Electrochemical Biosensors for Antioxidants

C H A P T E R

4 Electrochemical Biosensors for Antioxidants Juan Jose´ Garcı´a-Guzma´n1, David Lo´pez-Iglesias1, Mariana Marin2, Cecilia Lete2, Stelian Lupu3,*, Jose´ Marı´a Palacios-Santander1,* and Laura Cubillana-Aguilera1,* 1

Institute of Research on Electron Microscopy and Materials (IMEYMAT), Department of Analytical Chemistry, Faculty of Sciences, Campus de Excelencia Internacional del Mar (CEIMAR), University of Cadiz, Campus Universitario de Puerto Real, Polı´gono del Rı´o San Pedro, Puerto Real-Cadiz, Spain, 2 Institute of Physical Chemistry “Ilie Murgulescu” of the Romanian Academy, Bucharest, Romania, 3 Department of Analytical Chemistry and Environmental Engineering, Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania

4.1 INTRODUCTION An antioxidant can be defined from a chemical point of view as “any substance that, when present in low concentrations compared to that of an oxidizable substrate, significantly delays or inhibits the oxidation of that substrate” [1,2]. In other words, antioxidants reduce the damage caused by reactive oxygen species (ROS; superoxide anion: O2
2, hydroxyl:
OH, peroxyl: ROO
, alkoxyl: RO
radicals, hydrogen peroxide: H2O2, hypochlorous acid: HClO, ozone: O3, and singlet oxygen: 1 O2), either being free radicals by themselves or causing the generation of other free-radical

species. In fact, antioxidants act by preventing the formation of these kinds of substances, scavenging them, or by promoting their decomposition [3]. From a physiological point of view, the role of antioxidants seems to be directly related to damage prevention of cellular components (DNA mutations, malignant transformations, etc.) as a consequence of chemical reactions directly involving oxygen and/or ROS. Typically, some peroxidase and dismutase enzymes (catalase and tyrosinase, and superoxide dismutase, respectively), some vitamins (C or ascorbic acid, E or α-tocoferol), metal ions (selenium) carotenoids (β-carotene), and polyphenols (flavonoids, anthocyanins, etc.)

*Corresponding authors.

Advanced Biosensors for Health Care Applications DOI: https://doi.org/10.1016/B978-0-12-815743-5.00004-4

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© 2019 Elsevier Inc. All rights reserved.

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4. ELECTROCHEMICAL BIOSENSORS FOR ANTIOXIDANTS

[46], among others, are well-known antioxidants, being capable of counteracting the damaging effects of oxidation. A possible classification of the major antioxidants according to their origin, that is, exogenous (daily intake) or endogenous (synthesized by the organism itself, or “first line of defense”), can be found in Fig. 4.1 [7]. The mechanism of action of antioxidants, which can act at different steps of the oxidative radical process, can be summarized as [7]: In 2 In-In
1 In
Initiation In
1 L 2 H ! In 2 H 1 L
kiLH

L
1 O2 ! L 2 OO
kperox

Propagation

L 2 OO
1 L 2 H ! L 2 OOH 1 L
kp

2L 2 OO
! ½L 2 OO 2 OO 2 L Termination kt

½L 2 OO 2 OO 2 L-Nonradical species 1 O2 As can be seen, the mechanism implies the successive steps of initiation, propagation, and chain termination and can be described by taking into consideration the lipid peroxidation in cell membranes or foodstuffs [8]. Nevertheless, endogenous antioxidant defenses are insufficient to prevent damage completely. This first line of defense is typically composed of superoxide dismutase and H2O2-removing enzymes, metal-binding proteins and lowmolecular weight scavengers, such as glutathione, uric acid, bilirubin, coenzyme Q, α-lipoic acid, and melatonin, among others (see Fig. 4.1) [9]. Thus diet-derived antioxidants (also called exogenous antioxidants) like vitamins and polyphenols are critical in maintaining health [10]. These antioxidants can usually be found in vegetables [11,12] and fruits [13] and products derived from them, such as juice, wine, beer, coffee, tea, and others [14], and also in cosmetics [1517]. Recently, much

