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Application of Enzyme Biosensors in Analysis of Food and Beverages

Article in Food Analytical Methods · December 2012

DOI: 10.1007/s12161-011-9222-4

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Food Anal. Methods (2012) 5:40–53
DOI 10.1007/s12161-011-9222-4

Application of Enzyme Biosensors in Analysis of Food

and Beverages
Rastislav Monosik & Miroslav Stredansky & Jan Tkac &
Ernest Sturdik

Received: 28 November 2010 / Accepted: 22 February 2011 / Published online: 5 March 2011
# Springer Science+Business Media, LLC 2011

Abstract The importance of analyses of different parame- Introduction

ters in food products and monitoring of a production
process requires quick and reliable analytical methods and The quality control during manufacturing process and
devices. For this purpose, biosensors can be a suitable testing of qualitative parameters of food materials and final
option, whereas most of the current quality control food products is a very important task for manufacturers
techniques are time consuming, expensive, and unpractical. and hygiene inspections. Duration and accuracy of an
In this paper, we describe biosensors developed for analysis analysis plays a key role in this case. One option how to
of different components present in food samples, namely, perform an analysis of microbial contamination or impor-
glucose, fructose, sucrose, lactose, lactic, malic, acetic, tant production parameters is to use biosensor devices. The
ascorbic, citric and amino acids, ethanol, glycerol, and use of biosensors in foods can be broadly divided into two
triglyceride. Biosensors showed desirable sensitivity, selec- groups: enzyme sensors for food components, and immu-
tivity, and response time required for various applications. nosensors for pathogenic microbes and pesticides. They are
They are often designed to avoid interference from used in the food industry to obtain proximate analysis,
components present in a complex sample to be analyzed. nutritional labeling, determination of pesticide residues,
naturally occurring toxins and anti-nutrients, processing
Keywords Biosensors . Food analysis . Enzymes . Analysis changes, microbial contamination, enzymatic inactivation,
and biochemical oxygen demand of wastes (Venugopal
2002). Food industry and biotechnology are the fields
where biosensor applications recently penetrated though not
as intensively as in the field of medical diagnostics
(Dzyadevych et al. 2008). One of the reasons can be that
R. Monosik (*) : E. Sturdik while in the medical area the main matrices are blood,
Institute of Biochemistry, Nutrition and Health Protection, serum, or urine, in the food industry sector, there are more
Faculty of Chemical and Food Technology, types of samples and variations in their compositions. This
Slovak University of Technology,
makes the process of biosensor design, unification, and
Radlinskeho 9,
812 37, Bratislava, Slovak Republic optimalization of measurement conditions more difficult.
e-mail: rastislav.monosik@stuba.sk A biosensor is an analytical device, which converts a
biological response into an electrical signal. It consists of
M. Stredansky
two main components: a bioreceptor or a biorecognition
Biorealis Ltd,
Dubravska cesta 9, element, which recognizes the target analyte and a
841 04, Bratislava, Slovak Republic transducer, which converts the recognition event into a
measurable electrical signal (Velusamy et al. 2010). As a
J. Tkac
biorecognition system, enzymes, antibodies, DNA, micro-
Institute of Chemistry, Slovak Academy of Sciences,
Dubravska cesta 9, organism, etc. can be used. These elements are capable of
845 38, Bratislava, Slovak Republic recognizing their specific analytes and also to regulate the
Food Anal. Methods (2012) 5:40–53 41

specificity and sensitivity of the device. A majority of electrode was used to determine the glucose content in real
biosensors existing today use three types of transducers for samples. Results were in a good agreement with the
converting the action of the bioreceptor molecule into a conventional measurement method (Alp et al. 2000). For
measurable signal. These are mainly amperometry based on the determination of glucose in soluble coffee, Mattos and
H2O2 or O2 measurement, potentiometry based on pH or Areias developed a biosensor electrode consisted of a thin
pIon measurement, photometry utilizing optical fibers, and film of ferric hexacyanoferrate (Prussian Blue) electro-
calorimetric biosensors measuring the change in tempera- deposited on the glassy carbon electrode with immobilized
ture (Cock et al. 2009). The most commonly used class of glucose oxidase on a Nafion® polymer layer. Linear
biosensors are electrochemical-based ones (Chaubey and calibration in the range from 0.15 to 2.50 mM with a
Malhotra 2002). detection limits of approximately 0.03 mM has been
The purpose of this review was to describe biosensors obtained. The system is able to handle about 60 samples
for analyses of the most important food components with per hour and is very stable and suitable for industrial
potential applicability in real sample analysis for a routine measurements of glucose present in instant coffee
commercial use in the food industry. (Mattos and Areias 2005).
Svorc et al. prepared three types of glucose biosensors
using different transducers based on five various solid
Biosensors for Analysis of Important Food Components binding matrices (SBMs). The highest sensitivity showed a
biosensor using tetrathiafulvalene as a mediator reaching
Glucose value of 17.1 μA mM−1 cm−2. The glucose biosensors
based on the transducer with cholesteryl myristate SBM
The concentration of glucose is an important indicator in and ferrocene mediator were used for determination of
the food industry for quality and process control. Thus, glucose in wine samples. The results were in a good
glucose biosensors are widely applied in the monitoring of agreement with those obtained by an enzymatic kit (Svorc
fermentation products and in dairy, wine, beer, and sugar et al. 1997). System that uses screen-printed electrodes to
industry (Mao 2008). The majority of glucose amperomet- simultaneously detect D-glucose and L-lactate has been
ric biosensors are based on enzymes that consume oxygen developed by Sato and Okuma. They immobilized glucose
and produce hydrogen peroxide (oxidase enzymes). Mea- and lactate oxidase on a carbon working electrode. Ferricy-
suring of O2 consumption or H2O2 production is performed anide ions, which are electrochemically oxidized at a low
during the catalytic reaction using the substrate of interest, voltage, were chosen as a mediator. A linear range was found
e.g., analyte. The general equations of the mentioned over a range of 1–100 mM (D-glucose) and 1–50 mM (L-
principles are: lactate). Biosensor was applied in the analysis of beverages
prepared after fermentation with lactic acid bacteria and a
D
Glucose þ O2










→ D
gluconicacid þ H2 O2
Glucose oxidase
good agreement with high-performance liquid chromatogra-
ð1Þ phy (HPLC) results was obtained. Using the proposed
method, assays were completed within 5 min (Sato and
H2 O2 þ ½Medred

Okuma 2006). Blanes et al. developed a packed immobilized






→½Medox þ H2 O
Peroxidase
ð2Þ
enzyme reactor (IMER) and integrated it to a capillary
A flow injection optical fiber biosensor for glucose electrophoresis (CE) microchip. Glucose was detected above
based on luminol electrochemiluminescence was described. 100 μM range using particles modified with glucose oxidase
The sol–gel method is introduced to immobilize glucose packed at the end of the separation channel. The present
oxidase (GOx) on the surface of a glassy carbon electrode. microchip design can be used with or without the particles,
Glucose could be quantified in the concentration range and just by changing the material used to pack the IMER,
between 50 μM and 10 mM with a detection limit around different analytes can be detected. The applied procedure
26 μM. The proposed method can be applied for the involved the separation of the target analyte by a CE, which
determination of glucose in soft drink samples (Zhu et al. is then coupled to a post-column IMER that produced H2O2,
2002). Alp et al. used an amperometric probe-type glucose which was finally detected at the surface of a working
sensor with Pt working electrode and an Ag/AgCl reference electrode. Additions of fructose showed no effect on either
one polarized at +650 mV. The results showed that the peak position or the peak magnitude of glucose. The
cellulose acetate membranes treated with amylamine were microchip-CE-IMER was used to quantify glucose in
the most convenient structures to establish a single- carbonated beverages with a good agreement to other reports
membrane recognition layers. The linearity and response (Blanes et al. 2007). An extensive summary of glucose
time of this electrode were found to be up to 320 mM of biosensor constructions and their applications can be found
glucose and 500 s, respectively at pH 4. Finally, the in a review written by Wang (Wang 2008).
42 Food Anal. Methods (2012) 5:40–53

