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，并且自其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的早期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，导致有关衍生数据缺乏可重复性和准确性的主要批评。虽然荧光染料、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改进，但微阵列基底本身仍然是一个简单的玻璃表面。在这个建议中，我们描述了如何更换玻璃表面与一个特殊用途的光学传感器可以提供质量控制信息的interspot和intraspot密度的微阵列斑点，目前完全缺乏从微阵列分析，同时放大用于量化杂交DNA的荧光标记的强度。通过提供斑点变异性的信息，（代表微阵列分析中的主要误差来源），同时增加弱表达基因检测的信噪比（微阵列平台目前面临的劣势相比，其他定量基因表达平台），拟议的项目代表了微阵列技术的根本进步。用于提供这些特征的光学换能器是2维光子晶体（PC）表面，其被设计成提供光学共振，所述光学共振使得能够对沉积的微阵列斑点进行高分辨率无标记成像检测，以及对常用的微阵列荧光染料的高达550倍的增强检测灵敏度。PC是由一个大面积的nanoreplica塑料基板上的成型工艺，连接到标准的玻璃显微镜载玻片与现有的点样机器人，杂交室和检测仪器的兼容性。最近，大面积PC表面已经由伊利诺伊州的坎宁安集团开发为多功能光学换能器，其可以被设计为在任何期望的波长处产生窄波长电磁谐振，其特征在于渐逝地延伸到PC表面上的介质中的高强度场。与吸附的生物分子的光学共振的相互作用的结果在一个高度本地化的位移的共振波长，用于量化的密度吸附的材料，而不使用荧光标记，使标记的无图像的沉积的DNA微阵列斑点被测量与4 - 5 μ m的空间分辨率超过PC包括一个传统的微阵列载玻片的整个表面。PC表面也可以被设计成使得光学共振与用于激发荧光染料的激光的波长一致，从而使用称为增强荧光（EF）的效应，相对于将在普通玻璃微阵列载玻片上发生的强度增加荧光输出强度。EF效应已被证明导致使用市售微阵列激光扫描仪器检测到的荧光信号增加约50倍，但当PC被设计为还在荧光团的发射波长处并入光学共振时，可以进一步增强，导致灵敏度增加额外的10倍。在拟议的努力中，我们计划首次应用二维PC表面，将光学共振的无标记检测和EF斑点基因表达微阵列。无标记共振将用于量化沉积的DNA斑点的密度可变性，从而提供目前使用斑点阵列的研究人员不可用的质量控制工具。DNA斑点的无标记图像将用于量化斑点间和斑点内密度变异性，提供将用于从进一步分析中消除缺陷斑点的信息，或作为标准化来自后续荧光测量的检测信号的手段。EF共振将被应用于增强Cy 5标记的杂交DNA的输出，使得基因表达分析能够以较低的样品浓度进行，并且能够在比以前可能的更低水平下观察基因表达。该项目将使电气工程教师（在NSF资助下开发PC和EF技术）与作物科学教师（管理NSF大豆功能基因组学中心）之间的合作成为可能，从而使该技术能够进行全面测试并开发用于大型阵列。该方法的益处将在7680元件基因阵列上进行统计学量化，该基因阵列具有足够的芯片间和芯片内重复和对照，以量化从每个独立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.