Biosensors 3 (1987/88) 101-115

A Microelectronic Conductimetric Biosensor

L. D. Watson", P. Maynard", D. C. Cullen", R. S. Sethi ", J. Brettle"

and C. R. Lowe'"
"The Biotechnology Centre. Universit y of Cambridge. Downing Street. Cambridge CB2 3EF
(Great Britain)
bPlessey Research Caswell Ltd. Allen Clark Research Centre. Caswell. Towcester,
Northants NN 128EQ (Great Britain)

(Received 2 March 1987; revised version received 14April 1987; accepted 19June 1987)

ABSTRACT

The fabrication and operation of a microelectronic conductim etric

biosensor is described . The device monitors the change in solution
conductance occasioned by the catalytic action ofenzymes immobi/ised o ver
a planar conductance cell comprising serpentined and interdigitated metal
conductor tracks. The output of the instrument was linear over a 3 min
period on addition ofurea to a sample cell overlaid with immobilised urease.
The responses to any given urea concentration were reproducible to within
approximately :t: 1%. The device responds to urea present in serum sam ples.

Key words : Biosensor, conductance, conductimeter, urease, immobilised

enzyme, microelectronic resistor array.

INTRODUCTION

The last few years have seen unprecedented interest in the development of
analytical devices for the detection, quantification and monitoring of
biological analytes (Lowe, 1984, 1985). It is now generally appreciated that
inexpensive and reliable sensors for monitoring key metabolites, gases or
ions in the ward , surgery , home, outpatients department and central
laboratory are necessary for the delivery of effective patient care (Turner &

"To whom correspondence should be add ressed.

101
Biosensors0265-928X/87/$03· 50 © Elsevier Applied Science Publishers Ltd , England , 1987.
Printed in Great Britain
102 L. D. Watson, P. Maynard, D. C. Cullen, R. S. Sethi, J. Brettle, C. R. Lowe

Pickup, 1985). It is possible to construct specificand sensitive biosensors for

the detection and estimation of a number of biochemically and clinically
significant analytes by coupling the catalytic powers of enzymes or enzyme'
systems to appropriate transducers such as ion selective (Boitieux et ai.,
1979, 1981) or gas sensing electrodes (Aizawa et ai., 1979; Tipton et ai.,
1981), field effect transistors (Caras & Janata, 1980), thermistors (Mosbach
& Danielsson, 1981), optoelectronic devices (Goldfinch & Lowe, 1984) or
amperometric electrodes (Cass et ai., 1984;Albery et ai., 1986).
An alternative measuring principle which is widely applicable to
biological systems is the exploitation of solution conductance. Many
chemical reactions produce or consume ionic species and thereby alter the
overall electrical conductivity of the solution. The monitoring of solution
conductance was originally developed as a method of determining chemical
reaction rates but, more recently, it has been applied to enzyme-catalysed
reactions (Hanss& Rey, 1971;Lawrence, 1971;Lawrence & Moores, 1972),
notably, the urease-catalysed hydrolysis of urea (Chin & Kroontje, 1961;
Hanss & Rey, 1971)and the assay of proteases (Lawrence & Moores, 1972),
oxidases (Dumontier & Hanss, 1974), amidases and peptidases (Hill &
Tomalin, 1982). However, as the resistance of a solution is determined by
the migration of all ions that are present, conductimetric measurements are
often considered to be relatively non-specific. This paper describes the
development and operation of an inexpensive, rapid and accurate con-
ductance biosensor which circumvents this lack of specificity by monitoring
the change in conductance occasioned by the catalytic action of enzymes
immobilised proximal to a planar micro-electronic conductance cell of
defined geometry. The performance characteristics of the device are
assessed.

