Advance in Glucose Biosensors
Fall 1999
Clark and Lyons designed the first enzyme electrode to determine glucose based
on the strategy of combining the specificity of a biological system with the
simplicity and sensitivity of electrochemical transducer. Since three decades,
the search for an ideal glucose biosensor continues to be one of the main
motivation in this research field. The refinement of electrochemical approaches
for glucose sensitivity has occupied many research groups. Every year there
are lots of papers in glucose biosensor published. In this review article,
I am trying to cover the advance in glucose biosensor in 1999.
The most common strategies for glucose detection can be partitioned into the
following groups: those employing glucose oxidase; those using a dehydrogenase
enzyme or those relying on an inorganic catalyst for oxidation of glucose
or fluorescence due to the combination of fluorescein and glucose. Most papers
found by the literature search appear to use glucose oxidase(GOx) to oxidize
one of the anomers of glucose. Examples of papers falling into this category
discuss topics such as optimization of immobilization chemistry; new mediators
for transferring charge from the flavin redox center in GOx to an electrode
surface; interactions between GOx and conducting polymers; new strategies
for "wiring" the active site of GOx to the electrode surface and
techniques for eliminating or correcting for interference, etc.
Amperometric enzymatic electrodes based on GOx, which generates hydrogen peroxide
in the presence of oxygen and glucose, are most widely used (1):
Glucose + O2 +H2O======Gluconic acid + H2O2
Then H2O2 is reduced at -600mv vs Ag/AgCL.
These devices are designed either for monitoring hydrogen peroxide formation
or oxygen consumption. However, hydrogen peroxide transducers often suffer
from electrochemical interference by oxidable species in a complex matrix
such as serum. The electrode oxidizes these interferents as well as hydrogen
peroxide, which results in a current response with a positive error. Moreover,
biosensors based on oxygen consumption are affacted by the variation of oxygen
concentration in ambient air. In order to overcome these drawbacks, different
strategies have been developed.
Extensive efforts have been devoted for minimizing the error of electroactive
interference. One useful strategy is to design electrocatalytic transducers
at which the overvoltage for hydrogen peroxide redox reaction is greatly lowered.
In particular, metallized carbon biosurfaces and Prussian-Blue coated carbon
electrodes have been showed to be very useful for lowering the operational
potential and minimizing contributions from oxidizable constituents. Wang
et al developed an enzyme nanosensor, based on a carbon fiber cone nanoelectrode
modified by codeposition of Prussian blue and glucose oxidase (2). The new
sensor displayed a low-potential electrocatalytic detection of the enzymaticlly
liberated hydrogen peroxide, along with good reproducibility and high selectivity.
An operating potential of -0.1v(vs Ag/AgCL) yielded the highest selectivity
towards glucose, with no interference from ascorbic acid. Joseph Wang et al
(3) also reported a glucose biosensor which coimmobilize cupric hexacyanoferrate
and glucose oxidase with the interior of carbon paste electrode. The cupric
hexacyanoferrate catalyst has been showed to be advantageous over Prussian-Blue,
particularly due to its stable operation at PH 7.4 and this biosensor is highly
selective and fast responding. Wang et al(4) also developed a highly selective
disposable glucose biosensor based on the dispersion of cupric-hexacyanoferrrate
and glucose oxidase within a screen-printable carbon ink. Their operation
conditions eliminate the need for an anti-interference membrane and greatly
simplify the sensor fabrication with the one-step dispersion of the enzyme
and electrocatalyst. Lin and coworkers prepared a cobalt(II) hexacyanoferrate
base biosensor by codeposition of an enzyme, together with electrochemical
formation of a cobalt(II)hexacyanoferrate compound electrochemically(5). This
compound possesses the catalytic property of reducing hydrogen peroxide to
water at the operating potential of 0.0v vs. Ag/AgCL. No response was observed
from the addition of either 2*10(-4)M galactose, acetaminophen, ascorbic acid,
uric acid, cysteine, tyrosine, dopamine, or 1,4-dihydroxyquinone in the absence
and/or in the presence of 5*10(-4)M glucose. This unique interference-independent
feature is attributed to the low overvoltage characteristic of cobalt(II)hexacyanoferrate.
Lin et al (6) also used Chromium hexacyanofferate to develop a catalytic electrochemical
biosensor. There are two interferences observed at the detection of glucose
such as ascorbic acid(1.48%) and cysteine(2.78%) in the presence of 5.5mM
glucose. However, no other interference was observed from the addition of
other interference such as uric acid, acetaminophen, tyrosine and galactose.
