Photonic Crystal Surfaces for Label-Free Detection and Fluorescence Amplification

说明（由申请人提供）：本修订申请是根据通知号 NOT-OD-09-058 提交的（通知标题：NIH 宣布恢复法案基金可用于竞争性修订申请）。修订申请在先前资助的 R01 资助机会 PAR 07 070 申请中添加了两个新的具体目标，题为“用于无标记检测和荧光放大的光子晶体表面：在 DNA 微阵列中的应用”，作者：B.T.坎宁安、L.沃德金和G.博莱罗。原始赠款 (PHS 1R01 GM086382) 包括 2009 年 1 月 1 日至 2012 年 12 月 31 日的资金。此次修订为该项目增加了一位新的 PI——来自太平洋西北国家实验室 (PNNL) 的 Richard Zangar 博士，其中包括国家认可的蛋白质微阵列专业知识。 DNA和蛋白质微阵列能够同时评估数千个基因或数十种癌症生物标志物的相对表达水平，并且自最初引入以来发展迅速。因此，微阵列现在是识别毒性和疾病的早期遗传或表达蛋白质生物标志物的最优选技术之一。微阵列研究的结果可能受到许多技术和仪器因素的影响，导致对衍生数据缺乏再现性和准确性的重大批评。尽管荧光染料、表面化学、点样机器人、杂交室、检测仪器和数据分析工具都经历了实质性的发展和完善，但微阵列基底本身仍然是一个简单的玻璃表面。在本提案中，我们描述了如何用专用光学传感器替换玻璃表面，从而提供目前微阵列分析中完全缺乏的微阵列点间和点内密度的质量控制信息，同时放大用于量化杂交DNA或蛋白质的荧光标记的强度。通过提供斑点变异性信息（代表微阵列分析中的主要误差源），同时提高检测弱表达基因（微阵列平台目前与其他定量基因表达平台相比处于劣势）或血清中浓度<1 ng/ml的蛋白质生物标志物的信噪比，该项目代表了微阵列技术的根本性进步。用于提供这些功能的光学传感器是二维光子晶体 (PC) 表面，旨在提供光学共振，从而能够对沉积的微阵列点进行高分辨率无标记成像检测，并将常用微阵列荧光染料的检测灵敏度提高高达 550 倍。该 PC 是通过塑料基板上的大面积纳米复制成型工艺制造的，塑料基板连接到标准玻璃显微镜载玻片上，以便与现有的点样机器人、杂交室和检测仪器兼容。 最近，伊利诺伊州坎宁安集团开发了大面积 PC 表面作为多功能光学传感器，可设计为在任何所需波长下产生窄波长电磁共振，其特点是高强度场可瞬间延伸到 PC 表面的介质中。光学共振与吸附的生物分子的相互作用导致共振波长的高度局部化，该共振波长用于量化吸附材料的密度，无需使用荧光标记，从而能够在包含传统微阵列载玻片整个表面的 PC 上以 4 µm 空间分辨率测量沉积微阵列点的无标记图像。 PC 表面还可以设计为使光学共振与用于激发荧光染料的激光波长一致，从而利用称为增强荧光 (EF) 的效应，相对于普通玻璃微阵列载玻片上出现的强度增加荧光输出强度。事实证明，使用市售微阵列激光扫描仪器时，EF 效应可使检测到的荧光信号增加约 50 倍，但当 PC 设计为在荧光团发射波长处纳入光学共振时，EF 效应还可进一步增强，从而使灵敏度额外提高 10 倍。 在提出的额外具体目标中，我们计划首次应用二维 PC 表面，该表面结合了光学共振，用于无标记检测和蛋白质微阵列 EF。无标记共振将用于量化沉积的蛋白质捕获点的密度变异性，从而提供目前使用斑点阵列的研究人员无法使用的质量控制工具。点的无标记图像将用于量化点间和点内密度变异性，提供的信息将用于消除进一步分析中的缺陷点，或作为对后续荧光测量中检测到的信号进行标准化的方法。 EF 共振将用于增强血清中 Cy5 标记的杂交蛋白质生物标志物的输出，从而能够以较低的样品浓度进行生物标志物检测和定量，并能够以比以前更低的水平观察蛋白质表达。 公共健康相关性：拟议项目旨在开发一个技术平台，用于提供高灵敏度的无标记生物分子检测，并在大面积、塑料基纳米结构表面（称为“光子晶体”）上大幅放大荧光输出。目标是将光子晶体表面整合到 DNA 和蛋白质微阵列载玻片中，以提供阵列点的无标记质量控制，并能够更轻松地检测和识别血清中低浓度的低表达水平基因或癌症蛋白质生物标志物。该项目与开发基于基因和蛋白质的诊断测试相关，这些测试准确、可靠，并且能够检测低浓度的分析物。

DESCRIPTION (provided by applicant): This revision application is submitted in response to Notice Number NOT-OD-09-058 (Notice Title: NIH Announces the availability of Recovery Act Funds for Competitive Revision Applications). The revision application adds two new Specific Aims to a previously funded R01 application for Funding Opportunity PAR 07 070, entitled "Photonic Crystal Surfaces for Label-Free Detection and Fluorescence Amplification: Application to DNA Microarrays," by B.T. Cunningham, L. Vodkin, and G. Bollero. The original grant (PHS 1R01 GM086382) includes funding for 1/1/09 through 12/31/12. The revision adds a new PI to the project - Dr. Richard Zangar from Pacific Northwest National Laboratory (PNNL) to include nationally recognized expertise in protein microarrays. DNA and protein microarrays are capable of simultaneously evaluating the relative expression levels of thousands of genes or dozens of cancer biomarkers, and have developed rapidly since their initial introduction. As a result, microarrays are now one of the most preferred technologies for identifying early genetic or expressed protein biomarkers of toxicity and disease. The outcome of microarray studies can be affected by many technical and instrumental factors, resulting in major criticism regarding lack of reproducibility and accuracy of the derived data. Although fluorescent dyes, surface chemistries, spotting robots, hybridization chambers, detection instruments, and data analysis tools have all undergone substantial development and refinement, the microarray substrate itself remains as a simple glass surface. In this proposal, we describe how replacement of the glass surface with a special-purpose optical transducer can provide quality control information on the interspot and intraspot density of microarray spots that is currently completely lacking from microarray analysis, while simultaneously amplifying the intensity of fluorescent labels used to quantify hybridized DNA or protein. By providing information on spot variability, (representing a major source of error in microarray analysis), while at the same time increasing the signal-to-noise ratio for detection of