However the practical application of enzyme sensors based on the measurement of pH
changes in an enzymatic membrane is limited by several factors. These factors that
complicate the response are the buffer capacity of the sample, which itself is also a
function of pH, the pH-dependent enzyme kinetics and the fact that the products of
the enzyme reaction may be weak protolytes so that the amount of H+
or OH-
ions
produced per mole of converted substrate also depends on pH. On the other hand, the
pH of more alkaline solutions will be decreased by the same molecules. As a result of
these factors, the response of ISFET-based enzyme sensors is strongly non-linear and
the dynamic range depends on the composition of the sample solution. The general
conclusion must be that an elaborate calibration procedure is required and that the
practical value of these sensors is limited.
In subsequent research it was demonstrated that by coulometric control of the pH
inside the enzymatic membrane, it is possible to overcome these problems. The pHstatic
enzyme sensor measures the pH inside the membrane with an ISFET and
controls it through the generation of protons or hydroxyl ions at a noble metal
electrode, spaced closely around the ISFET gate area as shown in figure 10(a). The
acidic or alkaline products of the enzyme reaction are thus continuously neutralised.
The generating current needed to maintain the pH at a constant level now becomes the
output signal of the sensor. It is linearly related to the substrate concentration and
independent of the buffer capacity of the sample. In fact, an electrochemical pHactuating
mechanism, the current-controlled electrolysis of water, is used to
compensate the biochemical pH actuator, i.e. the enzyme, converting substrate
molecules and thus generating pH change.
The response of an enzyme-modified ISFET is generally pH dependent. However, if
the pH is kept constant, the sensitivity may also be considered to be constant. This
sensitivity, the change in the steady-sate ISFET output voltage Vout per change in
substrate concentration [S], is given by