-8的疏水性，我们在纤维嵌入SU-8后，通过原子层沉积15施加一层薄薄的氧化硅。在刚性基材上应用纤维进行生物分子/染料递送利用了 Davern 等人 6 中描述的镊子攻丝的冲击力以及 Morgan 等人 7 中描述的植物上和芯片上方法。柔性VACNF薄膜通过首先将薄膜上的DNA或染料液滴半干燥，如Morgan等人7的芯片方法，然后使用小型化妆涂抹器16,17在弯曲的植物表面上滚动薄膜，将柔性VACNF薄膜应用于弯曲的植物表面。图 1 描述了将刚性和柔性基材中的纤维应用于植物的各种方法。

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			<td>13&#34; x 13&#34; White 1/4-fold heavy duty Brawny industrial shop towel 70Ct</td>
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			<td>690535</td>
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			<td>Fischer Scientific</td>
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			<td>4&#34; Lid</td>
			<td>Entegris</td>
			<td>H22-401-0615</td>
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			<td>4&#34; tray</td>
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			<td>H22-40-0615</td>
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			<td>Acetone</td>
			<td>Fischer Scientific</td>
			<td>A18-4</td>
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			<td>Acetone used in the cleanroom at ORNL</td>
			<td>JT Baker</td>
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			<td>Apples</td>
			<td>Grocery store</td>
			<td>No product number</td>
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			<td>Arabidopsis thaliana&#160;</td>
			<td>Seeds of accession Columbia from the laboratory of Professor Jean Greenberg at the University of Chicago</td>
			<td>No product number</td>
			<td></td>
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			<td>Carbon direct current plasma enhanced chemical vapor deposition machine</td>
			<td>Oak Ridge National Laboratory&#160;</td>
			<td>Custom-built</td>
			<td></td>
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			<td>Cobham Green lettuce&#160;</td>
			<td>Seeds from the laboratory of Professor Richard Michelmore at the University of California, Davis</td>
			<td>No product number</td>
			<td>Butterhead lettuce</td>
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			<td>Fluorescein dye</td>
			<td>Sigma Aldrich</td>
			<td>F2456-2.5G</td>
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			<td>Gel-Pak</td>
			<td>&#160;AD-23C-00-X4</td>
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			<td>Heidelberg DWL 66 direct-write lithography tool&#160;</td>
			<td>Heidelberg</td>
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			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institues of Health&#160;</td>
			<td>No product number</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproponal (IPA) used in the cleanroom at ORNL</td>
			<td>Doe and Ingalls</td>
			<td>CMOS Grade 9079-05</td>
			<td></td>
		</tr>
		<tr>
			<td>JEOL 9300FS 100kV electron beam lithography system</td>
			<td>JEOL</td>
			<td>8100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>Kimberly-Clark Professional 34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Kord-Valmark disposable polystyrene petri dish</td>
			<td>VWR</td>
			<td>11019-554</td>
			<td></td>
		</tr>
		<tr>
			<td>Layout Editor&#160;</td>
			<td>&#160;juspertor GmbH</td>
			<td>No product number&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 710 confocal microscope</td>
			<td>Zeiss</td>
			<td>No product number</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 800 confocal microscope</td>
			<td>Zeiss</td>
			<td>No product number</td>
			<td></td>
		</tr>
		<tr>
			<td>Make-up applicator&#160;</td>
			<td>Amazon</td>
			<td>G2PLUS</td>
			<td>500 PCS Disposable Micro Applicators Brush for Makeup and Personal Care (Head Diameter: 1.5 mm)- 5 x 100 PCS</td>
		</tr>
		<tr>
			<td>Merlin field emission scanning electron microscope</td>
			<td>Zeiss</td>
			<td>Merlin</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBK/IPA&#160; (methyl isobutyl ketone/isopropanol) (1:3)</td>
			<td>Microchem</td>
			<td>M089025</td>
			<td></td>
		</tr>
		<tr>
			<td>Onions</td>
			<td>Grocery store</td>
			<td>No product number</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxford FlexAl atomic layer deposition</td>
			<td>Oxford</td>
			<td>FlexAl</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 495 A4</td>
			<td>Microchem</td>
			<td>M130004</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 950 A4</td>
			<td>Microchem</td>
			<td>M230004</td>
			<td>Can dilute down to A2</td>
		</tr>
		<tr>
			<td>Polyethylene terephthalate (PET)</td>
			<td>Amazon</td>
			<td>KS-6304-21-11</td>
			<td>Type D Clear PET (Polyester) Sheet .0005&#34; Thick x 27&#34; Width x 10 Ft Length 1 pc</td>
		</tr>
		<tr>
			<td>Precision tweezers</td>
			<td>Aven Inc.&#160;</td>
			<td>18032TT</td>
			<td></td>
		</tr>
		<tr>
			<td>pUBQ10:YFP-GW</td>
			<td>Arabidopsis Biological Resource Center</td>
			<td>CD3-1948</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon etcher (used for descum)&#160;</td>
			<td>Oxford</td>
			<td>Plasmalab</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon rubber kit</td>
			<td>Smooth-On Inc</td>
			<td>Ecoflex 00-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>Pure Wafer</td>
			<td>4N0.001-.005SSP-INV</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Brewer Sciences</td>
			<td>Model 100CB</td>
			<td></td>
		</tr>
		<tr>
			<td>SPR 955cm 0.7&#160;</td>
			<td>Megaposit</td>
			<td>10018314</td>
			<td></td>
		</tr>
		<tr>
			<td>Strawberries</td>
			<td>Grocery store</td>
			<td>No product number</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2015</td>
			<td>Microchem</td>
			<td>SU-8 2000 Series</td>
			<td>Toxic. Handle with care. Wear chemical goggles, chemical gloves and suitable protective clothing when handling SU-8 2000 resists. Do not get into eyes, or onto skin or clothing.</td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>Microchem</td>
			<td>SU-8 2000 Series</td>
			<td>Handle with care. Wear chemical goggles, chemical gloves and suitable protective clothing when handling SU-8 2000 resists. Do not get into eyes, or onto skin or clothing.</td>
		</tr>
		<tr>
			<td>Suss MicroTec contact aligner</td>
			<td>Suss MicroTec</td>
			<td>MA6/BA6</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microscope</td>
			<td>Phenom XL</td>
			<td>used for checking Ni catalysts after metal deposition</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermionics VE-240 e-beam evaporator</td>
			<td>Thermionics</td>
			<td>VE-240</td>
			<td></td>
		</tr>
	</tbody>
</table>

