Carboxyl-terminated dendrimer-coated bioactive interface for protein microarray: High-sensitivity detection of antigen in complex biological samples
Carboxyl-terminated dendrimer-coated bioactive interface for protein microarray: High-sensitivity detection of antigen in complex biological samples
复制标题
DOI：
10.1021/la063717u
复制
发表时间：
2007-05-08
期刊：
影响因子：
3.9
通讯作者：
Too, Heng-Phon
中科院分区：
文献类型：
--
作者：
Ajikumar, Parayil Kumaran;Kiat, Jin;Too, Heng-Phon
蛋白质微阵列是一种很有前途的工具，可以在疾病诊断和药物发现等领域实现高通量蛋白质组学筛选。蛋白质微阵列发展的一个关键方面是优化阵列的表面化学，以达到检测细胞裂解液和其他复杂生物混合物中蛋白质所需的高灵敏度。在本研究中，利用玻璃表面涂覆以羧基（PAMAM-COOH）结尾的聚（丙烯亚胺）树状大分子，制备了具有最小非特异性细胞蛋白吸附的高密度抗体阵列。与惰性peg修饰表面相比，端羧基树突状分子修饰表面具有几乎相似的非特异性细胞蛋白吸附。此外，树突表面可用于反应的多个功能位点促进了抗体的高密度固定和生物分析物的有效捕获。我们测试了各种分子在抗体固定后阻断或失活活性羧基表面的能力，以进一步减少非特异性结合。发现短聚乙二醇（NH2-d4-PEG-COOH）可以显著提高检测的信噪比，从而提高灵敏度。通过接触角和原子力显微镜测量，表征了各种表面的性能和功能质量。研究了非特异性蛋白质吸附和蛋白质固定化作为树突状分子世代的功能，以及从缓冲液（1 pM）和复杂细胞裂解液（10 pM）系统中捕获抗原的敏感性。我们详细的实验研究证明了一种制备高蛋白质负载和低非特异性蛋白质结合表面的简便方法，用于开发高灵敏度蛋白质微阵列。
Protein microarrays are promising tools that can potentially enable high throughput proteomic screening in areas such as disease diagnosis and drug discovery. A critical aspect in the development of protein microarrays is the optimization of the array's surface chemistry to achieve the high sensitivity required for detection of proteins in cell lysate and other complex biological mixtures. In the present study, a high-density antibody array with minimal nonspecific cellular protein adsorption was prepared using a glass surface coated with a poly(propyleneimine) dendrimer terminated with carboxyl group (PAMAM-COOH). The carboxyl-terminated dendrimer-modified surface has almost similar nonspecific cellular protein adsorption when compared to an inert PEG-modified surface. In addition, the multiple functional sites available for reaction on the dendrimer surface facilitated high-density immobilization of antibodies and efficient capture of bioanalytes. Various molecules were tested for their ability to block or deactivate the reactive carboxyl surface after antibody immobilization to further reduce the nonspecific binding. A short oligoethylene glycol (NH2-d4-PEG-COOH), was found to significantly improve the signal-to-noise ratio of the assay, resulting in higher sensitivity. The properties and functional qualities of the various surfaces were characterized by contact angle and AFM measurements. Nonspecific protein adsorption and protein immobilization as a function of dendrimer generations and sensitivity of antigen capturing from a buffer (1 pM) as well as from the complex cell lysate (10 pM) system were examined. Our detailed experimental studies demonstrated a facile method of preparing surfaces with high protein loading and low nonspecific protein binding for the development of high sensitivity protein microarrays.