Hello,

I am trying to make a biosensor to measure my analyte concentration in real time using FEIS with the following protocol:

1. Pt surface cleaning: acetone (rinse) - immediately IPA (sonication 1') - MilliQ (sonication 1') - UV ozone 30'

2. 1mM 11-MUA prepped freshly (3:1 ethanol:water) immersed for 3h @RT. Rinsed w/ copious ethanol +MilliQ - N2 blowdry gently

3. Activation by 5mM EDC/NHS (50mM MES pH 6.0) for 1h @RT, rinse w/ ethanol+milliq+N2 blowdry

4. Immersion in aptamer solution (w/ amine group on one end) for 3h @RT - rinse w/ only water -N2 blowdry gently

5. Blocking unreacted EDC/NHS by 1mM ethanolamine (50mM HEPES pH 8) - wash w/ milliq - N2 blowdry gently

The problem is when I measure impedance, I don't get increasing charge transfer resistance (Rct) when I increase analyte concentration. I clearly have low Rct when I have clean surfaces and very high Rct after 11-MUA which significantly drops when I add my aptamers. I suspect the homogenous thiol layer will have defects, holes, pinholes, etc after my aptamer addition, or my aptamers didn't bind my thiol at all. Can it be that their negative charges make them less prone to bind to each other? Will a high salt concentration buffer cancel out my charges? If you have any protocols or suggestions, I would be happy to hear them.