using vertically aligned carbon nanofiber arrays on rigid or flexible substrates for delivery of biomolecules and dyes to plants

将生物分子和不透水染料输送到完整的植物中是一项重大挑战。纳米材料是将DNA递送至植物的新兴工具。尽管这些新工具令人兴奋，但它们尚未得到广泛应用。在刚性基材（背衬）上制造的纳米材料特别难以成功应用于弯曲的植物结构。本研究描述了微加工垂直排列的碳纳米纤维阵列并将其从刚性基板转移到柔性基板的过程。我们详细介绍并演示了这些纤维（在刚性或柔性基材上）如何用于瞬时转化或染料（例如荧光素）递送至植物。我们展示了如何将 VACNF 从刚性硅衬底转移到柔性 SU-8 环氧树脂衬底以形成柔性 VACNF 阵列。为了克服SU-8的疏水性，柔性薄膜中的纤维涂有一层薄薄的氧化硅层（2-3nm）。为了将这些纤维输送到弯曲的植物器官，我们将 1 μL 染料或 DNA 溶液液滴沉积在 VACNF 薄膜的纤维侧，等待 10 分钟，将薄膜放在植物器官上，并使用滚动运动的拭子将纤维驱动到植物细胞中。通过这种方法，我们已经在具有曲面的植物器官中实现了染料和DNA的递送。

Watch this Scientific Journal Video about Unlocking Plant Transformation by Innovating with Carbon Nanofiber Arrays at JoVE.com

作者聚焦：通过使用碳纳米纤维阵列进行创新来解锁工厂转型 

在这里，我们描述了微加工垂直取向碳纳米纤维 （VACNF）、将 VACNF 转移到柔性基材上以及将 VACNF 应用于植物的刚性和柔性基材以进行生物分子和染料递送的方法。

在刚性或柔性基材上使用垂直排列的碳纳米纤维阵列，将生物分子和染料输送到植物

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to ...

我们正试图展示一种使用碳纳米纤维阵列将染料或DNA递送到植物中的通用方法。许多植物对传统的转化方法很顽固。我们的方法可以在柔性刚性基板上使用碳纳米纤维阵列来解决这个问题。我们的技术快速且用户友好。它几乎可以应用于任何植物物种或植物器官。它还可用于递送其他试剂，如染料和蛋白质。

using-vertically-aligned-carbon-nanofiber-arrays-on-rigid-or-flexible

Research

JoVE Journal

Bioengineering

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

1.3K 次观看.  The University of Chicago. 我们正试图展示一种使用碳纳米纤维阵列将染料或DNA递送到植物中的通用方法。许多植物对传统的转化方法很顽固。我们的方法可以在柔性刚性基板上使用碳纳米纤维阵列来解决这个问题。我们的技术快速且用户友好。它几乎可以应用于任何植物物种或植物器官。它还可用于递送其他试剂，如染料和蛋白质。

视频: 作者聚焦：通过使用碳纳米纤维阵列进行创新来解锁工厂转型 

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

柔性神经微探针的使用附加与Biodissolvable胶粘剂刚性加劲肋插入

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

科研

生物工程

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案:二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

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.

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

纤维素碳纤维溶胶单向冷冻干燥制备 Microhoneycomb 石柱: 方法和外延

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...