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Analyst, Decwnhei- 1996, Vol. 121 ( I 751-I 758)

1751

Rigid Carbon-Polymer Biocomposites for

Electrochemical Sensing*

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A Review

Salvador Alegret
Grup dc Sensors i Biosensors, Departament de Qui'mica, Uniwrsital Autdnoma
de Barcelona, 08293 Bellatei-ra, Catalonia, Spain

This paper reviews the use of biocomposite materials in

the construction of amperometric biosensors. These rigid
composites are formed by dispersing graphite particles in
assorted polymers (especially epoxy resins). These
composites are bulk-modified biologically (adding
enzymes and cofactors) and chemically (blending
mediators and catalysts).
Keywords: Biocomposites; conducting composites; rigid
c*arbon-polyniei-hioconiposites; amperometric biosensor;
elrc~ti.ochemic.alseiisor; reijien)

immobilization techniques are being tried for the mass production of these devices.
In this context, the present review covers recent work in the
field of amperometric biosensors based on new types of
materials known as hiocomposites. These materials are formed
by rigid conductive composites based on carbon-polymer
matrices where the biological material (enzymes) as well as
other modifiers (cofactors, mediators, catalysts, additives, etc.)
are jointly bulk-immobilized.

Conducting Composites
Introduction
Most strategies in analytical chemistry today call for complex
instrumentation and considerable support, including special
laboratory facilities and highly skilled personnel. Chemical
sensors are a key element of novel strategies applied to
analytical instrumentation. Sensors and sensor-based devices
provide original solutions without the need for complex
instruments or a huge support infrastructure. Chemical sensors
are devices that are small, robust, portable and easy to use.
Additionally, they do not need reagents to operate and they can
yield reliable information continuously.
A chemical sensor has two distinctive parts: a selective
recognition component (receptor) and an element (transducer)
that converts the primary signal produced by the receptor during
the recognition event into a more useful secondary signal. The
nature of the primary signal can be thermal, mass, electrochemical or optical and usually has to be transduced to an
electrical signal. This secondary electrical signal contains the
codified chemical information from the sample.
Several disciplines have to converge in the design of these
devices. The design of sensors with biological recognition
components such as enzymes, immunological species, chemoreceptors and DNA strands is receiving great attention
nowadays. The chemical selectivity shown by these biocomponentc is very high. Sensors of this kind are known as
hiosc~nsors.Biosensor science and technology use physical and
chemical immobilization procedures to couple biological recognition elements to appropriate transduction devices. Generally,
the biological material is fitted on the surface of transducers
using complex and wet immobilization procedures. However,
these procedures are seldom suitable for mass production.
Amperometric biosensors, usually formed by biologically
surface-modified voltammetric electrodes, are gaining increasing importance owing to their high reliability, robustness and
sencitivity. I Efforts continue to increase the quality of the
electrochemical response. Additionally, new materials and
' Prcscnted 'it the 6th European Conference o n Electroanalysis, Durham. March 25-29.

1996.

A composite is formed by the combination of two or more

phases of different nature. Each phase maintains its individual
traits, but the mixture may show new physical, chemical or
biological properties. If one of the phases is an electrical
conductor, the overall electrical properties of the conducting
composite will be determined by the nature, the relative content
and the distribution of each phase. Electrical resistance depends
on the connectivity of the conductor particles in the matrix of
the composite. Several conductimetric chemical sensors are
based on the disruptive action of organic vapours on the
conducting filaments of the material.2J Simple and inexpensive
all-solid-state potentiometric sensors have been developed by
replacing the metal substrate with a graphite+poxy or a metalepoxy composite. These composites are mouldable before
curing so sensors of different shapes and sizes can be
constructed. Ion-selective membranes adhere better to these
materials and the resulting devices are simple and inexpensive,
show prolonged lifetimes4.5 and the quality of their response is
acceptable for analytical applications. An extensive review of
ion-selective electrodes based on conducting epoxy composites
appeared recently.6

Conducting Composites for Amperometric Sensing

The polymer gives the biocomposite a certain physical,
chemical or biological stability. The biocomposite acquires
particular electrochemical traits from the distribution of the
conductive phase in the bulk and, consequently, on the surface
of the biocomposite. Carbon materials (graphite, carbon black,
etc-.) are ideal conductive phases for composites used in
amperometric sensors. These materials have a high chemical
inertia and show a wide range of working potentials. They also
have a low electrical resistance (approximately 10-4 52 cm) and
a crystal structure responsible for low residual currents.
If the surface of a macroelectrode is reduced, the signal and
the associated noise also diminish. In microelectrodes, according to Oldham,7>8the perimeter of the sensing surface has a
greater influence on the signal. By means of this edge effect,
non-linear diffusion is established and the quality of the signal
is enhanced. This enhancement is shown by a higher signal-tonoise ratio and lower detection limits. These features and their

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1752

Analyst, December 1996, Vol. 121

inherent small size have raised interest in microelectrodes.