They believe that the low overvoltage of the cluster resulted from the proper
selection of the two metal centers as well as the bridging ligands in the
mixed-valence cluster. Thus, the properly designed molecular clusters possess
limited interference from the easy oxidable compounds such as ascorbic acid,
catecholamines and uric acid. They also proposed the sensing mechanism as
following:
H2O Catalyst(Ox)
Glucose Glucose Oxidase(Ox) H2O2 Catalyst(Red)
Glucose acid Glucose Oxidase (Re) O2
Another glucose biosensor prepared by the deposition of Iridium and glucose oxidase on glassy carbon transducer was reported by Rodriguez et al (7). The strong electrocatalytic action of iridium towards hydrogen peroxide allows fast glucose quantification at very low potentials where the interference of easily oxidizable compounds such as ascorbic and uric acids is minima.
Another avenue to minimize interference is to cover the surface with permselective polymer films, which are being increasingly frequently electrosynthesized over enzyme electrodes in order to suppress or minimize interference from endogenous electroactive species in biological samples. The ability to control the enzyme's immobilization conditions, the electropolymerization site(the electrode surface) and variables such as the films permeability and thickness have fostered the construction of polymer coated biosensors. Specifically, polypyrrole(PPy, a conducting polymer) and poly(o-phenylenediamine)(oPPD, a non-conducting polymer)are currently widely used as supported for immobilizing enzymes and/or charge transfer mediators, which are efficiently protected from otherwise potentially severe interferences.
A variety of polymeric materials have been employed for discriminating
between such electrochemical interferents and hydrogen peroxide. Although
anionic polymers like Nafion have been reported to be effective for eliminating
anionic interferents, they are rather ineffective for restricting the transport
of uncharged molecules. Membranes that show permselectivity based on the solute
size would be more useful; thee molecular weight of hydrogen peroxide(=34)
is much smaller that that of each interferent(>120). However, the use of
these membranes often increases the response time of enzyme electrode. The
enzyme/polyion complex membrane was effective in restricting the transport
of the electrochemical interferents, whereas the analyte permeated easily
to undergo the enzymatic reaction. But an electrode based on the GOx-containing
polyion complex matrix didn't show a high ratio of glucose response to interferential
response, owing to the suppression of the analytical diffusion in the membrane.
Garjonyte et al(8) prepared amperometric glucose biosensors by immobilization
of glucose oxidase into an oPPD film by simple one-step electropolymerization
procedure. Ascorbate shows diminished interference with glucose. Cosnier,
S.(9) and coworkers reported the electrochemical immobilization of glucose
oxidase within polypyrrole films electrogenerated on mesoporous TiO2 films.
The porosity of anatase TiO2 layer allows the diffusion of amphiphilic pyrrole
ammonium tetrafluoroborate and its electrochemical oxidation at the underlying
SnO2 surface. The determination of glucose is carried at -0.15v (vs.Ag/AgCL)
via the selective reduction of hydrogen peroxide at the TiO2 surface.
Vidal et al(10) constructed a thin-layer amperometric sensor with layers of
PPy and/or oPPD electropolymerized over TTF-TCNQ paste electrodes. The polymers
formed strongly adhered, reproducible membranes, which resulted in improved
enzyme stability and electrode selectivity. The selectivity against the electroactive
interferents. Ascorbic acid(AA) and uric acid(UA) is substantially improved
relative to bare electrodes as a result of the molecular exclusion properties
of PPy and oPPD in both mono-layer and bi-layer coated sensors. The proposed
enzyme biosensors are highly stable and reproducible when they were used to
determine glucose in synthetic serum samples.
The potential advantages of electrosynthesizing oPPD over carbon paste electrodes(CPEs)
are improved stability of the resulting enzyme sensor, the ability to affect
the electrocatalytic oxidation of hydrogen peroxide, the high permselectivity
and reproducibility, the ability to screen out electroactive interferents
and avoiding electrode fouling and compatibility with flow injection and column
liquid chromatography operation. Optimization of inorganic/bioorganic matrix
for the development of new glucose biosensor membranes was reported by Polard
in 1998.
