We are trying to build an electrochemical sensor using the EDC/NHS linker on a screen-printed gold electrode to detect human serum albumin.
We are able to observe the impedance change from the bare electrode to EDC/NHS linker layer without a problem, but when we add antibody and antigen on the electrode the impedance decreases.
Based on what we read in the literature, adding antibody should increase the impedance, so I am very confused.
I would really appreciate any advice you could provide us regarding what we could try to solve this issue.
our general protocol is as follows:
1. Prepare electrode
2. incubate in MPA for 30 min
3. incubate in EDC/NHS for 30 min
4. put antibody on the working electrode and incubate in the fridge overnight
5. put HSA on the electrode and incubate 1 hr
between each step we rinse the electrode and measure impedance in Ferricyanide/ Ferrocyanide
What kind of buffers did you use in the different steps? Do you have data about the composition of your antibody solution?
Michael G. Weller
Hello Dr. Weller,
We are using PBS buffer for the steps after the linker is developed. It is 100mM phosphate buffered saline that includes 2.7mM potassium chloride and 137mM sodium chloride with pH7.4
Before antibody incubation, we use PBS once for pre-cleaning process.
Antibody comes with 100M phosphate-buffered saline pH 7.4 and containing 15mM sodium azide.
Both antibody and HSA are diluted in the same PBS that I mentioned above.
The sodium azide might interfere with the NHS chemistry. Try to exchange the buffer. The pH of 7.4 is a little bit low. A pH of 8 is often seen as optimal. In addition, be aware that the concentration of the antibody needs to be higher than you might expect. Most researchers use a MES buffer of about pH 5 for the EDC/NHS step.
Thank you for the advice! I will definitely try with different buffer and antibody concentration.
Dear Seohyun Shim
After the immobilization of the antibody, the impedance should be increase.
You can follow this article
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Seohyun Shim did you find a solution? I'm currently experiencing a similar issue. We suspect it might be due to excess thiol not being washed off properly.
Seohyun Shim I currently experienced the same issue as you did. Any idea why it happened? Looking forward to your reply. Thanks.
We couldn't find the solution to our issue and moved on to using a different linker (DSP in DMSO) for our experiment. I'm sorry I can't be of much help