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.

中文翻译：

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正常离子强度缓冲液中小分子结合动力学的电荷敏感光学检测

大多数无标记检测技术检测分子的质量，因此它们的灵敏度随着分子量而降低，这使得检测小分子具有挑战性。为了满足这一需求，我们开发了一种电荷敏感光学检测 (CSOD) 技术，该技术通过光纤检测电荷而不是分子的质量。然而，分子的有效电荷随着缓冲离子强度的增加而降低。出于这个原因，以前的 CSOD 使用稀释的缓冲液，这可能会影响测量的分子结合动力学。在这里，我们展示了一种能够在正常离子强度缓冲液中检测分子结合动力学的技术。开发了一个 H 形样品井，以增加感应区域的电流密度，以补偿由于在正常离子强度缓冲液中的离子筛选造成的信号损失，同时保持电极处的低电流密度以最小化电极反应。此外，使用琼脂糖凝胶覆盖电极，以防止电极反应产生的气泡进入传感区域。通过这种新设计，我们测量了正常缓冲液中 G 蛋白偶联受体 (GPCR) 与其小分子配体之间的结合动力学。我们发现在普通缓冲液中测量的亲和力比在稀释缓冲液中测量的更强，这可能是由于稀释缓冲液中相同的带电配体和受体之间的静电排斥力更强。通过这种新设计，我们测量了正常缓冲液中 G 蛋白偶联受体 (GPCR) 与其小分子配体之间的结合动力学。我们发现在普通缓冲液中测量的亲和力比在稀释缓冲液中测量的更强，这可能是由于稀释缓冲液中相同的带电配体和受体之间的静电排斥力更强。通过这种新设计，我们测量了正常缓冲液中 G 蛋白偶联受体 (GPCR) 与其小分子配体之间的结合动力学。我们发现在普通缓冲液中测量的亲和力比在稀释缓冲液中测量的更强，这可能是由于稀释缓冲液中相同的带电配体和受体之间的静电排斥力更强。