The approaches outlined above work well for large targets (e.g. bacteria or genes) because the sensor molecules are de-aligned upon binding to them. This format can also be used for smaller targets where there are multiple binding sites per target.
However, for smaller targets (e.g. proteins, peptides, haptens or other biomarkers) the binding of the target does not de-align the sensor molecules sufficiently. In these cases, we use a competition assay format in which the sensor is bound to a structure that mimics the target. This mimic may be either another sensor that has the target on it or e.g. a large bead that has the target on it.
This means that in the absence of the target the sensor is de-aligned (the opposite to the large target format). In the presence of the target the binding to the mimic is competed out, this releases the sensor molecule so it aligns and gives a change in the signal (Figure 2 below).
Long thin sensor molecules (phage) are de-aligned under flow in a liquid sample because they are bound to e.g. another sensor which has the small target on it. In the presence of the target in the sample the interaction causing de-alignment is competed out and the sensors can align. Alignment of the sensor molecules depends on the amount of target present. In this example the target is a small molecule. The degree of alignment (and therefore the amount of target) can be measured by LD. This works by measuring the amount of light (polarized parallel and perpendicular to the flow direction) that passes through the sample. We measure this using a hand-held reader.