weakly expressed genes (where microarray platforms currently face a disadvantage compared to other quantitative gene expression platforms) or protein biomarkers in serum at concentrations <1 ng/ml, the proposed project represents a fundamental advance in microarray technology. The optical transducer used to provide these features is a 2-dimensional photonic crystal (PC) surface that is designed to provide optical resonances that enable high resolution label-free imaging detection of deposited microarray spots and up to 550x enhanced detection sensitivity of commonly used microarray fluorescent dyes. The PC is fabricated by a large-area nanoreplica molding process on plastic substrates that are attached to standard glass microscope slides for compatibility with existing spotting robots, hybridization chambers, and detection instruments. Recently, large area PC surfaces have been developed by the Cunningham Group at Illinois as multifunctional optical transducers that can be designed to produce narrow-wavelength electromagnetic resonances at any desired wavelength, featuring high intensity fields that extend evanescently into the media on the PC surface. The interaction of the optical resonance with adsorbed biomolecules results in a highly localized shift of the resonant wavelength that is used to quantify the density of adsorbed material without the use of fluorescent labels, enabling label-free images of deposited microarray spots to be measured with 4 ?m spatial resolution over a PC comprising the entire surface of a conventional microarray slide. A PC surface may also be designed so that the optical resonance coincides with the wavelength of a laser used to excite a fluorescent dye, thereby increasing the fluorescent output intensity relative to the intensity that would occur on an ordinary glass microarray slide, using an effect called Enhanced Fluorescence (EF). The EF effect has been shown to result in ~50x increase in the detected fluorescence signal using commercially available microarray laser scanning instruments, but can be further enhanced when the PC is designed to also incorporate an optical resonance at the emission wavelength of the fluorophore, resulting in an additional 10x gain in sensitivity. In the proposed additional specific aims, we plan for the first time to apply 2-dimensional PC surfaces that incorporate optical resonances for both label-free detection and EF to protein microarrays. The label-free resonance will be utilized to quantify the density variability of deposited protein capture spots, thereby providing a quality-control tool that is not currently available to researchers using spotted arrays. The labelfree images of spots will be used to quantify interspot and intraspot density variability, providing information that will be used to eliminate defective spots from further analysis or as a means for normalizing the detected signal from subsequent fluorescent measurements. The EF resonance will be applied to enhance the output of Cy5-labeled hybridized protein biomarkers in serum, enabling biomarker detection and quantification to be conducted with lower sample concentrations and the ability to observe protein expression at lower levels than has previously been possible. PUBLIC HEALTH RELEVANCE: The proposed project seeks to develop a technology platform for providing high-sensitivity label-free detection of biomolecules and substantial amplification of fluorescence output on large-area, plastic based nanostructured surfaces called "photonic crystals." The goal is to incorporate photonic crystal surfaces into DNA and protein microarray slides to provide label-free quality control of array spots and the ability to more easily detect and identify genes with low expression levels or protein biomarkers for cancer at low concentrations in serum. The project is relevant for the development of gene-based and protein-based diagnostic tests that are accurate, reliable, and able to detect analytes at low concentration.