However, the low currents produced call for complex and
expensive instrumentation. If small sensors are not required, an
alternative is to build carbon fibre arrays separated by an
insulating matrix and connected in parallel.9
The signal produced by this macroelectrode formed by a
carbon fibre ensemble is the sum of the signals of the individual
microelectrodes. The size of the resulting signal is equivalent to
the signal produced by a carbon rod of the same active surface
but showing the signal-to-noise ratio of a microelectrode.
The construction of these ensembles is difficult. However, an
equivalent device can be constructed when a composite is made
of small conductive particles dispersed in a polymer matrix.
Additionally, these devices are easier to build.
The selectivity and sensitivity of an amperometric sensor are
greatly enhanced if the surface is modified with certain
chemical and biological species. One of the key advantages of
composite-based sensors is the ease of bulk modification
compared with the modification of the surface of a pure
conductor, which is usually complex and costly.
Conductive composites are modified easily because of their
plasticity before curing. Modifying fillers can be blended into
the matrix, conferring new abilities on the resulting composite.
These new abilities include immobilization of reagents involved
in the electrochemical reaction, electrocatalysis, preferential
preconcentration and surface structuring.

Soft versus Rigid Conductive Composites

Adams'O proposed the use of soft carbon pastes to build
amperometric transducers. These pastes are built by mixing an
inert conductor (e.g., graphite powder) with a non-conducting
liquid (e.g., paraffin oil, silicone, Nujol). This insulating liquid
has a specific viscosity and the paste has a certain consistency.
The resulting devices are easy to prepare and inexpensive and
can be coupled to simple instruments.
However, these pastes have limited mechanical and physical
stability, especially in flow systems. Additionally, the pastes are
dissolved by some non-polar electrolytic solvents, leading to a
deterioration of the signal. The general degradation of these
devices occurs quickly and has limited their use to the research
laboratory. Reviews on chemically' 1 and biologically12 modified carbon paste electrodes have appeared recently.
On the other hand, amperometric sensors and biosensors
based on rigid composites do not show the problems mentioned
above. Further, the fabrication of these devices can be adapted
for mass production at a low cost.

Rigid Carbon-Polymer Biocomposites

Rigid Carbon-Polymer Matrices
Creasy and co-workers reported the copolymerization of
styrene with divinylbenzene (a cross-linking agent) and vinylferrocene (a modifier), using carbon black' 3 (semigraphitic
carbon particles) or carbon fibre9314 as a conductor. This was the
basis for the construction of chemically bulk-modified electrodes. These devices showed better physical properties (the
sensing surface was renewable by polishing) and enhanced
chemical traits (they were stable in organic solvents) compared
with carbon paste electrodes. Wang and co-workers used a
commercially available graphite epoxy resin (Grade RX, Dylon,
Cleveland, OH, USA) to build chemically and biologicallyI6
bulk-modified electrodes. The use of this commercial composite rendered the Fabrication of the sensors easier, quicker and
more reproducible than the procedure proposed earlier by
Creasy and Shaw.9 The approach followed by Wang and coworkers for the preparation of rigid biocomposites was the first
report concerning this procedure and these materials. It has been
adapted in our laboratories using a non-conducting epoxy (Epo-

Tek H77, Epoxy Technology, Billerica, MA, USA), graphite

powder (Merck, Darmstadt, Germany) (particle size below
50 pm), biological materials and additives. All these elements
are mixed to build a particular biocomposite. l 7 , l x Our procedure
has been expanded to include other polymer matrices such as
silicone, polymethacrylate, polyester19 and polyurethane. All
these polymers can be prepared it? situ, they readily admit the
biological material and additives (catalysts, mediators, cofactors, etc.), they have a simple curing process and are
commercially readily available.
Graphite-Teflon electrodes were developed originally for
vol tammetric and amperometric applications .2",2l These materials with bulk-immobilized enzymes have served for the
development of biosensors.22-z4 In this particular instance, the
graphite and the powdered Teflon are mixed with the other
ingredients and the mixture is pressed to form pellets.