Fructose good stability characteristics, retained almost 40% of the

initial response after 8 h of continuous use with an initial
D-Fructose is a widely distributed monosaccharide found in sensitivity of 226 nA mM−1 and a response time of 75 s
three main forms in the food: as a free fructose (present in (Tkac et al. 2001, 2002). A fructose biosensor with a
fruits and honey), as a constituent of the disaccharide possible use for the determination of fructose in the real
sucrose, or as fructans – polymers of fructose usually in an samples of fruit juice, soft drinks, and honey based upon
oligosaccharide form such as inulin (present in some the enzyme FDH was developed by Trivedi et al. A
vegetables and wheat) (Rumessen 1992). Because of its polymer matrix of polyethylenemine and poly(carbamoyl-
higher sweetening ability compared to sucrose and glucose, sulphonate) hydrogel has been used for the immobilization
fructose is used as a diet sweetener in diabetic foods. of FDH on a platinum tip of a screen printed graphite
Enzyme electrodes for fructose determination are often electrode with a ferricyanide mediator as the electron
based on D-fructose-5-dehydrogenase (FDH) using an acceptor. The biosensor showed a good linearity over the
electron acceptor serving as an electrochemical mediator: range 3–13 μM with the detection limit of 0.65 μM
(Trivedi et al. 2009a). Another fructose biosensor working
D
Fructose þ ½Medox



















→
D
Fructose dehydrogenase
ð3Þ at a low potential (150 mV vs. Ag/AgCl) with very short
5
keto
D
fructose þ ½Medred response time <20 s was constructed by Montanez-Soto et
al. The device was designed by the incorporation of a
An improved amperometric biosensor based on a SBM tetrathiofulvalene-tetracyanoquinodimethane organic con-
composite transducer has been used for the determination ducting salt and an FDH enzyme, included in a polymeric
of D-fructose in some food samples. The enzyme, D- matrix of epoxy resin and a graphite powder. The linear
fructose dehydrogenase, was incorporated directly into a range achieved was from 0.01 to 0.3 mM with a detection
solid composite transducer containing both 2-hexadecanone limit of 5 μM. The biosensor was used to determine
as a SBM and chemically modified graphite. Ferricyanide fructose in high fructose syrups, and there were not
was used as a redox mediator, and the amperometric signals significant differences between these results and those
were linearly proportional to D-fructose concentrations in obtained by HPLC (Montanez-Soto et al. 2006). Tsujimura
the range 5×10−5–1×10−2 mol l−1. The use of chemically et al. developed a batch-type coulometric D-fructose
modified graphite by a mild oxidation step was shown to biosensor based on a direct electron transfer reaction of
improve the biosensor selectivity against anionic interfer- FDH adsorbed on a porous carbon electrode surface.
ents such as L-ascorbate. The assay of D-fructose by this Nanostructured carbon particle-modified electrodes were
electrode was not influenced by the presence of sugars or used to enhance the catalytic current density. This method
other interferents. The results agreed well with those is also applicable for the determination of several oligo/
obtained with the commercial kit (Stredansky et al. 1999). polysaccharides containing the D-fructose unit, in combina-
Bassi et al. immobilized FDH behind a thin non-conducting tion with specific hydrolases to yield D-fructose. An
electropolymerized film of 1,3-phenylenediamine-resorcinol. example was demonstrated by sucrose determination in
Two different types of electrochemical mediators, soluble which the electrode modified with FDH and invertase was
ferricyanide and the water insoluble tetracyanoquinodi- used as a working electrode. To address the problem of
methane, were applied as redox mediators for the electroactive interferences such as ascorbate, the electric
amperometric measurement of fructose. The minimum charge at the FDH-free electrode was subtracted from the
detection limit was 10 μM with a linear range up to total charge obtained at the FDH-adsorbed electrode. The
1.0 mM. The performance of the two mediators was also D-fructose concentrations in several beverages were suc-
compared in respect to the stability of the biosensor. Using cessfully determined with this method (Tsujimura et al.
the latter the proposed biosensor was applied for the 2009). Bhand et al. developed a highly selective,
determination of fructose in diluted honey samples. The interference-free biosensor for routine fructose monitoring
method correlated well with a chemical assay (Bassi et al. in food samples. The assay was based on the phosphory-
1998). lation D(−)fructose to fructose-6-phosphate by hexokinase
Tkac et al. applied ferrocene embedded membranes for and a subsequent conversion of fructose-6-phosphate to
the construction of a fructose biosensor by immobilization fructose-1,6-biphosphate by fructose-6-phosphate-kinase.
of FDH. The prevention of ferrocene leakage from an The heat liberated in the second reaction was monitored
electrode by a physical retention of the mediator in a matrix using an enzyme thermistor. The advantage of this
of cellulose acetate membrane was reported. Five types of biosensor was a selective measurement of fructose without
the cellulose acetate membranes were prepared, from which the need to eliminate glucose and use of an inexpensive
a biosensor comprising a membrane containing 1.8% of flow injection analysis (FIA)-based, mediator-free calori-
ferrocene and 0.05 % of Nafion® in the matrix showed metric measurement suitable for routine fructose analysis.
Food Anal. Methods (2012) 5:40–53 43

Linearity (0.5–6.0 mM) with a detection limit of 0.12 mM Lactose

was obtained (Bhand et al. 2010).
Lactose is the disaccharide present in milk at concen-
Sucrose trations of 2–8% (by weight), although the amount varies
among species and individuals. Its content is closely
Sucrose determination requires a multienzyme system in related to the quality of milk and dairy products. It is
which sucrose is first hydrolyzed by the enzyme invertase: hydrolyzed by the enzyme β-galactosidase to galactose
and glucose. Galactose can be further oxidized to
D
Sucrose þ H2 O





→D
fructose þ a
D
glucose
Invertase
galacto-hexodialdose:
ð4Þ b
galactosidase
D
Lactose þ H2 O










→ D
galactose þ D
glucose
Invertase together with GOx can be used for development ð5Þ
of a sucrose enzyme electrode, but there is a need to convert
α-D-glucose to its β-isomer, for which GOx is more specific.
For this purpose, the enzyme mutarotase is usually used. For D
galactose þ O2