antioxidant activity has been found in seaweed-associated bacteria as well, which could be of great interest for promoting blue economy and marine sustainability, since this kind of sea product might be used as food, for making drugs, and others as it is an issue still open for exploration with enormous potential [18]. The World Health Organization (WHO) advises that diets rich in fruit and vegetables promote good health. In fact, according to the WHO and supported by several research studies [1921], a daily intake of at least 400 g of fruit and vegetables is recommended to reduce the risk of cardiovascular diseases [22]. Another possibility might consist of the intake of dietary supplements or pharmaceuticals. However, rigorous research has not demonstrated antioxidant supplements or drugs to be beneficial in preventing diseases, mainly due to the synergistic effects with other substances present in fruit and vegetables, differences in chemical composition of the antioxidants in the supplements versus those in foods, specificity of some antioxidants for some diseases, the amount of the antioxidant intake, and others [2325]. In fact, no specific antioxidants have been recommended or offered by healthcare systems, neither have any been approved as therapy by regulatory agencies that base their decisions on evidencebased medicine. This is simply because, so far, despite many preclinical and clinical studies indicating the beneficial effects of antioxidants in many disease conditions, randomized clinical trials have failed to provide the evidence of efficacy required for drug approval [26]. Over the past decade, the key role of ROS species has been substantially evidenced in many fundamental cellular reactions. ROS may attack biological macromolecules, giving rise to protein, lipid, and DNA damage, cell aging and oxidative stress-originated diseases [27,28]. In fact, oxidative stress is suggested to be implied in the etiology of various disease states of an organism,

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Endogenous antioxidants

H N N H

N H

– O

N

O

HOOC

H3N +

HN

N H

NH2

CH3

H3C CH3

HS O

O NH

O

H

Nonenzymatic

O

Exogenous antioxidants (diet)

H N

CH3

HO OH

COOH

H

HO

O

H

O

CH3

O

HO

OH HO

Uric acid

Histidine

Glutathione

H N

HO

O

H3C

NH

HN

NH

HN

CH3

H3C

OH

O

Vitamin D

HO

OH

O

Vitamin C

O

O

H2N

Vitamin A

O

N

H2C

N H

CH3

OO H3C

Carnosine

CH3

HN

HN

H2C

Vitamin E

B Carotene

O

Bilirubin

Melatonin

O

OH HO

OH

HO HO

Enzymatic

OH O

Resveratrol

O O

CH3

H3C OH S

H3C

O

H

S

O

Alpha Lipoic acid Catalase

CH3

O

6–10

Coenzyme Q10 Peroxidase

Caffeic acid

Superoxide dismutase

Terpene

O

Flavone (Zn)

DNA-repair enzymes DNA-glycosylase Apurin/apyrimidinic endonuclease

FIGURE 4.1 Schematic classification of major antioxidants according to their exogenous or endogenous origin.

(Se)

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4. ELECTROCHEMICAL BIOSENSORS FOR ANTIOXIDANTS

being considered as crucial in the pathophysiology of common diseases including atherosclerosis, neurodegenerative diseases like Alzheimer’s, chronic renal failure, diabetes mellitus, and cancer [29]. Epidemiological studies have proven antioxidants ability to constrain the effects of ROS activity, and diminish the incidence of cancer and other degenerative diseases [7]. It is noteworthy to mention as well that scientists are also paying attention to reactive nitrogen species (RNS; nitroxyl anion, nitrosonium cation, higher oxides of nitrogen, S-nitrosothiols, and dinitrosyl iron complexes) as well as their implication in cell damage and death by inducing nitrosative stress at high concentration levels. RNS have been recognized as playing a crucial role in the physiologic regulation of many living cells, such as smooth muscle cells, cardiomyocytes, platelets, and nervous and juxtaglomerular cells. They possess pleiotropic properties on cellular targets after both posttranslational modifications and interactions with ROS [30,31]. In fact, both ROS and RNS, usually put together and known as reactive oxygen and nitrogen species (RONS), are considered to cause oxidative damage to biomolecules, contributing to the development of a variety of diseases [32,33]. Many different studies regarding the establishment of direct relationships between RONS and different illnesses have been conducted over the past few years affecting the liver [34], systemic autoimmune diseases [35], arthritis [36], a wide range of neurodegenerative diseases [37,38], immune system illnesses [9], and cardiovascular pathologies [39], among others. This chapter provides an overview of the current developments and achievements related to the use of electrochemical biosensors in the assessment of antioxidants in plants, food, and beverages, thereby critically showing their relationships with healthcare in most cases. The main reason for selecting electrochemical biosensors is due to their advantages versus other commonly used

