Detecting Protein–Ligand Interaction from Integrated Transient Induced Molecular Electronic Signal (i-TIMES)

Abstract

Quantitative information about protein–ligand interactions is central to drug discovery. To obtain the quintessential reaction dissociation constant, ideally measurements of reactions should be performed without perturbations by molecular labeling or immobilization. The technique of transient induced molecular electrical signal (TIMES) has provided a promising technique to meet such requirements, and its performance in a microfluidic environment further offers the potential for high throughput and reduced consumption of reagents. In this work, we further the development by using integrated TIMES signal (i-TIMES) to greatly enhance the accuracy and reproducibility of the measurement. While the transient response may be of interest, the integrated signal directly measures the total amount of surface charge density resulted from molecules near the surface of electrode. The signals enable quantitative characterization of protein–ligand interactions. We have demonstrated the feasibility of i-TIMES technique using different biomolecules including lysozyme, N,N′,N″-triacetylchitotriose (TriNAG), aptamer, p-aminobenzamidine (pABA), bovine pancreatic ribonuclease A (RNaseA), and uridine-3′-phosphate (3′UMP). The results show i-TIMES is a simple and accurate technique that can bring tremendous value to drug discovery and research of intermolecular interactions.

Charge-Sensitive Optical Detection of Small Molecule Binding Kinetics in Normal Ionic Strength Buffer

“…35 Both of the GPCR binding pairs we measured involve electrostatic interactions, since the reported binding site of GPR55 is hydrophilic, 43 and ADRB2 is partially hydrophilic. 44 In addition, the ionic screening effect also affects local pH value near the sensor surface, 45 which could also affect protein conformation and binding kinetics. Thus, it is not surprising that the affinity measured under normal and diluted PBS produces different values.…”