TY - JOUR
T1 - Charge-Sensitive Optical Detection of Small Molecule Binding Kinetics in Normal Ionic Strength Buffer
AU - Liang, Runli
AU - Ma, Guangzhong
AU - Jing, Wenwen
AU - Wang, Yan
AU - Yang, Yunze
AU - Tao, Nongjian
AU - Wang, Shaopeng
N1 - Funding Information:
Financial support from the National Cancer Institute of the National Institutes of Health under Award Number R33CA202834 is acknowledged. We also thank Prof. Heng Zhu and Prof. Prashant J. Desai at Johns Hopkins University for providing the VirD-GPCR samples. This work is dedicated to the memory of Dr. Nongjian Tao (1963-2020).
Publisher Copyright:
© 2021 American Chemical Society. All rights reserved.
PY - 2021/2/26
Y1 - 2021/2/26
N2 - Most label-free detection technologies detect the masses of molecules, and their sensitivities thus decrease with molecular weight, making it challenging to detect small molecules. To address this need, we have developed a charge-sensitive optical detection (CSOD) technique, which detects the charge rather than the mass of a molecule with an optical fiber. However, the effective charge of a molecule decreases with the buffer ionic strength. For this reason, the previous CSOD works with diluted buffers, which could affect the measured molecular binding kinetics. Here, we show a technique capable of detecting molecular binding kinetics in normal ionic strength buffers. An H-shaped sample well was developed to increase the current density at the sensing area to compensate the signal loss due to ionic screening at normal ionic strength buffer, while keeping the current density low at the electrodes to minimize the electrode reaction. In addition, agarose gels were used to cover the electrodes to prevent electrode reaction generated bubbles from entering the sensing area. With this new design, we have measured the binding kinetics between G-protein-coupled receptors (GPCRs) and their small molecule ligands in normal buffer. We found that the affinities measured in normal buffer are stronger than those measured in diluted buffer, likely due to the stronger electrostatic repulsion force between the same charged ligands and receptors in the diluted buffer.
AB - Most label-free detection technologies detect the masses of molecules, and their sensitivities thus decrease with molecular weight, making it challenging to detect small molecules. To address this need, we have developed a charge-sensitive optical detection (CSOD) technique, which detects the charge rather than the mass of a molecule with an optical fiber. However, the effective charge of a molecule decreases with the buffer ionic strength. For this reason, the previous CSOD works with diluted buffers, which could affect the measured molecular binding kinetics. Here, we show a technique capable of detecting molecular binding kinetics in normal ionic strength buffers. An H-shaped sample well was developed to increase the current density at the sensing area to compensate the signal loss due to ionic screening at normal ionic strength buffer, while keeping the current density low at the electrodes to minimize the electrode reaction. In addition, agarose gels were used to cover the electrodes to prevent electrode reaction generated bubbles from entering the sensing area. With this new design, we have measured the binding kinetics between G-protein-coupled receptors (GPCRs) and their small molecule ligands in normal buffer. We found that the affinities measured in normal buffer are stronger than those measured in diluted buffer, likely due to the stronger electrostatic repulsion force between the same charged ligands and receptors in the diluted buffer.
KW - G-protein-coupled receptors
KW - binding kinetics
KW - charge sensitive optical detection
KW - label-free
KW - normal ionic strength buffer
KW - small molecule
KW - virion display
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U2 - 10.1021/acssensors.0c01063
DO - 10.1021/acssensors.0c01063
M3 - Article
C2 - 32842724
AN - SCOPUS:85102322042
VL - 6
SP - 364
EP - 370
JO - ACS Sensors
JF - ACS Sensors
SN - 2379-3694
IS - 2
ER -