Total internal reflection focal molography (TIR-M)

Focal molography ("molography" in short) is a sensitive implementation of a diffractometric biosensor and has emerged as a new platform technology to study biomolecular interactions label-free in complex fluids and living cells. In contrast to established refractometric biosensors, in particular surface plasmon resonance, molography is almost insensitive to environmental noise, i.e. temperature gradients and nonspecific binding. Molography achieves this by modulating the analyte binding at a high spatial frequency and reads it out in Fourier space via diffraction of light at the bound molecules, i.e. molography applies the spatial lock-in principle for discrimination of the binding signal from disturbing effects on the sensor surface. In previous implementations of focal molography, the sensor was illuminated by a waveguide mode. While this arrangements has an outstanding resolution, it suffers from several drawbacks such as wavefront instabilities of the guided mode, the relatively high refractive index contrast at the waveguide interfaces and the manufacturing cost of waveguide and grating couplers. In this paper, we propose a simpler and more robust configuration for focal molography. Instead of a waveguide mode, it is based on darkfield illumination by total internal reflection (TIR) of a free space mode. We derive the coherent binding pattern, describe the fabrication process, show that its intensity distribution is as expected, derive the quantitative readout formula and perform a background and noise analysis. Real-time binding curves of streptavidin in buffer and concentrated bovine serum albumin solution show that TIR molography exhibits excellent resolution and robustness. •The paper present the most sensitive label-free biosensor (in terms of detected surface mass density).•A submicron scale affinity pattern (mologram) assembles a molecular hologram of analyte molecules and visualizes the specific binding event.•A new illumination method - total internal reflection - achieves higher stability than waveguide illumination methods.•The sensor is self-referencing and inherently robust against environmental noise and nonspecific binding.•The paper presents a comprehensive realization of a label-free biosensor.