ISFET as a Biosensor:
Addressing Practical Problems
B.LORA NARAYANA and R.E.C.JOHNSON
Instrumentation Group, Centre for Cellular and Molecular Biology, Hyderabad-500007, India
lora@ccmb.res.in, johnson@ccmb.res.in
Abstract
A measuring system was designed using an ion
sensitive field effect transistor (ISFET) to measure
changes in ion concentrations. While the ISFET
has certain drawbacks as a chemical sensor, it did
satisfy the primary requirements of sensing area
(for low volume samples), sensitivity and
adaptability that were called for. The paper is
aimed at providing an insight into the practical
difficulties encountered in employing the device for
biological measurements, and suggests a course of
action for circumventing some of the problems like
susceptibility to ambient light, noise etc. A
discussion of the probable sources of errors is also
presented.
Keywords: ISFET, pH, biosensor, sensitivity, drift
1 Introduction
Biosensors can have a variety of medical, industrial
and military applications. Ion-selective field effect
transistors are robust and sensitive devices to
monitor the changes in ion concentrations,
including protons, when they interact with the gate
surface. The ISFET could be made into a specific
device to detect a particular analyte by
incorporating enzymes or antibodies on the gate
surface. Hydrolytic enzymes that break down
specific analyte are used as biosensing element on
the gate surface. The action of the enzymes on the
analyte leads to changes in the ion concentration
that are detected by the FET device. Here, we have
made efforts to fabricate and optimise a circuit to
measure the potential changes occurring near a gate
surface of a commercial ISFET. Lipase, a
triglyceride hydrolysing enzyme, would be
incorporated on the gate surface to specifically
monitor the concentration of triglycerides in
biological fluids.
2 Background
pH is a measure of the ion concentration in a liquid
and is defined as the negative logarithm of the
Hydrogen ion concentration. It is measured in
terms of the potential given by the Nernst equation
(see also [1]):
1
E = E0 - (RT/nF)log(H+) (1)
where:
E = Energy level or electrode potential
E0 = Potential dependent on the reference electrode
R = Universal Gas Constant
T = Temperature (ºK)
n = Number of electrons released per atom
(oxidised/reduced)
F = Faraday constant
H+ = Ion concentration in moles per litre of
electrolyte
For Hydrogen ions at normal ambient conditions
(25ºC) the Nernst equation reduces to:
E = E0 + 0.059 pH (2)
Equation (2) predicts a potential gradient of 0.059
volts per pH.
3 Devices
3.1 ISFET
The ISFET [3] is one of the early products of
MEMS technology and its fabrication process is
closely similar to that of the metal oxide
semiconductor field effect transistor (MOSFET). It
only differs from the MOSFET in that the metal
gate is replaced with an ion sensitive membrane
such as SiO2, Al2O3, Ta2O5 etc., while an ionic
liquid acts as the gate. Electrical contact with the
gate is established indirectly using a reference
electrode [3]. Being a solid-state device, it offers
several advantages over conventional devices.
Figure 1 shows the structure of a typical ISFET and
fig.2 shows its surface topology as seen under a
microscope.
Fig.1 Structure of ISFET
 time and low short-term drift. The system was
expected to resolve pH variations in the order of
0.1pH, calling for a high sensitivity. The ISFET
satisfies these conditions, but demands elaborate
considerations in the interface circuitry.

5 Circuit design

5.1 Device characterisation

A commercially available ISFET device was used
for the purpose. Due to lack of data on this device
it was necessary to test and characterise the ISFET.
The circuit of fig.4 was used for plotting its volt-
ampere (V-I) characteristics. The LM334, a
variable current source from National Semi-
conductors was used to vary the drain-current of
Fig.2 Surface Topology of ISFET
the ISFET and a 7805 in conjunction with a
The ISFET was favoured over the conventional
glass electrode [1] for sensing pH, due to the
latter’s disadvantages of fragility, large sample
volume requirement, sluggish response and
cumbersome maintenance.

3.2 Reference Electrode

A Silver/Silver Chloride reference electrode of
cylindrical shape (dimensions: 30mm L x 10mm Ø)
was used for the gate connection. Ag/AgCl

Fig. 4 Circuit for tracing V-I characteristics

potentiometer was used for varying the gate-bias,

which was applied through an Ag/AgCl reference
electrode.
The V-I characteristics for various gate voltages
(Vref) at constant pH is shown in fig.5 and for
various pH values at constant Vref in fig.6, which
shows the pH dependency of VDS.
(a) Reference electrode (b) With ISFET
Fig. 3
600 Vref=0.5V
electrodes exhibit a lower voltage offset and better 500
Vref=0.3V
stability than other reference electrodes in the 400
Id(uA)

market. Figure 3(a) shows the reference electrode 300 Vref=0V

and (b) the same, when attached to the ISFET with 200
a rubber ‘O’ring. 100 pH=7
0
0 2 4 6 8 10
4 Design constraints
Vds(V)
Several constraints were imposed by the user-
Fig.5 ISFET Characteristics at constant pH
requirements: Sample volumes as low as 100 micro
litre dictated a sensing device of small geometry,
the intended kinetic studies required short response

