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

Area of Science:

• Biophysics
• Surface Science
• Optical Sensing

Background:

• Label-free quantification of molecular interactions is crucial for biosensing.
• Surface Plasmon Resonance (SPR) is a common technique, relying on refractive index changes near a surface.
• Total internal reflection's critical angle is also refractive index dependent, suggesting an alternative sensing mechanism.

Purpose of the Study:

• To develop and validate Critical Angle Reflection (CAR) imaging as a novel label-free method for quantifying molecular interactions.
• To compare CAR imaging's performance against SPR for various biomolecular detection applications.
• To explore CAR imaging's potential for applications beyond SPR's current capabilities.

Main Methods:

• Developed CAR imaging based on measuring reflectivity near the critical angle of total internal reflection.
• Performed CAR imaging on standard SPR imaging setups.
• Conducted side-by-side comparisons of CAR imaging and SPR for detecting proteins, nucleic acids, and cell-based interactions.

Main Results:

• CAR imaging demonstrated comparable performance to SPR for detecting proteins, nucleic acids, and cell-based interactions.
• CAR imaging successfully detected small molecule bindings and intracellular signals, extending beyond SPR's typical sensing range.
• CAR imaging offers tunable sensitivity and dynamic range, deeper vertical sensing, fluorescence compatibility, and broader optical/chemical flexibility.

Conclusions:

• CAR imaging is a versatile label-free technique for quantifying molecular interactions on glass surfaces.
• CAR imaging expands upon SPR capabilities, particularly for small molecule detection, whole cell-based assays, and simultaneous fluorescence imaging.
• The method's unique characteristics suggest significant potential for advancing biosensing and molecular interaction analysis.