→ D
galacto
D
galactose oxidase
ð6Þ
example, Surareungchai et al. developed a multi-enzyme
hexodialdose þ H2 O2
electrode obtained by a two-step immobilization of the
enzymes GOx, mutarotase, and invertase. GOx was entrap- For estimation of lactose in milk and its products,
ped in a poly-1,3-diaminobenzene film on a platinum Sharma et al. developed an amperometric lactose biosensor
electrode by electrochemical polymerization and a combina- by immobilizing galactosidase and galactose oxidase in
tion of mutarotase and invertase was cross-linked over the Langmuir–Blodgett films of poly(3-hexyl thiophene)/
electrode via bovine serum albumin and glutaraldehyde. The stearic acid. The enzyme electrodes showed linearity in
sucrose concentration was determined from hydrogen per- the range 1–6 g dl−1 for lactose and had a shelf life more
oxide oxidation at +0.7 V vs. Ag/AgCl. This immobilization than 120 days. The working electrode may be used for the
method minimized interference from ascorbic acid. A second estimation of lactose/galactose in food and biological fluids
electrode, for glucose determination only, was constructed (Sharma et al. 2004). There are also other papers describing
using an inactive invertase. The biosensor showed a good a construction of biosensors using these enzymatic princi-
agreement with the standard LC method for sucrose and ples (Eshkenazi et al. 2000; Sharma et al. 2007; Marrakchi
glucose analysis in soft drinks (Surareungchai et al. 1999). et al. 2008; Logoglu et al. 2006). Lukacheva et al.
The same principle is utilized in the work of Gouda et al. immobilized β-galactosidase into polyelectrolyte mem-
(Gouda et al. 2004) and Majer-Baranyi et al. (Majer-Baranyi branes with the use of organic solvents and perfluorosulfo-
et al. 2008) with similar performance of detection. nated polymer. The results of the analysis of milk whey in a
Another work described a conductometric biosensor for flow-injection system that included lactose biosensor based
sucrose determination using a complex three-enzyme (inver- on Berlin blue (as a signal transducer) and polyelectrolyte
tase, mutarotase, and GOx) containing membrane as a membranes correlated well with measurement data obtained
sensitive element immobilized on the conductometric inter- by a standard chromatographic technique (Lukacheva et al.
digitated planar electrodes. The biosensor was successfully 2007). Other paper described two types of amperometric
applied in practice (Soldatkin et al. 2008). Kennedy et al. biosensors based either on co-immobilization of two
constructed a flow injection analysis system for measuring of enzymes (galactose oxidase with peroxidase) or co-
sucrose using invertase and GOx. Enzymes were incorpo- immobilization of three enzymes (beta-galactosidase, ga-
rated into an active graphite paste mixed with tetracyano- lactose oxidase, and peroxidase). A graphite rod with
quinodimethane as a mediator. High sensitivity of 174± pre-adsorbed ferrocene was used as a working electrode.
8.7 nA mg−1 of sucrose made this sensor able to measure Glucose interference was avoided using galactose oxidase.
high concentrations of sucrose in sugarcane crops and The presence of beta-galactosidase greatly enhanced
industrial and fermentation processes, without dilution or the sensitivity of the biosensor, but its linear range
pre-treatment of the samples. Interference from organic was narrower compared to the biosensor without β-
acids, mainly ascorbic acid, which is oxidized at +350 mV galactosidase. Detection limit for lactose determination
vs. Ag/AgCl, was reduced in this system by lowering a from 44 to 339 μM were observed depending on the
working potential down to value of 100 mV vs. Ag/AgCl conditions and constructions used. Analyses of real samples
(Kennedy et al. 2007). Another biosensor for sucrose showed a good correlation with HPLC analysis (Tkac et al.
determination was constructed by Tsujimura et al. based on 2000a). Yang et al. co-immobilized β-galactosidase and
FDH and invertase and was used for analysis of sucrose in glucose oxidase in calcium alginate fiber and amine
several beverages (Tsujimura et al. 2009) (see Chapter 2.2). modified nanosized mesoporous silica (AMNMS) to
44 Food Anal. Methods (2012) 5:40–53

prepare a chemiluminescence (CL)-based flow-through determination by biosensors is typically based on the

biosensor for the determination of lactose. Enzyme activity following reactions:
and stability was increased by AMNMS with the cage
effect of the polymer. The relative CL intensity was linear L
Lactate þ O2












→ pyruvate þ H2 O2
L
Lactate oxidase
ð7Þ
with lactose concentration in the range of 0.08–4 μg ml−1
with a detection limit of 0.027 μg ml−1. It has been
L
Lactate þ NADþ ←L
Lactate
















→ pyruvate þ NADH þ Hþ
dehydrogenase
successfully applied to the determination of lactose in milk
(Yang et al. 2010). ð8Þ
Conzuelo et al. designed the bioelectrode based on the
use of a 3-mercaptopropionic acid self-assembled mono-
layer modified gold electrode on which the enzymes beta- NADH þ Hþ þ ½Medox
Diaphorase






→ ½Medred þ NADþ ð9Þ
galactosidase, glucose oxidase, peroxidase, and a mediator
tetrathiafulvalene (TTF) were coimmobilized by a dialysis A biosensor for the selective determination of L-
membrane. Beta-galactosidase catalyzed the hydrolysis of lactate in wine based on robust solid composite trans-
lactose, and the produced glucose was catalytically oxi- ducers was developed by Katrlik et al. Transducers
dized to gluconic acid and H2O2, which was reduced in the comprised of a solid binding matrix having hydrophobic
presence of peroxidase. The reduction of TTF at 0.00 V (vs. skeleton with the enzyme L-lactate dehydrogenase (LDH)
Ag/AgCl) gave rise to an amperometric signal proportional and diaphorase (DP) placed onto the transducer surface
to the lactose concentration. A linear calibration plot was covered by a dialysis membrane, which substantially
obtained for lactose from 1.5 to 1.2 mM with a limit of reduced interferences derived from easily oxidizable
detection of 0.46 μM. The biosensor showed a useful compounds of wine, e.g., polyphenols. As a mediator
lifetime of 28 days and was applied to the determination of ferricyanide was used. The results obtained by the
lactose in milk and other foodstuffs (chocolate, butter, biosensors were in a good agreement with those obtained
margarine, yogurt, cheese, and mayonnaise), and the results by a liquid chromatography (Katrlik et al. 1999). Another
obtained were validated using a commercial enzyme test kit paper described an amperometric lactate biosensor
(Conzuelo et al. 2010). based on a conducting polymer, poly-5,20-50,200-
For lactose biosensor the enzyme cellobiose dehydroge- terthiophene-30-carboxylic acid (pTTCA), and multi-
nase (CDH) was also utilized. CDH was immobilized on walled carbon nanotube (MWNT) composite present on
the solid spectrographic graphite electrode surface by a a gold electrode. LDH and the oxidized form of
simple physical adsorption, and the CDH-modified elec- nicotinamide adenine dinucleotide (NAD+) were subse-
trode was subsequently inserted into a wall-jet amperomet- quently immobilized onto the pTTCA/MWNT composite
ric cell integrated within a flow injection system film. The detection signal was amplified by the pTTCA/
(0.5 ml min−1). The biosensor had a detection limit for MWNT assembly with immobilized enzyme. The applica-
lactose of 1 μM, a sensitivity of 1,100 μA mM−1 cm−2, a bility of the biosensor in commercial milk and human
response time of 4 s, and a linear range from 1 to serum samples was demonstrated successfully (Rahman et
100 μM of lactose. The CDH-lactose sensor was al. 2009).
successfully used to quantify the content of lactose in Several biosensors based on immobilized lactate
pasteurized milk, buttermilk, and low-lactose milk, using oxidase (LOx) or LOx together with horseradish
the standard addition method (Stoica et al. 2006). In peroxidase were described (Gamella et al. 2010; Zaydan
another paper, two types of CDH (from Trametes villosa et al. 2004; Mazzei et al. 2007; Parra et al. 2006; Shkotova
and Phanerochaete sordida) were compared. Enzymes et al. 2008). Para et al. developed lactate biosensor for the
were immobilized on screen-printed carbon electrodes. determination of this analyte in wine and beer using two
The biosensors were able to detect lactose in a concentra- strategies of immobilization of Lox—a direct adsorption
tion range between 0.5–200 μM and 0.5–100 μM, and a covalent binding. In the presence of lactate using
respectively, with a limit of detection of 250 nM (Safina hydroxymethylferrocene as a redox mediator, biosensors
et al. 2010). obtained a clear electrocatalytic activity. The biosensor
characterization revealed a linear current response vs.
Lactic Acid lactate concentration up to 0.3 mM, with a detection limit
of 10 μM of lactate and a sensitivity of 0.77 μA mM−1.
In the food industry, the lactate level is an indicator of the Finally, biosensors were applied in the determination of
fermentative processes and is related to the freshness, lactate in wine and beer, and results were in an agreement
stability, and storage quality of several products such as with those obtained by an enzymatic-spectrophotometric
tomato sauces, fruits, juices, wine, and milk. Lactate assay kit (Parra et al. 2006). Shkotova et al. compared two
Food Anal. Methods (2012) 5:40–53 45