analytical techniques like chromatography or mass spectrometry. Advantages include simple instrumentation, no sample treatment, high specificity, low-cost, rapid response, sensitivity, relatively compact size, and ease of implementation to detect biomolecules [40,41]. The first part of the chapter (Section 4.2) is devoted to the methods and procedures for ROS determination, with an emphasis on the applications of enzymatic biosensors. The second part (Section 4.3) focuses on the determination of antioxidants, mainly polyphenols, in plants and foods, by using electrochemical biosensors based on oxidase enzymes. The functioning principle of electrochemical biosensors is described together with selected applications of various tyrosinase-based and laccase-based biosensors applied in the assessment of polyphenols in real samples. The analytical performances and advantages of electrochemical biosensors compared to classical analytical methodologies are discussed. Finally, the last part (Section 4.4) pays special attention to the analysis of polyphenols in the most internationally popular beverages such as wine, beer, coffee, tea, and juice by using electrochemical biosensors.

4.2 BIOSENSORS FOR THE DETERMINATION OF REACTIVE OXYGEN SPECIES Oxidative stress is a consequence of the imbalance between oxidative processes induced by ROS and the antioxidant capacity of aerobic organisms. As introduced earlier, ROS is a general term referring mainly to different radicals (HO
), superoxide radical anions (O22
), and molecules containing oxygen (e.g., H2O2) which are highly reactive. Along with RNS, they form the defense system that protects the human body against different viruses and/or bacteria. In a healthy body, ROS are usually trapped by different lowmolecular weight (vitamin C, glutathione,

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4.2 BIOSENSORS FOR THE DETERMINATION OF REACTIVE OXYGEN SPECIES

vitamin E, lipoic acid, etc.) and high-molecular weight (enzymes) antioxidants. However, antioxidants cannot scavenge excessive ROS and oxidative stress is installed concomitantly with its harmful effects on the human health [42,43]. The incomplete reduction of oxygen is the main factor generating ROS, whereas their overproduction and the pathological consequences can be determined by different conditions such as environmental pollution (water, air, and soil), radiation, stress, smocking, and others. Generally, ROS play a dual role in the living organisms being involved in both “good” biochemical processes (redox regulation, defense against viruses and bacteria, modulation of vasodilatation) and in the “bad” ones (inflammatory processes, arterial hypertension, cancers, neurological disorders, aging, diabetes mellitus, etc.) [1]. Nowadays, modern medicine achieves superior results concerning the quality of human health by bringing together prophylaxis and efficient treatments. Permanently connected to the fast scientific progresses, medicine has applied different approaches, technologies and materials in order to assess the effects of oxidative stress on human health by monitoring ROS in various biological systems. Fluorescence and chemiluminescence are the most employed methods for determination of ROS, but specificity and selectivity are their main disadvantages. Moreover, they are not well suited for in vivo determinations owing to the bulky apparatus and, therefore cannot provide accurate information for medical purposes. Electrochemical methods have attracted considerable attention due to their outstanding features like rapidity, sensitivity, selectivity, miniaturization, low costs, simplicity, and portability. The in situ monitoring of ROS by means of electrochemical tools is probably the most important achievement of medicine, knowing that ROS are present in low concentrations and for an extremely short period of time in biological environments.

