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

Abstract

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.

Human angiotensin I‐converting enzyme study by surface‐enhanced Raman spectroscopy

“…[ 14 ] Qualitative and quantitative analysis of low‐molecular analytes and proteins can be carried out using SERS spectra and multivariate statistics. [ 17–20 ]…”

“…[14] Qualitative and quantitative analysis of low-molecular analytes and proteins can be carried out using SERS spectra and multivariate statistics. [17][18][19][20] The first SERS studies of macromolecular substances were undertaken in the 1980s. [21,22] Their practical application was limited by the lack of theoretical understanding of the nature of the effect.…”

Raman Scattering-Based Biosensing: New Prospects and Opportunities

“…However, interest in measuring the intrinsic spectra of biomolecules is not diminishing because label-free detection provides information on the conformation and orientation of molecules and allows the analysis of molecular dynamic changes in bioprocesses and interactions between biomolecules and other compounds. To date, a successful application of the direct surface-enhanced Raman approach is confirmed in the detection of whole cells, bacteria, and proteins [ 80 , 118 , 119 , 120 ].…”

Individual Plasmonic Nanoprobes for Biosensing and Bioimaging: Recent Advances and Perspectives

“…[ 9,20 ] SERS spectrum and intensity are extremely sensitive to the local electromagnetic and chemical environments, thus SERS technique has ultrasensitive detection and identification for a molecule in close vicinity to the hot spots. [ 21–26 ]…”