Corresponding Author: Simona Carmen LitescuCentre of Bioanalysis, National Institute for [600737]

Chapter 17
*Corresponding Author: Simona Carmen Litescu—Centre of Bioanalysis, National Institute for
Biological Sciences Bucharest, 296 Splaiul Independentei, Bucharest 6, 060031, Romania.
Email: [anonimizat]
Bio‑Farms for Nutraceuticals: Functional Food and Safety Control by Biosensors
edited by Maria T eresa Giardi, Giuseppina Rea and Bruno Berra.
©2010 Landes Bioscience and Springer Science+Business Media.Biosensors for the Determination
of Phenolic Metabolites
Simona Carmen Litescu,* Sandra Eremia and Gabriel Lucian Radu
AbstractAntioxidants are groups of chemical substances, the most abundant being polyphenols,
mainly found in plants, fruits and vegetables. They include flavonoids, flavonoid deriva ‑
tives, polyphenols, carotenoids and anthocyanins. Currently, the nutritional quality of
many foodstuffs is guaranteed by the presence of antioxidant compounds. The importance of these chemicals as indicators and preservatives of nutritional quality makes necessary the develop‑
ment of accurate, versatile and rapid analytical tools necessary to detect their presence in many
foodstuffs and to assess their antioxidant efficacy. In this chapter, enzyme‑based biosensors such as monophenol monooxygenase (tyrosinase), catechol oxidase (laccase) and horseradish peroxidase
(HRP) are reviewed. Actually, these biosensors are the most commonly used for the detection of
polyphenols and flavonoids content.
Introduction
Biosensors are flexible, specific and accurate analytical tools for the detection of antioxidant
compounds. An antioxidant is defined as any compound that, when present in low concentration
compared to that of an oxidizable substrate, significantly delays or prevents the substrate oxidation.
The antioxidants present in the human body act as a defence against highly damaging chemical species such as the free radicals generated during cellular metabolism. The ability of polyphenols,
flavonoids and other molecules to scavenge free radicals is connected to the specificity of their
chemical structure resulting from the presence of aromatic rings. When these compounds react with a free radical the unpaired electron stemming from the radical is neutralized through the
delocalization over the aromatic ring. The captured electron is stabilized by the resonance effect
of the aromatic nucleus causing termination of the free radical chain reaction. In this context it should be underlined that polyphenolic compounds inhibit oxidation by means of a variety of mechanisms.
1‑4
The formal redox potential of polyphenols allows them to act towards free radicals both as
electron donors and as hydrogen donors.5 This behaviour is due to the fact that they can be oxidized
to phenoxyl radical releasing an electron or loosing a hydrogen atom from the OH group. The
phenoxyl radical stabilization is possible by means of H intramolecular bonds.6 The H donation
is possible thanks to the low bonding OH dissociation energy required to complete the reaction pathway.
7 Up to now, the highest reported antioxidant efficacy was obtained for polyphenols 1,2
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Biosensors for the Determination of Phenolic Metabolites
dihydroxy substituted on the aromatic ring. As mentioned above, this behaviour finds an explana ‑
tion by presupposing the stabilization of the radical by intramolecular H bonds.6
The term “polyphenols” comprises different subgroups of phenolic acids and flavonoids and
their derivatives. Assessing the polyphenols content involves a very important sampling step, which
consists in extracting the secondary metabolites from raw sources.
The extraction is performed using various plant sources such as leaves, roots, citrus fruits, grapes,
berries‑seeds etc. either as fresh or dried material. In the last decade, the use of in vitro cultivation has also made it possible to increase a plant’s capability to biosynthesize polyphenols. In particular,
it was discovered that, under specific conditions such as oxidative stress induced by high light il ‑
lumination or temperature, plants increase the production of secondary metabolites among which
are polyphenols. This allows researchers to control and raise the antioxidant production in in vitro
cultivation permitting the application of molecular biology techniques for large‑scale production of antioxidants and their use as food additives.
The main subclasses of flavonoids are the flavonols which include quercetin, kaempferol and
myricetin; the flavones which include apigenin, luteolin and tangeritin; the flavanones which include catechins, catechin gallates, naringenin and hesperetin; the isoflavones which include genistein, daidzen and glycitein.
3,8 Flavonoids and related structures are shown in Figure 1.