Biological Materials and Other Modifiers Immobilized in

Rigid Carbon-Polymer Matrices
The immobilization of some lyophilized en7ymes (oxidases,
peroxidases, dehydrogenases and cholinesterases) in rigid
carbon-polymer matrices has been reported (see Tables 1 4 ) . In
some instances, the enzyme is covalently bonded to graphitG2
or silica25 particles before blending it to the polymer matrix.
Different redox mediators and catalysts have been added to the
biocomposites in order to enhance their selectivity and
sensitivity. These modifiers may be substances related to
ferrocene,1"22.26
tetrathiafulvalene27
and
tetracyanoquinodimethane25 or a metallic catalyst such as gold and
palladium28-30 or platinum.31 Dehydrogenase biocomposites
have been produced featuring the nicotinamide adenine dinucleotide (NAD+) cofactor.32,33This has opened up the possibility of reagentless biosensors for alcohols and lactate.
An alcohol biosensor has been developed by confining dry
yeast to a graphite-epoxy matrix. '6 Eisenia hicyclis, an alga,
has also been immobilized in a graphite-epoxy matrix, forming
a composite used in bioaccumulation assays and in the
voltammetric measurement of metal ions. 1 6
In our laboratories, biocomposites based on immunospecies
immobilized in graphite-polymer matrices are being tried.
These biocomposites have a surface that may be regenerated by
polishing after each immunological assay.34

Preparation of the Biocomposites and Biosensor

Construction
The biocomposites are prepared very easily. The powdered
graphite is dispersed homogeneously by hand with the appropriate amount of polymer. According to Tallman and Petersen,x
these materials can be classified as dispersed composites since
the conductor particles have an equal opportunity to occupy any
point throughout the matrix.
The polymer material is activated when its components are
blended. The activation happens when a volatile fraction
evaporates or when a hardener, catalyst or initiator acts on the
resin. The resin may be epoxy, silicone, methacrylate, polyester
or polyurethane (see Table 1). The contents of the graphite, the
modifier (enzyme, catalyst, mediator) and the additives are
optimized for a particular polymer matrix. Graphite particles are
smaller than 50 pm.19 The goal is to achieve the maximum
electrical conductivity and the highest response quality with an
appropriate biocomposite rigidity. Graphite content may vary
from 20% (epoxy) to 60% m/m (silicone).I9 As mentioned
earlier, there is a commercial epoxy that already contains the
graphite. 16 The fraction of biological material may vary from
1 % (acetylcholinesterase)35 to 25% m/m (tyrosinase). l 6
The homogeneous mixture is introduced 2-3 mm into a tube
made of PVC, glass, etc. A metal disk coupled to a wire is used

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1753

Analyst, December 1996, Vol. 121

If the matrix is T e f l o n , 2 2 ~the

~ ~ granular polymer is mixed
with graphite in mass proportions of 7 + 3. The biological
material is previously immobilized on particles of graphite
powder22 or is homogenized with Teflon and graphite particles
at -20 OC.23 Once mixed, the material is pressed at 7000

to contact the composite inside the tube. The ensemble is left at

room temperature or slightly higher (40 "C) for one or more
days as needed by the curing of the polymer. When it is
hardened, the biocomposite is polished with abrasive papers of
decreasing grain size.

Table 1 Glucose biosensors based on rigid conducting biocomposites

vel'sus
Ag/AgCI/V
+1.15

P"
7.0

Linear
response
range
(mmol I-')
0.1-5

Epoxy (49)
(Epo-Tek H302)

+].I5

7.0

0.1-5

19

Graphite (49)

Methacrylate (49)
(Sealer-Healer 1540)

+1.1

7.0

0.2-5

19

GOD (2)

Graphite (62)

Silicone (36)
(Sellaceys)

+1.15

7.0

0.4-20

19

GOD (2)

Graphite (36)

Polyester (62)
(Resipol 9 144)

+1.1

7.0

0.1-5

19

GOD (2)

Graphite (60)

Polyurethane (38)

+1.15

7.0

0.05-5

This
work

GOD (20)
covalently
bound to
graphite

Graphite (10)

Teflon (70)
(7A Dupont)

+0.9
+0.8

7.4
7.4

2.5-30
0.2-1'-

22

GOD ( 1 5 )

Graphite (15.8)

Epoxy (63.0)
(Epo-Tek H77)

Gold (1 1.8) and

palladium (7.9)
1 ,l'-Dimethyl
ferrocene (26)

+0.9

7.0

0.0 1-2
1-10 gl-'-'-

28
29

+0.5
+0.3

7.4
6.5

Epoxy (63.0)
(Epo-Tek H77)

TTFl (19.7)

+O. 15

7 .0

1-6-1
0.1-2

16
26
27

Silicone (28)
(Sellaceys)

TTF, TCNQI: (70)

+0.2

7.0

0.1-5

Biocomposite components (% m/m)

c

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'"ippl

Mediator/
catalyst

Enzyme*

Carbon

Polymer

GOD (2)

Graphite (19)

Epoxy (79)
(Epo-Tek H77)

GOD (2)

Graphite (49)

GOD (2)

Graphite-epoxy (Dylon) (54)

GOD (20)
GOD (1.5)

Graphite (15.8)

GOD (2)

' Glucose oxidase (GOD) (100-200 U mg-I).

Flow-injection.

1 TTF =

Ref.
19

This
work

tetrahiafulvalene; TCNQ = tetracyanoquinodimethane.

