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

摘要9876651大卫A. Schultz加州大学圣地亚哥分校通过单分子标记和检测的高通量生物测定这项资助支持开发一种新的生物大分子超灵敏光学测定。 蛋白质或核酸靶分子将在1毫微微克的水平下检测。 这种灵敏度水平将允许在单个细胞中鉴定和定量所选的大分子。 这些检测的灵敏度、速度和准确度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稳健的和纳米尺寸的，但仍然具有非常大的光散射截面。 免疫标记的等离子体共振粒子（PRP）是理想的用于此目的。 PRP是小的（40-80纳米）金属颗粒，当以其等离子体共振频率激发时，其以非凡的效率弹性散射可见光。 它们价格便宜，无毒，并且有各种颜色可供选择。 在典型的白色光照射下从PRP散射的光是如此之大，以至于每个PRP都是单独可见的。 在适当的浓度下，视野中PRP的实际数量仅仅是与靶大分子结合的颗粒数量加上非特异性结合的任何颗粒。 可以使用CCD相机在毫秒内记录这种PRP场的图像。 这将允许快速和超灵敏地检测细胞中的靶大分子。 预期PRP免疫测定的商业化版本将允许低成本检测细胞中广泛的重要分子。 对于靶分子浓度非常低且靶扩增无益或不切实际的情况，这种免疫测定应该补充或取代标准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.