这项工作得到了澳大利亚研究理事会 （ARC） 发现计划 （DP150101065） 和 ARC 低级最终用户分析集成设备研究中心 （理想） （IH150100028） 的支持。

1. tBLM 电极制备
<ol><li>准备第一层单层涂层<ol>
<li>浸入一个新鲜溅出的金色图案电极显微镜滑动在乙烷溶液中，该溶液由 3 mM 1:9 比例的苯二甲醚- 四乙二醇-OH"垫片"分子（苯二甲醚由四个氧乙二醇垫片组成， 终止与OH组）和苯二硫化物（四乙酰二醇）n=2 C20-植物基"系"分子。这将创建可以固定双层的第一层涂层。 注:金电极通过蒸发100纳米，99.9995%黄金（5n5金）薄膜到定制25毫米x75毫米聚碳酸酯幻灯片20。</li>
		<li>在室温下用第一层孵化电极至少1小时。</li>
		<li>通过浸入大量超过30的纯乙醇来冲洗金电极。</li>
		<li>使用金电极滑梯与第一个单层直接为下一步或存储在一个充满纯乙醇的罐子。</li>
		<li>注意:为了确保第一层的完整性，请尽量减少与幻灯片黄金部分的任何直接接触</li>
	</ol></li>
	<li>组装第一个单层涂层幻灯片<ol>
<li>小心地从容器中取出一个胶质金电极滑梯，使用钳子，确保不会与 tBLM 形成的图案区域接触。 注意:注意识别存放黄金的幻灯片的侧面。</li>
		<li>空气干燥滑梯1 -2分钟，以消除任何残留乙醇。</li>
		<li>将金电极置于干燥表面，确保金电极正确定位，图案金面朝上。</li>
		<li>将透明胶粘层盖从薄层压板中剥离，并放置在 6 个通道上以定义每个井。</li>
		<li>使用压力辊在滑梯和透明粘合层之间释放任何空气，如图1A所示。 注:此步骤所需的时间需要由研究人员优化。在此协议中，时间范围为 2-3 分钟。</li>
		<li>尽快（在 1-2 分钟内）将第二个脂质双层引入组装的第一层涂层电极进行自组装，以避免损坏第一层。</li>
	</ol></li>
	<li>准备第二脂质双层<ol>
<li>在六口井滑梯中的第一口井中加入 6 μL 的 3 mM 脂质。不要让微管尖的边缘接触金表面，金表面会损坏电极上的系绳化学。 注:本工作使用的脂质混合物包括3 mM 70% zwitterionic C20 二苯二甲醚-甘油磷酸酯 （DPEPC） 和 30% C20 二苯二甲醚乙醚脂质 （GDPE） 与 3 mM 胆固醇-PEG-Biotin 混合在 50:1 摩尔比。</li>
		<li>将 6 μL 的脂质混合物引入其他油井，每口油井之间的间隙为 10。</li>
		<li>在室温下将每口井孵育整整2分钟，然后用PBS等缓冲器将脂质混合物交换到电极上。空间的时间添加和缓冲交换10秒分开，所以每口井孵育与脂质正好2分钟每个。</li>
		<li>再洗 3 次，用 50 μL 的 PBS 缓冲器 （pH 7.0） 清洗 3 次。请务必在电极上随时留下 50 μL 的缓冲。不要让电极干燥。 注:用这种方式（溶剂交换方法）将乙醇溶剂与溶剂分离，通过系绳化学快速形成固定在金电极上的单脂双层。</li>
	</ol></li>
	<li>使用电气阻抗光谱 （EIS） 测量测试 tBLM 形成<ol>
<li>将准备好的电极滑动插入交流阻抗光谱仪（例如 Tethapod）。确保光谱仪通过 USB 端口连接到运行软件的计算机。</li>
		<li>打开软件，单击设置并打开硬件。</li>
		<li>设置硬件设置，使用 25 mV 峰值到峰值交流激发。</li>
		<li>将频率设置在 0.1 到 10，000 Hz 之间，每十年设置两个步骤，用于快速阻抗措施按 OK。</li>
		<li>单击设置菜单并打开模型。</li>
