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.