Optical tweezers-controlled hotspot for sensitive and reproducible surface-enhanced Raman spectroscopy characterization of native protein structures

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.

Fig. 1. Illustration of the controllable SERS probe experiments.

a Schematic diagram of the optical tweezers-coupled Raman spectroscopic platform with microfluidic set-up. b Two trapping laser beams (red) to manipulate two AgNP-coated beads and one Raman probe beam (green) to detect signals from the gap between the two AgNP-coated beads. c The real-time camera image of two trapped AgNP-coated beads from the microscope. d SEM image of the gap between two AgNP-coated beads. The scale bar is 0.1 μm. e SEM image of AgNP-coated beads to show the uniform AgNP coating. The scale bar is 1 μm. All micrographs are representative images of three independent measurements.

Fig. 2. Creation and adjustment of the dynamic hotspot with in situ SERS measurements of 1% ethanol aqueous solution.

a The real-time camera images of the two AgNP-coated beads trapped at different distance. b SERS spectra of 1% ethanol aqueous solution with 1 s acquisition time when the two AgNP-coated beads approaching. c The intensity of the ethanol characteristic peak at 1458 cm⁻¹ as a function of the distance between the two AgNP-coated beads from beads approaching to beads separating. The reversible bead positions are illustrated as inset. d SERS spectra of 1% ethanol aqueous solution with 1 s acquisition time when the two AgNP-coated beads separating. Source data are provided as a Source data file.

Fig. 3. The spectroscopic characterizations of hemoglobin at its native states.

a SERS spectra of 100 nM hemoglobin in aqueous solution when the two AgNP-coated beads were trapped at different distance (50–10 nm). b SERS spectra of 100 nM hemoglobin solution under different Raman excitation power (5–100%). c The comparison between SERS spectra of 100 nM hemoglobin solution with 1 s acquisition time (blue) and the spontaneous Raman spectrum of 250 μM hemoglobin solution with 60 s acquisition time (black). d 3D stacking plot of SERS spectra of 100 nM hemoglobin solution obtained from AgNP-coated beads trapped at 20 nm with 1 s acquisition time. e, f The histograms of the intensities of the hemoglobin characteristic peaks at 1376 cm⁻¹ (mean = 4929.15 in arb. units, RSD = 14.63%) and 1594 cm⁻¹ (mean = 5391.41 in arb. units, RSD = 16.35%) across the 50 SERS spectra in (d), respectively. Source data are provided as a Source data file.

Fig. 4. The spectroscopic characterizations of lysozyme in its compact globular structure.

a The comparison between 50 SERS spectra of 1 μM lysozyme solution with 1 s acquisition time (bottom) and the spontaneous Raman spectrum of 1 mM lysozyme solution with 5 min acquisition time (top). b Histogram of the Amide I band distribution of the 50 SERS spectra of 1 μM lysozyme solution in (a), indicating the mean as 1655 cm⁻¹ with 0.1% RSD. c Illustration of the ensemble averaging from the spontaneous Raman measurement of lysozyme in the concentrated solution and the small-size sampling from the SERS measurements of lysozyme in the dilute solution. Source data are provided as a Source data file.

Fig. 5. The spectroscopic characterizations of an intrinsically disordered protein: alpha-synuclein.

a CD spectrum of 200 μM alpha-synuclein in aqueous solution. b Spontaneous Raman spectra of 2 mM (green) and 250 μM (blue) alpha-synuclein solution with 10 min acquisition time. c The comparison among three representative types of SERS spectra of 1 μM alpha-synuclein solution with 1 s acquisition time (blue, red, and black) and the SERS spectrum of 250 μM alpha-synuclein solution with 5 min acquisition time (purple). d Mapping of 200 SERS spectra of 1 μM alpha-synuclein solution obtained from two AgNP-coated beads trapped at 20 nm with 1 s acquisition time. The color bar shows the normalized intensities from low (dark blue) to high (red). e Illustration of the ensemble averaging from the measurement of alpha-synuclein at high concentration with long accumulation time and the small-size sampling from the measurements of alpha-synuclein at low concentration with short accumulation time. Source data are provided as a Source data file.