		<li>使用等效电路模型，将系绳金电极描述为连续的恒定相位元件，电阻器描述电解质缓冲器和并行电阻电容器网络来描述脂质双层，并按确定。</li>
		<li>按下"开始"按钮，开始实时测量膜电容 （Cm）和膜传导 （Gm）。Cm值的典型 tBLM 应在 12.5 nF 到 15.5 nF 的范围内为 10% 系绳化学21，22。</li>
		<li>运行协议并完成实验后，保存数据。</li>
		<li>用下一口井重复测量。</li>
	</ol></li>
</ol>2.激光照射
<ol><li>实验设置 注:为每个 tBLM 单独设置定制系统。<ol>
<li>在防光箱中执行实验，以最大限度地减少激光危险。</li>
		<li>使用光学表设置实验以减少不必要的振动。</li>
		<li>将阻抗读器（黄金幻灯片连接在哪里）放在 XYZ 舞台上，并提升使其位于激光源的路径上。</li>
		<li>使用粗细聚焦微观齿轮控制激光源的高度，达到适当的精度。</li>
		<li>沿着电极滑梯的纵向轴瞄准激光路径。 注意:始终佩戴合适的激光安全眼镜，保持良好的激光安全规程。</li>
		<li>在开始实验之前，允许选定的调谐激光器稳定下来。 注:实验设置的示意图在图2A中说明。</li>
	</ol>
</li>
	<li>激光和金电极的对齐 注:在开始之前，请始终使用功率计评估激光功率输出，以确保仅向 tBLM 输送非常低的功率。<ol>
<li>调整激光路径或电极的角度，使激光穿过覆盖电极的液体，在金表面均匀可见。</li>
		<li>通过在观察膜传导变化的同时，通过微调提高或降低激光束源来调整每个实验的激光束光位置。</li>
		<li>在没有电导变化时，锁定旋钮以固定激光路径的位置。 注:当激光与底层金电极相互作用时，将产生增加的膜传导值。因此，调整激光路径非常重要，因此不可能进行这种相互作用。</li>
	</ol>
</li>
	<li>样品制备<ol>
<li>准备激光束光对齐（膜传导没有变化），如图2所示，位置3。</li>
		<li>将感兴趣的 GNP（功能化或裸露）添加到 PBS 缓冲器中，当激光关闭时，TBLM 会沉浸其中。</li>
		<li>轻轻混合 TBLM 周围的 PBS 缓冲器三次，小心不要触摸电极。</li>
		<li>在室温下孵育5-10分钟。</li>
		<li>将激光打开以照射样品，使用图2、位置3中正确的对齐激光束光位置。</li>
		<li>使用 GNP 大小、形状和浓度与激光波长的适当组合。 注:设置波长的激光束应耦合到相应的GNP质子共振频率。</li>
		<li>记录连续测量电流（实时测量）。</li>
		<li>执行步骤 2.2.1 - 2.3.7，省略控制实验的 GNP 添加。</li>
	</ol>
</li>
</ol>3.统计数据分析和介绍
<ol><li>将数据导出到电子表格中。</li>
	<li>提取膜传导参数与时间。</li>
	<li>在以正确位置设置激光束灯后和引入 GNP 之前使用记录的数据。</li>
	<li>通过将测量的膜导电度除以基线膜导电性来使数据正常化。 注:这证实了引入辐照GNP引起的膜传导值的相对变化。</li>
	<li>将数据作为时间图（x轴）与规范化膜传导（y轴）。</li>
</ol>4.预测从辐照纳米粒子（热预测模型）中TBLM中产生的局部热量量
<ol><li>根据多布罗夫斯基23日解决辐射转移问题，以计算辐照纳米粒子溶液中的吸收辐射功率。</li>
	<li>通过将吸收辐射产生的热源纳入能量方程来计算热量生成。 注:有关辐照纳米粒子和纳米电极界面的tBLM中热生成数值分析的详细解释，请参阅19。</li>
</ol>