MATERIALS AND METHODS

Materials

Urease (urea amidohydrolase, EC 3.5.1.5, Jack beans), glutaraldehyde

(25% (vjv) solution), imidazole and bovine serum albumin (fraction V)
were purchased from Sigma (Poole, Dorset, UK). Urea, potassium chloride
and sodium chloride were all 'Analar' grade available from BDH (Poole,
Dorset, UK). All electrical components were from RS Components Ltd
(Corby, Northants, UK).
Potassium chloride calibration solutions were made up in 'super Q' water
(Millipore; 18 Mf] grade water) whilst all other solutions were made up in
5 mM imidazole-HCl buffer, pH 7·5.
 A microelectronic conductimetric biosensor 103

DeviceFabrication

The microelectronic conductance devices were fabricated at Plessey

Research Caswell Ltd (Towcester, Northants, UK) according to the
following principal regimes: thermal oxidation, metallisation, photolitho-
graphy and etching, sawing, chip bonding, wire bonding and encapsulation.
A silicon wafer was thermally oxidised in a furnace using a standard
process to provide ~ 550 nm thick silicon dioxide (SiOz) layer on its surface.
A multi-metal coating was deposited onto the oxidised silicon surface using
standard sputter deposition processes or thermal evaporation under
vacuum. A three metal scheme, comprising sequentially deposited layers of
Ti, Pt and Au was employed. The thickness ofthe Ti and Pt metal layers was
typically ~ 100nm whereas that of Au varied between 1000 and 3500 nm.
The serpentined and interdigitated parallel metal conductor tracks, 12~
3500 nm thick and 5000 nm wide, were produced by using appropriate
photolithographic processes and metal etching. The required metal patterns
were produced by etching metals through developed resist patterns either by
dry etching techniques such as ion-beam milling for removing all the metals
(Au, Pt and Ti) or by a combination of standard wet etching (Au and Ti) and
dry etching (Pt) techniques. The resist was subsequently removed by
dissolution in acetone or standard AZ resist remover (Hoechst, FRG). The
silicon wafer was finally subjected to a washing sequence involving
deionised water, acetone, methanol and isopropyl alcohol and sawed by a
standard process to produce discrete silicon chips containing three surface
devices each.
The chip was mounted and bonded on a standard 12-pin T05 header
either using epoxy bonding or a Au-Sn (80:20) eutectic solder. Connections
to the devices were made by thermocompression gold wire (thickness
~ 25J.tm) bonding between the device pads and the pin-heads in the
package. An open ended can was welded onto the header and the devices
encapsulated in epoxy to permit exposure of an appropriate number of
serpentined networks. A plan view of the complete device is presented in
Fig. l(a) whilst a more detailed illustration of the microcircuit surface is
shown in Fig. l(b).

Construction and Operation of theConductance Biosensor

Conventional AC conductimetric monitoring techniques were used to

reduce Faradaic processes, double-layer charging and concentration
polarisation at the microelectrode surface (Pungor, 1965). A monolithic
integrated circuit waveform generator (RS Components Ltd, RS8038CC)
was employed to generate a low distortion sine wave of 1 kHz frequency and
104 L. D. Watson. P. Maynard. D. C. Cullen. R. S. Sethi, J. Brettle, C. R. Lowe

(a)

(b)
Fig. 1. The microelectronic enzyme conductimeter: (a) top view of the silicon chip showing
the interdigitated serpentined networks bonded to a 12-pin open-top T05 package; (b) a plan
view of the microcircuit surface.
 A microelectronic conductimetricbiosensor 105

to mV peak-to-peak amplitude about 0 V across the interdigitated conduct-

ance cell. Amplification was achieved with a standard inverting operational
amplifier with gain constructed with a high input impedance FET opera-
tional amplifier (RS Components Ltd, CA3140) with a fixed feedback
resistor (10 Mil ± 1%) and the conductance cell as the input resistor. The
operational amplifier is a scaler/inverter with the voltage output directly
proportional to the cell conductance, C eeli. i.e.

VOU! = ------

The output signal from the amplifier was taken to an RMS-DC converter
integrated circuit (RS Components Ltd, AD563A) such that the output
voltage could be monitored on a chart recorder (Gould BS-272) and/or a
digital multimeter (Hewlett Packard 3468A).
The conductance biosensor could be operated in either a single cell or a
dual cell configuration comprising a reference and sample pair of inter-
digitated conductors. Two identical sine wave generator/inverting
operational amplifier circuits were constructed, as shown in Fig. 2, with the
outputs from the two reference and sample RMS-DC converters being

