Biospecific irreversible fishing coupled with atomic force microscopy for detection of extremely low-abundant proteins
Biospecific irreversible fishing coupled with atomic force microscopy for detection of extremely low-abundant proteins
复制标题
DOI：
10.1002/pmic.200800598
复制
发表时间：
2009-03-01
期刊：
影响因子：
3.4
通讯作者：
Zgoda, Victor
中科院分区：
文献类型：
--
作者：
Archakov, Alexander;Ivanov, Yurii;Zgoda, Victor
在缺乏蛋白质PCR类似物的情况下，浓度检测限（DL）成为真实的挑战。这个问题可以通过将生物特异性不可逆捕捞与原子力显微镜（AFM）相结合来解决。原子力显微镜提供了登记单个分子及其复合物的能力，而生物特异性捕鱼利用了分布在大量生物材料上的分析物分子与固定在芯片表面上的配体分子之间的仿射相互作用。捕捞可以以依赖Kd的可逆模式和不依赖Kd的不可逆模式进行。在这项研究中，DL的两个以前应用的蛋白质组学方法进行了测定和DL的一个新开发的分析方法进行了比较。第一种方法是在2-DE或LC分离蛋白质后对生物材料进行MS分析，其DL为10（-8）-10（-10）M。第二种方法，基于蛋白质组微阵列形式的分子相互作用的光学生物传感器分析，具有10（-9）-10（-10）M的DL。我们提出的结合生物特异性捕捞与AFM的方法使我们能够在可逆结合条件下达到10（-11）M的DL值，在不可逆结合条件下达到10（-16）M。
In the absence of an analog of PCR for proteins, the concentration detection limit (DL) becomes a real challenge. The problem may be solved by means of a combination of biospecific irreversible fishing with atomic force microscopy (AFM). AFM offers the ability to register individual molecules and their complexes, while biospecific fishing takes advantage of an affine interaction between analyte molecules spread over a large volume of biomaterial and ligand molecules immobilized on the chip surface. Fishing may be conducted in Kd-dependent reversible mode and in Kd-independent irreversible mode. In this study, the DLs of two previously applied proteomic approaches were determined and compared to the DL of a newly developed analytical method. The first approach, based on M S analysis of biomaterial after 2-DE or LC separation of proteins, attained a DL at the level of 10(-8)-10(-10) M. The second approach, based on the optical biosensor analysis of molecular interactions in the format of proteomic microarrays, had a DL of 10(-9)-10(-10) M. Our proposed method which combines biospecific fishing with AFM allowed us to attain DL values of 10(-11) M under reversible binding conditions and 10(-16) M under irreversible binding conditions.