Luminescent bioassays using immobilized bioconjugated quantum dots within fluidics systems

我们正在推出用于基因片段、多肽和蛋白质分析的荧光方法，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特性与依赖于微米尺度通道中非湍流流体流动的物理特性相结合来获得分析功能方面的优势。我们的工作一直在探索单链寡核苷酸修饰的量子点(QD)，作为使用荧光共振能量转移(FRET)进行生物检测的基础。当量子点激发附近的一种染料时，荧光强度就会增加，这种染料与双链DNA的形成有关，双链DNA是探针和靶链杂交的结果。不同量子点的混合物在多个波长发光，可以用单一的蓝光或紫外光激发。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，并建立固定的固相量子点探针薄膜系综，以提供更好的功能和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，以及紧密堆积的固定化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和流体流动环境中实现严格控制和样品处理的情况下，在阻止非特定吸附的同时提供高效的选择性结合。该提案描述了三个具体目标： 1.探索微流控通道作为量子点探针的平台 2.纸质QD探头平台的开发 3.改善信噪比和选择性，包括使用QD-FRET实施适配子 我们正在介绍一种利用微流控技术实现快速靶向寡核苷酸定量的新方法。该方法利用固相QD-FRET过程中发光的通道内空间分布进行量化。通过设计量子点的共轭结构，选择量子点的密度，以及对通道结构的限制，我们打算实现亚飞摩尔检测极限，结果在几秒到几分钟内完成。将利用所学知识的延伸来指导将QD探头固定在纸衬底内，从而提供一种不同形式的流体系统，以便在使用手机和数字平板电脑上的电子相机作为探测器时便于实际实施。我们将利用杂交敏感的荧光碱基，这些碱基被整合到寡核苷酸探针中，因此不需要标记目标寡核苷酸。适体的应用将把QD-FRET转导技术扩展到多肽和蛋白质靶标的分析。QD-探针膜的固相组装的一个优点是通过FRET使一个以上的受主与一个施主相互作用，从而大大放大了信号的大小。我们希望建立一个模型来更好地理解如何从这样的固相集合中改善信号增强。信号幅度的进一步改善将通过实施上转换纳米粒子(UCNP)来实现，该纳米粒子被包裹着量子点探针。被近红外(NIR)激光激发的UCNP可以发光来光学激发量子点。这将减少背景荧光和光学散射，同时保持量子点之间的协同效应，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-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.