Table 2 Rigid conducting biocomposite-based biosensors for phenol and phenolic substrates
Biocomposite components (5% m/m)
Eappl

Enzyme
Tyrosinase (7.5)

Carbon/
polymer

Catalyst

Graphite-epoxy
(Dylon) (92.5)

Substrate
Catechol'

vei'sus
Ag/AgCl/V
-0.2

PI3
(working
solution)
7.4
(methanol
50% v/v)

42

6.7

Phenolics
Phenol

-0.2

Phenolics
Catechol
Phenol
Catechol
Phenol

-0.1

-0.1

6.0
(acetonitrile
5-20% v/v)
(methanol
5-20% v/v)

30

Catechol
Phenol
Catechol

-0.1

6.0

30

-0.05

7.0
(methanol
10% v/v)

0.Y
1" 1

Graphite-epox y
( D y W (99)

Grapite-epoxy
(Dylon) (79)
Graphite (18)
Graphite-Teflon
(10-30%) (80.2)
Detection limit. 1 Flow iiijection.
Tyrosinase (1)
(2400 U nig-1)
Tyrosinase (1.8)
(3900 U mg-I)

Ref.
16

50-350

Dopamine
Mushroom
tyrosinase (5)
(6300 U mg- I )
Mushroom
tyrosinase (3
(12600 U)
Tyrosinase (1)
(2400 U mg

Linear
response
range
(pmol 1-I)

Gold (8)
Palladium ( 12)

43

6.0
0.04*,'
1 .OW.'

0.2-25'

23

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I754

Analyst, Decwnher 1996, Vol. 121

kg cm-2, producing 2 mm thick disks. These pellets are coupled

to a tube to form an electrode. According to Tallman and
Peterseqx these materials can be classified as consolidated
composites, since the conductor particles extend throughout the
matrix in a random, reticulated fashion with regions of pure
insulator and pure conductor.

Amperometric Biosensors Based on Rigid

Carbon-Polymer Biocomposites

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Glucose Biosensors
Several glucose biosensors based on biocomposites have been
reported (see Table 1). Glucose oxidase (GOD) has been used in
our laboratory as an enzyme model to study the biocatalytic
characteristics of rigid conducting biocomposites that feature
immobilized enzymes. This oxidase is compatible with matrices
of graphite and several polymeric materials such as epoxy
resins, polymethacrylate, silicone, polyester, polyurethane and
Teflon. These biocomposites have been applied to glucose
measurement based on the direct oxidation of the hydrogen
peroxide produced by the action of the enzyme [see Fig. 1 (A)].
This happens at extreme potentials (0.9-1.15 V versus Ag/
AgCI) (see Table I). When a graphite-polymer composite is
used, a shift towards more positive potentials is observed
compared with measurements realized with graphite or plati-

num electrodes.36337It is known that carbon electrodes that have

metal particles (Pt, Ru, Rh, Pd, etc-.)on their surface show great
catalytic action.3X.39The same happens when the metal is
dispersed in carbon pastes.3() The addition of catalysts (gold,
palladium) to a GOD graphite-epoxy biocomposite for the
oxidation of hydrogen peroxide increases the stability of the
signal and reduces the response time. Further, the oxidation
potential of hydrogen peroxide is lowered by 250 mV.l* This
decrease is also found in experiments with carbon rods where
Au-Pd was sputtered to the surface of the electrode."'
Therefore, metal bulk-modified composites represent more
viable alternatives than those surface-modified electrodes
produced by sophisticated technologies. However, the inclusion
of metal catalysts in the biocomposite does not hinder the action
of the usual interferents found in biological samples (ascorbic
acid, uric acid, etc.1.28 On the other hand, it has been observed
in our laboratory that this material retains the enzymic activity
in dry storage for more that 1 year.
The lower working potential and the higher quality of the
signal observed in biocomposi tes containing Au-Pd has
permitted the use of these materials in flow injection systems.
Biosensors with these materials have been used to monitor
glucose in fermentation processes.29
Artificial electron acceptors may be added to the biocomposite. These substances act as electron mediators between GOD

Table 3 Rigid conducting biocomposite-based biosensors for hydrogen peroxide and organic peroxide substrates
Biocomposite components (5% m/m)
E.'PPl

Enzyme

Carbon/
polymer

Horseradish
peroxidase (25)

Graphite-epox y
( D y W (75)

Mediator/
catalyst

l'f2YSI4.5

PH
mediator
(working
solution)

Substrate

Ag/AgCl/V

H202

-0.2

7.4
hexacyano-

Organic
peroxides

-0.2

7.4
o-pheny lene
diamine
7.4

Linear
re spon \e
range
(nimol I-')

Ref.
16

ferrate(1r)

Horseradish
peroxidase (1 5 )
(94 U mg-I)
Horseradish
peroxidase
covalently
bound
to graphite (16)

Graphite ( 10)
Teflon (70)

Horseradish
peroxidase

Graphite
Teflon

Ferrocene (4)

Detection limit.

