Ji Jin, O'Connell J Garland, Carter David J D, Larson Dale N
Technology and Engineering Center, Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Anal Chem. 2008 Apr 1;80(7):2491-8. doi: 10.1021/ac7023206. Epub 2008 Feb 29.
We have developed an integrated label-free, real-time sensing system that is able to monitor multiple biomolecular binding events based on the changes in the intensity of extraordinary optical transmission (EOT) through nanohole arrays. The core of the system is a sensing chip containing multiple nanohole arrays embedded within an optically thick gold film, where each array functions as an independent sensor. Each array is a square array containing 10 x 10 nanoholes (150 nm in diameter), occupying a total area of 3.3 mum x 3.3 mum. The integrated system includes a laser light source, a temperature-regulated flow cell encasing the sensing chip, motorized optics, and a charge-coupled detector (CCD) camera. For demonstration purposes, sensing chips containing 25 nanohole arrays were studied for their use in multiplexed detection, although the sensing chip could be easily populated to contain up to 20 164 nanohole arrays within its 0.64 cm2 sensing area. Using this system, we successfully recorded 25 separate binding curves between glutathione S-transferase (GST) and anti-GST simultaneously in real time with good sensitivity. The system responds to binding events in a concentration-dependent manner, showing a sharp linear response to anti-GST at concentrations ranging from 13 to 290 nM. The EOT intensity-based approach also enables the system to monitor multiple bindings simultaneously and continuously, offering a temporal resolution on milliseconds scale that is decided only by the camera speed and exposure time. The small footprint of the sensing arrays combined with the EOT intensity-based approach enables the system to resolve binding events that occurred on nanohole sensing arrays spaced 96 mum apart, with a reasonable prediction of resolving binding events spaced 56 mum apart. This work represents a new direction that implements nanohole arrays and EOT intensity to meet high-throughput, spatial and temporal resolution, and sensitivity requirements in drug discovery and proteomics studies.
我们开发了一种集成的无标记实时传感系统，该系统能够基于通过纳米孔阵列的超常光学传输（EOT）强度变化来监测多个生物分子结合事件。该系统的核心是一个传感芯片，其中包含嵌入光学厚金膜内的多个纳米孔阵列，每个阵列都作为一个独立的传感器。每个阵列是一个包含10×10个纳米孔（直径150 nm）的方形阵列，总面积为3.3μm×3.3μm。该集成系统包括一个激光光源、一个包裹传感芯片的温度调节流动池、电动光学器件和一个电荷耦合探测器（CCD）相机。为了演示目的，研究了包含25个纳米孔阵列的传感芯片在多重检测中的应用，尽管该传感芯片在其0.64 cm²的传感区域内可以轻松填充多达20164个纳米孔阵列。使用该系统，我们成功地实时同时记录了谷胱甘肽S-转移酶（GST）和抗GST之间的25条单独的结合曲线，灵敏度良好。该系统以浓度依赖的方式响应结合事件，在13至290 nM的浓度范围内对抗GST表现出急剧的线性响应。基于EOT强度的方法还使该系统能够同时连续监测多个结合，提供仅由相机速度和曝光时间决定的毫秒级时间分辨率。传感阵列的小尺寸与基于EOT强度的方法相结合，使该系统能够分辨在相距96μm的纳米孔传感阵列上发生的结合事件，并合理预测分辨相距56μm的结合事件。这项工作代表了一个新的方向，即利用纳米孔阵列和EOT强度来满足药物发现和蛋白质组学研究中的高通量、空间和时间分辨率以及灵敏度要求。
