Photonic Crystal Enhanced Fluorescence: Development of Sensors Structures and Det

描述(申请人提供)：临床上需要快速、多重和经济有效的检测基于可溶性蛋白质的癌症生物标记物。最近，光子晶体(PC)表面被证明是提高用于蛋白质微阵列生物标记物检测的荧光染料的输出和收集效率的一种有效方法。使用定量夹心荧光酶联免疫吸附分析(ELISA)形式，PC增强的荧光团激发和PC增强提取的组合效应被用来降低血浆中乳腺癌生物标记物的检测限，与在普通玻璃表面进行相同的检测相比，使得能够检测0.1-10pg/ml浓度范围的生物标记物，正如低丰度蛋白质所需的那样。在拟议的项目中，我们将为仪器设计开发几种创新的方法，这些方法将进一步降低基于PC的荧光ELISA分析的检测极限，同时将PC集成到微流控格式中，从而将分析体积降至最低，并允许在一滴血浆上执行分析过程。PC将由低自发荧光硅材料制造，其设计使高质量因数共振光学模式能够同时提供增强的激发和增强的荧光提取。该检测仪器将利用一种新的激光扫描方法，将激光照明最佳地耦合到PC中，并匹配共振耦合条件。拟议的努力旨在将光子晶体增强荧光(PCEF)技术在之前NIH资助下发展到原理证明阶段，并将其转化为临床应用。在我们之前的工作中，我们开发并首次展示了用于DNA微阵列和蛋白质微阵列的PC器件结构、制造方法和检测仪器。虽然在拟议的项目中将对一组乳腺癌生物标记物的检测进行评估，但该检测平台可以应用于其他癌症和疾病的多路生物标记物分析阵列。为了验证新的传感器和检测仪器，我们将检测添加到血浆中的生物标记物以建立校准标准，直接与传统的ELISA进行比较，然后定量检测已知Her2和雌激素受体状态的乳腺癌患者血液中的生物标记物浓度。将对一套生物信息学工具进行修改，以实现数据分析和解释的自动化。总体目标是开发一个高灵敏度、多重、快速和自动化的荧光微阵列ELISA检测平台，可以对一滴血浆进行生物标记物分析。

DESCRIPTION (provided by applicant): There is a clinical need for rapid, multiplexed, and cost-effective detection of soluble protein-based cancer biomarkers. Photonic crystal (PC) surfaces have been recently demonstrated as an effective approach for increasing the output and collection efficiency of fluorescent dyes that are used for protein microarray biomarker detection. Using a quantitative sandwich fluorescent enzyme-linked immunosorbent assay (ELISA) format, the combined effects of PC- enhanced fluorophore excitation and PC-enhanced extraction have been used to reduce the limits of detection of breast cancer biomarkers in plasma, compared to performing the same assay on an ordinary glass surface, resulting in the ability to detect biomarkers in the 0.1 - 10 pg/ml concentration range, as is required for low abundance proteins. In the proposed project, we will develop several innovative approaches for the instrument design that will further reduce the limits of detection for PC-based fluorescent ELISA assays, while integrating the PC into a microfluidic format that will minimize assay volume and allow the assay process to be performed on a droplet of plasma. The PC will be fabricated from low autofluorescence silicon materials, with a design that enables a high quality-factor resonant optical mode to simultaneously provide enhanced excitation and enhanced extraction of fluorescence. The detection instrument will utilize a novel laser scanning approach that optimally couples laser illumination into the PC and matches the resonant coupling condition. The proposed effort seeks to translate photonic crystal enhanced fluorescence (PCEF) technology developed to the proof-of-principle stage under previous NIH funding towards clinical applications. In our previous work, PC device structures, fabrication methods, and detection instruments were developed and first demonstrated for DNA microarrays and protein microarrays. While detection of a panel of breast cancer biomarkers will be evaluated in the proposed project, the detection platform can be applied to multiplexed arrays of biomarker assays for other cancers and diseases. To validate the new sensor and detection instrument, we will detect biomarkers spiked into plasma to establish calibration standards, directly compare against conventional ELISAs, and subsequently quantify biomarker concentrations in blood from breast cancer patients with known Her2 and estrogen receptor statuses. A suite of bioinformatics tools will be modified for automated data analysis and interpretation. The overall goal is to develop a highly sensitive, multiplexed, rapid, and automated assay platform for fluorescent microarray ELISA that can perform biomarker analysis on a droplet of plasma.