>-..... _~RMS
-DC

')-..... _~RMS
-oc

Chart
recorder

Fig. 2. Block diagram of the differential enzyme conduct imeter showing the sample and
reference cell circuitry comprising the Jowdistortion sine wave generator (5 ). the high input
impedance FET invert ing operational amplifier (lOA). the RMS-DC converter and the
differential amplifier (DA) .
106 L. D. Watson, P. Maynard. D. C. Cullen, R. S. Sethi, J. Brettle, C. R. Lowe

taken to a conventional differential amplifier and thence to the chart

recorder/digital multimeter. In the single cell mode the output voltage from
the RMS-DC converter was fed directly into the chart recorder. The device
was temperature equilibrated to 30 ± O·l°C by means of a glass water jacket
connected to a Julabo 20B heating/circulating water bath.

Instrument Calibration

The instrument was calibrated by connecting low temperature coefficient ,

± 1% tolerance , 0·25 W metal-film type resistors (4-27 kn) across the
sample cell electrodes in order to simulate solution conductance changes.
This calibration procedure permitted the output voltage of the conductance
biosensor to be related to the SI units (Siemens) for conductance.

Enzyme Immobilisation

Urease (urea amidohydrolase, EC 3.5.1.5) was immobilised over the sample

interdigitated network by carefully adding 10 ILl of a mixture of equal
volumes of enzyme (100 mg ml") , bovine serum albumin (100 mg rnl") and
glutaraldehyde (2,5% (v/v» solution , all made up in 5mM imidazole-HCl
buffer, pH 7·5 . An enzyme-albumin gel formed after 9-10 min at 20°C and
was washed exhaustively with 5 mM imidazole-HCI buffer, pH 7·5. The
urease-albumin gel thickness was approximately 1·5 mm.

Sample Measurement Protocol

The microelectronic conductance biosensor was initially calibrated against

known concentrations of urea . Imidazole-HCl buffer (5 mM, pH 7,5)
(180 ILl) was added to the device equilibrated to 30°C, whence, once a stable
baseline had been achieved (3G--60 s), a known concentration of urea (G-
100 mM) (20 ILl) was added. The output voltage from the differential
amplifier was monitored over a 3-4 min time period to ensure linearity.
When the conductimeter was used to monitor the levels of urea in human
plasma a modified buffer was used as the supporting electrolyte. The
instrument was calibrated in 5 mM imidazole-HCI buffer (pH 7,5),
containing 4 mM NaCl and 1·6 g litre." human serum albumin in order to
simulate the background conductance anticipated from a 25-fold diluted
human serum. Serum samples of known urea concentration were obtained
from Southampton General Hospital , diluted 25-fold in the imidazole-
NaCl-alburnin buffer and assayed for urea in the conductance biosensor.
Unknown clinical samples were determined by interpolation from a
calibration curve for urea produced in the same buffer.
 A microelectronic conductimetric biosensor 107

