Luminescent bioassays using immobilized bioconjugated quantum dots within fluidics systems

我们正在引入用于分析基因片段，肽和蛋白质的荧光方法，这些方法提供了在几秒钟到几分钟内选择性地确定少量多个标记的令人兴奋的能力。我们正在学习如何通过将纳米颗粒的光学性质与依赖于微米尺寸通道中的非湍流流体流动的物理性质相结合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（称为“QD探针”）修饰的量子点（QD）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，该染料与探针和靶链杂交产生的双链DNA的形成相关。在多个波长下发光的不同QD的混合物可以使用单个蓝色或紫外波长来激发。我们的特点是这样的多重方案结合QD-FRET检测寡核苷酸靶的混合物。我们建议继续探索量子点表面的修饰，并建立固定化固相量子点探针膜的集合，以赋予改进的功能并达到新的分析性能水平。 ** 拟议工作的目标是了解如何组装表面涂层，以及紧密包装的固定化QD探针如何在利用近场光学相互作用实现信号增强的配置中提供高效的选择性结合，同时阻止非特异性吸附，以及流体流动环境以实现严格控制和样品处理。本提案中描述了三个具体目标：*1。 探索微流体通道作为QD探针的平台 *2. QD探针纸基平台的开发 *3. 信噪比和选择性改善，包括使用QD-FRET实现适体 ** 我们正在引入一种新方法，通过使用微流体技术实现快速靶寡核苷酸定量。该方法利用来自固相QD-FRET过程的发光的通道内空间分布进行定量。通过量子点上的共轭设计，量子点探针密度的选择，以及对通道结构的限制，我们打算在几秒到几分钟内实现亚毫微微摩尔的检测限。所学到的知识的扩展将用于指导纸衬底内的QD探针的固定，提供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检测器时，该系统有助于实际实施。 我们将利用整合到寡核苷酸探针中的杂交敏感的荧光核碱基，使得不需要标记靶寡核苷酸。适体的实现将QD-FRET转导技术扩展到肽和蛋白质靶标的测定。 QD探针膜的固相组装的优点是由于多于一个受体与一个供体通过FRET的相互作用而使信号幅度显著放大。我们希望建立一个模型，以更好地了解如何提高信号增强从这样的固相合奏。信号幅度的进一步改善将通过实施涂覆有QD探针的上转换纳米颗粒（UCNPs）来实现。由近红外（NIR）激光激发的UCNP可以发光以光学激发QD。这将减少背景荧光和光学散射，同时保持QD之间的合作效应以实现信号放大。

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-probes*2. Development of paper-based platforms for QD-probes*3. Signal-to-noise and selectivity improvements, including implementation of aptamers using QD-FRET**We 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.