2
 700 pH4
600
500 pH7
Id(uA)
400 pH9.2
300
200
100 Vref = 0.5V
0
0 1 2 3 4 5 6 7 8 9 10
Vds(V)

Fig.6 ISFET Characteristics at constant Vref

Fig. 8 REF200 pinout

5.2 Prototype circuit-1
membrane is exposed to a liquid sample, any
A prototype circuit was built using TL071 opamps
variation in the drain-source voltage (VDS) due to
and LM334 current source ICs to drive the ISFET
pH variations causes IC5 to change the gate bias
in constant-current, constant-voltage mode as
(applied through a reference electrode), which
shown in fig.7. The circuit established satisfactory
counteracts the change in VDS, thereby maintaining
pH dependence of the device, but the variation was
a constant VDS. The operating point chosen was
not very linear, as can be seen in the graph of fig.10
VDS=0.5V, ID=200µA. The gate-drive required to
(labelled ‘ckt-1’)

Fig. 7 Prototype circuit-1

5.3 Prototype circuit-2

The final circuit designed for interfacing the ISFET
used the feedback topology and is shown in fig.9.
A current reference IC, REF200 was used for
maintaining a constant drain-current (ID) of 200µA.
The REF200 from Texas Instruments contains two
Fig. 9 Prototype circuit – 2
independent 100µA current sources and a current
mirror, and was specifically chosen for its very low
maintain a constant VDS is proportional to the pH
temperature coefficient and high precision as well
value, thereby providing the readout signal as well.
as ease of use. Figure 8 shows the pin-out of this
This signal is buffered by IC6 and is fed to IC8,
device. In the circuit of fig.9, IC3 presents a
which is a variable gain amplifier with adjustable
constant voltage to the drain pin and IC4 sinks a
offset. The final output was adjusted using the gain
constant source-current. IC5 operates in feedback
and offset, to read 100mV/pH, so that a 4½ digit
mode with the ISFET’s gate-source voltage (VGS)
DVM could be used to display the pH value
forming the loop. When the ISFET’s ion-sensitive
directly. The output of IC6 had a constant slope of

3
57.6mV/pH as shown by the graph of fig.10 Table 1. Measuring pH in low sample volumes
(labelled ‘ckt-2’). pH Sample pH Output
volume (mV)
2.2 ckt-2 (µL)
7 100 706
1.7 50 707
Vgs (V)

40 706
1.2 30 707
ckt-1 20 Unstable reading
0.7 10 Unstable reading
2 3 4 5 6 7 8 9 10 11 12 4 100 405
50 404
pH
40 405
30 Unstable reading
Fig.10 pH-mV curves for circuits of figs. 7 & 9 20 Unstable reading
10 Unstable reading
6 Results 9 100 905
50 905
The system was put through various tests to 40 906
evaluate its suitability for the actual application, 30 Unstable reading
and the results of these are presented below. 20 Unstable reading
10 Unstable reading

6.1 Low volume samples

A salient feature of the system is the ability to
measure the pH in very small sample volumes.
Sensing Area
6.2 Hysteresis and short-term drift
Figure 12 shows a study of the hysteresis effect.
The error introduced by changing the pH from 7 to
9 and back was less than 9mV, corresponding to
0.15pH. The plot also shows the short-term drift to
be less than 8mV over a ten-minute period.

2.05 pH9
2
1.95
Vout (V)

pH7
1.9
1.85 Error<9mV
1.8
1.75 pH4
Sample Droplet 1.7
0 20 40 60
Time (min)
Fig.11 Sensor with 50µL sample
Fig.12 Hysteresis and short-term drift
A common problem in biological measurements is
the extremely small quantities of samples available. 6.3 Long term drift
Therefore, one of the primary objectives of the
design was focussed on making measurements on A slow drift of 10mV was observed over a one-
small volume samples. The ISFET geometry was hour period, as shown in fig.13.
well suited for this, with an exposed sensing area of
2mm diameter, as can be seen in Fig 3b. By 1.12
positioning the reference electrode with its orifice 1.10
in proximity to the ISFET’s sensing area as shown
Vout (V)

1.08
in fig.11, the sample could be applied with a micro 1.06
litre pipette. Stable readings were obtained with 1.04
sample volumes as low as 50 micro litres. 1.02
1.00
Table 1 shows the readings obtained with 0 10 20 30 40 50 60 70
diminishing sample volumes. The direct pH Time (min)
readout signal from circuit-2 was used for this.
Fig.13 Drift characteristics