RESULTS

The determination of the electrical conductivity of solutions depends on the

accurate measurement of the electrical resistance between two electrodes of
defined geometry immersed in the conducting solution. In the differential
microelectronic conductance biosensor described here, the device
comprises identical pairs of interdigitated multi-metal electrodes fabricated
on a silicon substrate in a planar configuration (Fig I). The accompanying
instrumentation applies a low distortion sine wave of frequency I kHz and
of an amplitude of 10 mV about 0 V to the sample and reference cells and
amplifies the response via a standard inverting operational amplifier with
gain (Fig. 2). The output signal from this amplifier is converted into a DC
voltage via an integrated circuit RMS-DC converter. The instrument was
calibrated by relating the DC output of the RMS-DC converter to reciprocal
ohms (Siemens) by connecting ± 1% tolerance metal oxide resistors across
the sample pair of microelectrodes to simulate solution conductance
changes. Figure 3 shows that the output voltage from the RMS-DC
converter is linearly related to the calibration conductance with a
correlation coefficient of 0·998 deduced by linear regression analysis. The
calibration line does not pass through the origin, presumably because of a
DC offset in the operational amplifier (Op Amp lOA). However, the
conductimeter in the assay solution , was used to monitor the rate of change
of conductance in the assay solution , thereby eliminating this effect from the
measurements. This calibration curve suggests that the output voltage from
the RMS-DC converter will be linearly related to the change in ionic
strength generated during the initial period of an enzyme-catalysed
reaction , provided that the basal ionic strength of the buffered solution is
low enough to permit an adequate signal-to-background ratio. For this
reason, a buffer of low intrinsic conductance, imidazole, has been used
throughout for the measurements.
Preliminary experiments were conducted with soluble urease (final
concentration 20 U ml" (where IV = I /Lmol min" product formed at
25°C); 10 /Lg total protein) in order to confirm the capability of the
microelectronic device to monitor enzyme-catalysed reactions. Typical
responses of the urease-loaded conductance biosensor operating in single
cell mode to several concentrations of urea in 5 mM imidazole-HCl buffer
(pH 7'5) at 30°C are shown in Fig. 4. The responses demonstrate the stability
of the baseline and , subsequent to the addition of urea , the linearity of the
output of the instrument over a 5 min period . The respon ses to any given
concentration of urea were reproducible to within approximately ± 1%; for
example , at 2·5 mM urea , the output voltage of the RMS-DC converter was
24·0±0·21mV min-I, whilst at 7·5mM urea the output was
108 L. D. Watson, P. Maynard, D. C. Cullen, R. S. Sethi, J. Brettle, C. R. Lowe

1·4

1·2

io

0·8
>
~

J
~ ()'6
'5
Q.

8
O~

o 50 100 150 200 250

Conductanc~ (pS)

Fig. 3. Calibration curve for the microelectronic conductimeter in single cell mode of
operation. Output voltage of the RMS-DC converter plotted as a reciprocal function of
known resistances (± 1%) connected across the 'sample' pair of interdigitated electrodes.

34·8±O·39mVmin- 1 • The output voltage change per minute was hyper-

bolically related to the urea concentration, whence linear regression analysis
of the corresponding double reciprocal plot of substrate concentration
versus output voltage per minute, shown in Fig. 5, revealed an apparent Km
(urea) of 1·69 mM. The apparent Km value was of a comparable magnitude
to the value (3-5,1 mM) normally accepted for the soluble enzyme (Varner,
1960; Reithel, 1971). The kinetics of the enzyme urease are complex; the
values of K; for urease are strongly dependent on the buffer system chosen,
as well as the temperature, ionic strength and the pH at which the assay was
carried out.
The microelectronic conductimeter was operated in the single cell or
differential mode (Bourelly & Bourelly-Durand, 1965). An enzyme-loaded
albumin membrane was cast over the serpentined interdigitated 'sample'
 A mi croelectronic conductimetric biosensor 109

CUr..) mM

I I J
o 1 2 345
Time (min)

Fig. 4. Typical response curves of the rnicroconductirn etric sensor to urea concentrations in
the ran ge 0-] -7 -5 mMat 30°C in 5 mMimidazole buffer (pH 7·5) containing IO g ml- t urea.
no L. D. Watson, P. Maynard, D. C. Cullen, R. S. Sethi, J. Brettle, C. R. Lowe

400
o

~ 300
E
I
>
.s
Q)
In
c:
& 200
In
Ql
a:
...
iE
'fi
.gc 100
o
~
~

-2 o 2 4 6 B 10
1
1/(Ureal (mM- )

Fig. 5. Double reciprocal plot of conductimeter response (mV change in 5min) against
concentration of urea (mM)for urease in solution.

pair of electrodes but not over the 'reference' pair, such that subtraction of
the amplified signals emanating from the respective RMS -DC converters
continuously corrects for any background signal changes. This simple
expedient circumvents many of the problems associated with non-specific
variations in basal conductivity of the buffers and biological fluids in which
measurements are being made.
Jack bean urease was immobilised over the metal network by forming a
cross-linked enzyme-albumin membrane with glutaraldehyde (Avrameas,
1969). Figure 6 shows the output response of the operational amplifier in
mV min " as a function of urea concentration in the range of 0-1-10 mM. A
linear response for up to 3 min were obtained at all urea concentrations. A
double reciprocal plot (Fig. 7) of substrate concentration versus output
voltage change revealed an apparent K; (urea) of 6·34 mM for the
immobilised urease system and a maximal output voltage change at
saturation of approaching 700 mV min-I.
The immobilised urease conductimetric biosensor also responded to urea
present in serum samples. The instrument was calibrated in 5 mM
imidazole-HCI buffer (pH 7 '5) containing 4 mM NaCI and 1-6 g litre."
human serum albumin in order to simulate the background conductance
 A microelectronicconductimetric biosensor III