methods of LOx immobilization on the surface of used as an acidulant. Its determination using biosensors is
commercial SensLab platinum printed electrodes. The based on following reactions:
biosensor with LOx immobilized by a physical adsorption
L
Malate þ NADþ ←













→ oxalacetate þ NADH þ Hþ
Malate dehydrogenase
in a Resydrol polymer showed both narrower dynamic
range (0.004–0.5 mM lactate) and higher sensitivity ð10Þ
(320 nA mM−1) compared to the one based on the enzyme
immobilized in a poly(3,4-ethylenedioxythiophene) by an
Malic enzymeðpyruvic
malic carboxylaseÞ
electrochemical polymerization (0.05–1.6 mM and L
Malate þ NADPþ



























→ pyruvate
60 nA mM−1, respectively). The lactate content in wine þ CO2 þ NADPH þ Hþ
and in samples taken during wine production during ð11Þ
fermentation was analyzed, and the data obtained corre-
lated well with those provided by standard chromatogra- Malic acid determination by biosensors utilizing various
phy (Shkotova et al. 2008). Zanini et al. used a glassy enzymatic pathways involves malate dehydrogenase
carbon electrode modified with laponite/chitosan hydro- (MDH) and diaphorase (Katrlik et al. 1999; Gamella et al.
gels for LOx immobilization. Ferrocenemethanol was 2010; Prodromidis et al. 1996; Arif et al. 2002), malic
utilized as an artificial mediator. The biosensor showed a enzyme, and pyruvate oxidase (see reaction 15) (Gajovic et
very short response time lower than 5 s and a detection al. 1997) or even MDH with peroxidase (Mazzei et al.
limit of 3.8 μM (Zanini et al. 2011). 2007). Bucur et al. developed an amperometric biosensor
Other paper described a novel trienzyme sensor con- based on malate quinone oxidoreductase for monitoring of
structed by immobilization of salicylate hydroxylase (SHL), the malolactic fermentation of wines. The purpose of this
LDH, and pyruvate oxidase (PyOx) on a Clark-type oxygen study was to find a promising alternative for the develop-
electrode. The enzymes were entrapped in a poly(carba- ment of NAD-independent malic acid biosensors; however,
moyl)sulfonate hydrogel on a Teflon membrane (used to a successful analytical device required further improve-
avoid interferences). SHL catalyzes the irreversible decar- ments concerning the performance of the electrochemical
boxylation and the hydroxylation of salicylate in the mediator. Interferences due to non-specific oxidations were
presence of oxygen and NADH produced by LDH. SHL shown to be negligible when using phenazine methosulfate
and PyOx shift the equilibrium of dehydrogenation of (PMS) as a mediator (Bucur et al. 2006). Katrlik et al.
lactate by LDH to the product side by consuming NADH designed a biosensor for a selective determination of malic
and pyruvate. Dissolved oxygen acts as an essential co- acid using the same principles as was described in chapter
substrate for both PyOx and SHL during their respective 2.5, using MDH and diaphorase immobilized on SBM with
enzymatic reactions. The biosensor showed a linear range a soluble mediator ferricaynide (Katrlik et al. 1999). A
from 10 to 400 μM of lactate and a good agreement with a promising concept utilizing glassy carbon electrode modi-
commercial lactate testing kit was obtained in beverage fied by single-walled carbon nanotubes on which MDH
samples (Kwan et al. 2004). was directly adsorbed was designed by Arvinte et al.
Ballesta-Claver et al. prepared a chemiluminescence- Enzyme immobilization in Nafion membrane increased the
based one-shot biosensor tested for the analysis of lactate in biosensor stability, and a linear calibration curve was
yoghurt. The lactate recognition system was based on LOx obtained for L-malic acid concentrations between 0.2 and
and the transduction system consisted of luminol, peroxi- 1 mM at an applied working potential of +300 mV vs. Ag/
dase from Arthromyces ramosus, all immobilized in a AgCl. However, the biosensor should be tested using real
polyion complex membrane on a metallic aluminum samples to verify its applicability in the food industry
electrode. The measurement of the chemiluminescence (Arvinte et al. 2008). A comparison of two strategies for L-
was performed in a luminometer when 1 ml of the sample malic acid monitoring in wine was described by Gurban et
was injected into a conventional cell containing the al. A bi-enzymatic biosensor was based on the coupling of
disposable sensing membrane. The performance of biosen- LDH with NADH oxidase, while monoenzymatic systems
sor was validated by the results obtained using an were based either on LDH or MQQ. The bienzymatic
enzymatic reference procedure (Ballesta-Claver et al. biosensor showed sensitivity of 3.6 mA M−1 and a
2008). detection limit of 4.5 μM, while for the monoenzymatic
biosensor, the sensitivity was 1.1 mA M−1 with a detection
Malic Acid limit of 2.4 μM. The optimized biosensors were used for
determination of L-malic acid in different samples of wines.
Malic acid is present mainly in fruit and vegetables, juices, Even if MDH-based biosensors have been shown to be
and other commodities. This acid is a very important suitable tools for malic acid detection in wine, these
parameter characterizing quality of wine, and it is often systems require the addition of cofactors in the working
46 Food Anal. Methods (2012) 5:40–53

medium. To overcome this drawback, a cofactorless on the determination of consumption of dissolved oxygen. A
biosensor was designed based on the use of malate- linear response was observed from 5 μM to 1.2 mM of AA.
quinone oxidoreductase flavinic enzyme. Suitable media- Finally, the results of some plant and drug samples analyzed
tors that could be easily incorporated in this system are with the presented biosensor were compared with the
subject of further studies (Gurbanab et al. 2006). spectrophotometric method (Tillman reagent) used as a
reference (Sezginturk et al. 2010).
Ascorbic Acid
Acetic Acid
L-Ascorbic acid occurs naturally in many foods and is
frequently added to processed foods as an antioxidant. As Acetic acid is another key compound in the food industry,
the ascorbic acid content in food materials is an indicator produced during the fermentation and present in the final
of its freshness and nutritive value, rapid and accurate product (e.g., wine, soy sauce, and vinegar) (Castillo et al.
analysis of ascorbic acid is important (Rekha and 2004). The following biochemical principles used Mizutani
Narasimha 2010). Beyond its function in collagen forma- et al. for an acetic biosensor:
tion, ascorbic acid is known to increase absorption of
inorganic iron and to have essential roles in the metabo- Acetate þ ATP











→ acetyl
P þ ADP
Acetate kinase
ð13Þ
lism of folic acid, some amino acids, and hormones.
Phosphoenolpyruvate þ ADP←








