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Abstract
Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful tool to detect biomolecules in aqueous environments. However, it is challenging to identify protein structures at low concentrations, especially for the proteins existing in an equilibrium mixture of various conformations. Here, we develop an in situ optical tweezers-coupled Raman spectroscopy to visualize and control the hotspot between two Ag nanoparticle-coated silica beads, generating tunable and reproducible SERS enhancements with single-molecule level sensitivity. This dynamic SERS detection window is placed in a microfluidic flow chamber to detect the passing-by proteins, which precisely characterizes the structures of three globular proteins without perturbation to their native states. Moreover, it directly identifies the structural features of the transient species of alpha-synuclein among its predominant monomers at physiological concentration of 1 μM by reducing the ensemble averaging. Hence, this SERS platform holds the promise to resolve the structural details of dynamic, heterogeneous, and complex biological systems.
Highlights
Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful tool to detect biomolecules in aqueous environments
We introduce a convenient approach to visualize and control hotspots to provide consistently high SERS enhancements for the characterization of protein structures and conformational fluctuations at physiological concentration, by developing the optical tweezers-coupled Raman spectroscopy
Under microscopic visualization and precise manipulation, two aggregation of silver nanoparticle (AgNP)-coated beads were approached by optical tweezers to create tunable hotspots for efficient, reproducible, and convenient SERS measurements with single-molecule level sensitivity
Summary
The optical tweezers-coupled Raman microscope and the trappable SERS substrate. The home-built SERS platform combines dual-trap optical tweezers and Raman microscope. Under the visualization and manipulation by optical tweezers, the two AgNP-coated beads were approached at incremental distance and gradually adjusted Raman excitation power to optimize the SERS signal of 100 nM hemoglobin in aqueous solution in Fig. 3a and b. Histogram analysis of other spectral features is presented in Supplementary Fig. 11 These reproducible and stable spectra prove the consistent SERS enhancements in the parallel measurements when two AgNP-coated beads were trapped at a constant distance. All the peak assignments of the 1 μM alphasynuclein SERS spectra are summarized in Supplementary Table 216, since the subtle spectral features of alpha-synuclein acquired at physiological concentration could reveal the structural details of its transient species with great biological significance. Such direct identification of the structural variation of alpha-synuclein verifies that our sensitive SERS platform could reduce the ensemble averaging to reveal more structural information on IDP transient species, providing perspective to investigate the behaviors and functions of IDPs during complex biological processes
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