High-throughput Biological-assays Via Single Molecule Labeling and Detection

摘要：david a . schultz，加州大学圣地亚哥分校通过单分子标记和检测进行高通量生物测定该资助支持开发一种新的生物大分子超灵敏光学测定方法。无论是蛋白质还是核酸靶分子，都可以在1飞图水平检测。这种灵敏度水平将允许在单个细胞中对所选大分子进行鉴定和定量。这些检测的灵敏度、速度和准确性将通过实时体外研究进行优化。用于这种检测的报告分子必须是坚固的和纳米级的，但仍然具有非常大的光散射横截面。免疫标记等离子共振粒子（PRPs）是实现这一目的的理想方法。PRPs是一种小的（40-80纳米）金属颗粒，当被等离子体共振频率激发时，它会以非凡的效率弹性地散射可见光。它们便宜，无毒，并且有多种颜色可供选择。在典型的白光照射下，PRP散射的光非常大，以至于每个PRP都是单独可见的。在适当的浓度下，视场中PRPs的实际数量仅仅是与目标大分子结合的颗粒数量加上非特异性结合的颗粒数量。使用CCD相机可以在几毫秒内记录这种PRPs场的图像。这将允许在细胞中的目标大分子的快速和超灵敏的检测。预计商业化版本的PRP免疫测定将允许低成本检测细胞中广泛的重要分子。在靶分子浓度很低且靶扩增无效或不实用的情况下，这种免疫测定应补充或取代标准的ELISA或放射免疫测定。

ABSTRACT9876651David A. SchultzUniversity of California, San DiegoHigh-throughput Biological -assays Via Single Molecule Labeling and DetectionThis grant supports the development of a new ultrasensitive optical assay for biological macromolecules. Either protein or nucleic acid target molecules will be detected at the level of 1 femtogram. This level of sensitivity will allow the identification and quantification of the chosen macromolecule in a single cell. The sensitivity, speed, and accuracy of these assays will be optimized using a real-time in-vitro studies.The reporter molecule for such an assay must be robust and nano-sized yet still have a very large light-scattering cross-section. Immmunolabelled plasmon resonant particles (PRPs) are ideal for this purpose. PRPs are small (40-80 nanometer) metal particles that scatter visible light elastically with extraordinary efficiency when excited at their plasmon resonance frequency. They are inexpensive, non-toxic, and are available in a variety of colors. The light scattered from PRPs under typical white light illumination is so large that each PRP is individually visible. At appropriate concentration, the actual number of PRPs in a field of view is simply the number of particles bound to the target macromolecule plus any particles that are non-specifically bound. An image of such a field of PRPs can be recorded using a CCD camera within milliseconds. This will allow for the rapid and ultrasensitive detection of the target macromolecules in the cell. It is anticipated that commercialized versions of the PRP immunoassay will allow for low cost detection of a wide range of important molecules in the cell. Such an immunoassay should complement or replace standard ELISA or radio-immunoassays for cases where the concentration of the target molecules if very low and target amplification is not beneficial or practical.