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.

表面上分子相互作用的定量分析通常借助无标记技术实现，例如表面等离子体共振（surface plasmon resonance, SPR）。SPR的灵敏度源于其特性：SPR角对表面折射率变化具有敏感性。类似地，在另一界面光学现象——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，亦可实现表面折射率变化的定量检测。基于这一原理，我们开发了一种名为临界角反射（critical angle reflection, CAR）成像的方法，用于定量分析玻璃表面的分子相互作用。CAR成像可在SPR成像装置上完成。通过平行对照实验，我们证实CAR可覆盖SPR所能实现的绝大多数分子相互作用检测场景，包括蛋白质、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检测范围的小分子结合与细胞内信号事件。相较于SPR，CAR具备多项独特优势：可调谐的灵敏度与动态范围、更深的垂直检测范围、荧光兼容性、更宽泛的光波长与偏振选择范围，以及适配玻璃表面的化学修饰体系。我们预期，CAR将可拓展SPR在小分子检测、全细胞检测、同步荧光成像以及更广泛偶联化学应用中的能力。