<ol>
	<li>Her, S., Jaffray, D. A., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+for+applications+in+cancer+radiotherapy:+Mechanisms+and+recent+advancements.">Gold nanoparticles for applications in cancer radiotherapy: Mechanisms and recent advancements.</a> Advanced Drug Delivery Reviews. 109, 84-101 (2017).</li><li>Pissuwan, D., Valenzuela, S. M., Killingsworth, M. C., Xu, X., Cortie, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+destruction+of+murine+macrophage+cells+with+bioconjugated+gold+nanorods.">Targeted destruction of murine macrophage cells with bioconjugated gold nanorods.</a> Journal of Nanoparticle Research. 9 (6), 1109-1124 (2007).</li><li>Pissuwan, D., Valenzuela, S. M., Miller, C. M., Cortie, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+golden+bullet?+Selective+targeting+of+Toxoplasma+gondii+tachyzoites+using+antibody-functionalized+gold+nanorods.">A golden bullet? Selective targeting of Toxoplasma gondii tachyzoites using antibody-functionalized gold nanorods.</a> Nano Letters. 7 (12), 3808-3812 (2007).</li><li>Zhang, H. -. G., Mehta, K., Cohen, P., Guha, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermia+on+immune+regulation:+a+temperature's+story.">Hyperthermia on immune regulation: a temperature's story.</a> Cancer Letters. 271 (2), 191-204 (2008).</li><li>Gobin, 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=Near-infrared+resonant+nanoshells+for+combined+optical+imaging+and+photothermal+cancer+therapy.">Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy.</a> Nano Letters. 7 (7), 1929-1934 (2007).</li><li>Jackson, J. B., Halas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+Raman+scattering+on+tunable+plasmonic+nanoparticle+substrates.">Surface-enhanced Raman scattering on tunable plasmonic nanoparticle substrates.</a> Proceedings of the National Academy of Sciences. 101 (52), 17930-17935 (2004).</li><li>Emelianov, S. Y., Li, P. -. C., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustics+for+molecular+imaging+and+therapy.">Photoacoustics for molecular imaging and therapy.</a> Physics Today. 62 (8), 34 (2009).</li><li>Dimitriou, N. 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=Gold+nanoparticles,+radiations+and+the+immune+system:+Current+insights+into+the+physical+mechanisms+and+the+biological+interactions+of+this+new+alliance+towards+cancer+therapy.">Gold nanoparticles, radiations and the immune system: Current insights into the physical mechanisms and the biological interactions of this new alliance towards cancer therapy.</a> Pharmacology &amp; Therapeutics. 178, 1-17 (2017).</li><li>Mueller, P., Rudin, D. O., Tien, H. T., Wescott, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cell+membrane+structure+in+vitro+and+its+transformation+into+an+excitable+system.">Reconstitution of cell membrane structure in vitro and its transformation into an excitable system.</a> Nature. 194 (4832), 979 (1962).</li><li>Tamm, L. K., McConnell, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+phospholipid+bilayers.">Supported phospholipid bilayers.</a> Biophysical Journal. 47 (1), 105-113 (1985).</li><li>Plant, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+hybrid+bilayer+membranes+as+rugged+cell+membrane+mimics.">Supported hybrid bilayer membranes as rugged cell membrane mimics.</a> Langmuir. 15 (15), 5128-5135 (1999).</li><li>Sackmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+membranes:+scientific+and+practical+applications.">Supported membranes: scientific and practical applications.</a> Science. 271 (5245), 43-48 (1996).</li><li>Alghalayini, A., Garcia, A., Berry, T., Cranfield, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Tethered+Bilayer+Lipid+Membranes+to+Identify+the+Mechanisms+of+Antimicrobial+Peptide+Interactions+with+Lipid+Bilayers.">The Use of Tethered Bilayer Lipid Membranes to Identify the Mechanisms of Antimicrobial Peptide Interactions with Lipid Bilayers.</a> Antibiotics. 8 (1), 12 (2019).</li><li>Khan, M. S., Dosoky, N. S., Williams, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+lipid+bilayer+membranes+for+protein+studies.">Engineering lipid bilayer membranes for protein studies.</a> International Journal of Molecular Sciences. 14 (11), 21561-21597 (2013).</li><li>Urban, P., Kirchner, S. R., M&#252;hlbauer, C., Lohm&#252;ller, T., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+control+of+current+across+lipid+membranes+by+local+heating.">Reversible control of current across lipid membranes by local heating.</a> Scientific Reports. 6, 22686 (2016).