An optical plasmonic tweezer-controlled surface-enhanced Raman spectroscopy (SERS) platform developed by Hong Kong University of Science and Technology (HKUST) enables efficient, high-throughput, single-molecule characterization in solution. It has the potential to uncover hidden molecular mechanisms that can affect the health of people living with type 2 diabetes. Existing single-molecule approaches are limited to ultra-dilution or molecular immobilization because their diffraction-limited detection volume cannot be further reduced. These methods hinder the ability to study the dynamic actions of these molecules, necessitating the development of advanced single-molecule methods. In a recent breakthrough, the research team led by Jinqing Huang, assistant professor at HKUST’s department of chemistry, has successfully developed a single-molecule platform combining optical plasmonic manipulation and SERS measurement to reduce detection volume and elevate signal enhancement, enabling efficient and high-throughput single-molecule characterization to study pH-dependent amylin species at physiological concentrations. The optical-plasmonic, tweezer-coupled SERS platform addresses the challenge of characterizing a single molecule from heterogeneous mixtures in aqueous milieus. Since both optical-plasmonic trapping and SERS techniques are surface-sensitive, relying on nanostructured substrates, their integration overcomes the optical diffraction limit, confining the position of the plasmonic nanocavity and reducing the SERS active-detection volume for consistently high SERS enhancements. The platform can detect freely diffusing analytes at the single-molecule level without molecular immobilization or solution dilution. This makes it possible for the platform to identify various molecular behaviors and interactions in complex biological processes. To build the platform, the researchers constructed a plasmonic junction between two antigen nanoparticle-coated silica microbeads. They trapped an additional silver nanoparticle to form a dynamic nanocavity upon laser irradiation. The nanocavity surpasses the optical diffraction limit, enabling precise position control while minimizing detection volume and boosting SERS enhancements. Professor Jinqing Huang (front row) and members on her research team, including Xin Dai (left, second row), Vince St. Dollente Mesias (middle, second row), and Wenhao Fu (right, second row) at the Department of Chemistry at Hong Kong University of Science and Technology (HKUST). The team developed a technique to study molecular dynamics (MD) using plasmonic tweezers and surface enhanced Raman spectroscopy (SERS). Courtesy of HKUST. By switching the laser light between the on and off states, the researchers can control the optical-plasmonic trapping to modulate the assembly and disassembly of the dynamic nanocavity. This allows for high-throughput sampling and simultaneous SERS measurements. The antigen nanoparticle-coated silica microbead dimers are more stable than the conventional antigen nanoparticle assemblages in solutions. This makes the plasmonic junction easier to locate and observe on a standard microscope, further improving efficiency and reproducibility. The researchers used the single-molecule platform to detect various amylin species associated with type 2 diabetes. Amylin has the propensity to aggregate and form amyloid fibrils in type 2 diabetes patients. The molecular mechanism that causes aggregation remains unclear, due to the difficulty of detecting the heterogeneous amylin species in a dynamic mixture. The team used the on-and-off control of the laser light to probe various amylin species in mixtures at the single-molecule level. The plasmonic, tweezer-controlled SERS platform enabled the team to uncover the heterogeneous structures of pH-dependent amylin species and the amyloid aggregation mechanisms associated with type 2 diabetes. The researchers studied amylin under different physiological pH conditions by combining spectroscopic experiments and molecular dynamics (MD) simulations. Using the single-molecule platform, they gathered a statistically significant amount of SERS spectra underlying the structural features of various amylin species under two distinct physiological conditions — pH 5.5 and pH 7.4. They identified two types of low-populated amylin species at the early stage of amyloid aggregation in neutral pH. The team found that a slight shift in the equilibrium among different amylin species could drive irreversible amyloid development, even after the adjustment of pH from 7.4 to 5.5. The direct structural characterization of the amylin species within heterogeneous mixtures showed the effect of pH on the amylin species’ intra- and intermolecular interactions and provided insight into the mechanism behind pH-regulated amyloid aggregation in type 2 diabetes. “Our single-molecule platform can acquire a large amount of SERS spectra as molecular snapshots, comparable to those obtained through MD simulations,” Huang said. “By statistically analyzing the structural details at the single-molecule level, we are able to reconstruct the bulk properties and gain unique insights into the population and probability of specific molecule types within the heterogeneous mixture.” By removing ensemble averaging, single-molecule techniques can discern the signal of individual molecules to reveal previously unseen details that can increase scientific understanding of heterogeneous molecular systems. Huang believes that the single-molecule platform from HKUST has the potential to uncover hidden mysteries in complex biological systems. The research was published in Nature Communications (www.doi.org/10.1038/s41467-023-42812-3).