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4.2.1 Biosensors for Hydroxyl Radical Hydroxyl radical (HO
) results from Fenton reactions of different transition metal ions such as Fe(II), Cu(II), and Cr(III) with H2O2 and/or from different biochemical reactions normally occurring in the human body. Owing to its short half-time, HO
is one of the most aggressive ROS, being involved in lipid peroxidation, inflammatory processes, neurological disorders induced by damaging of dopaminergic neurons, hyperthermia, arterial hypertension, and others. The monitoring of HO
is very important especially for clinical diagnostic purposes, but is challenging due to its high reactivity. One approach to track HO
in both in vivo and in vitro biological environments is the electron spin resonance (ESR). The employment of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (hydroxy-TEMPO) as a spin probe [44] allowed to monitor HO
in the brain of mice and to assess their damaging effect toward dopaminergic neurons. 5,5-Dimethyl N-oxide pyrroline (DMPO), another spin probe, was used to achieve the ESR signal in the presence of HO
[45]. An optical sensor obtained by immobilization of nitrophenol on fiber-optic material enabled sensitive determination of HO
by reflectance spectroscopy for clinical analyses purposes and showed good stability over time (more than 6 months in conditions of dry storage) [46]. On the other hand, different fluorescence methods have been developed for HO
sensing in biological environments. The hybrid phenothiazine platform-based cyanine dye (MPT-Cy2) used as fluorescent probe for HO
determination in living cells and bacteria has proved good selectivity over other ROS and was employed for in vivo monitoring of HO
released in zebra fish in the presence of TiO2NPs under sunlight-like illumination conditions [47]. Another fluorescence sensor for HO
was based on coumarin-modified

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cyclodextrin derivatives and allowed sensitive and specific determination of HO
in water solution and yeast cells [48]. The rapid progress in nanotechnology and the outstanding features of nanoparticles have encouraged their application in the development of fluorescence biosensors systems for HO
. Thus, gold nanoclusters protected by bovine serum albumin (AuNCs-BSA) and conjugated with 2-[6-(40 -hydroxy)phenoxy-3Hxanthen-3-on-9-yl]benzoic (HPF) were successfully tested for HO
determination in living cells [49]. The fluorescence biosensor showed a linear response in the range 1150 μM (R2 5 0.998) and a detection limit of 0.68 μM, with no interferences from other ROS or biologically active compounds usually present in biological samples. Another fluorescence nanobiosensor was obtained by immobilization of single-stranded DNA onto gold nanoparticles (AuNPs) surface via strong amide bonds [50]. The fluorescence intensity increased linearly with HO
concentration from 0.1 to 50 μM, the detection limit being 42 nM. The dyes employed in fluorescence measurements can promote potential damage toward human health or can generate false-positive results, thus becoming less attractive for determinations in biological environments. Electroanalytical approaches have received increasing attention thanks to their attractive features such as low cost, fast and simple analyses, possibility of miniaturization, and others. Meanwhile, the sensitivity and selectivity of the determinations can be optimized by choosing the appropriate electroanalytical method and sensing materials. A major problem encountered when electrochemical methods are employed for measurements in biological systems is the fouling of the electrode surface caused by the adsorption of either analyte or other compounds present in the sample. In order to avoid this situation different redox mediators can be immobilized onto electrodes

surface. The resulting modified electrodes are less prone to lose their electroactivity and generally show superior analytical performance over unmodified electrodes. An amperometric sensor obtained by the deposition of doped polyaniline (PANI) onto a gold electrode showed a linear response toward HO
. The sensor response, that is, electrical conductivity of PANI, changed with HO
concentration and was linear in the range from 0.2 to 0.8 μM [51]. Different biologically active compounds such as enzymes, nucleic acids, proteins, and others have been employed for the construction of biosensors. Over the past few decades, biosensors have received increasing attention for biomedical applications as they enable to perform the clinical analysis and to monitor in small sample volumes with good sensitivity and selectivity. Different electrochemical enzymeless biosensors have been obtained by immobilization of flavonoid compounds on the surface of an optically transparent electrode. Thus, quercitin (quer), primuletin (prim), and morin (mor) deposited on the surface of (3-aminopropyl)triethoxysilane(APTES) fluorine-doped tin oxide (FTO) electrode leads to voltammetric biosensors that allowed the rapid determination of HO
with low detection limits, that were 2, 3, and 5 nM, respectively, (see Table 4.1) and good selectivity with respect to other potential interfering ROS [52]. DNA-based biosensors for HO
have been developed starting from the harmful potential of ROS toward nucleic acids. The construction of these biosensors involves the immobilization of nucleic acids either on the surface of an unmodified electrode or onto an electrochemical transducer previously modified with a redox mediator. When nucleic acids have been adsorbed onto carbon-based electrodes, the resulting DNA-biosensors showed good sensitivity toward HO
and was also successfully applied in order to assess the

