Single-molecule calorimeter and free energy landscape

Abstract

The precise measurement of thermodynamic and kinetic properties for biomolecules provides the detailed information for a multitude of applications in biochemistry, biosensing, and health care. However, sensitivity in characterizing the thermodynamic binding affinity down to a single molecule, such as the Gibbs free energy (Gb), enthalpy (Hb), and entropy (Sb), has not materialized. Here, we develop a nanoparticle-based technique to probe the energetic contributions of single-molecule binding events, which introduces a focused laser of optical tweezer to an optical path of plasmonic imaging to accumulate and monitor the transient local heating. This single-molecule calorimeter uncovers the complex nature of molecular interactions and binding characterizations, which can be employed to identify the thermodynamic equilibrium state and determine the energetic components and complete thermodynamic profile of the free energy landscape. This sensing platform promises a breakthrough in measuring thermal effect at the single-molecule level and provides a thorough description of biomolecular specific interactions.

Label-Free Imaging of Nanoscale Displacements and Free-Energy Profiles of Focal Adhesions with Plasmonic Scattering Microscopy

“…This experiment demonstrates that PSM can quantify the nanoscale displacement and free-energy profiles of biological substances in a label-free manner without using the gold nanoparticle labels, which are usually required by the traditional SPR microscopy. 33,34 The effective spring constant represents the curvature (second derivative) of the free energy near equilibrium, reflecting the restoring force of the system back to equilibrium, which can be used to quantify how far the observed state is away from the thermal equilibrium. Considering that the living cells operate out of thermodynamic equilibrium at the molecular scale, 41 the effective spring constant can be employed as an excellent indicator to evaluate the binding conditions of focal adhesions during their migrations.…”

“…19 In addition, the bright spot created by the analyte in the PSM image exhibits a quasi-Airy pattern, thus allowing one to employ the two-dimensional Gaussian fitting, which has been widely used for superresolution fluorescence microscopy, 32 to analyze the PSM images for super-resolution localization of focal adhesion sites and tracking of their nanoscale lateral diffusions. We demonstrate that by tracking the nanoscopic diffusion of proteins on the antibody-modified surface, which has been well studied with gold nanoparticle labels using traditional SPR microscopy, 33,34 PSM can quantify lateral diffusions of singleanalyte−substrate interactions and measure the free-energy profiles, which determine the molecular binding strength and rate. Cell Culture.…”

Single‐Entity Electrochemical Detection of As‐Prepared Metallic and Dielectric Nanoparticle Stochastic Impacts in a Phosphonium Ionic Liquid

“…Metal and dielectric NPs, however, are of interest owing to their unique catalytic [5] and optical [24–27] properties. Movement of metal and dielectric NPs through solution under Brownian motion results in stochastic impacts at the UME surface which are recorded as ‘spikes’ typically in the current‐time ( i‐t ) domain, i. e ., with chronoamperometry (CA).…”

Rapid Regulation of Local Temperature and Transient Receptor Potential Vanilloid 1 Ion Channels with Wide-Field Plasmonic Thermal Microscopy

“…(3) W-PTM records the light from the top of the gold surface, making it possible to integrate with fluorescence detection approaches, whose signals are massively dissipated through the gold surface in traditional SPR systems. 19,20 To investigate the local heating behavior on W-PTM, polymers with low critical solution temperature (LCST) were chosen due to their phase transition and nonphotobleaching features. 14,15 Moreover, a wide range of responsive temperatures (i.e., LCST) can be easily achieved by applying the salt effect to a specific polymer.…”