Photonic Crystal Surfaces for Label-Free Detection and Fluorescence Amplification

描述(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，自最初引入以来发展迅速。因此，DNA微阵列现在是识别毒性和疾病的早期生物标志物的最受欢迎的技术之一。微阵列研究的结果可能会受到许多技术和仪器因素的影响，导致人们对所得数据缺乏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，但微阵列衬底本身仍然是一个简单的玻璃表面。在这项建议中，我们描述了用特殊用途的光学换能器替换玻璃表面如何提供关于微阵列斑点的点间和斑点内密度的质量控制信息，而这是目前微阵列分析完全缺乏的，同时放大用于量化杂交DNA的荧光标记的强度。通过提供有关斑点可变性的信息(这是微阵列分析中的一个主要误差来源)，同时提高检测弱表达基因的信噪比(与其他定量基因表达平台相比，微阵列平台目前面临劣势)，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，从而实现对沉积的微阵列斑点的高分辨率无标签成像检测，并将常用微阵列荧光染料的检测灵敏度提高高达550倍。PC是通过在塑料基板上进行大面积纳米复制成型工艺制造的，塑料基板附着在标准玻璃显微镜载玻片上，以与现有的点样机器人、杂交室和检测仪器兼容。最近，伊利诺伊州的坎宁安集团开发了大面积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电磁共振，具有高强度场，逐渐延伸到PC表面的介质中。光学共振与被吸附的生物分子的相互作用导致用于在不使用荧光标记的情况下定量吸附材料密度的共振波长的高度局域移动，使得能够在包括传统微阵列载玻片整个表面的PC上以4 5m的空间分辨率测量沉积的DNA微阵列斑点的无标记图像。PC表面也可以被设计成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)的效应来相对于普通玻璃微阵列载玻片上出现的强度来增加荧光输出强度。使用商用的微阵列激光扫描仪器，EF效应已被证明导致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结合光学共振时，可以进一步增强，从而导致额外的10倍的灵敏度增加。在拟议的工作中，我们计划首次将结合了光学共振的二维PC表面应用于斑点基因表达微阵列，以进行无标记检测和EF。无标记共振将被用来量化沉积的DNA斑点的密度变异性，从而提供一种目前使用斑点阵列的研究人员无法获得的质量控制工具。DNA斑点的无标记图像将用于量化斑点间和斑点内密度的变异性，提供的信息将用于从进一步分析中消除缺陷斑点，或作为从后续荧光测量中检测到的信号归一化的手段。EF共振将被应用于提高Cy5标记的杂交DNA的产量，使基因表达分析能够在更低的样本浓度下进行，并能够在比以前更低的水平上观察基因表达。该项目将促进电气工程教员和作物科学教员之间的合作，前者在NSF资助下开发了PC和EF技术，后者管理NSF大豆功能基因组中心，从而使该技术能够针对大型阵列进行全面测试和开发。该方法的好处将在一个7680元素的基因阵列上进行统计量化，该阵列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试验台，因为它不需要处理人类DNA和人类衍生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.