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TABLE 4.1 Analytical Performances of Different Sensors and Biosensors for HO
Determination Sensor

Technique

Linear Range (µM)

Detection Limit (µM)

References

MPT-Cy2

Fluorescence

110

1.16

[47]

AuNCs-HPF

Flourescence

1150

0.68

[49]

AuNPs-DNA

Fluorescence

0.150

0.042

[50]

Au/PANI

Amperometry

0.20.8

0.2

[51]

quer/APTES-FTO

SWV

00.05

0.002

[52]

mor/APTES-FTO

0.003

prim/APTES-FTO

0.005

AuNPs/MCH/DNA1/Au

SWV

510000

3

[56]

MBs-DNA-Ag

ASV

0.054

0.01

[57]

APTES-FTO, ((3-aminopropyl)triethoxysilane-fluorine doped tin oxide) electrodes; ASV, anodic stripping voltammetry; AuNCs, gold nanoclusters; AuNPs, gold nanoparticles; HPF, 2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid; MBs, magnetic beads; MCH, 6-mercaptohexanol; mor, morin; MPT-Cy2, cyanine dye based on a hybrid phenothiazine platform; PANI, polyaniline; prim, primuletin; quer, quercetin; SWV, square wave voltammetry.

antioxidant capacity of different antioxidants [5355]. The outstanding features of nanomaterials have encouraged their application in DNA-biosensor development in order to improve their analytical performances. The voltammetric response of the DNA-Au biosensor was enhanced after its subsequent modification by DNA-functionalized AuNPs. An increment of the linear response in the range from 5 μM to 10 mM and a lower limit of detection (eight times) have been obtained when DNA1/Au biosensor (DNA1: 5_-SH(CH)6-GGT CCG CTT GCT CTC GC-30 ) was replaced by DNA2-AuNPs/MCH/DNA1/Au (DNA2: 5_-SH-(CH)6-CGG GCG AGA GCA AGC GGA-30 ) for the assessment of HO
, where MCH is 6-mercaptohexanol [56]. The enhancement of electroanalytical nanoparticlebased DNA-biosensor performances is due to a more effective surface area that AuNPs show against their bulk homologue. Another selective nanoparticle-based biosensor for HO
was obtained by functionalization of DNA-immobilized magnetic beads (MBs) with

silver nanoparticles (AgNPs) [57]. The working principle of the MBs-DNA-AgNPs consisted in the oxidation of the nucleic acid and the breakage of the strands from DNA-nanobiosensor followed by AgNPs detaching from the nanobiocomposite surface. The as-“released” AgNPs were subsequently detected by the anodic stripping voltammetry (ASV) technique, proving the peak current to be proportional to the concentration of HO
. Thus, the linear response range (0.054 μM) and the limit of detection (10 nM) were significantly improved by choosing appropriate electroanalytical method and biosensor components. These features together with good reproducibility and selectivity of MBsDNA-AgNPs electrochemical biosensor against the competing ROS species strongly recommend its application for HO
quantification and for the assessment of antioxidant capacity for different antioxidants. Table 4.1 summarizes the main results concerning the analytical performance of different sensors and biosensors for HO
determination.

