系绳双层脂膜，以监测金纳米粒子和脂膜之间的热传递
1School of Life Sciences,University of Technology Sydney, 2ARC Research Hub for Integrated Devices for End-user Analysis at Low-levels (IDEAL), Faculty of Science,University of Technology Sydney, 3School of Mechanical and Manufacturing Engineering,The University of New South Wales, 4Surgical Diagnostics Pty Ltd., 5Institute for Biomedical Materials and Devices,University of Technology Sydney
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. tBLM 电极制备 准备第一层单层涂层 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…
Representative Results
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<s…
Discussion
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。
<p class="jove_content" fo:keep-together.within…Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 30 nm diameter streptavidin-conjugated gold nanoparticles | Cytodiagnostics | AC-30-04-05 | This is a streptavidin-conjugated GNPs product ready for use |
| 30 nm diameter bare gold nanoparticles | Sigma-Aldrich | 753629 | This is a bare GNPs product ready for use |
| Cholesterol-PEG-Biotin (MW1000) | NANOCS | PG2-BNCS-10k | Dissolved in highly pure ethanol |
| C20 Diphytanyl-Glycero-Phosphatidylcholine lipids | SDx Tethered Membranes Pty. Ltd. | SDx-S1 | 1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol |
| Benzyl-disulfide-tetra-ethyleneglycol-OH | SDx Tethered Membranes Pty. Ltd. | SDx-S2 | Spacer molecules |
| Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl | SDx Tethered Membranes Pty. Ltd. | SDx-S2 | Tethered molecules |
| 532 nm green laser continuous light | OBIS LS/OBIS CORE LS, China | ND-1000 | The power of this laser was ~135 mW |
| tethaPod EIS reader | SDx Tethered Membranes Pty. Ltd. | SDx-R1 | A reader of conductance and capacitance on six channels simultaneously |
| tethaPlate cartridge assembly | SDx Tethered Membranes Pty. Ltd. | SDx-BG | Materials to attach the slide with electrodes to the flow cell cartridge |
| Clamp and slide assembly jig | SDx Tethered Membranes Pty. Ltd. | SDx-A1 | Materials to attach the slide with electrodes to the flow cell cartridge |
| Lipid coated coplanar gold electrodes | SDx Tethered Membranes Pty. Ltd. | SDx-T10 | Coplanar gold electrodes are made from 25 mm x 75 mm x 1 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% |
| tethaQuick software | SDx Tethered Membranes Pty. Ltd. | SDx-B1 | Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes |
| 99.9% Pure ethanol | Sigma-Aldrich | 34963 | Absolute, 99.9% |
| Phosphate buffered saline (PBS) | Sigma-Aldrich | P4417 | pH 7 |
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Cite This Article
Alghalayini, A., Jiang, L., Gu, X., Yeoh, G. H., Cranfield, C. G., Timchenko, V., Cornell, B. A., Valenzuela, S. M. Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes. J. Vis. Exp. (166), e61851, doi:10.3791/61851 (2020).