In our last blog we discussed the electrical basis for square wave voltammetry. In this blog we will focus on the chemistry part of electrochemical.
The powder depicted above is methylene blue (MB). This is a redox agent, meaning that this molecule is an oxidation and a reduction agent. In square wave voltammetry performed in an aqueous solution (buffer) without a reducing agent, there will be no signal. There needs to be a redox agent present, and moreover the redox agent must be proximal to the electrode surface in order to have an effect.
This is the key element of this type of analysis the presence of a target molecule affects the proximity of MB to the electrode surface. To achieve this, aptamers make a much better choice than antibodies. Aptamers have the intrinsic capacity to flex, to alter their structure upon binding to a target. Antibodies are too big, their active binding areas are likely to be too far from the surface of the electrode, and they are not intrinsically flexible. How many times have you seen an IgG molecule not drawn as a ‘Y’?
There are at least four ways that we can use aptamers to modulate an MB signal in the presence of a target molecule
- Free MB
- Aptamer Allosteric shift
- Antisense negative signal displacement
- Antisense positive signal displacement
In the first example we add free MB to the reaction, MB will intercalate into DNA and thus there is potential for displacement upon target binding. This is a very simple approach, and the level of the signal can be optimized with the concentration of free MB.
Example 2 is a classic example of an aptamer that folds differently when bound to the target. This is not my favourite approach as it requires that the aptamer both binds to the target with high affinity and specificity and undergoes an allosteric shift when doing so. This is a lot to ask of evolution. A key advantage of aptamers is that we know their sequences. This means that we can engineer antisense molecules to module the needed allosteric shift upon binding.
In example 3, we show how this can be done with the MB on the antisense, where target binding will result in a decrease in signal.
In example 4, MB is conjugated to the aptamer and the competing antisense keeps it off the surface. If the antisense is displaced by target binding then voila, we increase signal.
In our next blog we will discuss electrical impedance spectrometry (EIS).