Biosensors Used in the Determination of Polyphenols
The development of accurate, sensitive and specific methods for the quantitative determination
of polyphenols is a challenging task for researchers. T o increase the simplicity and performance
level of polyphenols determination, highly sensitive, fast and selective methods able to replace
classical methods such as high‑performance liquid chromatography, up to now the most successful and accurate method, are required.
Biosensors are a sub‑group of chemical sensors capable of operating directly in complex matrices
to detect analytes and to ensure, at the same time, the requirements of accuracy, sensitivity and
Figure 1. Chemical structures of main antioxidant polyphenols.
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236 Bio‑F arms for Nutraceuticals
selectivity which are the reason for their extensive use in recent years. Biosensors allow quantitative
and semi‑quantitative analysis based on the use of a biological recognition element (biochemical
receptor), which is in direct and spatial contact with a transductor element.
Biosensors can be classified according to the type of active biological component involved in
the mechanism, the mode of signal transduction or a combination of these two aspects. The choice of the biological material and the transducer depends on the sample properties and the type of
physical variable to be measured.
The type of bio‑component determines the degree of selectivity or specificity of the biosen ‑
sor. The recognition elements are divided in three groups: bio‑catalytic, bio‑affinity and hybrid receptors.
Bio‑Catalytic Receptors
Bio‑catalytic receptors can be mono or multi‑enzyme systems, whole cells systems (using mi ‑
croorganisms such as bacteria, fungi, eukaryotic cells and yeasts) and cells organelles and plant or
animal tissue slices‑based systems. The biosensors using microorganisms, plant or animal tissue as
bio‑components offer the advantage of not requiring extraction and purification procedures that are time‑expensive and very laborious, however, because of their high selectivity,
9 enzyme‑ based
biosensors are the most commonly used sensing agents.
Bio‑Affinity Receptors
The affinity‑based biosensors may be based on antibodies or nucleic acids chemo‑receptors.
These species provide highly selective interactions with specific ligands leading to thermodynami ‑
cally very stable complexes. Antigen‑antibody complexes may be coupled to every type of transduc ‑
tor element, but, generally different active substances are used as labelling compounds to increase the detectable signal. Among these are enzymes, fluorescent compounds, electrochemically active
substances, radionuclides and avidin‑biotin complexes.
10,11
Hybrid Receptors
Hybrid receptors are based on nucleic acid chemo‑receptors. The deoxyribonucleic acid (DNA)
structure is a double helix consisting of two polynucleotide strands, each strand being constituted of a polymeric chain containing the nucleobases adenine, thymine, cytosine and guanine. Hybrid
receptors are made using a unique well‑defined sequence of nucleic acid bases. The detection, in
this way, becomes highly specific and selective because the hybridization and the recognition oc ‑
curs only in presence of the complementary DNA fragment.
Most biosensors devoted to the determination of phenolic metabolite content use biocata ‑
lytic receptors and electrochemical‑based transducers, the measurements being performed by
an amperometric system. In this section some of these biosensors are presented emphasizing the
performance criteria achieved during biosensor development. The general criteria to evaluate
biosensors performance are based on the IUPAC
11 requirements and are based on the calibration
characteristics ‑as sensitivity, working at linear concentration range, detection and quantitative
determination limits‑ and selectivity, reliability, steady‑state, transient response times, reproduc ‑
ibility, stability and lifetime.
When the determination of the phenols content in a sample is performed, the total amount of
polyphenols is generally detected rather than each of them individually, since the overall response
is the most important.12
The term ‘total phenolics’ refers to all phenols that are responsible for the total antioxidant
capacity of a specific sample.
The electrochemical reaction used in the amperometric detection of polyphenols is based
on two subsequent steps: first, at the electrode surface, the substrate (polyphenol) is oxidised by means of a bio‑catalyst (enzyme), in the presence of oxygen. Successively, the regeneration of the
enzyme to its original oxidation state occurs, carried out by the electron transfer from a suitable compound (for example, phenols or flavonoids in their reduced form).
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Biosensors for the Determination of Phenolic Metabolites
The most commonly used biocatalysts in the determination of phenolics content belong mainly
to two enzymes classes, phenoloxidases and peroxidases.
