  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.