350

300

250

200

..
c
°E
> 150
E
<I

100

50

2 4 6 8 10
(ureal (mM)

Fig. 6. Calibration curve for urea in 5 mM imidazole- HCI buffer (pH 705) at 3lrC in the
differential mode conductimeter with immobilised urease.

anticipated from 2S-fold diluted human serum. Serum samples of known

urea concentrations were obtained from a major hospital laboratory,
diluted 25-fold in the imidazole-NaCl-albumin buffer and assayed for urea
in the conductance biosensor. Figure 8 shows that there was a linear
relationship (correlation coefficient> 0,99) between the urea concentra-
tions determined with the microelectronic device and those obtained from
the major hospital laboratory .

DISCUSSION

Many enzymes catalyse reactions which lead to overall changes in solution

conductance and thereby display great potential as sensing elements in
conductirnetric biosensors. Enzyme catalysis can create conductance
changes by generating additional charged species (hydrolases , amidases), by
112 L. D. Watson, P. Maynard, D. C. Cullen, R. S. Sethi, J . Brettle, C. R. Lowe

150 0

c
"E
'I
>
E 100
Ql
'"
C
0
Cl.
'"
Ql
a::
t
q;
E
-fi 50
:l
"tl
C
0
!:2

-2 o 2 4 6 8 10

l/[Ureal (mM -')

Fig. 7. Doubl e reciprocal plot of conductimeter response (mV change in I min) against
concentration of urea (mM) for immobilised urease .

separation of unlike charges (decarboxylases, dehydratases), by proton

migration (esterases, proteases) , by changes in the degree of association of
ionic species (kinases) or by a change in the size of charge-bearing groups
(phosphatases, sulphatases , nudeases). The enzyme conductimeter, like
the enzyme thermistor (Mosbach & Danielsson, 1981), thus provides a most
useful and versatile addition to the analytical methods currently available.
The microelectronic device and instrumentation described here is relatively
inexpensive, robust and easy to use and appears to respond to urea with
more than adequate sensitivity and precision.
The two principal sources of experimental error encountered in the
development of the device were the precision by which the conductance
measurements could be made and the elimination of temperature effects
(Robinson & Stoke s, 1959; Hill & Tomalin, 1982). Errors in conductance
measurements were minimised experimentally in the present device by
tuning the frequency of the low distortion sine wave generator to eliminate
capacitance effects and by applying a very low amplitude signal to ensure
negligible electrochemical effects. The temperature coefficient of con-
ductance of electrol ytes in water is almost invariably positive and of a
magnitude of about 2%OC- 1 (Willard et al. , 1974). The small sample
volume s employed in the present study obviate the requirement for the long
 A microelectronic conductimetric biosensor 113

3 o

it 2
.s
c 0
E 0
:..
::::l

0-f-""T""""T""""T""--r--r--r---r---r---r-....,................................-,-.....,...............,........,...---.
o 2 3 4

mM Ur~a (m icrocooductimetric
sensor )

Fig. 8. Co mpa riso n of urea con centrations in human serum determ ined with the micro -
co nd uctime tric biosensor and those clinical samples determi ned at a major hospit al
lab orator y.

thermal equilibration times observed by others (Bourelly & Bourelly-

Durand, 1965; Lawrence & Moores, 1972). The microelectronic conducti-
metric biosensor has been used to measure urea with a typical accuracy of
± 1%. It is expected that this miniaturised device and associated instrument-
ation will find application in the analysis of urea for in vitro blood analysis, in
renal surgery and in dialysis mon itoring .

ACKNOWLEDGEMENT

This work was supported in part by the Science and Engineering Research
Council.
114 L. D. Watson, P. Maynard, D. C. Cullen, R. S. Sethi, J. Brettle, C. R. Lowe

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