Photonic Crystal Surfaces for Label-Free Detection and Fluorescence Amplification

描述（由申请人提供）：DNA微阵列能够同时评估数千个基因的相对表达水平，并且自最初引入以来发展迅速。因此，DNA微阵列现在是鉴定毒性和疾病早期生物标志物的最首选技术之一。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可重复性和准确性的主要批评。尽管荧光染料、表面化学、斑点机器人、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，但微阵列衬底本身仍然是一个简单的玻璃表面。在本建议中，我们描述了如何用专用光学传感器替换玻璃表面，以提供目前微阵列分析完全缺乏的关于微阵列斑点的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的荧光标记的强度。通过提供现场变异性的信息（这是微阵列分析中的主要误差来源），同时增加检测弱表达基因的信噪比（微阵列平台目前与其他定量基因表达平台相比面临劣势），拟议的项目代表了微阵列技术的根本进步。用于提供这些功能的光学换能器是二维光子晶体（PC）表面，其设计用于提供光学共振，从而能够对沉积的微阵列斑点进行高分辨率无标签成像检测，并且可将常用微阵列荧光染料的检测灵敏度提高550倍。PC是通过大面积纳米复制成型工艺在塑料基板上制造的，塑料基板附着在标准的玻璃显微镜载玻片上，与现有的斑点机器人、杂交室和检测仪器兼容。最近，伊利诺斯州的Cunningham小组开发了大面积PC表面作为多功能光学换能器，可以设计成在任何所需波长产生窄波长电磁共振，具有高强度场，可瞬时扩展到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域化位移，用于在不使用荧光标记的情况下量化吸附材料的密度，使沉积的DNA微阵列点的无标记图像能够在包含传统微阵列载玻片整个表面的PC上以45米的空间分辨率进行测量。还可以设计PC表面，使光学共振与用于激发荧光染料的激光的波长一致，从而使用一种称为增强荧光（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。使用商用微阵列激光扫描仪器，EF效应已被证明可使检测到的荧光信号增加约50倍，但当PC被设计为在荧光团的发射波长处也包含光学共振时，可以进一步增强，从而使灵敏度增加10倍。在本文中，我们计划首次将二维PC表面应用于斑点基因表达微阵列，该二维PC表面包含光学共振，用于无标记检测和EF。无标记共振将用于量化沉积DNA点的密度变异性，从而提供一种质量控制工具，这是目前使用斑点阵列的研究人员无法获得的。DNA斑点的无标记图像将用于量化斑点间和斑点内的密度变异性，提供信息，用于消除进一步分析中的缺陷斑点或作为对随后荧光测量中检测到的信号进行归一化的手段。EF共振将用于提高Cy5标记的杂交DNA的输出，使基因表达分析能够在较低的样品浓度下进行，并且能够在较低水平下观察基因表达，这是以前可能的。该项目将使电气工程学院（在美国国家科学基金会的资助下开发PC和EF技术）和作物科学学院（管理美国国家科学基金会大豆功能基因组学中心）之间的合作成为可能，从而使该技术能够在大型阵列上得到充分的测试和开发。该方法的好处将在7680个元件的基因阵列上进行统计量化，具有足够的芯片间和芯片内复制和控制，以量化从每个独立的PC传感器功能获得的灵敏度和质量控制增益。由此产生的能力将广泛适用于利用微阵列进行人类、动物和植物基因表达分析的广泛科学研究。大豆基因阵列分析被选为新传感器技术的理想测试平台，因为它不需要使用人类DNA和人类来源的测试样本的安全性和批准协议。

DESCRIPTION (provided by applicant): DNA microarrays are capable of simultaneously evaluating the relative expression levels of thousands of genes, and have developed rapidly since their initial introduction. As a result, DNA microarrays are now one of the most preferred technologies for identifying early 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. 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), 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 DNA microarray spots to be measured with 4 5m 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 effort, we plan for the first time to apply 2-dimensional PC surfaces that incorporate optical resonances for both label-free detection and EF to spotted gene expression microarrays. The label-free resonance will be utilized to quantify the density variability of deposited DNA spots, thereby providing a quality- control tool that is not currently available to researchers using spotted arrays. The label-free images of DNA 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 DNA, enabling gene expression analysis to be conducted with lower sample concentrations and the ability to observe gene expression at lower levels than has previously been possible. The project will enable collaboration between faculty in Electrical Engineering, who developed the PC and EF technology under NSF funding, and faculty in Crop Science, who manage the NSF Soybean Functional Genomics Center, thus allowing the technology to be fully tested and developed for large arrays. The benefits of the method will be statistically quantified on a 7680-element gene array with sufficient inter-chip and intra- chip replicates and controls to quantify sensitivity and quality control gains obtained from each independent PC transducer function. The resulting capability will be broadly applicable across a wide range of scientific research that utilizes microarrays for human, animal, and plant gene expression analysis. Analysis of soybean gene arrays was selected as an ideal testbed for the new sensor technology, as it will not require the safety and approval protocols for working with human DNA and human-derived test samples. 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 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. The project is relevant for the development of gene-based diagnostic tests that are accurate, reliable, and able to identify genes at low concentration.