Physics and Chemistry - Basis of Biotech. Vol 7

341 Pages·2002·0.467 MB·English

Amperometric enzyme-based biosensors for application in food and beverage industry
Table 3. Bioelectrochemical characteristics of peroxidase modified electrodes.
Enzyme Sensitivity Linear range Detection
(µA mM-1)a (µM) limit (nM)b
HRP 55±12 0.5-130 35±15
R = 0.9981
-
TOP 37±6.2 0.5-470 81 ±68
R = 0.9958
SPP 236 38 0.5-220 25±10
R = 0.9988
acalculated as the ratio between Imax and KM app best imated for 2S/N
The obtained results indicated the possibility of using some of the newly isolated
peroxidases (e.g. SPP) insteadofHRP inbi-enzyme sensor designs, especially in cases
when the concentration of the target analyte is low, and therefore low detection limit
and high sensitivities are crucial.
4.2. AMINE OXIDASE-BASED BIOSENSORS FOR MONITORING OF FISH
FRESHNESS
Biogenic amines can act as possible biomarkers for control of food products [35-37].
Putrescine, cadaverine, tyramine and histamine are the most known compounds in this
class, their concentration being a good indicator of fish, meat, and cheese freshness [38-
42]. Biogenic amines are generally produced by microbial decarboxylation of
corresponding amino acids and their toxicological significance in food products is still
unclear. However, they can cause severe effects, such as headache and facial flushing,
even when consuming very small amounts of infested fermented beverages and/or food
[43, 44]. Many enzymatic methods have been developed for measuring biogenic
amines in blood, biological tissues and food products [45-51], most of them being
based on amine oxidase (AO) which catalyses the following reaction (1):
R-CH2-NH2 + H2O + O2 → R-CHO + H2O2 + NH3 (1)
We recently reported on the development, characteristics, and application of
amperometric graphite electrodes based on a newly isolated and characterised AO, both
in a mono- [30] and bi-enzymatic (co-immobilised AO and HRP) design [3 1], either in
the presence or in the absence of an electrochemical mediator (Os-based redox
polymer). The grass-pea AO used during this work is a newly described copper-
containing enzyme, which besides the metal ions also contains an organic cofactor with
a quinoid structure (topa quinone) in its catalytic site [52, 53], showing the possibility
of transferring electrons directly to the graphite electrode [32], The working principle
of the mediated mono- and bi-enzyme biosensor architectures is presented in schemes 5
and 6, respectively.
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Scheme 5. Electron-transfer pathway for a mono-enzyme histamine electrode.
Scheme 6. Electron-transfer pathway for a bi-enzyme histamine electrode.
Mono- and bi-enzyme electrodes were prepared following previously published
protocols [30, 3 I]. Briefly, unmediated electrodes were prepared by placing a defined
amount of A0 (6 µ1 of 5 mg/ml in phosphate buffer at pH 7.2) or 6 µl of a premixed
solution of AO and HRP (80 % AO and 20 % HRP; AO, as above, HRP 1.25 mg/ml in
phosphate buffer pH 7.2). When integrating these enzymes in redox polymers, a
premixed solution of AO, PVI dmeOs and PEGDGE (66.3 % AO, 27 % PVI dmeOs
13 13
and 6.7 %PEGDGE, w/w %) was added on the top of the electrodes. Electrodes were
cured overnight at 4 °C. The working potential ofthe mono-enzyme electrodes was
+200 mV vs. Ag/AgCl while bi-enzyme electrodes were operated at -50 mV vs.
Ag/AgCl, regardlessusing adirectoramediatedelectron-transfermechanism.
Generally, the bi-enzymatic electrodes showed considerably improved sensitivity
(e.g. 0.073 A/Mcm2 for histamine) and lower detection limits (e.g. 0.33 µM for
histamine, 2S/N) as compared with the mono-enzymatic ones (0.007 A/Mcm2 and 2.2
µM, respectively), especially in the presence of the electrochemical mediator (optimum
composition of the sensing film; 49 %AO, 12 % HRP, 19.5 Y PVI dmeOs and 19.5 %
O 13
PEGDGE,W/W%).
The optimisedredox hydrogel integratedbi-enzyme biosensorcouldbe also applied
for the measurement of biogenic amines in extracts of fish samples stored in different
conditions (at 4 and 25 °C, respectively).
