Critical angle reflection imaging for quantification of molecular interactions on glass surface

Quantification of molecular interactions on a surface is typically achieved via label-free techniques such as surface plasmon resonance (SPR). The sensitivity of SPR originates from the characteristic that the SPR angle is sensitive to the surface refractive index change. Analogously, in another interfacial optical phenomenon, total internal reflection, the critical angle is also refractive index dependent. Therefore, surface refractive index change can also be quantified by measuring the reflectivity near critical angle. Based on this concept, we develop a method called critical angle reflection (CAR) imaging to quantify molecular interactions on glass surface. CAR imaging can be performed on SPR imaging setups. Through a side-by-side comparison, we show that CAR is capable of most molecular interaction measurements that SPR does, including proteins, nucleic acids and cell-based detections. In addition, we show that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing range. CAR exhibits several distinct characteristics over SPR, including tunable sensitivity and dynamic range, deeper vertical sensing range, fluorescence compatibility, broader wavelength and polarization of light selection, and glass surface chemistry. We anticipate CAR can expand SPR capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry.

Methods
The dataset include binding kinetics data measured by CAR and SPR imaging techniques.

表面分子相互作用的定量分析通常可通过无标记技术实现，例如表面等离子体共振（Surface Plasmon Resonance，SPR）。SPR的灵敏度源于其核心特性：SPR角度对表面折射率变化具有极高敏感性。与之类似，在另一种界面光学现象——全内反射中，临界角同样与折射率相关。因此，通过测量临界角附近的反射率，同样可以实现表面折射率变化的定量分析。基于这一原理，我们开发了一种名为临界角反射成像（Critical Angle Reflection，CAR）的方法，用于定量分析玻璃表面的分子相互作用。临界角反射成像可在SPR成像装置上开展实验。通过平行对照实验，我们证实CAR可完成SPR所能实现的绝大多数分子相互作用检测，涵盖蛋白质、核酸以及基于细胞的检测。此外，我们发现CAR能够检测超出SPR传感范围的小分子结合反应与胞内信号。相较于SPR，CAR具备多项独特优势：灵敏度与动态范围可调、垂直传感深度更深、可兼容荧光成像、可选择的光波长与偏振范围更广，且可适配玻璃表面化学修饰。我们预计，CAR将拓展SPR在小分子检测、全细胞检测、同步荧光成像以及更广泛的偶联化学领域的应用能力。
方法
本数据集包含通过临界角反射成像与表面等离子体共振成像技术测得的结合动力学数据。