Detection of particles and hemolysis was also explored with this approach 18, 19. Indeed, previous studies have shown that 10 −6 refractive index change can be resolved on the glass-water interface by measuring the reflectivity near the critical angle 17. Because the critical angle is also refractive index dependent, therefore, the critical angle shift should be a measure of the refractive index change near the glass surface. Inspired by this principle, we thought that the molecular interactions induced refractive index changes can also be detected on bare glass when the incident angle is close to the critical angle. By measuring the resonance angle shift, we can measure molecular interactions on the surface that changes the local refractive index. For example, in SPR, the reflectivity of the light is significantly reduced at a specific incident angle when the light energy resonates with the surface plasmons in the gold film, and the resonance angle is highly sensitive to the refractive index changes near the surface. The sensitivity of most label-free optical biosensors originates from the response of the probe light to the refractive index change on the sensor surface 16. It is desirable to develop a simple and sensitive technique for molecular interaction measurements. Reflectometry based on measuring phase shift can directly quantify binding kinetics on a cover glass 14, 15, but owing to the instrumentation complexity and moderate sensitivity, it is not as competitive as SPR. However, these sensors are made through microfabrication and still labor-consuming. The gold film is not required for glass-based biosensors, such as interferometers 8, 9, 10, microring and microsphere resonators 11, 12, 13. To generate SPR on the surface, the sensor chip (glass slide) must be coated with a metal film (often gold), which is laborious and increases the operation cost. Owing to the sharp response to the refractive index change on the surface, the high sensitivity of SPR enables the detection of biomolecules 3, small molecules 4, viruses 5, and cells 6, 7. Surface plasmon resonance (SPR) is the most widely used label-free technique in pharmaceuticals and research laboratories for measuring molecular binding kinetics 1, 2, 3.

Molecular interactions are ubiquitous in biological systems and important to the understanding of molecular biology and drug discovery. We anticipate CAR can expand SPR′s capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry. CAR exhibits several distinct characteristics, including tunable sensitivity and dynamic range, deeper vertical sensing range, fluorescence compatibility, broader wavelength and polarization of light selection, and glass surface chemistry. In addition, we show that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing range. Through a side-by-side comparison, we show that CAR is capable of most molecular interaction measurements that SPR performs, including proteins, nucleic acids and cell-based detections.

CAR imaging can be performed on SPR imaging setups. Based on this concept, we develop a method called critical angle reflection (CAR) imaging to quantify molecular interactions on glass surface. Therefore, surface refractive index change can also be quantified by measuring the reflectivity near the critical angle. Analogously, in another interfacial optical phenomenon, total internal reflection, the critical angle is also refractive index dependent. The sensitivity of SPR originates from the characteristic that the SPR angle is sensitive to the surface refractive index change. Quantification of molecular interactions on a surface is typically achieved via label-free techniques such as surface plasmon resonance (SPR).

Angle of reflection