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Amperometric enzyme-based biosensors for application in food and beverage industry
Figure 7. Monitoring of biogenic amines in fish sample extracts stored in different
conditions. The total amine concentration is expressed in histamine equivalents.
Internationally established regulation accept a maximum level of histamine in fish of
200 - 400 mg/kg [36]; as seen, the sample stored at room temperature already attained
this value after 4 days of storage, showing thus, that the developed electrode is effective
to screen food samples (detection limit: 0.33 µM for histamine, 0.17 µM putrescine and
cadaverine, respectively at a sample throughput of 30 samples/hour). Interestingly, the
different sensor designs display different selectivity patterns [30, 3 13. The selectivity of
the mono-enzymatic electrode for the biogenic amines decreased according to the row:
histamine>>agmatine> spermidine > ethylenediamine >putrescine > cadaverine > Z,
E-2-butene-1,4-diamino dihydrochloride. While the selectivity of the bi-enzymatic
electrodes was following the order: putrescine > spermidine > cadaverine > histamine >
agmatine > Z, E-2-butene-1,4-diamino dihydrochloride > ethylenediamine. By
comparing these selectivity patterns it is obvious that using both types of electrodes it is
not only possible to determine the sum of the biogenic amines, but also histamine alone
with high specificity.
4.3. ALCOHOL BIOSENSORS BASED ON ALCOHOL DEHYDROGENASE
The importance of aliphatic alcohols and especially ethanol determination in clinical,
industrial, food and environmental analysis, coupled with the generally high selectivity
displayed by biosensors, resulted in the development of redox enzyme-based
atnperometric alcohol electrodes [54-56]. Alcohol oxidase [56-59], NAD+ -dependent
alcoholdehydrogenase [60-62], PQQ-dependentalcohol dehydrogenase [33, 63] arethe
most studied enzymes in this context, all displaying inherent advantages and
disadvantages. Alcohol oxidase is catalytically active for a range of short chain
aliphatic alcohols including methanol, whereas NAD+-dependent alcohol
dehydrogenase is more specific for primary aliphatic and aromatic alcohols other than
methanol [64] but requires the addition of its soluble cofactor that complicates the
analysis system.
The present examples describes the development of biosensors based on a newly
isolated quinohaemoprotein alcohol dehydrogenase from Gluconobacter sp. 33which
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contains several cofactors (haem and pyrroloquinolin quinone /PQQ, denoted QH-
ADH). Three different biosensor designs have been tested and optimised: (i) Type I
electrodes were based on the enzyme simply adsorbed on the electrode surface (direct
electron transfer) (see scheme 7a), (ii) Type II electrodes integrated the enzyme into an
Os-modified redox polymer (mediated electron transfer) (see scheme 7b), and (iii) Type
III electrodes entrapped the same enzyme into a conducting polymer network
(polypyrrole) (see scheme 8).
Scheme 7a Electron-transfer pathway for Type I QH-ADH electrodes.
Scheme 7b. Electron-transfer pathway for Type II QH-ADH electrodes.
Scheme 8. Electron-transfer pathway for Type Ill QH-ADH electrodes.
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Amperometric enzyme-based biosensors for application in food and beverage industry
The ethanol sensors were prepared as follows: Type I, screen printed graphite
electrodes were coated with 1 µL ofQH-ADH (2.5 mg/ml in 50 mM sodium acetate
buffer, at pH 6.0 and 100 mM KC1) and dried overnight at 4 °C in refrigerator. Type II,
screen printed graphite electrodes were prepared by placing the same amount of a
premixed solution of QH-ADH, redox polymer and crosslinker/PEGDGE (36 % QH-
ADH, 57 % PVI dmeOs and 7 % PEGDGE, w/w % When preparing type III
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electrodes, QH-ADH has been entrapped into a polypyrrole film during its
electrochemical-induced formation following a potential-pulse profile as previously
described [33]. A solution containing 2.5 mg/ ml QH-ADH, 100 mM pyrrole and 100
mM KCl was used for the electrochemical formation of the conducting film by
applying 30 potential pulses from 950 mV (Is) to 350 mV (10s).
The calibration plots for ethanol obtained for the Type I and Type II electrodes
operated at +300 mV vs. Ag/AgCl showed typical Michaelis Menten profiles (see
figure 8). However, Type II electrodes were characterisedby increased sensitivity due
to a more efficient electron-transfer pathway in the presence of the polymer-bound Os-
mediator. The response ofthe optimised redox hydrogel-based biosensor was linear in
the range 5 - 100 µM and displayed a detection limit of0.97 µM ethanol (defined as
2S/N ratio).