→ pyruvate þ ATP
Pyruvate kinase
Consequently, the determination of ascorbic acid (AA) in
various natural and prepared foods, drugs, and physiolog- ð14Þ
ical fluids is very essential (Akyilmaz and Dinckaya
1999). L-Ascorbate is oxidized on dehydroascorbate:








Pyruvate þ phosphate þ O2 ←Pyruvate


→ acetylphosphate
oxidase

2L
ascorbate þ O2













→ 2L
dehydroascorbate

L
Ascorbate oxidase
þ CO2 þ H2 O2
þ 2H2 O ð15Þ
ð12Þ
The concentration of acetic acid was determined by a
Wang et al. bound ascorbate oxidase (ASOx) to poly- combination of a FIA with amperometric trienzyme biosensor
maleimidostyrene to form stable ASOx micelle structure in a detection. The biosensor was prepared by immobilizing acetate
polystyrene (PS) membrane. This micellar membrane was kinase (AK), pyruvate kinase (PK), and PyOx on a poly
coated on both aminated glassy carbon electrode and gold (dimethylsiloxane) (PDMS)-coated electrode. The oxygen
electrode for the amperometric detection of AA based on the consumption was monitored using the PDMS-coated electrode
consumption of oxygen. A good linear range was observed without the interference from the PyOx reaction product,
in the concentration range from 5 μM to 0.4 mM at an hydrogen peroxide. Thus, the biosensor-based system could be
applied potential of −500 mV vs. Ag/AgCl. Interferences used for the determination of acetic acid from 0.05 to 20 mM
from the reducing agents can be avoided because the with a sampling rate of 20 h−1 and was stable for a month. The
detection was conducted at a cathodic potential (Wang et FIA system could be successively applied in analysis of
al. 2008). Other papers report the development of a micro- wines (Mizutani et al. 2003). Mieliauskiene et al. in the
plate calorimetric biosensor for fast ascorbic acid quantifica- similar system used LDH instead of PyOx. Enzymes were
tion. The biosensor was based on a microplate differential immobilized into a poly(ethyleneglycole) diglycidyl ether
calorimetry (MiDiCal) technology in which the heat gener- (PEGDGE) film also containing Brilliant Cresyl Blue as an
ation, due to the exothermic reaction between AA and electrochemical mediator. The amperometric biosensor mea-
ASOx, is differentially monitored between two neighboring sured the current decrease caused by the diminution of
wells of an integrated circuit-built wafer. A severe discrep- NADH concentration at a modified graphite electrode. The
ancy was found between expected and observed biosensor optimized biosensor showed a linear response for acetate in
readings. Linear range was from 2.4 to 350 mM with a limit the range of 0.2–8 mM with a detection limit of 0.13 mM
of detection of 0.8 mM. Validation experiments on fruit juice (calculated as S/N=3). The acetate content in different
samples, food supplements, and a pain reliever supplemented samples (wine and vinegar) could be reliably quantified with
with ascorbic acid correlated well with HPLC reference the described biosensor (Mieliauskiene et al. 2006).
measurements (Vermeir et al. 2007). Sezginturk et al. used
zucchini (Cucurbita pepo) tissue homogenate, which Citric Acid
contained ASOx. The tissue was crosslinked with gelatine
using glutaraldehyde and fixed on a pretreated teflon Citric acid is present in numerous natural products.
membrane. The principle of the measurements was based Several fresh fruits such as lemons and limes owe their
Food Anal. Methods (2012) 5:40–53 47

acidic taste due to the presence of the citrate anion. Citric (Friedman 1999). D -Amino acids are also generally
acid is also an additive in the food industry, mainly as a considered to be important markers of bacterial contam-
preservative and an acidulant. Citrate lyase (CL) is an ination of the food products. D-Amino acid oxidase
unstable enzyme, but some papers describing biosensor (DAAO) is a peroxisomal enzyme containing FAD as a
utilizing this enzyme were published (Prodromidis and cofactor that is expressed in a wide range of species from
Karayannis 2002). Prodromidis et al. proposed a method yeasts to human, but not in bacteria nor in plants
based on a sequence of reactions involving thiamine (Pollegioni et al. 2007). Its function is to catalyze the
pyrophosphate (TPP) and the enzymes CL, oxaloacetate oxidative deamination of D-isomer of amino acids to the
decarboxylase (OACD), and PyOx, according to the corresponding 2-oxoacid and ammonia. During this
following scheme: step, the FAD is reduced, and the catalytic cycle is
completed through its reoxidation by O2 to yield hydrogen
Citrate







→ oxaloacetate þ aceticacid
Citrate lyase
ð16Þ peroxide.

D
aminoacid þ O2 þ H2 O














→ 2
oxoacid

D
Aminoacid oxidase
Oxaloacetate


















→pyruvate þ CO2
Oxaloacetate decarboxylase
ð17Þ
þ NH4 þ þ H2 O2
The method is based on the action of the enzymes CL,
ð18Þ
OACD, and PyOx, which convert citric acid into H2O2
with the latter being monitored amperometrically with a An amperometric and a colorimetric biosensor to detect
H2O2 probe. A multi-membrane system, consisting of a and quantify D-amino acids selectively was constructed
cellulose acetate membrane for the elimination of inter- using DAAO from Rhodotorula gracilis. The biosensor
ferants, an enzymic membrane and a protective polycar- responded linearly with D-alanine concentration within the
bonate membrane were placed on a Pt electrode and used range of 0.2–3 and 0.1–1 mM for the amperometric (at a
with a fully automated flow injection manifold. Interfer- working potential of 400 mV vs. Ag/AgCl) and colorimet-
ence from various compounds present in real samples was ric system, respectively (Sacchi et al. 1998). Rosini et al.
minimized. Calibration graphs were linear over the range developed a biosensor based on DAAO mutants, which
0.01–0.9 mM of pyruvate, 0.015–0.6 mM of oxaloacetate, responded to all (neutral, acidic, and basic) D-amino acids
and 0.015–0.5 mM of citrate. A 8–10% loss of the initial employing the flavoenzyme D-amino acid oxidase from the
activity of the biosensor was observed after 100–120 yeast R. gracilis. To produce a device in which the D-amino
injections (Prodromidis et al. 1997). Maines et al. acid composition did not alter the results, both the wild-
coimmobilized (or immobilized separately) these enzymes type and a number of mutants obtained by rational design
on different types of high protein binding membrane such and directed evolution approaches were used. The
as mixed cellulose ester. The relative optimum concen- Amberzyme-immobilized T60A/Q144R/K152E and
trations of several activators (divalent cations) and M213G mutants were identified as the best choice: Their
cofactors, such as FAD and TPP, were investigated with response showed only a limited dependence on the solution
the probe assembled as a pyruvate biosensor. An extended composition when at least 20% of the D-amino acid is made
linearity up to 100 mM of citric acid was achieved up of D-alanine. The entire D-amino acid content was
(Maines et al. 2000). Ribeiro et al. prepared several determined using an amperometric biosensor based on a
selective membranes for citrate electrodes, having distinct screen-printed electrode, with a detection limit of 0.25 mM
mediator solvents and, in some cases, p-tert-octylphenol and a mean response time of 10–15 min (Rosini et al.
as an additive. Pharmaceutical preparations, soft drinks 2008). Wcislo et al. immobilized DAAO on a graphite
and beers were analyzed under conditions that enabled working electrode of a screen-printed strip modified with
simultaneous pH and ionic strength adjustment (pH=3.2, Prussian Blue and Nafion layers. The electrode was
ionic strength=10−2 mol l−1), and the results acquired modified with carbon nanotubes to enhance the signal
agreed well with the used reference method (Ribeiro et al. magnitude. A fast linear response was observed for D-
2002). alanine in the concentration range from 5 to 200 μM, and
an excellent enantioselectivity toward D-amino acids was
Amino Acids discovered. D-Amino acids were detected in fruit juices and
some milk samples. The results were in a good agreement
The determination of D-amino acids in food is a very with those obtained by capillary electrophoresis measure-
important task because the presence of D-amino acids in ments (Wcislo et al. 2007).
food is associated with a decrease in protein digestibility, Some papers described biosensors for determination
thus affecting the bio-availability of essential amino acids of L-amino acids. An amperometric biosensor for rapid
and seriously impairing the nutritional value of the food determination of the concentration of L-amino acids was
48 Food Anal. Methods (2012) 5:40–53