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4.2.2 Biosensors for Superoxide Anion Radical Superoxide anion (O22
) is an intermediate of molecular oxygen reduction. O22
and together with other ROS has an important contribution to immune response of living systems. On the other hand, O22
is very unstable and can interact rapidly with different biologically active compounds and/or other radicals containing oxygen, leading to different pathological effects. Hence, the reaction of O22
with NO
is responsible for vasoconstriction [58] whereas that with NO produces peroxynitrite, which is involved in neurodegenerative diseases [59]. Under normal physiological conditions O22
appears at low concentrations owing to its efficient scavenging by endogenous antioxidants. The overproduction and/or accumulation of O22
, when the concentration is expected to be in the range of 10261027 M [60], leads to oxidative stress and harmful effects on human health including cancer, neurodegenerative diseases, arterial hypertension, and others. In other words, the border between the physiological and pathological conditions is a function of O22
concentration, location, and duration. An efficient assessment of health status and/or disease progress assumes permanently monitoring of O22
by clinical analysis and various imaging methods that involve the use of sensitive, selective, and biocompatible analytical tools for the detection of O22
. One approach to detect O22
in the vascular wall is chemiluminescence, achieving a low detection limit (20 nM) with negligible cellular toxicity. However, this approach shows some limitations regarding the concentration of probe and existence of antioxidants in the sample. The fluorescence generated in the presence of dyes such as dihydroethidium (DHE), its derivatives, [61,62], and 20 ,70 -dichlorodihydrofluorescein [63] has been successfully applied for detection of O22
in vascular cells. The fluorescence

nanosensor obtained by microemulsion polymerization [64] and the nanobiosensor developed by immobilization of horseradish peroxidase (HRP) and superoxide dismutase (SOD) [65] have showed good sensitivity and specificity toward O22
quantification. However, the electrochemical methods are very attractive for clinical monitoring due to their specific features that allow the rapid and selective determination of O22
both in small volumes of samples or onsite during physiological and/or pathological processes in living systems. Biosensors are powerful analytical devices for the determination of very reactive species in biological samples, both in vivo and in vitro, owing to their specificity, fast response, and small size affording spatial and time resolution. Selective electrochemical biosensors have been designed by employing enzymes, proteins, or permeable membranes deposited on the surface of different conducting materials and applied to O22
monitoring either for clinical analysis or imaging purposes. Cytochrome c (Cyt c) is a small redox protein that significantly increases the electrochemical response when immobilized onto electrode surfaces. A nonenzymatic multilayer structure very sensitive to O22
was obtained by alternating deposition of Cyt c and poly(aniline(sulfonic acid)) (PASA) onto Au wire electrodes [66]. Under optimum conditions (six layers), the nonenzymatic Au/PASA/Cyt c electrochemical biosensor showed a linear response in the range of 0.41.5 μM O22
and a sensitivity of 0.398 A/mol cm2. The amperometric biosensor obtained by covalent immobilization of Cyt c onto an array of Au electrodes allowed the determination of O22
produced by stimulation of A172 human glioblastoma cells with a sensitivity of 10.3 nA/μM cm2 and no interferences from ascorbic acid and H2O2 [67]. Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of O22
to O2 or H2O2 and plays an important role in the defense systems of living organisms by ROS




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4.2 BIOSENSORS FOR THE DETERMINATION OF REACTIVE OXYGEN SPECIES

scavenging. Because of its specificity and high reactivity toward O22
, SOD has become very attractive in the development of sensitive and selective biosensors. A third-generation biosensor obtained by immobilization of SOD onto a Au electrode modified by cysteine (Au/Cys) was employed for the amperometric determination of O22
with good sensitivity and no interferences from H2O2, uric acid, ascorbic acid, and DOPA C [59]. The entrapment of SOD within polypyrrole (PPY) matrix during electropolymerization of the monomer onto a platinum (Pt) electrode was used to obtain an amperometric microbiosensor with good analytical performances toward O22
[60]. Thus, the wide linear response range and the detection limit of 15 nM together with the small size have determined the use of Pt/PPY/SOD microbiosensors in the quantification of O22
in abdominal aorta of dogs after stimulation. New enzymatic biosensors with good electrocatalytic properties starting from the outstanding properties of the nanomaterials have been also reported. The covalent bonding of SOD to Fe3O4 nanoparticles followed by their immobilization onto the Au surface allowed to achieve a detection limit of 11.5 nM for O22
[68]. Furthermore, three different nanostructured Au electrodes (pyramidal, spherical and rod-like) were modified with SOD. They were successfully applied for the amperometric determination of O22
over a wide range of concentrations and with low-detection limits, depending on the applied potential [69]. Another selective approach enabling the assessment of O22
in ovarian cancer cells was based on the immobilization of the biomimetic enzyme-like Mn3(PO4)2 on TiO2 nanoneedles by using Nafion membranes. The synergistic effects generated by employing biomimetic enzymes and highly conductive TiO2 have provided superior performances to the sensor such as a wide linear range (5 3 10271.5 3 1023 M) and a low detection limit of 170 nM [70].

