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Surface-Enhanced Raman Scattering for Direct Protein Function Investigation: Controlled Immobilization and Orientation

Ma, Hao, Tang, Xiaofan, Liu, Yawen, Han, Xiao Xia, He, Chengyan, Lu, Hui, Zhao, Bing
Analytical chemistry 2019 v.91 no.14 pp. 8767-8771
NADH dehydrogenase, Raman spectroscopy, adenine, affinity chromatography, amino acid sequences, biotechnology, cytochrome c, drug design, ligands, mitochondria, polypeptides, retinoic acid, silver
Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in protein identification and quantification. However, the poor spectral reproducibility, originating from random protein immobilization on SERS substrates, still makes it challenging for SERS to probe protein functions without any extrinsic Raman labels. Here, in our study, spacer molecules between proteins and SERS substrates are optimized for both biocompatible protein immobilization and Raman scattering enhancement. We have accordingly prepared iminodiacetic acid (IDA)-functionalized silver substrates, which are used for capturing His-tagged proteins via nickel–imidazole coordination. The controlled immobilization enables excellent SERS spectral reproducibility as evidenced by 6 polypeptides. Furthermore, the interactions between two model proteins, Erv1C (C-terminal domain of flavine adenine dinucleotide-dependent mitochondrial cytochrome c reductase Erv1) and AFP (alpha-fetoprotein), and their ligands Cyt c (cytochrome c) and ATRA (all-trans-retinoic acid) are examined, respectively. The results indicate that the IDA-functionalized silver substrates enable controlled protein immobilization and allow label-free protein function investigation by SERS. As a proof-of-concept study, the proposed functionalized SERS-active substrates combined with immobilized metal-affinity chromatography will be useful for mechanism studies on protein–ligand interactions, which is crucially important for understanding the structural basis of protein functional versatility and will contribute to the fields of drug design and biotechnology.