Quantifying Ligand–Protein Binding Kinetics with Self-Assembled Nano-oscillators

Measuring ligand–protein interactions is critical for unveiling molecular-scale biological processes in living systems and for screening drugs. Various detection technologies have been developed, but quantifying the binding kinetics of small molecules to the proteins remains challenging because the sensitivities of the mainstream technologies decrease with the size of the ligand. Here, we report a method to measure and quantify the binding kinetics of both large and small molecules with self-assembled nano-oscillators, each consisting of a nanoparticle tethered to a surface via long polymer molecules. By applying an oscillating electric field normal to the surface, the nanoparticle oscillates, and the oscillation amplitude is proportional to the number of charges on the nano-oscillator. Upon the binding of ligands onto the nano-oscillator, the oscillation amplitude will change. Using a plasmonic imaging approach, the oscillation amplitude is measured with subnanometer precision, allowing us to accurately quantify the binding kinetics of ligands, including small molecules, to their protein receptors. This work demonstrates the capability of nano-oscillators as an useful tool for measuring the binding kinetics of both large and small molecules

Label-Free Multimetric Measurement of Molecular Binding Kinetics by Electrical Modulation of a Flexible Nanobiolayer

Most label-free techniques rely on measuring refractive index or mass change on the sensor surface. Thus, it is challenging for them to measure small molecules or enzymatic processes that only induce a minor mass change on the analyte molecules. Here, we have developed a technique by combining Surface Plasmon Resonance sensing with an Oscillating Biomolecule Layer approach (SPR-OBL) to enhance the sensitivity of traditional SPR. In addition to the inherent mass sensitivity, SPR-OBL is also sensitive to the charge and conformational change of the analyte; hence it overcomes the mass limit and is able to detect small molecules. We show that the multimetric SPR-OBL measurement allows for sensing any changes regarding mass, charge, and conformation, which expands the detection capability of SPR

Label-Free Multimetric Measurement of Molecular Binding Kinetics by Electrical Modulation of a Flexible Nanobiolayer

Most label-free techniques rely on measuring refractive index or mass change on the sensor surface. Thus, it is challenging for them to measure small molecules or enzymatic processes that only induce a minor mass change on the analyte molecules. Here, we have developed a technique by combining Surface Plasmon Resonance sensing with an Oscillating Biomolecule Layer approach (SPR-OBL) to enhance the sensitivity of traditional SPR. In addition to the inherent mass sensitivity, SPR-OBL is also sensitive to the charge and conformational change of the analyte; hence it overcomes the mass limit and is able to detect small molecules. We show that the multimetric SPR-OBL measurement allows for sensing any changes regarding mass, charge, and conformation, which expands the detection capability of SPR

Probing Single Molecule Binding and Free Energy Profile with Plasmonic Imaging of Nanoparticles

Measuring binding between molecules is critical for understanding basic biochemical processes, developing molecular diagnosis, and screening drugs. Here we study molecular binding at the single molecule level by attaching nanoparticles to the molecular binding pairs. We track the thermal fluctuations of the individual nanoparticles with sub-nanometer precision using a plasmonic scattering imaging technique and show that the fluctuations are controlled by the molecular binding pairs rather than by the nanoparticles. Analysis of the thermal fluctuations provides unique information on molecular binding, including binding energy profile, effective spring constant, and switching between single and multiple molecular binding events. The method provides new insights into molecular binding and also allows one to differentiate nonspecific binding from specific binding, which has been a difficult task in biosensors

Probing Single Molecule Binding and Free Energy Profile with Plasmonic Imaging of Nanoparticles

Measuring binding between molecules is critical for understanding basic biochemical processes, developing molecular diagnosis, and screening drugs. Here we study molecular binding at the single molecule level by attaching nanoparticles to the molecular binding pairs. We track the thermal fluctuations of the individual nanoparticles with sub-nanometer precision using a plasmonic scattering imaging technique and show that the fluctuations are controlled by the molecular binding pairs rather than by the nanoparticles. Analysis of the thermal fluctuations provides unique information on molecular binding, including binding energy profile, effective spring constant, and switching between single and multiple molecular binding events. The method provides new insights into molecular binding and also allows one to differentiate nonspecific binding from specific binding, which has been a difficult task in biosensors

Three-Dimensional Tracking of Tethered Particles for Probing Nanometer-Scale Single-Molecule Dynamics Using a Plasmonic Microscope

Three-dimensional (3D) tracking of surface-tethered single particles reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in the axial direction, for measuring the dynamics of biomolecules with a spatial scale of several nanometers. Here, we present a plasmonic imaging technique that can track the motion of ∼100 tethered particles in 3D simultaneously with sub-nanometer axial precision and single-digit nanometer lateral precision at millisecond time resolution. By tracking the 3D coordinates of a tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzymes. We further show that the particle motion pattern can be used to identify specific and nonspecific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level

Study of Small-Molecule–Membrane Protein Binding Kinetics with Nanodisc and Charge-Sensitive Optical Detection

Nanodisc technology provides membrane proteins with a nativelike lipid bilayer and much-needed solubility and enables in vitro quantification of membrane protein binding with ligands. However, it has been a challenge to measure interaction between small-molecule ligands and nanodisc-encapsulated membrane proteins, because the responses of traditional mass-based detection methods scale with the mass of the ligands. We have developed a charge-sensitive optical detection (CSOD) method for label-free measurement of the binding kinetics of low molecular mass ligands with nanodisc-encapsulated membrane proteins. This microplate-compatible method is sensitive to the charge instead of the mass of a ligand and is able to measure both large and small molecules in a potentially high-throughput format. Using CSOD, we measured the binding kinetics between peptide and small-molecule ligands and a nanodisc-encapsulated potassium ion channel protein, KcsA-Kv1.3. Both association and dissociation rate constants for these ligands are obtained for the first time. The CSOD results were validated by the consistency of the values with reported binding affinities. In addition, we found that CSOD can tolerate up to 3.9% dimethyl sulfoxide (DMSO) and up to 10% serum, which shows its compatibility with realistic sample conditions

Probing Single Molecule Binding and Free Energy Profile with Plasmonic Imaging of Nanoparticles

Measuring binding between molecules is critical for understanding basic biochemical processes, developing molecular diagnosis, and screening drugs. Here we study molecular binding at the single molecule level by attaching nanoparticles to the molecular binding pairs. We track the thermal fluctuations of the individual nanoparticles with sub-nanometer precision using a plasmonic scattering imaging technique and show that the fluctuations are controlled by the molecular binding pairs rather than by the nanoparticles. Analysis of the thermal fluctuations provides unique information on molecular binding, including binding energy profile, effective spring constant, and switching between single and multiple molecular binding events. The method provides new insights into molecular binding and also allows one to differentiate nonspecific binding from specific binding, which has been a difficult task in biosensors

Probing Single Molecule Binding and Free Energy Profile with Plasmonic Imaging of Nanoparticles

Measuring binding between molecules is critical for understanding basic biochemical processes, developing molecular diagnosis, and screening drugs. Here we study molecular binding at the single molecule level by attaching nanoparticles to the molecular binding pairs. We track the thermal fluctuations of the individual nanoparticles with sub-nanometer precision using a plasmonic scattering imaging technique and show that the fluctuations are controlled by the molecular binding pairs rather than by the nanoparticles. Analysis of the thermal fluctuations provides unique information on molecular binding, including binding energy profile, effective spring constant, and switching between single and multiple molecular binding events. The method provides new insights into molecular binding and also allows one to differentiate nonspecific binding from specific binding, which has been a difficult task in biosensors