It's quite likely that the methylene blue (MB) is indeed staining the surface of your Ag/AgCl reference electrode. MB is a positively charged molecule and can adsorb onto surfaces, especially after repeated use.

Is the electrode still reusable?

Yes, in many cases, the reference electrode can still be reusable, but it's crucial to address the MB contamination. Here's why and what to consider:

Potential Shift: The primary function of a reference electrode is to provide a stable and known potential. If MB is adsorbed onto the surface, it could alter the surface potential of the Ag/AgCl electrode. This could lead to a shift in your overall electrochemical measurements, affecting the accuracy and reproducibility of your DNA detection.

Kinetic Hindrance: A layer of adsorbed MB might also hinder the necessary ion exchange at the electrode surface, potentially increasing the impedance of the reference electrode.

Signal Fluctuation at Attomolar Concentrations: You're right to be concerned about signal fluctuations at such low concentrations. Even a small change in the reference electrode's potential or impedance can become significant when measuring minute amounts of analyte. The MB contamination could introduce noise or baseline drift, making it harder to reliably detect attomolar DNA levels.

Is there any specific drawback to the signal?

Yes, as mentioned above, the potential drawbacks of your signal include:

Shifted Potentials: Your cyclic voltammetry or other electrochemical measurements might appear shifted along the potential axis.

Increased Noise and Baseline Drift: The adsorbed MB could contribute to unwanted electrochemical reactions or impede the stability of the reference potential, leading to a noisier signal.

Reduced Sensitivity: If the reference potential is unstable, it can indirectly affect the sensitivity of your DNA biosensor.

Poor Reproducibility: Over time, the amount of MB adsorbed might change, leading to inconsistent results between different measurements.

Is it possible to remove the stain without damaging the electrode?

Yes, it is often possible to remove the MB stain without damaging the Ag/AgCl reference electrode. Here are a few methods you can try:

Thorough Rinsing: Start with extensive rinsing with copious amounts of high-purity water (Milli-Q water). Sometimes, weakly adsorbed MB can be removed this way.

Gentle Sonication: If rinsing alone doesn't work, try gentle sonication in high-purity water for a short period (e.g., 5-10 minutes). Be careful not to sonicate too vigorously, as this could damage the electrode.

Electrochemical Cleaning: You can try applying a potential sweep or holding a potential that might desorb the MB. However, you need to be cautious not to oxidize or reduce the Ag/AgCl layer. A mild potential sweep within the stable window of your electrolyte might be worth trying, but research the electrochemical behavior of MB on Ag/AgCl first.

Mild Chemical Cleaning (Use with Caution):

Dilute Acid or Base: Very briefly dipping the electrode in a very dilute acid (e.g., 0.1 M HCl) or base (e.g., 0.1 M NaOH), followed by thorough rinsing with water, might help. However, prolonged exposure can damage the Ag/AgCl layer, so this should be done cautiously and for a very short duration.

To Nik Humaidi Nik Zulkarnine and Monika Antil

Hello, Dear Nik,

Hi, Dear Monika Antil, since you seem to be affiliated with a tech company or institution dealing with (pH-meters and - evtlly. specific-) such sensitive electrodes would you mind to add your opinion about

-i) Ethanol (80-100%) (eventually just only wiping carefully with a soft &wet lab paper towel and thorough rinsing then with A.(bi)dest/Ultrapure), or

after -first of all - emptying the electrode reference solution (NaCl solution?) IN the Ag/AgCl2 electrode, followed then by treatment either with

-ii) mixture of -say- (1 Part 1%) acetic acid (or - frankly spoken - vinegar) : (1 Part) saturated 'Vit C' = Ascorbic acid (as a reducing agent) solution, or

-iii) a solution of glucose and sodium hydroxide... ?

After application (as short as possible) thorough washing with A.(bi)dest. / Ultrapure and (also after thoroughly rinsing inside) refilling the reference electrolyte solution in the electrode...and calibrating again against suited calibration buffer.

(Unfortunately I don't remember the very cleaning procedure I did years ago in my lab 100% ...)

Otherwise I'd recommend also to search the website or ask the electrode provider (in this case it seems to be BAS Inc. TOKYO JAPAN ) how their proposal for satisfying electrode cleaning would be.... as I have seen, the(se) manufacturers offer not only cleaning protocols but also 'Cleaner'/'Cleaning solutions' (only as ONE example out of many see: https://cdn.hach.com/7FYZVWYB/at/gb8x74hzktb7kb7bt4qqnr/LIT2781.pdf

Good luck, W.H.M.

Hello, Dear Nik,

The Ag/AgCl reference electrode (RE-1B) can still be used after being stained with methylene blue, but the dye may slightly affect its stability and cause potential drift, especially when you're working at attomolar DNA levels. According to the ALS Japan RRDE-3A manual (pp. 16–17), you should rinse the electrode with deionized water and store it in 3 M KCl—avoid using strong organic solvents or scrubbing, as that can damage the AgCl layer. Even mild surface contamination can shift the reference potential and introduce signal noise.

If the signal remains unstable after cleaning, replacing the electrode is best.

Please read: chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.als-japan.com/dlc/manual/rrde_manual/rrde-3a3.0_manual_en.pdf

Good luck,

Electrochemical cleaning. Apply a potential to the electrode in a suitable electrolyte solution to electrochemically clean the surface.

If the stain persists, you may need to consult the electrode's manufacturer for specific cleaning recommendations.

Have you compared the voltammogram of your Ag/AgCl electrode using a platinum electrode? Generally, if the potential window doesn't shift and you still have the same working window, it might not affect your measurements. However, with 1 M nitric acid, methylene blue is usually removed.