</li><li>Palankar, 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=Nanoplasmonically-induced+defects+in+lipid+membrane+monitored+by+ion+current:+transient+nanopores+versus+membrane+rupture.">Nanoplasmonically-induced defects in lipid membrane monitored by ion current: transient nanopores versus membrane rupture.</a> Nano Letters. 14 (8), 4273-4279 (2014).</li><li>Bendix, P. M., Reihani, S. N. S., Oddershede, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurements+of+heating+by+electromagnetically+trapped+gold+nanoparticles+on+supported+lipid+bilayers.">Direct measurements of heating by electromagnetically trapped gold nanoparticles on supported lipid bilayers.</a> ACS Nano. 4 (4), 2256-2262 (2010).</li><li>Plaksin, M., Shapira, E., Kimmel, E., Shoham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+transients+excite+neurons+through+universal+intramembrane+mechanoelectrical+effects.">Thermal transients excite neurons through universal intramembrane mechanoelectrical effects.</a> Physical Review X. 8 (1), 011043 (2018).</li><li>Alghalayini, A., 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=Real-time+monitoring+of+heat+transfer+between+gold+nanoparticles+and+tethered+bilayer+lipid+membranes.">Real-time monitoring of heat transfer between gold nanoparticles and tethered bilayer lipid membranes.</a> Biochimica et Biophysica Acta (BBA)-Biomembranes. , 183334 (2020).</li><li>Moradi-Monfared, S., Krishnamurthy, V., Cornell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+machine+biosensor:+construction,+predictive+models+and+experimental+studies.">A molecular machine biosensor: construction, predictive models and experimental studies.</a> Biosensors and Bioelectronics. 34 (1), 261-266 (2012).</li><li>Hoiles, W., Gupta, R., Cornell, B., Cranfield, C., Krishnamurthy, V. <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+tethers+on+artificial+cell+membranes:+A+coarse-grained+molecular+dynamics+study.">The effect of tethers on artificial cell membranes: A coarse-grained molecular dynamics study.</a> PloS One. 11 (10), 0162790 (2016).</li><li>Cranfield, C. G., 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=Transient+potential+gradients+and+impedance+measures+of+tethered+bilayer+lipid+membranes:+pore-forming+peptide+insertion+and+the+effect+of+electroporation.">Transient potential gradients and impedance measures of tethered bilayer lipid membranes: pore-forming peptide insertion and the effect of electroporation.</a> Biophysical Journal. 106 (1), 182-189 (2014).</li><li>Dombrovsky, L. 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> Radiation heat transfer in disperse systems. , (1996).</li><li>Cornell, B. A., Braach-Maksvytis, V., King, L., Osman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biosensor+that+uses+ion-channel+switches.">A biosensor that uses ion-channel switches.</a> Nature. 387 (6633), 580 (1997).</li><li>Maccarini, 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=Nanostructural+determination+of+a+lipid+bilayer+tethered+to+a+gold+substrate.">Nanostructural determination of a lipid bilayer tethered to a gold substrate.</a> The European Physical Journal E. 39 (12), 123 (2016).</li><li>Beugin-Deroo, S., Ollivon, M., Lesieur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayer+stability+and+impermeability+of+nonionic+surfactant+vesicles+sterically+stabilized+by+PEG-cholesterol+conjugates.">Bilayer stability and impermeability of nonionic surfactant vesicles sterically stabilized by PEG-cholesterol conjugates.</a> Journal of Colloid and Interface Science. 202 (2), 324-333 (1998).</li><li>Kendall, J. K., 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=Effect+of+the+Structure+of+Cholesterol-Based+Tethered+Bilayer+Lipid+Membranes+on+Ionophore+Activity.">Effect of the Structure of Cholesterol-Based Tethered Bilayer Lipid Membranes on Ionophore Activity.</a> ChemPhysChem. 11 (10), 2191-2198 (2010).</li><li>Jiang, W., Kim, B. Y., Rutka, J. T., Chan, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-mediated+cellular+response+is+size-dependent.">Nanoparticle-mediated cellular response is size-dependent.</a> Nature Nanotechnology. 3 (3), 145 (2008).</li><li>He, C., Hu, Y., Yin, L., Tang, C., Yin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+particle+size+and+surface+charge+on+cellular+uptake+and+biodistribution+of+polymeric+nanoparticles.">Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles.</a> Biomaterials. 31 (13), 3657-3666 (2010).</li><li>Wang, F. X., 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=Surface+and+bulk+contributions+to+the+second-order+nonlinear+optical+response+of+a+gold+film.">Surface and bulk contributions to the second-order nonlinear optical response of a gold film.</a> Physical Review B. 80 (23), 233402 (2009).</li></ol>

