SST: Microtubule-Based Immunosensors

这项研究将探索使用生物分子马达作为一种全新的、特定于分子的传输机制的基础，这种机制可能在生物分子传感器中有强大的应用。这项研究将生物分子马达运输与免疫传感策略结合在一起，这种策略实际上允许人们对传感器分子进行编程，从液体溶液中捕获特定的生物分子，然后将它们携带到固定的检测窗口。这种传感器的工作原理可以概括如下：将液体样本放入含有抗体衍生的微管溶液的储存池中，外加氧气清除剂三磷酸腺苷。然后加入标记的(或酶标记的，或结合珠粒的)抗体。微管、目标分析物和标记抗体之间发生快速(非扩散限制)和高度特异的“夹心式”结合，并将柱塞放置在储液器的顶部，按压以将该混合物注入微流控芯片的空腔中。在这个空腔内，微管将自动移动(使用溶液中无所不在的三磷酸腺苷作为燃料)，但在编程方向上朝向芯片上的检测窗口，在那里它们将集中进行LED/光电二极管荧光的高灵敏度检测。微管定向编程是利用微流控芯片内表面上的CytopTM(旋转式氟聚合物)图案化薄膜来实现的。该膜的工作原理既允许有选择地形成动蛋白图案，又通过提供机械导向器来引导微管沿着所需的转位路径。进行了原理验证实验，并展示了基于这一概念的基本传感器。关于利用这些现象的传感器的特异性、速度和灵敏度的许多问题仍有待回答。研究计划是测试这种基于微管的免疫分析方法对各种抗体的通用性，并在各种微型设备中表征这种方法的效率和速度。统计研究将利用基于图像处理的微管移位的“粒子跟踪”研究来表征和优化移位路径。还将研究和优化集中器设计，以便在尽可能短的时间内从简单、集成的LED/光电二极管检测系统获得尽可能高的信号。还将进行微管在流体剪切下的稳定性以及传感器性能对温度等控制不佳变量的依赖关系的实验。

0428090HasselbrinkThis research will investigate the use of biomolecular motors as the basis for a completely novel, molecule-specific transport mechanism that could have powerful applications inbiomolecule sensors. This research combines biomolecular motor transport with an immosensing strategy that literally allows one to program sensor molecules to grab specific biomolecules from a liquid solution, and then carry them to a fixed detection window. The operation of such a sensor can be summarized as follows: a liquid sample is placed in a reservoir containing a solution of microtubules derivatized with antibodies, plus ATP, an oxygen scavenger. Labeled (or enzymelinked, or bead-bound) antibodies are then added. Fast (non- diffusion limited) and highly specific "sandwich"-type binding between microtubules, target analyte, and labeled antibody occurs, and a plunger is placed atop the reservoir and pressed to inject of this mixture into a cavity in a microfluidic chip. Within this cavity, the microtubules will translocate autonomously (using ATP omnipresent in the solution as fuel), but in a programmed direction towards a detection window on the chip, where they will concentrate for highly sensitive detection by LED/photodiode fluorescence. Microtubule directional programming is achieved using a patterned film of CytopTM (spin-on fluoropolymer) on the microfluidic chip inner surfaces. The film works by both allowing for selective patterning of kinesin, and by providing mechanical guides that direct the microtubules along the desired path of translocation. Proof-of-principle experiments have been performed, and demonstrated a rudimentary sensor based on this concept. Numerous questions regarding the specificity, speed, and sensitivity of sensors exploiting these phenomena remain to be answered. The research plan is to test the generality of this microtubule-based immunoassay approach to a variety of antibodies, and to characterize the efficiency and speed of this approach in a variety of microdevices. Statistical studies will characterize and optimize translocation paths using image processing-based "particle tracking" studies of microtubule translocation. Concentrator designs will also be studied and optimized so that the highest possible signal may be obtained from simple, integrated LED/photodiode detection systems in the shortest possible time. Experiments on the robustness of microtubules under fluid shear, and dependencies of sensor performance on poorly-controlled variables such as temperature, will also be conducted.