Hello everyone
It is a while I am working on electrochemical assay development using SPEs and I noticed in many papers that the reference is an Ag Pseudo electrode or Ag/AgCl printed Pseudo electrode (Not covered with chloride saturated polymer layer.
I also see some famous commercial brands of SPE production also produce it with pseudo reference electrodes.
As per my knowledge, a Pseudo ref is used when we are working in a non aqueous environment and we have to have internal standards of ferrocene or other alternatives. but in case of for example amperometry, how it is used in bioassays? when we have two different blood samples with different concentration of Cl, the potential of the reference is different, how the sensor is working? what is the logic behind this simplification?
I wish more electrochemical sensor developers asked themselves this question - it is a source of unreliability in sensors that won't get developed past research publication. The pseudo-reference electrode will definitely be affected by the Cl in your samples, and in many cases it will change progressively every second it is exposed to your electrolyte: see Article Measuring the impact on impedance spectroscopy of pseudo-ref...
These "reference" pads are typically screen printed inks of Ag and AgCl. So they'll need a fixed chloride ion concentration to give a stable interfacial potential. This may be in your sample, but I wouldn't bank on it. Furthermore, many ions can complex Ag+ and certainly protein will help strip the Ag+, foul the surface and definitely alter the stability of the interfacial potential. You may be able to deposit a gel with KCl or NaCl which will work in many circumstances.
How serious this is depends on the application. For amperometry, the working electrode is typically used in the diffusion limit and there may be a few hundred millivolts where the current is essentially independent of potential. Here the reference electrode performance may not be critical. In any case you can record a quick i-V response to identify the diffusion-limited region.
Potentiometry is a different matter altogether. There is no quick way of testing your reference electrode in use and the anlytical data- concentration- depends exponentially on potential.
In general, stability to better than a few millivolt for longer than 1-2 hr is a major challenge.
Anne Sawhney
Thanks for your answer. I am not very familiar with the EIS but for me this simplification is a reason of error since as you mentioned the potential of electrode is not fixed. I will read the paper. Thanks
Danny O'Hare
I appreciate your answer. I am working on amperometric sensors. I usually functionalize the electrode, get a CV curve with and without analyte and apply the peak potential on working electrode since i think will result in the best sensitivity. As I understood from your sentences, if I have a pseudo reference and I am using amperometry, I can find the potential range in which current is diffusion limited by getting a CV, apply a potential in middle of this range and be sure that this wont affect my assay. This is doable but I will have lower sensitivity, Is that correct?
You also mentioned stripping ( is it the same with corrosion?) electrode which I never thought about that, I wondered is this issue related to the nature of redox reaction we measure on the working electrode? As you described it, I would expect it if we have a reduction on working electrodes and is not an issue of concern when we measure the current of oxidation reaction on working electrode. Is that correct?
Again I appreciate your help.
Ameneh Mohammadnezhad The peak potential in CV is where the electrode reaction starts to be limited by diffusion, so your approach is essentially what I suggested. In steady state experiments (microelectrodes, rotating disc etc) the diffusion limited region is a plateau, rather than a peak. Sensitivity is complex- it is rare these days for signal to be limited by noise, but there will always be larger currents for transient techniques (CV, DPV, SWC, chronoamperometry) since the concentration boundary layer is smaller at shorter times.
For the stripping of Ag+ from the reference electrode, this is a chemical process. Ag+ is small and can form complex ions with amino acids and probably other biological spectator species. Exposed Ag/ AgCl inks or other deposits are therefore not stable in protein solutions. This happens independently of whether the electrode reaction is happening or not. You can see if this is a problem in your set-up by recording the internal pseudo-reference potential versus e.g. a SCE with a salt bridge- you'll need a high impedance voltmeter or a second commercial potentiostat set to measure OCP. In that second set-up, the internal reference will need to be the working electrode. Reference electrode drift is a more serious problem than is often realised.
Danny O'Hare Thanks for clarifying those concepts. Yes, I planned to measure the pseudo ref potential in my samples matrix(whole blood). I ordered a low profile ag/agcl and I wanted to measure the potential of the pseudo ref in two electrode system dip in sample matrix versus Ag/Agcl. The salt bridge will be the permeable membrane of low profile Ag/AgCl, Is there any difference between my plan and your recommendation to measure the drift?
Again thanks for your consideration and guidance.
@ameneh this sounds like an excellent plan. If, as is likely, you observe drift, consider adding a polymer plus KCl to protect the reference or at least slow down its degradation.
Danny O'Hare I will do that, thanks. I really appreciate your attention and help.
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