作者宣布了以下经济利益/个人关系，这可能被视为潜在的竞争利益:布鲁斯·康奈尔教授是外科诊断SDx系绳膜有限公司的科学和技术总监。

该协议描述了使用tBLM模型与平面电极基板结合水平激光对齐设置，使实时电阻抗记录响应激光照射金纳米粒子。此处介绍的 EIS 记录方法构建了一个最少的实验列表，用于记录整个膜的离子电流变化，这与耦合激光和金纳米粒子相互作用产生的热量相对应。本协议中的关键步骤是激光路径向双层脂膜周围的缓冲器的仔细和精确对齐。
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辐照金纳米材料的高温性能为感染和肿瘤提供了一类新的微创、选择性、有针对性的治疗方法。可以用激光加热的纳米粒子被用来选择性地摧毁患病细胞，并为选择性药物输送提供手段。加热质子纳米粒子光热化现象的后果是对细胞膜的损害。流体脂质双层膜被认为是接受此类治疗的细胞特别脆弱的部位，因为内膜蛋白的变性以及膜损伤也可能导致细胞死亡4，因为许多蛋白质在那里维持细胞膜的离子潜在梯度。虽然确定和监测纳米级传热的能力对研究和应用辐照GNP1、5、6、7、评估和理解GNP与生物膜之间的分子相互作用以及嵌入式GNP在生物组织中的激光诱发加热现象的直接后果至关重要， 尚未完全阐明8。因此，彻底了解辐照GNP的体温过高过程仍然是一个挑战。因此，开发一个模拟细胞自然环境的纳米材料电极接口，可以提供一种手段，对生物系统中辐照金纳米粒子的传热特性进行深入研究。
原生细胞膜的复杂性是理解细胞中辐照GNP相互作用的重大挑战之一。已开发出各种人造膜平台，提供接近简单的生物模仿版本的天然脂膜结构和功能，包括，但不限于，黑色脂膜9，支持平面双层膜10，混合双层膜11，聚合物缓冲脂质双层膜12和系绳双层脂膜13。每个人造脂膜模型在模仿天然脂膜方面具有明显的优点和局限性。
本研究利用tBLM模型，将脂质膜涂层电极作为评估金纳米粒子和脂膜相互作用的传感器。基于tBLM的生物传感器检测方案提供了固有的稳定性和灵敏度13作为系绳膜可以自我修复，不像其他系统（如膜形成的补丁夹或脂质体），其中只有少量的膜损伤导致其崩溃15，16，17，18。此外，由于 tBLM 的尺寸为 mm2，因此背景阻抗的幅度比贴片夹式记录技术低，从而能够记录由于纳米粒子相互作用而导致的基底膜离子通量的变化。因此，本协议可以对比受约束的GNP对膜传导的变化，这些GNP是由其功耗低至135 nW/μm2的激光激发的。
此处介绍的系统为确定设计和开发热疗法所需的精确激光参数、粒子大小、粒子涂层和成分提供了一种敏感且可重复的方法。这对改进新兴的光热疗法至关重要，并为生物系统内的详细传热机制提供了有价值的信息。提交的协议是基于先前公布的工作19。协议大纲如下:第一部分定义 tBLM 形成:第二部分概述了如何构建设置和对齐激发激光源:最后一部分说明了如何从电阻抗光谱数据中提取信息。

