KD is not the same as LOD

In Biosensors, K_D is not the same as LOD. Clients often ask if is it possible to obtain a limit of detection (LOD) for a target that is lower than the K_D of the probe. The answer is yes, here we explain why.

Let’s say that the desired LOD for a target is 1 nM. This means that we need to be able to detect the presence of the target when it is present in a sample at this concentration. Let’s consider this from the target’s perspective, what proportion of a target at a concentration of 1 nM is bound by probes that have varying binding affinities?

The figure below provides the binding trajectories for an aptamer binding to 1 nM target for varying K_D values of the aptamer with 100 nM of aptamer immobilized on the biosensor.

There are several things to note with this result:

1 ) Equilibrium is achieved relatively rapidly, more rapidly the weaker the aptamer binds to the target.

2) At a K_D equivalent to the LOD (1e-9) almost 100% of the target is bound. At a K_D 100X weaker (1e-7) 50% of the target is bound.

The difference described in point 2 above sounds significant, but to understand sensitivity we need to flip the perspective from the target to the aptamer. It is the aptamer that we are relying on for signal not the target. We need to be able to distinguish the signal provided by the aptamer/target complex in the presence of 1 nM target versus the absence of this signal in the absence of target. From this perspective we can rewrite point 2 above as follows:

3) At a K_D equivalent to the LOD (1e-9), 1% of the aptamer present in the sensor is complexed with target and providing a signal. At a K_D 100X weaker (1e-7) ,0.5% of the aptamer present in the sensor is providing a signal.

When the problem is considered as described in point 3 the difference in binding affinity is not as large. This clearly shows that the basis of the LOD is much more heavily influenced by the sensitivity of the signal that the aptamer/target complex provides. The binding affinity of the aptamer matters but the signal measured by the biosensor matters much more.