Department of Biomedical Informatics, Asia University, Taichung 41354, Taiwan.
Anal Biochem. 2012 Apr 1;423(1):26-35. doi: 10.1016/j.ab.2011.12.043. Epub 2012 Jan 8.
The development of protein chips has suffered from problems regarding long-term protein stability and activity. We present a protein sensor surface for immunodetection that is prepared by a DNA-directed protein immobilization method on a mixed self-assembled monolayer (SAM). By this approach, an immobilized single-stranded DNA (ssDNA) surface can be transferred/modified into a protein chip by flowing in ssDNA-conjugated protein when the protein chip measurement is needed. Therefore, the long-term stability of the protein chip will not be a problem for various applications. We tried various compositions for the SAM layer, the length of the ssDNA spacer, the end-point nucleotide composition, and the processes of ssDNA immobilization of the SAM for an optimized condition for shifting the DNA chip to a protein chip. The evaluations were made by using surface plasmon resonance. Our results indicated that a 50:1 ratio of oligo(ethylene glycol) (OEG)/COOH-terminated OEG and DNA sequences with 20mer are the best conditions found here for making a protein chip via a DNA-directed immobilization (DDI) method. The designed end-point nucleotide composition contains a few guanines or cytosines, and ssDNA immobilization of the SAM by dehybridizing immobilized double-stranded DNA (dsDNA) can improve the hybridization efficiency.
蛋白质芯片的发展一直受到长期蛋白质稳定性和活性问题的困扰。我们提出了一种用于免疫检测的蛋白质传感器表面，该表面是通过 DNA 指导的蛋白质固定化方法在混合自组装单层 (SAM) 上制备的。通过这种方法，当需要进行蛋白质芯片测量时，可以将固定化的单链 DNA（ssDNA）表面通过流动 ssDNA 偶联的蛋白质转导/修饰成蛋白质芯片。因此，蛋白质芯片的长期稳定性不会成为各种应用的问题。我们尝试了各种 SAM 层组成、ssDNA 间隔物的长度、末端核苷酸组成以及 ssDNA 在 SAM 上的固定化过程，以优化将 DNA 芯片转换为蛋白质芯片的条件。评估是通过使用表面等离子体共振进行的。我们的结果表明，OEG/COOH 端基 OEG 的 50:1 比例和 20 个碱基的 DNA 序列是通过 DNA 指导的固定化 (DDI) 方法制作蛋白质芯片的最佳条件。设计的末端核苷酸组成包含几个鸟嘌呤或胞嘧啶，通过解杂交固定化的双链 DNA（dsDNA）来固定 SAM 上的 ssDNA 可以提高杂交效率。
