Hello, I hope everything is going well. I have been working on enzyme based sensors for a while. I don't know your particular sensing platform but to begin we must consider that even in solution there is a point in what increases in substrate concentration will not generate significant changes in analytical response because the system reaches saturation, the phenomenon is well addressed in any analytical chemistry book with an enzyme kinetics section (I recommend the Lehninger's biochemistry book). We can think like if enzymes becomes kind of the limiting reagent because you have an excess of substrate and a fixed enzyme number. If you want to increase the linear response range maybe considering to add more enzymes in your sensor can be a good try. On another hand, non enzymatic sensors have also a linear response range and a saturation regime is reached in some point because you have limited surface area or limited analyte diffusion, in this case you could try to improve diffusion of the analyte to the surface or increase electrochemical surface area itself.

L. Miguel. Hernandez Parra i am working with pt nanoparticles and GOx using GCE. I have varied the concentration of GOx multiple times and it seems the system reaches saturation after about 4 or 5 addition of glucose. Because i am trying to achieve a r squarare value of 0.99 when concentration response is plotted against concentration

Yaasir Olanrewaju Greetings, if you are looking for a 0.99 correlation value in a wider concentration range you could try plotting the analytical response versus the logarithm of the substrate concentration.

L. Miguel. Hernandez Parra is there a theoretical basis for that? (using current response vs logC)

Yaasir Olanrewaju Thanks for sharing the data and context.

From your attached amperometric plot, it looks like you're doing a stepwise glucose addition (likely in 1 mM increments), and you're observing a diminishing response after the 5th or 6th addition. The current plateaus or increases very slowly after that — which is a common issue in enzyme-based electrochemical glucose sensors.

What Could Be Causing the Non-Linear Amperometric Response?

Here are the most probable reasons based on your experiment and the plot:

1. Enzyme Saturation (GOx reaches Vmax)

• GOx (glucose oxidase) follows Michaelis-Menten kinetics.
• After a certain glucose concentration (typically >4–6 mM), the enzyme becomes saturated and the rate (current) no longer increases linearly.
• This is biochemical, not instrumental — and it's expected.

What to do:

• If you need a linear response beyond 5–6 mM, increase GOx loading (more enzyme on electrode).
• Or, optimize the enzyme immobilization layer to reduce diffusion barriers.

2. Oxygen Limitation (especially for 1st-gen GOx sensors)

• GOx catalyzes: Glucose + O₂ → Gluconolactone + H₂O₂
• If dissolved O₂ in the buffer becomes limiting (typically after several additions), reaction rate drops.
• This often causes the plateau you’re seeing after repeated glucose additions.

What to do:

• Saturate your buffer with O₂ before measurement.
• Use mediators like ferrocene, Prussian Blue, or [Os(bpy)₃] to shuttle electrons instead of relying on O₂ (i.e., shift to 2nd- or 3rd-gen sensor design).

3. H₂O₂ Accumulation or Electrode Fouling

• If your electrode detects H₂O₂ oxidation, it can cause surface fouling over time, reducing sensitivity.
• H₂O₂ is reactive and can damage enzyme or electrode surface.

What to do:

• Use low potential redox mediators to avoid direct H₂O₂ oxidation.
• Include washing steps or flow-cell setup for continuous measurement.

4. Diffusion Limitation at the Electrode

• If the active layer (GOx or sensing material) is thick or non-uniform, glucose may not reach the enzyme efficiently after repeated additions.
• This gives rise to slow current increases or saturation.

What to do:

• Use thinner enzyme layers.
• Optimize crosslinker or matrix (e.g., chitosan, Nafion, PVA) to improve mass transport.

5. Instrumental or Experimental Setup Factors

• Stirring may be insufficient, leading to local glucose depletion near the electrode.
• Electrode area might be small, amplifying limitations at higher concentrations.

What to do:

• Use magnetic stirring during measurement.
• Ensure proper calibration and response time in your i-t setup (yours looks good with 0.1 s interval though).

Tips for Getting a Linear Amperometric Response

• Immobilize optimal amount of GOx with good dispersion (e.g., with BSA + glutaraldehyde or chitosan).
• Use mediators instead of relying on O₂ (e.g., ferrocene derivatives, PB-modified electrodes).
• Test in oxygen-saturated buffer if no mediator is used.
• Maintain stirring or use a flow-cell setup.
• Keep enzyme layer thin and homogeneous to reduce diffusion barriers.
• Use low detection potentials (avoid H₂O₂ oxidation at >0.6 V if possible).

Optional Troubleshooting Steps:

• Plot current vs. glucose concentration to confirm non-linearity and identify saturation point.
• Try control experiment with known mediator to isolate enzymatic bottleneck.