Since phenolic‑derivatives are suitable substrates for oxidases, electrodes modified with tyrosi ‑
nase, laccase, peroxidase and cellobiose dehydrogenase have been developed to detect the phenolic
compounds. All these proteins belong to the class of ‘oxidase’ enzymes in which phenols work
as electron donors. Laccase and tyrosinase are the most commonly used biological recognition
elements in polyphenolics biosensors. The main difference between the two enzymes consists in their specificity: while tyrosinase catalyses only ortho‑substituted phenols (polyphenols), laccase
is able to act as efficient bio‑catalyst for a wider class of polyphenols.
13
Laccase‑Based Biosensors
Laccase is a cuproprotein belonging to the small group of enzymes named blue oxidases14 able
to catalyze the oxidation of various aromatic compounds.
This behaviour can be explained on the basis of the peculiar affinity of laccase towards oxygen
that makes it an efficient catalyst when acting as electron acceptor.15 Laccase is also able to oxidise
many nonphenolic compounds.16 Moreover, it catalyzes the hydrogen atom removal from hydroxyl
group of either ortho ‑ or para‑substituted mono‑ and poly‑polyphenolic substrates.17,18 The rationale
supporting the use of laccase as biomediator in phenols determination is linked to the extremely high sensitivity of this enzyme towards phenolic compounds.
A phenolic substrate, in the presence of laccase, undergoes one‑electron oxidation leading to
the formation of an aryl‑oxy radical that can be converted to a quinone in a second step of the oxidation. The principle of polyphenol detection which uses a laccase‑based amperometric biosen ‑
sor is briefly represented in Figure 2.
The catalytic features of laccase are, as expected, highly dependent on the laccase source. The
highest catalytic activity has been found in laccase from fungal sources.
Immobilisation of laccase for use as bio‑recognition element has been attempted on different
solid supports like graphite,
19 redox hydrogel on glassy carbon,20 carbon paste,21 carbon fibres,22
polyethersulphone membranes23 or platinum.24
A biosensor based on the immobilisation of laccase derived from the fungus Coriolus V ersicolor
on polyethersulphone membranes fixed on Pt–Ag supports was reported by Gomes24 and ap ‑
plied to the determination of polyphenols, flavonoids (caffeic acid, gallic acid, catechin, rutin,
trans ‑resveratrol, quercetin) and anthocyanidins (malvidin) from complex samples. The developed
Figure 2. Basis of the detection of polyphenols using laccase–based amperometric biosensors.
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238 Bio‑F arms for Nutraceuticals
biosensor achieved a limit of detection of 1.0  × 10–6 mol L–1, a linearity range from 2.0 to 14.0  × 10–6
mol L–1, high sensitivity (0.0566 mA/mol L–1) and reproducibility (R.S.D.  < 10%) when catechin
and caffeic acid were used as substrates.
Wilkołazka et al13 reported a laccase‑based biosensor for the detection of catechin hydrate,
epicatechin, epicatechin gallate, prodelphinidin and caffeic acid. The enzyme was immobilised
by physical adsorption on the surface of a graphite electrode. The electrodes were inserted in a
flow‑injection cell and the obtained sensitivities were between 57.92 nA/ µmol L–1 and 7.81 nA/
µmol L–1 depending on the substrate used, with limits of detection ranging between 0.56  µmol L–1
and 2.44 µmol L–1.
Gamella et al25 have developed a laccase‑based biosensor for the determination of polyphenol
index in wines. The estimation of the polyphenol index was performed both in batch and flow
injection conditions. The enzyme was immobilised by cross‑linking with glutaraldehyde onto a
glassy carbon electrode, while caffeic acid and gallic acid were used as standard compounds.
T yrosinase‑Based Biosensors
The tyrosinase biosensors are restricted to the monitoring of phenolic compounds with at
least one free ortho ‑position. Tyrosinase catalyses two different oxygen‑dependent reactions that
occur consecutively: the o‑hydroxylation of monophenols to o‑diphenols (cresolase activity) and
the subsequent oxidation of o‑diphenols to o‑quinones (catecholase activity).26,27
Carralero Sanz et al28 have reported the development of a tyrosinase biosensor based on the
immobilisation of the enzyme onto a glassy carbon electrode (GCE) modified with electrodepos‑
ited gold nanoparticles. The GCE was modified with gold nanocrystals after previous polishing
and rinsing. The modification was performed by immersion of the electrode into HAuCl 4 and
applying a potential of –200 m V for 1 min. Then tyrosinase was added onto the modified elec ‑
trode surface. The developed biosensor was applied for the estimation of the content of phenolic compounds in beverages.
