When your aim is to inhibit the reaction between the enzyme and substrate reaction, then keep the optimal enzyme loading for given substrate concentration (you need to check by varying the enzyme amount at constant substrate concentration) and try to check the inhitory eefect on the conductance of the sensor.

In your case, the loading amount of enzyme is more crucial.

in my opinion there will be lots of trial-and-error work involved. Immobilized enzyme would behave differently compared to enzyme in solution for sterical reasons. Logically, the immobilized enzyme molecules should be accessible and available for the substrate to approach it and react. I'd suggest to run a series of parallel experiments, keeping the substrate constant and in the range in which you want to detect it, and using different concentrations of immobilized enzyme. After you narrow the enzyme concentration down, keep it constant, and vary the substrate to find the detectable substrate range. The buffer composition, the temperature and the EC parameters are likely to affect the reaction too, so they should be constant. Always use a related but "wrong" substrate as a negative control. Good luck!

In my opinion the correct answer to your question depends on two factors: (1) the overall quantities of inhibitors to be detected, relative to the effective concentration of the immobilized enzyme, and (2) the expected inhibition mechanism, e.g. "competitive" (very common), "noncompetitive" (also quite common) or "uncompetitive" (very rare).

Enzyme load:

In solution (as opposed to immobilized) enzyme kinetics, and in the absence of inhibitors, the reaction rate usually follows the Michaelis-Menten equation:

v = [E]o kcat [S]o/([S]o + Km).

This tells us that the reaction rate increases exactly proportionally with the enzyme concentration: the higher the enzyme, the higher the reaction rate. From linearity of the M-M equation (with respect to [E]o) we can expect that progressively higher enzyme load on the sensor might increase overall sensitivity, which is a good thing, I suppose. The only situation where very enzyme load would not be beneficial is if the concentration of inhibitors to be detected by the sensor happened to be significantly lower than the "effective concentration" of the immobilized enzyme. Under those circumstances even if all inhibitor were bound to the enzyme, the majority of enzyme molecules on the sensor would not be inhibited and the decrease in signal caused by inhibition would be too low to detect, as you suggested. So it all depends on what is the relative magnitude of the expected inhibitor concentration range and the "effective concentration" of the immobilized enzyme.

Substrate concentration:

Many inhibitors are kinetically competitive with the relevant substrates. If so, a substrate concentration very much hither than the Km will "compete out" the inhibitor and there will be very little inhibitory effect. More precisely the "apparent inhibition constant" for a competitive inhibitor will increase (i.e., there will be less inhibition) at progressively higher substrate concentration according to this formula:

Ki(app) = Ki (1 + [S]o/Km) ... competitive

So at [S] = 10 x Km the "apparent" inhibition constant Ki(app) is 11 times higher than the "true" inhibition constant Ki. Now for a noncompetitive inhibitor it so happens that the substrate concentration has no effect on the "apparent" Ki:

Ki(app) = Ki ... noncompetitive

So the observed inhibitory activity (potency) of a noncompetitive inhibitor is always the same regardless of the amount of substrate in the assay. Long story short if you expect to detect competitive inhibitors, I would use as little substrate as possible such that you still have good overall sensitivity. For noncompetitive inhibitors the substrate concentration should probably be chosen only on the basis of the overall sensitivity of the sensor assay. Uncompetitive inhibitors are special cases, so we don't need to discuss them.

Total concentration of enzyme and inhibitor vs. the Ki:

One issue that comes into play in sensor-based assays of enzyme inhibition is the overall concentration of both partners, enzyme and inhibitor, relative to the Ki. If both [E] and [I] are well below the Ki, then there will be very little inhibitory effect no matter what else is going on. Please remember that for the binary equilibrium,

E + I EI Ki = [E]eq [I]eq / [EI]eq

the concentration of free enzyme [E] in terms of the total or analytic concentration [E]o is given by

[E] = 0.5 ([E]o - [I]o - Ki + sqrt [([E]o - [I]o - Ki)^2] + 4 [E]o Ki)

You can use this equation to figure out expected free/total ratio, [E]/[E]o, at different enzyme and inhibitor concentrations relative to the Ki. If you do that, you'll see that if both [E]o and [I]o are well below the Ki then there will be very little inhibitory effect because the [E]/[E]o ratio will be very close to unity. The upshot is that if at all possible I would concentrate the sample if it is reasonable to expect that the total inhibitor concentration in the original sample, [I]o, is well below the inhibition constant, Ki.

If you don't like equations, as most people don't, you can use the DynaFit software to study problems like this. In DynaFit you just type E + I EI and the software will derive the equations for you:

http://www.biokin.com/dynafit/

Hope this helps...

It is simply a matter of inhibitor type. If your inhibitor is competitive, I suggest your substrate concentration is around the Km value of the enzyme. In case of using a non-competitive inhibitor, better use the highest substrate concentration to have the maximal enzyme activity.

One more thing i want to know that it is reported that use of high concentration buffer results in high conductivity of solution (in mS/cm), that create high background signal. So they used 10mM buffer concentration (instead of 250mM) and got results in microS/cm. But when i tried at lower buffer concentration (20mM), i got very low activity, while the optimized buffer concentration of 0.5M give better activity (but results in mS/cm). So can i report my data in mS/cm, instead of microS/cm OR will I have to use lower buffer conc.?

I would suggest papers published by Arkhypova V.N et al on alkaloids and ISFETs will be very helpful - potentiometry and conductometry are very similar.. You should remember only, inhibition mechanism and inhibition constants can be measured only for reversible inhibitors - you cannot do this for irreversible inhibitors. Two facts that prove reversibility are - independence of the inhibition level on time of exposure with inhibitor and decrease of inhibition effect with increase of the substrate concentration.

In my opinion 'type of inhibitor' and 'substrate concentration' is more important. As mentioned by Petr Kuzmic, sometimes substrate itself may inhibit the enzyme beyond a certain concentration. So you should check the optimum substrate concentration.

Inhibitor concentration is also a major issue in your case.