0.0

Butan-2-one
peroxide
H202
Butan-2-one
peroxide
Ferrocene

Horseradish
peroxidase (1 5 )
(90 U mg- I )
mixed with
human serum
albumin ( 5 )

Horseradish
peroxidase (2)
(318 U mg-I)
Horseradish
peroxidase (2)
Horseradish
peroxidase ( I .9)

H202

0.0

H202
-0.1
Butan-2-one

Hz02
-0.25
B utan-2-one
peroxide

7.4
(reversed
micellar
media)
7.4

22

1-60 pmol 1
1-100 pmol I-]

24

?-0.02

48

'!-0.05

'?-(I. I

?- 1

H Z 0 2

-0.35

Graphite (20)
Epoxy (78)
Graphite ( 19.6)
Epoxy (76.6)

H202

-0.3

H202

-0.05

injection.

22
* I

' L O .I

Graphite (1 9)
EPOXY (79)

-1 Flow

2.5 pmol 1 I '

20-200 pmol 1
3.0pmol I-'*

7.4
(acetonitrile
90% v/v)

Cumene
peroxide
terr-Butyl
peroxybenzoate
tel-r-Buty1
hydroperoxide

Platinum (1.9)

47

0.005-0.5

19

7.0

0.03-7

31

7.0

0.09-9

31

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Analyst, December 1996, Vol. 121

and the electrode [see Fig. l(B)] and include 1,l'-dimethylfewoceneI6.'6 and tetrathiafulvalene.'7 The addition of these mediators permits the use of working potentials in the range 0.5-0.15
V. The action of interferents is greatly reduced at these working
potentials. In the biocomposite modified with tetrathiafulvalene, ascorbic acid interference is reduced by 90% and the
detection of uric acid is negligible.27

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Phenol Biosensors
Biocomposites featuring tyrosinase have been used in biosensors for
(see Table 2). In this enzyme
system, the species produced electrochemically (catechol) is
also the enzyme substrate (see Fig. 2). This amplifies the
electrochemical response.44 That is the reason for the low
detection limits found in these biosensors (see Table 2).
However, Onnerfjord et al.,43 using tyrosinase-based rigid
biocomposites, found detection limits higher by one to two
orders of magnitude than those produced by thyrosinase
biosensors based on carbon pastes. If gold and palladium
particles are introduced into the biocomposite, an increase in
current is achieved.30

1755

On the other hand, the products of the enzyme reaction

(quinones) are highly unstable in water. Furthermore, they
polymerize quickly into polyphenols that block the enzyme, and
may passivate the electrode. Wang et al.42 reported a 4%
decrease in the response of a tyrosinase biosensor after 10
successive discontinuous measurements of 1 X 10-5 mol 1-1
phenol samples. This decrease was explained as being due to
slow fouling of the measuring surface by the products of the
reaction. This deleterious effect may be minimized by working
in flow ~ y s t e m s ~ or
~ 3renewing
~3
the surface of the biocomposite
by polishing. Tyrosinase keeps its biocatalytic action when
confined to graphite+poxy matrices for moderate periods of
times (3% decrease in 10 d with the device in dry storage at
4 "C). However, the biocatalytic activity could be regained after
polishing (7040% of the original activity for catechol).4' Wang
et al.42 proposes that this stabilizing effect may be due in part to
the protective action of the epoxy matrix, not unlike the reported
effect in non-aqueous media.4"46
This tyrosinase-graphite-epoxy biocomposite has been used
to measure phenols in a partially aqueous medium containing
50%16 or 5-20%30 v/v methanol and 5-20% v/v acetonitrile.")
Amperometric biosensors incorporating tyrosinase-graphite-

Table 4 Biosensors for bilirubin, alcohols, lactate and pesticides, based on rigid conducting biocomposites
Biocomposite components (% m/m)
Carbon/
polymer

Enzyme
Bilirubin
oxidase ( 5 )
Horseradish
peroxidase ( 5 )

Ethanol

+0.6

7.4
hexac yanoferrate( 11I )
NAD+

NAD+ ( 10)

Alcohols
Ethanol
Ally1 alcohol
Propan- 1-01
Butan- 1-01
Propan-2-01

+0.7

7.4

NAD+ (12)

Lactate

+0.7

7.4

Acety lthiocholine

+0.7

7.0

5-120 pmol I-'

20 pg I-]*
carbofuran
27 pg I-'
paroxon
20 pg 1 - 1
carbaryl
22 pg I-'
dichlorvos
2.5-1 00 pmol 12.2 pg 1 - 1 '
carbofuran
27.5 pg 1-1
paroxon
2.0 pg I-'
carbaryl
5-100 pmol I- I