developed using L-amino acid oxidase (LAAO) immobi- alcohol oxidase or dehydrogenase catalyzing the following
lized by gel entrapment with poly(carbamoyl) sulfonate reactions:
hydrogel. The broad substrate range of LAAO allowed
Ethanol þ NADþ














→ acetaldehyde þ NADH þ Hþ
Alcohol dehydrogenase
this biosensor to be flexible in applications. The artificial
sweetener, aspartame, was determined by coupling of ð19Þ
LAAO with pronase (Kwan et al. 2002). LAAO attached
to a polyion complex membrane was prepared on a Ethanol þ O2










→ acetaldehyde þ H2 O2
Alcohol oxidase
ð20Þ
glassy carbon (GC) electrode. Polystyrene sulfonate and
poly-L-lysine solutions were successively placed on the A combination of alcohol oxidase (AOx) together with
GC electrode to prepare a polyion complex membrane. horseradish peroxidase (HRP) and ferrocene as a mediator
The enzyme electrode was used for the detection of L- incorporated into reticulated vitreous carbon-epoxy resin
amino acids, while +1 V vs. Ag/AgCl was applied to the electrode matrices was described for alcohol measurement
base electrode to detect enzyme-produced hydrogen (Pena et al. 2002). Another way of immobilization this bi-
peroxide. For the L-phenylalanine, the lower detection enzymatic combination is the use of electrodeposition
limit was 5 μM, and a linear response range was up to paints (EDPs) with a first layer integrating HRP within an
1 mM with a response time of 40 s. Response to Os-complex modified EDP in order to assure fast electron
other amino acids, such as L-leucine and L-methionine transfer between the enzyme and the electrode surface. On
was almost of the same magnitude as the one for L- the top of this layer, AOx was entrapped within an EDP
phenylalanine. These results indicate that the electrode layer, thus assuring fast substrate diffusion within the
could be used for the L -amino acid detection such as L- hydrogel layer concomitantly with a stabilization of the
phenylalanine, L-leucine, and L-methionine (Yabuki et al. enzyme (Smutok et al. 2006). Shkotova et al. developed a
2001). Basu et al. developed a monosodium glutamate biosensor based on amperometric transducer and AOx
(MSG) biosensor made by co-immobilizing L-glutamate immobilized in Resydrol polymer for ethanol detection.
oxidase and L-glutamate dehydrogenase (LGLDH). Re- Electrochemical deposition of the polymer film has been
generation of MSG by a substrate recycling provided an achieved by applying the potentiostatic pulse profile
amplification of the biosensor response. Higher signal consisting of 20 consecutive pulses at +1900 mV for 0.3 s
amplification was found in the presence of ammonium and at −300 mV for 5 s. The minimal detection limit was
ion. The biosensor was used to determine MSG in the 3.5.10−2 (%, v/v) of ethanol. The biosensor developed has
range of 0.02–3.0 mg l−1. Linearity was obtained from showed its potential for ethanol detection in alcoholic
0.02 to 1.2 mg l−1 in the presence of ammonium ion beverages (Shkotova et al. 2006).
(10 mM) and NADPH (2 mM), but in the absence of Amperometric biosensors based on pyrroloquinoline
LGLDH, a detection limit of MSG was confined to quinone alcohol dehydrogenase (PQQ-ADH), have been
0.1 mg l−1. The electrode was used for over 50 measure- developed for the determination of ethanol. Amperometric
ments, and the activity of the enzyme-immobilized biosensors based on PQQ enzymes are attractive due to
membrane was tested over a period of 60 days (Basu et their oxygen independence without requirement for having
al. 2006). Tani et al. utilized carbon nanotube gel, which a soluble cofactor and because, in some cases, a direct
was composed of a mixture of single-walled carbon electron transfer between their active centre and suitable
nanotube (CNT), an ionic liquid, and a thermostable D- electrodes was achieved, thus making the construction and
proline dehydrogenase (D-ProDH) immobilized on the an application of biosensors based on such enzymes more
electrode for the determination of D-amino acids in wine simple. The enzyme has been integrated in redox hydro-
and vinegar samples. When a critical comparison with gels using an Os complex-modified non-conducting
CNT, Ketjen Black, and carbon powder was also carried polymer employed as the electrochemical mediator and
out, the CNT/D-ProDH immobilized electrode showed the PEGDGE as the cross-linking agent. The substrates were
highest sensitivity and the lowest detection limit for D- measured via oxidation of the Os (II/III) mediator at
proline (Tani et al. 2009). +200 mV vs. Ag/AgCl (Niculescu et al. 2003a). Other
biosensor assembly (conductometric) for the determina-
Ethanol tion of short-chain primary aliphatic alcohols was pre-
pared through immobilization of AOx and bovine liver
The determination and control of ethanol is important in catalase in a photoreticulated poly(vinyl alcohol) mem-
brewing, winemaking, and distilling industries. Tax regula- brane at the surface of interdigitated microelectrodes. The
tion requires also exact determination of ethanol content, sensitivity was maximal for methanol (0.394 μS μM−1)
especially in spirits (Prodromidis and Karayannis 2002). and decreased with an increased alcohol chain length. The
Ethanol biosensors are based mainly on immobilized response was linear up to 75 μM for methanol, 70 μM for
Food Anal. Methods (2012) 5:40–53 49

ethanol, and 65 μM for 1-propanol with a limit of modification of a glassy carbon electrode. The selectivity
detection down to 0.5, 1, and 3 μM, respectively. The of the whole G. oxydans cell biosensor was greatly
bi-enzymatic biosensor was successfully applied to the enhanced by the size exclusion effect of a cellulose acetate
determination of ethanol in different alcoholic beverages membrane. The biosensor was successfully used in an
with no significant interferences observed (Hnaien et al. offline monitoring of ethanol with achieved detection limit
2010). Alcohol biosensors based on conducting polypyrrole, of 0.85 μM during batch fermentation by immobilized
poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4- Saccharomyces cerevisiae cells with an initial glucose
ethylenedioxypyrrole) were constructed by Turkarslan et al. concentration of 200 g L−1 (Tkac et al. 2003).
AOx was immobilized during electropolymerization of the
monomers in sodium dodecylsulfate and phosphate-buffered Glycerol
electrolysis medium. The highest activity was observed for
the PEDOT/AOx biosensor. The alcohol contents in distilled In foods and beverages, glycerol serves as a humectant,
beverages (vodka, dry gin, whisky, and raki) determined by solvent, and sweetener and may help preserve foods. It is
the biosensor was in a good correlation with the chromatog- also used as a filler in commercially prepared low-fat foods
raphy results (Turkarslan et al. 2010). For analysis of (e.g., cookies) and as a thickening agent in liqueurs.
methanol–ethanol mixtures, a portable bienzymatic analyti- Glycerol is a secondary fermentation product of alcoholic
cal system was developed, which consisted of two bio- secondary fermentation, contributing to the viscosity and
sensors, one based on ADH that responded only to ethanol smoothness of a wine, with a favorable effect on the taste
and the second one based on AOx that responded to both (Compagnone et al. 1998). Glycerol determination by an
methanol and ethanol. The transducers were screen-printed amperometric system can be based on the enzymatic
electrodes modified with mediators (Meldola blue for ADH reactions:
and co-phthalocyanine for AOx). The AOx biosensor was
Glycerol þ NADþ