4
6.4 Evaluation for use as biosensor
The device was tested to detect triglycerides.
Hydrolysis of triglycerides at 10 mM (Potassium
phosphate buffer 5mM, pH7.6) was monitored in
5ml volume in the presence of Bacterial lipase (40
µg /ml). The mV signal was followed after the
addition of lipase. The change in pH as a function
of time is shown in the graph of fig.14.
1.89
1.88
1.87
Vout (V)
appropriately. It also requires to be stored in 3M
NaCl solution.
7.2 Circuit-related
7.2.1 Drift, noise and EMI
Circuit-related problems include drift associated
with operational amplifiers and susceptibility to
electromagnetic interference. The OP05 and TL071
operational amplifiers were selected for low drift,
low noise, high input impedance and low bias-
current specifications. Figure 12 shows the overall

1.86 drift and hysteresis of the circuit and ISFET

1.85 combined. They were sufficiently low and did not
1.84
1.83 interfere with the intended measurements.
1.82
1.81 The effect of electro-magnetic interference has
11.9
0
1.0
2.0
3.0
4.0
5.5
6.5
7.5
8.0

been minimised by impedance considerations in the

Time (min)
Fig.14 Detecting Triglycerides hydrolysis
A variation of 40mV was recorded over a 10-
minute period. This establishes the viability of
adapting this device for detecting triglycerides. A
suitable method to immobilise the enzyme
molecules on the sensing surface is presently under
study.
circuit topology, layout geometry in the printed
circuit board layout and power supply decoupling.
However, a suitable enclosure has not been
fabricated at the time of this writing. All the
measurements presented here have been taken with
the circuit board in open condition. A proper
enclosure with pertinent shielding and grounding
scheme is expected to improve the performance
further.
7.2.2 Repeatability

A problem of repeatability on a long-term basis

7 Addressing practical difficulties
As the measurement calls for a high degree of
precision, all possible sources of error must be
considered and either eliminated or minimised. The
following is an account of the problems that were
encountered and the methods used to address them
categorically:
7.1 Sensor-related
7.1.1 Ambient light and electrostatic fields
During initial measurements the ISFET showed a
strong sensitivity to ambient light, and was also
found to be susceptible to electrostatic fields in its
vicinity. Both these effects could be almost
completely eliminated by externally connecting the
Substrate pin to the Source pin of the device to
bleed off the charge accumulation on the substrate
material.
7.1.2 Reference electrode issues
was noticed: the slope of the pH was constant, but
the absolute value of the mV reading showed
deviations when the instrument was switched on
after an interval of more than a day. Complete
elimination of this problem would involve
complicated steps, such as a reference FET of
geometry identical to the ISFET that is fabricated
on the same substrate. As this was beyond our
scope, the problem was circumvented by replacing
the offset trim-pot R11 (Fig.9) with a multi-turn
potentiometer that could be used for adjusting the
output to read 700mV with a standard pH7 buffer
prior to the measurement session.
7.2.3 Temperature compensation
Even though temperature compensation is also an
important consideration in pH measurements, it
was not implemented on this system for the
following reasons: (a) all the measurements were
intended to be carried out in controlled ambient
conditions of the laboratory and (b) a calibration
was to be done before each set of measurements.

The Ag/AgCl reference electrode (used for the 7.3 Usage-related

gate-connection) is susceptible to frit clogging and
electrolyte-loss and therefore should be handled These include calibration errors, contamination
issues and human errors in buffer preparation.

5
Special care is needed on the part of the end-user in
all stages of the measurement to eliminate these
errors.
8 Instrumentation
The following instruments were utilised in the
design and evaluation phase:
• Fluke-45 dual display bench-top
multimeter
• Fluke-105B Scope Meter
• Hinditron86 - 4½digit DMM
• Meco 3½ digit DMM
• Pacific-PDC20 Component Development
System
• Genrad-1657 RLC Digibridge
Figure 15 shows the instrumentation set-up used
during initial stages of the design.
Fig. 15 Instrumentation setup
Conclusion
A measuring circuit was built with a commercial
ISFET as the sensing element. A linear, near-
Nernst sensitivity of 57.6 mV/pH was achieved in
the range 4pH to 9pH. The speed of response,
short-term drift and hysteresis were measured to be
within the limits required by end-use objectives.
Stable readings on sample volumes as low as 50µL
could be achieved. The viability of using the device
as a biosensor for detecting triglycerides was also
established. However, the enzyme (lipase) has to be
immobilised on the ISFET’s sensing surface for
actual utility as biosensor and is currently under
study.
Acknowledgements
The authors wish to thank Dr.Madhusudhan Rao,
Scientist, CCMB and his group for providing
valuable inputs pertaining to the research
applications as well as providing the necessary
standards and test samples. The support and
encouragement from Shri. Sekar Ramachandran
and Shri. G.G. Kingi of Instrumentation Group,
CCMB is also acknowledged.
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