Graphite-epox y
(Dylon) (90)

Yeast (20)
(Sac i hul-omyes
cel-r\~i.siuP)

Lactate
dehydrogenase (6)
(148.7 U mg-I)

Graphite-epoxy
(Dylon) (82)

Acetylcholine
aterase ( 12)
( 1 120 U mg-1)

Graphite ( 1 7.6)
Epoxy (70.4)

Acetylcholine
esterdse
covalently
bound
to silica (2)

Graphite (18)
Epoxy (7 1 )

TCNQ (9)t

Acetylthiocholine

+0.3

7.5

Butyrylcholine
esterase
covalcntly
bound
to silica (2)

Graphite (18)
Epoxy (7 1

TCNQ (9):i

Butyrylthiocholine

+0.3

7.0

* Detection limit.

Linear
response
range

Substrate
Bilirubin

Cofac lor/
mediator

Yeast alcohol
Graphite-epoxy
dehydrogenase (7.5)
(Dylon) (82.5)
(350 U mg-I)

P"
mediator
(working
solution)
7.4
hexacyanoferrate( 1 1 )

E"Wl
versus
Ag/AgCl/V
-0.2

4-1 00 pmol I-

16

32
0 4 . 4 rnmol 1- I
(-5.8 mmol I-'
0-8.2 mmol 1-1
0-1 1.7 mrnol 113-32 mmol 1- I
0.08 mmol 1-1
33
0.5-20 mmol 1-11

22.1 pg 1 - 1 carbofuran
2.8 pg 1-1
paroxon
3.6 ug I-'
chlorfenvinphos

Flow injection.

TCNQ = 7,7,8,8-tetracyanoquinodimethane.

Ref.
49

35

25

25

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Teflon biocomposite have been developed for the detection of

catechol.23 Studies of the operational stability of the biocomposite response in organic media (10% v/v methanol or
acetonitrile) were carried out in the flow injection mode. Better
stability was achieved in methanol.23
Tyrosinase shows poor selectivity with respect to phenol
substrates. The reported selectivity sequences for different
tyrosinase biocomposites show inconsistencies among themselves42.43 and with conventional tyrosinase biosensors. The
hydrophobic nature of the graphite-epoxy resin modifies the
selectivity sequence.

Peroxide Biosensors
Horseradish peroxidase (HRP) has been immobilized in rigid
carbon-polymer matrices. This is the basis for the development
of sensors for hydrogen peroxide and small organic peroxides.
The first reports in literature follow the approach shown in
Fig. 3(A). Reducing agents, such as hexacyanoferrate(I1) ion16
or o-phenylenediamine,47 are added to the solution to regenerate
the enzyme to its reduced form. In this way, the oxidized form
of these mediators can be detected at lower voltages (-0.2 V
versus Ag/AgCl) than those used for the direct detection of
hydrogen peroxide (see Glucose Biosensors section). Peroxidase has been immobilized with the mediator ferrocene in
graphite-Teflon mat rice^.^^,^^ This opens up the possibility of
developing reagentless sensors, capable of working at potentials
around 0.0 V versus Ag/AgCl. These devices simplify the
measurement process as they function as direct sensors that do
a-D-glucose

It

p-D-glucose

ox

~~

Bilirubin Biosensor

GOD 2H+
6-gluconolactone

H2 0

The co-immobilization of HRP and bilirubin oxidase in a

graphite-epoxy composite and the addition in solution of
ferrocene as a mediator complete the construction of a bilirubin
biosensor,49 as seen in Fig. 4 and Table 4. The rapid passivation
induced by the adsorption of bilirubin or biliverdin calls for
frequent polishing of the surface of the biosensor. The
renewable surfaces associated with graphite-polymer biocomposites lend themselves well for this task.

2H'

\red 1

D-gluconate + H+

ox

Fig. 1 Reaction sequences for the amperometric detection of glucose

using biocomposite electrodes: (A) GOD-graphite-polymer biocomposite;
and (B) GOD-mediator-graphite-polymer biocomposite.
I

Biocomposite

2H'

2Hi

not require additional reagents. The rapid response shown by

these biosensors makes them ideal for flow applications.zz
The biocomposite HRP-ferrocene-graphite-Teflon is stable
in a medium of acetonitrile water (9 + 1 v/v). Biosensors based
on this composite have been applied to the determination of
hydrophobic organic peroxides in this mediumz2 and in
reversed micellar media.24
Biocomposites based on HRP-graphite-epoxy have been
used to prepare mediatorless biosensors where direct electron
transfer takes place between the active sites of the enzyme and
the graphite particles when the substrate is present 19,31,48 [see
Fig. 3(B)]. We have observed31 that the addition of platinum
particles in these biocomposites permits one to work with a
lower potential than the optimum working potential of unmodified HRP-graphite-epoxy biocomposite electrodes (see Table
3).
The trend of sensitivity for mediatorless biocomposites4~
is in accordance with data obtained for o-phenylenediaminemediated HRP-carbon paste electrodes47 : hydrogen peroxide >
butan-2-one peroxide > tert-butylperoxy benzoate > cumene
peroxide > tert-butyl hydroperoxide.
The response and surfxe-to-surface reproducibility have
been improved by mixing HRP with human serum albumin
(HSA).48 This can be attributed to the stabilizing effect of HSA
on HRP during the curing process of the graphite-epoxy resin
(reaction of epoxy with amino groups), similarly to glutaraldehyde inactivation of pure enzymes due to a cross-linking
reaction with amino groups.48