For the detection of polyphenols in tea, Abhijith
29 constructed a tyrosinase biosensor based
on the immobilisation of the enzyme onto a Clark‑oxygen electrode membrane cross‑linking the
protein with glutaraldehyde. The principle of the biosensor was the enzymatic transformation of
polyphenols during oxygen consumption. The change in the dissolved oxygen amount depended on the concentration of catechins in the sample. The oxygen consumption at the electrode con ‑
sumes electrons, resulting in an electrochemical signal that is proportional to the concentration of polyphenols in the sample.
A multilayer tyrosinase based‑biosensor sensor was described by Schuhmann.
30 A redox dye
was covalently bound to an electrogenerated poly‑ ω‑carboxyalkylpyrrole layer which was covered
by a second layer of polypyrrole incorporating the enzyme. This multilayer configuration was able
to prevent electrode fouling caused by the polymerization of quinone derivatives.
A tyrosinase biosensor was also developed by Liu et al31 based on the immobilisation of tyrosi‑
nase in a positively charged Al 2O3 sol‑gel membrane onto a glassy carbon electrode. It was found
that Al 2O3 sol‑gel has two functions: the hydrophilic, porous and positively matrix provides a
friendly microenvironment for the enzyme to retain its functional activity and also acts as an ef ‑
fective promoter for the electron transfer between o‑quinone and the electrode.
A mediator‑free phenol biosensor was developed by Li and coworkers.32 Tyrosinase, was
adsorbed on the surface of the ZnO nanoparticles by electrostatic interactions and subsequently immobilized onto a glassy carbon electrode surface via a chitosan film. The phenolic compounds
(catechol, p‑cresol and phenol) were determined by the direct reduction of bio‑catalytically generated quinones at –200 m V.
Recently, amperometric phenol‑oxidases sonogel carbon based biosensors were developed for
the determination of polyphenols in complex samples.
33 The detection limit for caffeic acid in
the case of the laccase biosensor was of 0.06  µmol L–1 and the linear range 0.04‑2  µmol L–1. The
enzymatic solution was mixed with glutaraldehyde and then modified with 0.5% bovine serum
albumin (BSA).
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Biosensors for the Determination of Phenolic Metabolites
A biosensor based on horseradish peroxidase (HRP) and DNA immobilized onto
silica–titanium has also been reported by Mello and Kubota34 and applied to determination of
polyphenolic compounds in vegetable samples. The biosensor performance characteristics reported
against chlorogenic acid exhibited a linear response range from 1 to 50 mmol L–1, applied a potential
–50 m V versus Ag/AgCl, biosensor sensitivity—expressed as current density on concentration‑ is about 181 nA/mmol L
–1 cm–2 and the obtained detection limit of 0.7 mmol L–1. The biosensor
response compared to the Folin–Ciocalteau method proved the suitability of the biosensor for the quantitative analysis of the total polyphenol in the tested plant extract samples.
Conclusion
Biosensors designed and developed by immobilisation or co‑immobilization of one or two
phenol‑oxidase enzymes on the surface of solid supports—gels, graphite, printed inks, conduc ‑
tive metals etc.—exhibit very encouraging performances when applied to the determination of
polyphenols. Depending on their construction, the devices show reasonable stability and working lifetime even in complex sample matrices (beverages—beer, wines, food and food raw materials).
As exemplified above, biosensors for the determination of polyphenols based on laccase and tyro ‑
sinase are versatile and work well both in batch and in flow analysis. When flow injection analysis
is performed the determination sensitivity improves, reaching detection limits of 560 nmol L
–1.
This level of sensitivity is comparable to chromatographic analysis (high‑performance liquid
chromatography or gas‑chromatography).
Moreover, the use of polyphenol‑oxidases based biosensors to assess ‘total phenol content’ from
plant extracts and foodstuff ensures a higher selectivity compared to the classical Folin Ciocalteu method. The former method, unlike the second is, in fact, exempt from the interferences caused
by other compounds (e.g., sugars, ascorbic acid) occurring in plant material.
35
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