Principles for sensitive and robust biomolecular interaction analysis - The limits of detection and resolution of diffraction-limited focal molography

Abstract

Label-free biosensors enable the monitoring of biomolecular interactions in real-time, which is key to the analysis of the binding characteristics of biomolecules. While refractometric optical biosensors are sensitive and well-established, they are susceptible to any change of the refractive index in the sensing volume caused by minute variations in composition of the sample buffer, temperature drifts and nonspecific binding to the sensor surface. Refractometric biosensors require reference channels as well as temperature stabilisation and their applicability in complex fluids such as blood is limited by nonspecific bindings. Focal molography does not measure the refractive index of the entire sensing volume but detects the diffracted light from a coherent assembly of analyte molecules. Thus, it does not suffer from the limitations of refractometric sensors since they stem from non-coherent processes and therefore do not add to the coherent molographic signal. The coherent assembly is generated by selective binding of the analyte molecules to a synthetic binding pattern - the mologram. Focal Molography has been introduced theoretically and verified experimentally in previous papers. However, further understanding of the underlying physics and a diffraction-limited readout is needed to unveil its full potential. This paper introduces refined theoretical models which can accurately quantify the amount of matter bound to the mologram from the diffracted intensity. In addition, it presents measurements of diffraction-limited molographic foci. These improvements enabled us to demonstrate a resolution in real-time binding experiments comparable to the best SPR sensors, without the need of temperature stabilisation or drift correction and to detect small molecules label-free in an endpoint format. The presented experiments exemplify the robustness and sensitivity of diffractometric sensors.