Figure 8. Calibration curvesfor ethanol obtainedfor Type I (QH-ADH, o) andII (QH-
ADH-PVI13dmeOs-PEGDGE, ) electrodes. Experimental conditions: batch system,
applied potential+300 mV vs. Ag/AgCl.
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Type III electrodes were based on a direct electron-transfer between the polypyrrole
(PPy) entrapped QH-ADH and a platinised Pt-electrode or glassy carbon via the
conducting-polymer network [33]. These electrodes displayed very different
bioelectrochemical characteristics as compared with Type I and II ones, as discussed
below more in details.
Figure 9. Calibration curves for ethanol obtained for Type III electrodes in the presence
(o)orabsence( )ofQH-ADH. In the absence ofQH-ADH, bovine serum albumin was
used. Experimental conditions: batch system, applied potential +300 m V vs. Ag/AgCl.
The multi-subunit enzyme has been integrated into the polymer film in an active
conformation, demonstrated by the current generated in the presence of ethanol and
phenazine methosulphate (PMS) as a free-diffusing redox mediator (constant-potential
amperometry). Although the diffusion of the mediator into the QH-ADH/PPy film may
be slow due to the properties and morphology of the conducting-polymer network, a
current-concentration curve could be obtained saturating at about 5-6 mM of ethanol.
Addition of ethanol - in the absence of any free-diffusing redox mediator - up to
concentrations of 100 mM, gave rise to an unexpected increase of the steady-state
current. This observed current was attributed to a possible internal electron-transfer
pathway involving the different enzyme-integrated redox sites (PQQ and haem),
located in the different subunits of the enzyme. It was therefore anticipated that the
alcohol is primarily oxidised via the PQQ-site, which might be regenerated in a
subsequent step by heam reduction of the haem units located in subunits I and II.
Control experiments using the enzyme adsorbed at a platinised Pt-surface did not show
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Amperometric enzyme-based biosensors for application in food and beverage industry
a significant catalytic current and hence clearly demonstrated the efficient electron-
transfer pathway via the conducting polymer chains. Since a direct electron transfer
implies by definition, that the redox reaction should occur close to the formal potential
ofthe involved active site, cyclic voltammograms have been recorded in oxygen-free
50 mM acetate buffer containing different ethanol concentrations. The increase of a
redox wave at potentials of +190 mV (haem oxidation) supported this hypothesis. The
co-operative action of the enzyme-integrated prosthetic groups - PQQ and haem- is
assumed to allow this electron-transfer pathway from the enzyme’s active site to the
conducting polymer backbone. This unusual electron-transfer pathway leads to an
accentuated increase of the Kapp-value (up to about 100 mM in dependence from the
polymer-film thickness and the electrode material [33]) and hence to a significantly
increased linear detection range of an ethanol sensor based on this enzyme.
By changing the electrode material from Pt to glassy carbon a similar electron-
transfer pathway via the conducting polymer chains could be obtained. In figure 9, the
related calibration plot recorded on glassy carbon type III electrodes for increasing
ethanol concentrations is shown, characterising the described ethanol sensor by a linear
range of up to about 25 mM, a sensitivity of 4.54 µA/M, and an apparent K aPPof 80.86
M
mM.
Comparing the calibration curves obtained with the studied electrode types one can
see the great flexibility regarding the linear range offered by the different
immobilisation methods. The presented alcohol biosensors fulfil requirements for very
different K values, from 62 µM (electrodes type I) and 170 µM (electrodes type 11) up
m
to 80mM(electrodes type IIIbasedonglassycarbon)and 100mM(electrodestype III
based on platinum). The three different ethanol sensors clearly demonstrated the
different electrochemical characteristics one can obtain using various sensor
architectures, and thus the possibility of their tailoring for a particular application.
5. Enzyme-based amperometric biosensors for monitoring in different
biotechnological processes
Although, as mentioned, biosensors are not yet widely spread in food and beverage
industry, a number of enzyme-based electrodes have already been successfully applied
to monitor fermentation processes, the biosensors being an essential part of a process
control system. It has to be accentuated that the biosensors themselves, are only parts of
the entire analysis system, where other components for sampling, for elimination of
contamination, for sample transport, etc. are of equal importance. A detailed discussion
of this subject is, however, beyond the scope of this chapter.