2H+

Fig. 2 Reaction sequence for the amperometric detection of phenol and

phenolic substrates using a tyrosinase-graphite-polymer biocomposite.

Alcohol Biosensors
The co-immobilimtion of alcohol dehydrogenase (ADH) and
NAD+ in a graphite-epoxy matrix has allowed the development
of reagentless alcohol biosensors32 (see Table 4), following the
scheme shown in Fig. 5(A). This type of biosensor shows a
rapid decrease of the signal on continuous use owing to a
fouling of the biocomposite, incomplete recycling of NAD+NADH system or a loss of this cofactor. If the surface is
polished, the initial activity is restored reproducibly. ADH from
yeast, used in these biocomposites, oxidizes primary alcohols
quickly (with the exception of methanol). Secondary alcohols
are also oxidized but more slowly. The sensitivity sequence of
these biosensors (i.e.,ethanol > ally1 alcohol > butan-1-01 >
propan- 1-01 > propan-2-01) is slightly different to the sequence
observed for the same enzyme in solution.32

0,

biliverdin

Fig. 3 Reaction sequences for the amperometric detection of hydrogen

peroxide and organic peroxide substrates using biocomposite electrodes:
(A) mediated HRP-graphite-polymer biocomposite (mediator in solution
or in biocomposite); and (B) mediatorless HRP-graphite-polymer biocomposite.

2H+

2H'

Fig. 4 Reaction sequence for the amperometric detection of bilirubin

using a biocomposite electrode: mediated bilirubin oxidase (BOX)horseradish peroxidase (HRP)-graphite-polymer biocomposite (mediator
in solution).

View Article Online

Analyst, December 1996, Vol. 121

In the first paper in which Wang and Varughese16 singled out

polishable and robust biological electrode surfaces, they
reported a graphite-epoxy biocomposite containing dry yeast.
These materials had enzyme activity blended into the rigid
conductive composite. The resulting alcohol biosensor worked
in a buffered medium containing the cofactor and hexacyanoferrate(II1) as a mediator [see Fig. 5(B)].

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Lactate Biosensor
Following the strategy mentioned earlier [see Fig. 5(A)], lactate
dehydrogenase (LDH) and the cofactor NAD+ have been
immobilized in rigid matrices consisting of graphite and
epoxy33(see Table 4). The resulting reagentless biosensors may
experience a rapid decrease in sensitivity, as found in ADHNAD-graphite-epoxy biocomposites. Owing to the high working potential (0.70 V versus Ag/AgCl) needed for the
regeneration of NAD+, these biosensors show significant
interferences from several species such as acetaminophen,
ascorbic acid and uric acid. These biosensors have a fast
response and are suitable for continuous-flow measurements. In
flow injection procedures, where the sample is briefly in contact
with the sensor, passivation effects are much less noticeable
than in discontinuous measurements.33

Pesticide Biosensors
Organophosphorus and carbamate pesticides have been determined with biosensors based on biocomposites containing
acetyl~holinesterase.2~~~~
This enzyme hydrolyses both its
natural substrate and thiocholine esters. The hydrolysis of
acetylthiocholine produces thiocholine. This electroactive species is detectable at a potential of 0.7 V versus Ag/AgCl applied
to the biocomposite. Fig. 6(A) shows the biosensor response to
the substrate. This response is inhibited if organophosphorus
and carbamate pesticides are present. This enzyme inhibition is
irreversible, calling for the renewal or the reactivation of the
enzyme content in the electrode surface either by replacing
more enzyme or by regenerating it with special reagents.
Biosensors based on rigid biocomposites are an attractive
proposition here, since this enzyme reloading is achieved by
simple polishing of the biosensor surfxe. If the mediator TCNQ

Biocomposite

Fig. 5 Reaction sequences for the amperometric detection of substrates

(S) as alcohols or lactate using biocomposite electrodes: (A) dehydrogenase-NAD-graphite-polymer biocomposite; and (R) mediated dehydrogenase-graphite-polymer biocomposite (cofactor and mediator in
solution).

