Luminescent bioassays using immobilized bioconjugated quantum dots within fluidics systems

我们正在引入荧光方法来分析基因片段、多肽和蛋白质，这些方法提供了令人兴奋的能力，可以在几秒钟到几分钟内选择性地确定少量的多个标记。我们正在学习如何通过结合纳米粒子的光学特性和依赖于微米尺度通道中非湍流流体流动的物理特性来获得分析功能的优势。我们的工作一直在探索用单链寡核苷酸修饰的量子点（QDs）（称为“量子点探针”）作为使用荧光共振能量转移（FRET）操作的生物测定的基础。当QD激发附近的染料时，荧光强度增加，该染料与探针和目标链杂交产生的双链DNA形成有关。在多个波长发光的不同量子点的混合物可以用单一的蓝色或紫外线波长激发。我们已经描述了这种结合QD-FRET的多路方案，用于检测寡核苷酸目标的混合物。我们建议继续探索量子点表面的修饰，并构建固定化的固相量子点探针薄膜，以赋予改进的功能并达到新的分析性能水平。所提出的工作的目标是了解如何组装表面涂层，以及如何紧密包装固定量子点探针可以提供高效的选择性结合，同时阻断非特异性吸附，在配置中利用近场光相互作用实现信号增强，以及流体流动环境实现严格控制和样品处理。本建议描述了三个具体目标：微流控通道作为量子点探针平台的探索2。qd探针纸质平台的开发信噪比和选择性的改进，包括使用qd - fret适配体的实现。我们正在引入一种新的方法，通过使用微流体技术实现快速的目标寡核苷酸定量。该方法利用固相QD-FRET过程的通道内发光空间分布进行量化。通过对量子点的共轭设计、量子点探针密度的选择以及通道结构的限制，我们打算实现亚飞摩尔的检测极限，并在几秒到几分钟内得到结果。扩展所学知识将用于指导在纸基板内固定量子点探针，提供一种不同形式的流体系统，当使用手机和数字平板电脑上的电子相机作为探测器时，有助于实际实施。我们将利用整合到寡核苷酸探针中的杂交敏感荧光核碱基，这样就不需要标记目标寡核苷酸了。适配体的实现将把QD-FRET转导技术扩展到肽和蛋白质目标的检测。量子点探针薄膜的固相组装的一个优点是由于一个以上的受体与一个供体的FRET相互作用的信号量级的实质性放大。我们希望建立一个模型来更好地理解如何从这种固相集成中提高信号增强。信号量级的进一步改善将通过上转换纳米粒子（UCNPs）的实施来实现，这些纳米粒子被涂覆在量子点探针上。被近红外（NIR）激光激发的UCNPs可以发光以光学激发量子点。这将减少背景荧光和光散射，同时保持量子点之间的合作效应，实现信号放大。

We are introducing fluorescence methods for analysis of gene fragments, peptides and proteins that offer the exciting capability to selectively determine low quantities of multiple markers in only seconds to minutes. We are learning how to attain advantages in analytical functionality by combining optical properties of nanoparticles with physical properties that are dependent on non-turbulent fluid flow in channels of micrometer dimension. Our work has been exploring quantum dots (QDs) decorated with single-stranded oligonucleotides (termed “QD-probes”) as the basis for bioassays that operate using fluorescence resonance energy transfer (FRET). A fluorescence intensity increase occurs when the QD excites a nearby dye that is associated with the formation of double-stranded DNA that results from hybridization of probe and target strands. Mixtures of different QDs luminescing at multiple wavelengths can be excited using a single blue or ultraviolet wavelength. We have characterized such multiplexed schemes combining QD-FRET for detection of mixtures of oligonucleotide targets. We propose to continue to explore modifications of the surface of QDs, and to build ensembles of immobilized solid-phase QD-probe films, to impart improved functionality and attain new levels of analytical performance. The goal of the proposed work is to learn how surface coatings can be assembled, and how closely packed immobilized QD-probes can offer highly efficient selective binding while blocking non-specific adsorption, in a configuration that makes use of near-field optical interactions to achieve signal enhancement, and a fluidics flow environment to achieve stringency control and sample processing. Three specific objectives are described in this proposal:1. Exploration of microfluidic channels as platforms for QD-probes2. Development of paper-based platforms for QD-probes3. Signal-to-noise and selectivity improvements, including implementation of aptamers using QD-FRETWe are introducing a new approach to achieve rapid target oligonucleotide quantification by using microfluidic technology. The method makes use of in-channel spatial distribution of luminescence from solid-phase QD-FRET processes for quantification. By design of conjugation on QDs, selection of QD-probe density, and constraints on channel structures, we intend to achieve sub-femtomole detection limits with results in seconds to minutes. Extension of what is learned will be used to guide the immobilization of QD-probes within paper-substrates, providing for a different form of fluidics system that facilitates practical implementation when using electronic cameras found on cell phones and digital tablets as detectors. We will make use of hybridization-sensitive fluorescent nucleobases that are integrated into oligonucleotide probes so that labeling of target oligonucleotide is unnecessary. Implementation of aptamers will extend the QD-FRET transduction technology to assays for peptide and protein targets. An advantage of solid-phase assembly of QD-probe films is a substantial amplification of signal magnitude due to interaction of more than one acceptor with one donor by FRET. We wish to establish a model to better understand how to improve signal enhancement from such solid-phase ensembles. Further improvement in signal magnitude will be achieved by implementation of upconverting nanoparticles (UCNPs) that are coated with QD-probes. UCNPs that are excited by near infrared (NIR) lasers can luminesce to optically excite the QDs. This will reduce background fluorescence and optical scattering, while maintaining cooperative effects between the QDs to achieve signal amplification.