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

Abstract

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.

Figure 1.. Principle and setup of Nano-oscillators.

(a) The Nano-oscillators are assembled on a gold surface and driven into oscillation by an alternating electric field which is applied via a three-electrode system, where the gold surface, a Pt coil, and a Ag/AgCl wire serve as the working, counter, and reference electrode, respectively. The oscillation is tracked with a plasmonic imaging setup. A drug perfusion system is used to flow ligand or buffer into the system for binding kinetics measurement. (b) Each Nano-oscillator is a particle tethered to the gold surface by DNA linkers. The particle is functionalized with protein molecules which can bind the ligand molecules introduced into the system. The binding or unbinding of the ligand molecules induces charge change of the particle, leading to oscillation amplitude change. (c) The oscillation of Nano-oscillators are recorded by camera as an image sequence, from which the oscillation amplitude of each individual Nano-oscillator is determined. (d) The binding curves are obtained by measuring the oscillation amplitude change of each Nano-oscillator, and the association rate constant k_a, dissociation rate constant k_d, and the equilibrium constant K_D are determined from the binding curves.

Figure 2.. Oscillation of Nano-oscillators and detection limit.

(a) Nano-oscillators (540 nm silica nanoparticles and 245 nm DNA linkers) are driven into oscillation by an applied potential and the images are recorded. A video showing the oscillation of Nano-oscillators can be found in Supporting Information. The bottom panel shows four snapshot images of an individual Nano-oscillator (marked by the red squared) during different phases of an oscillation cycle, where the image intensity reflects the particle-gold surface distance. The buffer is 6 mM PBS, pH = 7.4. (b) Particle-gold surface distance (red) of the Nano-oscillator squared in (a) and applied potential with frequency 5 Hz (black). (c) FFT of particle-gold surface distance showing a pronounced peak at 5 Hz, and the height of the peak is the oscillation amplitude (red). The detection limit is 1.5 nm as determined by the oscillation amplitude at 5 Hz without applied potential (black).

Figure 3.. Validation of motion equation.

(a) The Randles circuit was used to model the system, where U₀, R_s, R_p and C_dl represent the applied potential, solution resistance, double layer resistance, and double layer capacitance, respectively. (b) Potential dependence of oscillation amplitude. Frequency: f = 5 Hz. Buffer: 6 mM PBS and pH = 7.4. (c) Frequency dependence of oscillation amplitude. Applied potential: U₀ = 300 mV. Buffer: 6 mM PBS and pH = 7.4.

Figure 4.. Measuring binding kinetics with Nano-oscillators.

(a) Sensor response curves of BSA – anti-BSA binding at different concentrations. Solid curves are global fitting of the data to the first order kinetics. The grey curve shows control experiment by introducing 7 nM anti-BSA to streptavidin coated Nano-oscillators. Applied potential: U₀ = 0.4 V, f = 5 Hz. Buffer: 6 mM PBS and pH = 7.4. (b) Sensor response curves of KcsA-Kv1.3 – 1 binding. The grey curve shows control experiment by introducing 3 μM 1 to empty Nanodisc coated Nano-oscillators. (c) Sensor response curves of KcsA-Kv1.3 – ShK binding. The grey curves marked with control 1 and control 2 represent control experiments by introducing 1 nM ShK and 670 nM IgG to empty Nanodisc coated Nano-oscillators, respectively. In figure (b) and (c), the applied potential is U₀ = 0.4 V and f = 5 Hz. Buffer: 3 mM Nanodisc buffer and pH = 7.4. The data for each concentration in (a), (b) and (c) are averaged over at least 5 individual Nano-oscillators.

Figure 5.. Multiplexed detection with Nano-oscillators.

(a) Multiple Nano-oscillators were measured simultaneously with a prism based plasmonic imaging setup. (b) KcsA-Kv1.3 – 1 binding kinetics was measured with 20 individual Nano-oscillators (marked in (a)) in parallel, and the curves were fit with the first order kinetics model. The concentration of 1 injected into the system was 10 nM. (c)-(e) Distributions of k_a, k_d, and K_D obtained from the fittings of 20 Nano-oscillators in (b).

Figure 6.. Factors that affect the performance of Nano-oscillators.

(a) Distributions of the average particle-substrate distance of free particles (without DNA linker) on the gold surface coated with neutral spacer (red) and negative spacer (blue), where the solid curves are Gaussian fitting to the data. Particle diameter, 5 μm; recording time, 5 seconds; buffer, 6 mM PBS, pH = 7.4. (b) Dependence of oscillation amplitude on salt concentration. Oscillation amplitude increases with salt concentration before reaching the maximum value at ~2 mM, which is due to increased conductivity of the solution. Further elevating salt concentration leads to significant ionic screening effect, thus reducing the oscillation amplitude. Applied potential: U₀ = 0.3 V, f = 5 Hz. Buffer pH = 7.4. (c) Dependence of oscillation amplitude on pH. U₀ = 0.3 V, f = 5 Hz, and the buffer is 6 mM PBS, pH = 7.4. For (b) and (c), at least 5 Nano-oscillators were measured to generate the error bar.