In amperometric biosensors, immobilization of the enzymes is
also very important. Conventional methods of enzyme immobilization include
covalent binding, physical adsorption or cross-linking to a suitable carrier
matrix. Braun(11) first demonstrated the possibility of protein immobilization
in a sol-gel silica matrix. The low temperature sol-gel process represents
an attractive avenue for the immobilization of biological entities in connection
with the development of new biosensors, because the porous inorganic sol-gel
matrix possesses physical rigidity, chemical inertness, high photochemical,
biodegradable and thermal stability and experience negligible swelling in
both aqueous and organic solutions
Most of the sol-gel modified biosensors are based on enzymes trapped in a
silica matrix. Li et al(12) developed an amperometric mediated glucose biosensor
based on a sol-gel derived carbon composite material. Glucose oxidases and
the mediator vinylferrocene were immobilized within the porous, rigid and
organically modified silica network in the composite material. The organic
group in the silica network controls the hydrophobicity of the electrode surface
and thus limits the wettability of the electrode surface. The glucose biosensor
can be renewed easily in a reproducible manner by a simple polishing step
and it has a long operational lifetime. Li and his coworkers (13) developed
a new type of Al2O3 derived glucose biosensor. The enzyme electrode comprises
glucose oxidase immobilized in the AL2O3 sol-gel matrix on a platinized glassy
carbon electrode which possesses a number of advantages over conventional
platinum electrodes. The porous platinum particle matrix provides not only
a large surface area for higher enzyme loading but also a desirable microenvironment
for the electronic signal. The GOx entrapped in a platinized platinum particle
matrix is much more stable than that immobilized on a Pt disc electrode. Thus,
the life time of the biosensor is much longer than that of a Pt disc based
sensor. Enhancement of glucose biosensor sensitivity by addition of silver
sols was reported by Tang et al(14). For the optimum conditions, the current
response of the enzyme electrode containing hydrophobic silver gels for 10mmol/L
glucose was enhanced to 18200nA as compared with the electrode without silver
gels with 280nA current response. An overview is presented of the state-of
-the-art of electrochemical biosensors employing sol-gel materials by Wang(15).
The various advantages of biogels for amperometric biosensing are discussed,
along with common designs of sol-gel derived bioelectrodes, recent advances
and trends, and future prospects.
A novel means to establish electrical contact between the redox centers of enzymes and the electrode surfaces is based on a reconstitution approach. Reconstitution of enzyme or proteins with semiartificial cofactors was applied to generate semi-synthetic proteins of new functionalities. A recent research activity that reconstitution of apo-flavoenzymes on relay-FAD functionalitied electrodes yield electrically contacted enzyme electrode was reported by Eugenii Katz(16). The resulting electrically wired glucose axidase demonstrates very efficient electrical communication with an electrode surface. The reconstitution process on the electron-FAD-mono-layer modified electrode results in an integrated enzyme electrode capable to stimulate the bioelectrocatalysed oxidation of glucose with an extremely high efficiency.
The other common enzymatic route reported for glucose detection
involves the use of any one of several dehydrogenase enzymes. Laurinavicius
et al(17) designed an oxygen insensitive glucose biosensor based on PQQ-dependent
glucose dehydrogenase without special precautions the sensitivity of the biosensor
with the immobilized enzyme was slow. Sensitivity can be increased more than
100 times in the presence of soluble mediators such as phenazine methosulphate.
An advantage of the biosensor based on PPQ-glucose dehydrogenase is good linearity
at low glucose concentrations due to the elimination of oxygen influence.
A few research groups have undertaken studies of non-enzymatic methods for
detection of glucose. For example, Russell et al(18) prepared a fluorescence-based
glucose biosensor using concanavalin A and Dextran encapsulated in a poly(ethylene
glycol) hydrogel. In the absence of glucose, tetramethylrhodamine isothiocyanate
concanavalin A (TRITC-Con A) binds with FITC-dextran, and the FITC fluorescence
is quenched through fluoresence resonance energy transfer. Competitive glucose
binding to TRITC-Con A liberates FITC-dextran, resulting in increased FITC
fluorescence proportional to the glucose concentration.
However, compared to amperometric glucose biosensors, little work has been
done on potentiometric glucose biosensors and the microfabricated conductometric
glucose biosensors based on thin films interdigitated electrodes. The conductometric
biosensors are constructed in a simple way, rugged and relatively, cheap,
there is no need of any reference electrode and the voltage can be rather
small to substantially decrease the power consumption and to reduce safety
risks when used in living organisms. However, apart from a number of essential
advantages, the conductometric glucose biosensors have also disadvantages
such as a dramatic decrease of the biosensor response with increasing buffer
capacity of a sample solution, and the oxygen limitation.
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