Devising Self-Assembled-Monolayers for Surface-Enhanced Infrared Spectroscopy of pH-Driven Poly-l-lysine Conformational Changes

Abstract

Surface-enhanced infrared absorption spectroscopy (SEIRA) is applied to study protein conformational changes. In general, the appropriate functionalization of metal surfaces with biomolecules remains a challenge if the conformation and activity of the biomolecule shall be preserved. Here we present a SEIRA study to monitor pH-induced conformational changes of poly-l-lysine (PLL) covalently bound to a thin gold layer via self-assembled monolayers (SAMs). We demonstrate that the composition of the SAM is crucial. A SAM of 11-mercaptoundecanonic acid (MUA) can link PLL to the gold layer, but pH-driven conformational transitions were hindered compared to poly-l-lysine in solution. To address this problem, we devised a variety of SAMs, i.e., mixed SAMs of MUA with either octanethiol (OT) or 11-mercapto-1-undecanol (MUoL) and furthermore SAMs of MT(PEG)4 and NHS-PEG10k-SH. These mixed SAMs modify the surface properties by changing the polarity and the morphology of the surface present to nearby PLL molecules. Our experiments reveal that mixed SAMs of MUA-MUoL and SAMs of NHS-PEG10k-SH-MT(PEG)4 are suitable to monitor pH-driven conformational changes of immobilized PLL. These SAMs might be applicable for chemoselective protein immobilization in general.

A combined approach of surface passivation and specific immobilization to study biomolecules by ATR-FTIR spectroscopy1

“…Those self-assembled monolayers (SAMs) are highly ordered two-dimensional structures, that form spontaneously on a variety of surfaces [25]. Besides using SAMs to passivate surfaces, they can also be used to immobilize proteins and also other biomolecules specifically at the crystal surface and thereby enhance the local surface concentration [8,14,15,23,24,28]. Thus, good signal-to-noise ratios are achieved while only low sample concentrations are required for measurements.…”

Immobilization approaches can affect protein dynamics: a surface-enhanced infrared spectroscopic study on lipid–protein interactions

“…Effect of the gold surface Protein immobilization on gold surfaces might influence IR band frequencies when SEIRA spectra are compared to conventional ATR measurements. 27,41 Thus we used SEIRA spectra of αS in solution placed directly on a non-modified gold film (Fig. 3) as reference for our studies with immobilized αS.…”

“…SEIRA measurements were performed by utilizing a single reflection unit using a Si prism IRE as reported before. 27 A thin SEIRA-active film with gold nanostructures was deposited on a Si crystal by application of a deposition method described in details elsewhere. [27][28][29] In brief, the surface of the Si prism was polished and covered with 40% w/v NH 4 F for 1 min to remove the residual oxide layer and to terminate the surface with hydrogen.…”

“…Immobilization can be achieved by modification of the gold layer with self-assembled monolayers (SAMs) of linker molecules. 27,[30][31][32] SAMs are considered to provide a stable and reproducible approach for the immobilization of molecules. SAMs of polyethylene glycol (PEG) have been utilized in several studies to modify a surface for specific protein immobilization.…”

“…[34][35][36][37] In a previous SEIRA study, we have devised SAMs of mixed NHS-PEG-SH (10 kDa) (NHS: N-hydroxysuccinimide) and MT (PEG) 4 (methyl-and sulfhydryl-terminated PEG) for immobilization of poly-L-lysine (PLL) to gold. 27 In this work, we used SAMs of 5 kDa NHS-PEG-SH (Nanocs) and MT(PEG) 4 (Thermo Scientific), thereby we reduced the length of the NHS-PEG-SH linker molecules to ≈30 nm and facilitated protein immobilization closer to the gold surface leading to more enhancement of the absorbance signal. MT(PEG) 4 are only 1.58 nm long, and were applied as inactive spacers to passivate the gold surface against non-specific interactions between αS and the gold film.…”