Biosensors have been considered for this application mainly because of their
versatility offering the possibility of easy and automated on-line monitoring, replacing
thus, off-line analysis which involved manual sampling and sample handling steps.
Biosensors proved to be the right tools and therefore have been also used to develop
feedback control strategies [84-90]. The implementation of the biosensor in the
technological line is made usually in one of the following ways:
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(cid:127) the target substrate is directly-detected (in-situ) in the fermenter. The practical
requirements for an in-situ biosensor, such as: sterilisation possibility,
adequate measuring range, resistance to membrane fouling, have so far not
been entirely met, which has precluded the widespread application of this
approach [84]. Due to these requirements, measurements in an external flow
stream or in on-line systems are more often applied. However, a mediated
amperometric glucose biosensor for the in-situ monitoring of a pulse-fed
baker’s yeast cultivation on defined medium was already reported [85]. The
biosensor displayed an improved stability (4 days of continuous use) and
extended working range (up to 20 g/I). Also, an autoclavable glucose
biosensor was used to monitor in-situ the fed-batch fermentation of
Escherichia coli [ 84].
(cid:127)
the target substrate is sampled via a flow-injection system and is detected
using an amperometric biosensor. A split-stream flow-injection analysis
system was described for the simultaneous determination of glucose and L-
glutamine in serum-free hybridoma bioprocess media. In this approach the
system assayed 12 samples/h with a linear response to glucose in the range of
0.03 to 30 mM [86].
(cid:127)
the target substrate is sampled from the fermenter using a microdialysis system
and is detected subsequently with a biosensor housed in a flow-injection
system, The microdialysis system provides a cell-free dialysate while the flow-
injection system permits a high sampling rate. Such a system was used to
monitor glucose and lactate (up to 70 mM) in lactic acid fermentation of
Lactobacillus delbrueckii. The sensor system monitored glucose and lactate
concentrations during a 24 h long fermentation process, without any
interfering signals, as confirmed with a conventional (colorimetric) method
[87]. The same fermentation process was also monitored by coupling the
microdialysis sampling with a flow-through electrochemical cell housing both
a glucose and a lactate biosensor. The system was characterised by a sampling
frequency of 15 h-1 and a delay between sampling and detection of less than 3
minutes. Obtained results were confirmed with a standard off-line analysis
using HPLC [88]. An interesting study compared the characteristics of such
analytical systems with those obtained for an off-line system, based on manual
sampling and clean-up, and column liquid chromatography in combination
with refractive index detection [89].
(cid:127)
the target substrate is sampled via an automated analyser, passed through an
oxidase-immobilised mini-reactor, monitoring the produced hydrogen
peroxide by amperometry. Such a system was combined with a column
switching valve downstream from the injector for monitoring of glucose,
ethanol and glutamate during the fermentation of aged fish sauce in a
fermenter loaded with Torulopis versatiles-immobilisedbeads [90].
An example from our laboratory illustrates the monitoring of glucose and ethanol
during the fermentation process of Tokay wine (see Fig. 10). Commercially available
glucose and ethanol biosensors were purchased from SensLab (Leipzig, Germany),
integrated into an on-line sampling and detection system (OLGA, Institut Air
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Bioanalytik, Göttingen, Germany [9 1]) and their characteristics were evaluated and
compared with those obtained using reagentless biosensors developed in our
laboratories (see section 4.3).
As seen from the figure 10 the used sequential-injection analyser with integrated
biosensors was able to follow the decrease of glucose and simultaneous increase of the
ethanol concentration in the expected way.
Figure 10. Monitoring of glucose and ethanol during the fermentation process of Tokay
wine (see text for more details)
6. Conclusions
Many integrated biosensors fulfil the requirements for their analytical applications in
food and beverage industry. Their bioelectrochemical characteristics (sensitivity,
selectivity, and stability) combined with their simplicity in use and relative cheapness
forecast a wide spread of these analytical tools in the field of production and control of
various foodstuff and beverages.
Acknowledgements
The authors thank the following organisations for financial support: the European
Commission (Contract No. IC 15CT96-1008 and IC 15-CT98-0907), the Swedish
Council for Forestry and Agricultural Research (SJSF), and Swedish National Board for
Industrial and Technical Development (NUTEK).
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