→ dihydroxyacetone
Glycerol dehydrogenase
able to quantify both analytes in mixtures that contain
methanol between 3 and 70 mM and ethanol ranging from þ NADH þ Hþ
15 to 110 mM. Interferences due to non-specific oxidations ð21Þ
from common oxidizable compounds like gallic acid and
ascorbic acid were smaller in the case of a transducer based Glycerol þ ½Medox



















→ dihydroxyacetone
PQQ
glycerol dehydrogenase
on Meldola blue. The analytical system was successfully
tested using real samples, non-alcoholized beer spiked with þ ½Medred
ethanol or methanol and a falsified rose wine (Bucur et al. ð22Þ
2008).
SBMs were used for construction of robust alcohol Glycerol þ ATP









→ L
glycerolphosphate þ ADP
Glycerol kinase

biosensors using alcohol dehydrogenase and diaphorase ð23Þ

placed on the surface of the SBM-based transducer
containing NAD+ or the one with all components incorpo-
L
Glycerolphosphate
rated directly into the transducer. The use of various
þ O2



















→ dihydroxyacetonephosphate
L
Glycerol phosphate oxidase
mediators (organic dyes, vitamin K3, ferricyanide, and
ferrocene) and methods of biosensor construction were þ H2 O2
studied. The biosensors were used for the determination of
ð24Þ
ethanol in samples of wine, resulting in a good agreement
with data determined by photometric measurements (Katr- Álvarez-González co-immobilized the enzyme glycerol
lik et al. 1998). A microbial amperometric biosensor for the dehydrogenase (GDH) and its cofactor NAD+ in a carbon
measurement of ethanol in FIA was constructed by Valach paste electrode using an electropolymerized layer of
et al. The system used bacteria Gluconobacter oxydans nonconducting poly(o-phenylenediamine). After partial
attached on the surface of a combined (glassy carbon and oxidation of the immobilized NAD+, the modified electrode
Ag/AgCl reference) electrode mounted into the flow cell. allowed the amperometric detection of the NADH
The parallel colorimetric glucose determination was used to enzymatically obtained at an applied potential above 0 V
eliminate a non-specific glucose response. The response of (Ag/AgCl). The resulting biosensor showed a fast and
the biosensor for ethanol was linear in the range from linear response to glycerol within the concentration range
10 μM to 1.5 mM. Ethanol concentrations obtained were in of 1.0−100 μM with a detection limit of 0.43 μM. The
a good correlation with a gas chromatography reference biosensor was applied to the determination of glycerol in a
method (Valach et al. 2009). Tkac et al. prepared a plant-extract syrup, what resulted in a good agreement with
ferricyanide-mediated microbial biosensors by surface those obtained by standard spectrophotometric method
50 Food Anal. Methods (2012) 5:40–53

(Alvarez-Gonzalez et al. 2000). Niculescu et al. constructed analysis of natural samples was tested with dry white and
biosensors based on FIA with GDH either co-immobilized red wines as samples, and the method was validated by a
with phenazine methosulphate (PMS) or cross-linked to an spectrophotometric enzyme assay (Ghica and Brett 2006).
Os-complex-modified poly(vinylimidazole) redox polymer Goriushkina et al. analyzed the efficiency of using glycerol
(PVI13dmeOs) using PEGDGE. The GDH/PMS biosensor oxidase preparations and chose an electrochemical poly-
was characterized by a linear range from 0.01 to 1 mM of merization of glycerol oxidase in a polymer poly(3,4-
glycerol and a detection limit (calculated as ratio S/N=3) of ethylenedioxythiophene) as the most effective method of
0.9 μM. The redox hydrogel-based biosensors showed the glycerol oxidase immobilization on the surface of an
same dynamic range, but improved biosensors character- amperometric biosensor. The developed glycerol biosensor
istics, i.e., a sensitivity, a detection limit of 0.1 μM, and a was characterized with a linear range of 0.05–25.6 mM and
signal loss of only 20% after 15 h of operation under the a minimum detection limit of 0.05 mM of glycerol, and the
same conditions. The optimized biosensor configurations biosensor exhibited 75% of the initial activity after 15 days
were further used for analysis of glycerol in wine of storage. The biosensor was tested using wine samples
(Niculescu et al. 2003b). In another work, the same authors with satisfactory results (Goriushkina et al. 2010).
constructed a glycerol biosensor based on PQQ-glycerol
dehydrogenase using the same system as mentioned above Triacylglyceride
in the case of ethanol biosensor (Niculescu et al. 2003a).
Two different biosensor configurations were designed by Determination of triacylglycerides by biosensors is often
Gamella et al. The first one was based on the glycerol performed by its hydrolysis and after this an amount of
dehydrogenase/diaphorase (GDH/DP) bienzyme system, glycerol released is detected. Pundir et al. described a
and the second one used glycerol kinase/glycerol-3- method for construction of an amperometric triacylglycer-
phosphate oxidase/peroxidase (GK/GPOx/HRP). Both en- ide biosensor using polyvinylalcohol (PVA) membrane
zyme systems were immobilized together with the mediator bound enzymes. A mixture of commercial lipase, GK,
tetrathiafulvalene on a 3-mercaptopropionic acid self- glycerol-3-phosphate oxidasem, and horseradish peroxidase
assembled monolayer modified gold electrode using a was co-immobilized onto PVA membrane through glutar-
dialysis membrane. The first biosensor showed a very good aldehyde coupling. The minimum detection limit was
stability—the GDH/DP biosensor still exhibited 87% of the 0.21 mM. Among various serum substances tested, only
original sensitivity after 51 days of use, while the GK/ cholesterol caused slight interference (20%) (Pundir et al.
GPOx/HRP biosensor exhibited 46% of the original 2010). A similar construction was done by Narang et al.
response after 8 days. Calibration graphs for glycerol with (2010). Tkac et al. developed a biosensor utilized non-
linear range from 1.0 to 20 or from 1.0 to 10 μM of specific lipase isolated from Candida rugosa and intact G.
glycerol were obtained with GDH/DP and GK/GPOx/HRP oxydans cells, containing membrane-bound GDH. The
biosensors, respectively. The calculated detection limits sensor prepared from G. oxydans cells showed a detection
were 0.4 and 0.31 μM, respectively. The biosensors were limit of 20 μM, linear range up to 2 mM and a response
applied to the determination of glycerol in different wines, time of 84 s (90% of steady state). A calibration curve
and the results correlated well with those provided by a linear up to 12 mM was obtained for triolein samples (Tkac
commercial enzyme kit (Gamella et al. 2008). et al. 2000b). A promising concept for triglyceride
Ghica and Brett designed a bienzymatic biosensor for biosensor based on nanoparticles was proposed by Ganjlai
glycerol based on co-immobilization of two enzymes, et al. Lipase and glycerol dehydrogenase enzymes were
glycerol kinase (GK) and glycerol-3-phosphate oxidase. immobilized onto the CeO2 nanoparticles and multiwalled
The GK phosphorylates glycerol to glycerol-3-phosphate. carbon nanotubes placed on a glassy carbon electrode by
The enzymes were immobilized by a crosslinking with the aid of nafion. The detection method was based on fast
glutaraldehyde on carbon film electrodes with poly(neutral Fourier transform continuous cyclic voltammetry in a flow
red) as a mediator of the enzymatic reaction. Glycerol, as injection system. Under optimal detection conditions, the
well as glycerol-3-phosphate, was determined in an linear response was in the range of 1–100 mg l−1 with a
amperometric mode at −350 mV vs. saturated calomel detection limit of 0.5 mg l−1. The biosensor showed an
electrode. The linear response range to glycerol was acceptable reproducibility and a good stability, but com-
directly dependent on the concentration of adenosine-5′- parison with reference analytical method was not performed
triphosphate (ATP). With 3 mM of ATP in the measured (Ganjali et al. 2010). Ben Rejeb et al. used a Prussian Blue
electrolyte, glycerol was determined in the range 5– modified screen-printed electrode as a substrate for glycerol
147 μM. A monoenzymatic glycerol-3-phosphate biosensor dehydrogenase and NADH oxidase immobilization. The
was also characterized with a working range from 20 to glycerol biosensor was used for determination of lipase
700 μM. An application of the bienzymatic biosensor for activity. The system was challenged against an olive or
Table 1 Miscellaneous biosensors with a potential to be used in the food industry with some characteristics (Rahman et al. 2009)