<table><tbody><tr><td>30 nm diameter streptavidin-conjugated gold nanoparticles</td><td>Cytodiagnostics</td><td>AC-30-04-05</td><td>This is a streptavidin-conjugated GNPs product ready for use</td></tr><tr><td>30 nm diameter bare gold nanoparticles</td><td>Sigma-Aldrich</td><td>753629</td><td>This is a bare GNPs product ready for use</td></tr><tr><td>Cholesterol-PEG-Biotin (MW1000)</td><td>NANOCS</td><td>PG2-BNCS-10k</td><td>Dissolved in highly pure ethanol</td></tr><tr><td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-S1</td><td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td></tr><tr><td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-S2</td><td>Spacer molecules</td></tr><tr><td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&amp;nbsp;</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-S2</td><td>Tethered molecules</td></tr><tr><td>532 nm green laser continuous light</td><td>OBIS LS/OBIS CORE LS, China</td><td>ND-1000</td><td>The power of this laser was ~135&amp;nbsp;mW&amp;nbsp;</td></tr><tr><td>tethaPod EIS reader</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-R1</td><td>A reader of conductance and capacitance on six channels simultaneously</td></tr><tr><td>tethaPlate cartridge assembly</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-BG</td><td>Materials to attach the slide with electrodes to the flow cell cartridge</td></tr><tr><td>Clamp and slide assembly jig</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-A1</td><td>Materials to attach the slide with electrodes to the flow cell cartridge</td></tr><tr><td>Lipid coated coplanar gold electrodes</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-T10</td><td>Coplanar&amp;nbsp; gold electrodes are made from 25 mm x 75&amp;nbsp;mm x&amp;nbsp;1&amp;nbsp;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&amp;nbsp;</td></tr><tr><td>tethaQuick software</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-B1</td><td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td></tr><tr><td>&amp;nbsp;99.9% Pure ethanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>34963</td><td>Absolute,&amp;nbsp; 99.9%</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td>pH 7</td></tr></tbody></table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

在这里，我们报告了一项协议，使用用金电极组装的系绳双层脂质膜（tBLMs）进行电化学，调查辐照金纳米粒子（GNPs）和双层脂质膜之间的热传递。辐照改性GNP，如链球菌素结合的GNP，嵌入含有目标分子（如生物素）的tBLM中。通过使用这种方法，辐照GNP和具有相关实体的双层脂膜之间的传热过程由水平聚焦激光束进行介导。热预测计算模型用于确认 tBLM 中的电化学诱导导电导变化。在所使用的特定条件下，检测热脉冲需要金纳米粒子与膜表面的特定附着，而未绑定的金纳米粒子则无法引起可测量的反应。该技术作为强大的检测生物传感器，可直接用于设计和开发热疗法策略，从而优化激光参数、颗粒大小、粒子涂层和成分。

可以创建 tBLM 的黄金基板显示在图 1中。实验设置的示意图在图2中提出。
如图1A所示，平面金电极由25毫米×75毫米×1毫米聚碳酸酯基底基板制成，并配有图案黄金阵列。透明胶粘层定义了六个单独的测量室。平面金电极允许激光直接照射到tBLMs膜上。电极阵列的每个井都包含一个圆形工作电极（区域:0.707 cm<sup...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

这项工作概述了一项协议，以实现从激光辐照的金纳米粒子到tBLM的热传递的动态、非侵入性监测。该系统结合阻抗光谱，用于实时测量 TBLM 的传导变化，并配有水平聚焦激光束，驱动金纳米粒子照明，用于热生产。

系绳双层脂膜，以监测金纳米粒子和脂膜之间的热传递

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

tethered-bilayer-lipid-membranes-to-monitor-heat-transfer-between

Research

JoVE Journal

Biology

2.6K 次观看.  University of Technology Sydney. 这项工作概述了一项协议，以实现从激光辐照的金纳米粒子到tBLM的热传递的动态、非侵入性监测。该系统结合阻抗光谱，用于实时测量 TBLM 的传导变化，并配有水平聚焦激光束，驱动金纳米粒子照明，用于热生产。

视频: Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

的模板导演电浆黄金纳米管的合成与可调谐红外吸收

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

科研

化学

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

生物工程

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

生物化学

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

纳米巴支持的脂质双层系统，用于体外研究膜曲率传感蛋白

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

温度控制组件和液滴接口比莱耶的特征

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...