@
H,O + thiocholine

Fig. 6 Reaction sequences for the amperometric detection of thiocholine

esters using biocomposite electrodes: (A) cholinesterase-graphite-polymer
biocomposite; and (JS) cholinesterdse-mediator-graphite-polymer biocomposite. The enzymic hydrolysis of thiocholine esters is inhibited by the
presence of some pesticides.

1757

(7,7,8,8-tetracyanoquinodimethane)25 is added to the biocomposite [see Fig. 6(B)], thiocholine can be detected at a
potential of 0.3 V versus Ag/AgCl, curtailing the effect of
interferents. Biocomposites made of butyrylcholinesteraseTCNQ-graphite-epoxy have been prepared for the measurement of butyrylthiocholine at this same potential25 (see Table
4).

If the origin of the cholinesterase (electric eel, horse serum

and bovine erythrocytes) is altered, serious inconsistencies are
noted in the biocomposites prepared due to the leakage of the
enzyme. A satisfactorily reproducible response is attained only
with acetylcholinesterase from bovine erythrocytes. If the
enzyme is immobilized on silica particles for stability, good
reproducibility is attained regardless of the origin of the
enzyme. This step does not alter the curing process of the
biocomposite.25

Conclusions
Biosensors based on rigid polymer-graphite composites are a
recent development and examples of their design and application are still scarce in the literature (see Tables 1 4 ) . However,
several advantageous qualities of biocomposites based on rigid
graphite-polymer mixtures can be envisaged from the present
review.
The preparation procedure for these biocomposites is simple
and involves dry chemistry techniques for the most part. In
some cases, the enzyme has first to be immobilized on some sort
of support particles.
Before curing, these biocomposites are highly mouldable.
This permits the easy construction of amperometric sensors of
various shapes (cylindrical, planar, tubular, flow-through, etc.),
and sizes. After curing, these materials are very stable from a
mechanical point of view. The surface is stable, rigid and
polishable and can be drilled or otherwise altered mechanically.
The components of the sensing surface can be controlled by
defining their content in the bulk. The presence of enzymes,
cofactors, mediators, additives, etc., on the sensing surface can
be tailored by adjusting their content in the bulk of the
biocomposite.
Biosensors prepared with the techniques described here have
great biological stability. The biocomposite acts as an impervious reservoir for the biologically active components. The
decrease in sensitivity on the surface is recovered by a simple
polishing procedure. Each new surface yields reproducible
results if all the individual components of the biocomposite are
dispersed homogeneously in the bulk.
Epoxy resins and Teflon are employed as polymer matrices
because they are well known materials. They provide chemical
stability and the resulting biosensors can be used in partially
aqueous media (methanol-water, acetonitrile-water, etc.).
The morphology, size and distribution of the conducting
particles define the behaviour of the biosensor as a microelectrode array. These microelectrode arrays or ensembles show
efficient mass transport and a better electrochemical response
(high signal-to-noise ratio, low detection limits, fast response
times).
The resulting biosensors are suitable for flow systems
because of these electrochemical, chemical, mechanical and
biological features.
Finally, the preparation of the biocomposites and the
construction of the biosensors are inexpensive.

Final Remarks
Surface characterization is a key point in understanding the
function of modified electrodes. This knowledge is useful in the
design of surface microstructures suitable for the construction

View Article Online

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1758

of more selective sensors. Surfaces of the type reviewed here

have not been studied thoroughly. Scanning tunnelling microscopy (STM) has been useful in the study of graphite distribution
in the surface of an ADH-NAD-graphite-qoxy
biocomposite,32 but this technique cannot produce useful information
about the other non-conductive components of the material.
The biosensors reviewed here (see Tables 1-4) have been
constructed manually in cylindrical shapes. Thick-film technologys" may be the fastest, most reproducible and economical
way of mass producing biosensors. Screen printing and ink-jet
printing techniques have shown great potential in this respect.
These procedures have been used for the sequential deposition
of the layers on the device (conductor. receptor, mediator,
permselector, insulator, etc.). Using the biocomposites described here, these methods can be transformed into one-step
processes.s1-s4 In this fashion, the printing process becomes
simpler and more reproducible. Biocomposites can be rendered
more fluid and applied as inks for these methods of mass
production. The coupling of the biocomposites mentioned here
and printing processes for the production of biosensors have
great promise. This work is in progress in our laboratories.
All the biocomposites reviewed have been developed for
using in amperometric devices. However, recently a novel
potentiometric biosensor based on a peroxidase-graphiteepoxy biocomposite has been r e p ~ r t e d . ~With
'
this approach a
challenging field is envisaged.
Financial support from the Commission of the European
Communities, Environment and Climate Programme (EVSVCT94-0407) and the Interministerial Commission for Science
and Technology (CICYT), Madrid, is gratefully acknowledged.

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Paper- 6103420K
R e c e i i d M q 17, 1996
Accepted Jiirze 13, I996