Analyte Bioelement Transducer Characteristics Application References

Cholesterol Cholesterol oxidase Spectrofluorometric LR, 0.07–7.5 mM; DL, 70 μM Butter (Wu and Choi 2003)
Cholesterol oxidase and esterase Amperometric LR, 2.65–403 g dl−1 Fish (Yoneyama et al. 2009)
Inulin Fructose dehydrogenase and inulinase Amperometric LR, 5–100 μM; DL, 0.66 μM Chicory and prebiotic food (Manso et al. 2008)
Galactose Galactose oxidase Amperometric LR, 0.5–3 g dl−1 Synthetic milk (Sharma et al. 2006)
Methanol Alcohol oxidase/horse radish Amperometric LR, 100–800 μM; DL, 20 μM Beer, wine, liquor (De Prada et al. 2003)
peroxidase
Sulfite Sulfite oxidase Amperometric LR, 4–750 ppm; DL, 4 ppm Water (Abass et al. 2000)
Food Anal. Methods (2012) 5:40–53

Xanthine Xanthine oxidase Amperometric LR, 0.2–10 μM; DL, 0.1 μM Fish (Gao et al. 2009)
Xanthine Xanthine oxidase, superoxide Fluorescent DL, 20 nM Meat and fish freshness (Salinas-Castillo et al. 2008)
dismutase and peroxidase
Hypoxanthine Xanthine oxidase Amperometric LR, 1–400 μM; DL, 800 nM Fish freshness (Hu et al. 2000)
Histamine Monoamino oxidase Amperometric LR, 10–200 mg kg−1 Fish, meat, sauerkraut, beer, (Lange and Wittmann 2002)
dairy products, wine
Tyramine Tyramine oxidase Amperometric LR, 10–200 mg kg−1 Fish, meat, sauerkraut, beer, (Lange and Wittmann 2002)
dairy products, wine
Putrescin Diamine oxidase Amperometric LR, 5–200 mg kg−1 Fish, meat, sauerkraut, beer, (Lange and Wittmann 2002)
dairy products, wine
Oxalate Oxalate oxidase/horse radish Amperometric LR, 0.1–2.0 mM; DL, 0.09 mM Spinach (Perez et al. 2001)
peroxidase
Organo phosphates and Acetyl cholin esterase/Butyryl Photothermometric DL, 0.2 ng ml−1 Salad, onion (Pogacnik and Franko 2003)
carbamate pesticide cholinesterase
Nitrite Cytochrome c nitrite reductase Amperometric LR, 0.25–50 μM Fresh water (Silveira et al. 2010)
Histidine, cadavarin, Diamine oxidase Amperometric LR, 0.03–3 μM Fish (soul, rainbow trout) (Male et al. 1996)
putrescin
Caffeine Cells of Pseudomonas alcaligenes Amperometric LR, 0.1–1 mg ml−1 Instant tea and coffee (Babu et al. 2007)
MTCC 5264
3′5′-cyclic phosphodiesterase Potentiometric LR, 0–4 mg ml−1; DL, 0.6 mg l−1 Coffee (Pizzariello et al. 1999)
Glutamate Glutamate oxidase Amperometric DL, 0.5 mM Tomato foods (Pauliukaite et al. 2006)
Urea Urease Potentiometric DL, 2.5×10−5 mol l−1 Milk (Trivedi et al. 2009b)
Acrylamide Hemoglobin Voltametric DL, 1.2×10−10mol l−1 Potato chips (Stobiecka et al. 2007)

The table is an updated and completed version of a previously published one

LR linear range, DL detection limit
51
52 Food Anal. Methods (2012) 5:40–53

sunflower oil real samples in order to detect fatty acids, and Basu AK, Chattopadhyay P, Roychudhuri U, Chakraborty R (2006)
Biosens Bioelectron 21:1968
the results were compared with those provided either by the
Ben Rejeb I, Arduini F, Amine A, Gargouri M, Palleschi G (2007)
manufacturer or by reference methods with a good Anal Chim Acta 594:1
agreement (Ben Rejeb et al. 2007). Bhand SG, Soundararajan S, Surugiu-Wärnmark I et al (2010) Anal
Chim Acta 1:668
Blanes L, Mora MF, Do Lago CL, Ayon A, Garcia CD (2007)
Electroanalysis 19:2451
Biosensors for Other Food Components Bucur B, Mallat E, Gurban AM et al (2006) Biosens Bioelectron
21:2290
Several other biosensors successfully used for the determi- Bucur B, Radu GL, Toader CN (2008) Eur Food Res Technol
226:1335
nation of important analytes in real samples are summarized
Castillo J, Gaspar S, Leth S et al (2004) Sens Actuat B: Chem 102:179
in the Table 1. They are not as often the object of research Chaubey A, Malhotra BD (2002) Biosens Bioelectron 17:441
as biosensors described in previous sections, but they can Cock LS, Arenas AMZ, Aponte AA (2009) Chilean J Agric Res
be used for notable analytical procedures, such as food 69:270
Compagnone D, Esti M, Messia MC, Peluso E, Palleschi G (1998)
safety monitoring or final products control. Biosensors
Biosens Bioelectron 26:875
were used for analysis of both liquid and solid food Conzuelo F, Gamella M, Campuzano S, Ruiz MA, Reviejo AJ,
samples. Pingarron JM (2010) J Agric Food Chem 58:7141
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Acknowledgement This work was supported by the Slovak Bioelectron 13:181
Research and Development Agency (project VMSP-P-0073-09) and Katrlik J, Pizzariello A, Mastihuba V, Svorc J, Stredansky M, Miertus
the Agency of the Ministry of Education, Science, Research and Sport S (1999) Anal Chim Acta 379:193
of the Slovak Republic for the Structural Funds (project ITMS Kennedy JF, Pimentel MCB, Melo EHM, Lima-Filho JL (2007) J Sci
26240220040). Food Agric 87:2266
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