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4.2.3 Biosensors for H2O2 H2O2 is another ROS with mild oxidant power. It can be involved in physiological processes such as redox signaling or immune defense system, and can be responsible for different pathologies like cancer, myocardial infarction, or Alzheimer’s disease. Its dual role is a consequence of its concentration, location, and/or accumulation in target organs. In the human body, H2O2 can result as a response of different cells to various bacteria and viruses or by O22
scavenging in the presence of SOD. Catalase and peroxiredoxin are the enzymes responsible for maintaining H2O2 under physiological levels, thus avoiding the occurrence of pathological effects. The quantification of H2O2 in different biological samples (tissues, cells, plasma, serum, urine) is an attractive approach to assess the human health and/or the related diseases progression. Optical methods such as fluorescence [7174] and chemiluminescence [75,76] are sensitive to H2O2 and have been the first ones employed for its determination and for the assessment of the antioxidant activity for different compounds. Despite their sensitivity, these methods have become less attractive as are not selective and usually employ dyes with potential harmful effects. H2O2 is a redox active molecule, and therefore electrochemical methods are well-suited for its monitoring. The continuous efforts and scientific achievements in the field of materials science have enabled the development of new sensing electrochemical tools with superior activity toward H2O2, that were employed not only for in vitro analysis, but also for in vivo monitoring of H2O2 released from living cells. Various electrochemical sensors for H2O2 determination have been reported in the literature and an increased prevalence of electrochemical methods within the methodology dedicated to H2O2 monitoring was observed

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over the past few decades. The main concerns regarding the electrochemical detection of H2O2 are the potential interference of other biocompounds existing in biological samples and/or the fouling of the electrode surface that can affect the accuracy of measurements. There are different ways to overcome these drawbacks and to obtain reliable results such as the selection of appropriate approach (e.g., determination by electrochemical reduction instead of oxidation) and the employment of redox mediators and/or different permselective membranes. The electrochemical reduction of H2O2 takes place at negative potentials, where the others compounds present in the sample are not electroactive. Moreover, the use of small-sized sensing tools (providing spatial and temporal resolution) and redox mediators showing electrocatalytic activity have enabled new achievements in medicine. Thus a platinized-carbon microelectrode has allowed the monitoring of H2O2 released by cells under physical polarization and has also served to highlight when no H2O2 is developing in the case of singleimmunostimulated macrophage cells [77]. Nanosized carbon-based materials deposited on different conductive surfaces have been successfully employed in the development of sensitive electroanalytical tools for H2O2 determination in biological systems. The indium tin oxide electrode (ITO) modified in a first step by single-walled carbon nanotubes (SWCNTs) and by osmium bipyridine in a second step, was used for the monitoring of H2O2 released in macrophage cell (RAW 264.7) under bacterial stimulation [78], without interferences from other ROS, and offered new insights regarding the mechanism of the immune response inside the cells. The amperometric sensor obtained by immobilization of multiwalled carbon nanotubes (MWCNTs) onto platinum black electrodes was employed to acquire information concerning the level of H2O2 in

aging of nervous central systems [79] thanks to the possibility to cross the cell membrane. Since its discovery in 2004, graphene oxide (GO) has attracted much attention due to the outstanding properties such as electrical conductivity and low weight. The electrical conductivity of GO increases by chemical or electrochemical reduction, and the additional modifications of the resulting reduced graphene (RGO) lead to different sensors and biosensors for H2O2 monitoring. The functionalization of RGO by CuS allowed a linear response of the resulted sensor (CuS/RGO/ GCE) on a wide range of concentration (51500 μM) and a detection limit of 0.27 μM, without interferences from other biologically active compounds present in the biological fluids [80]. The sensor was tested with good results in human serum, urine and HeLa cells. Following the same line of action, the biosensor obtained by immobilization of Cyt c onto the surface of RGO/GCE was used for the selective amperometric detection of H2O2 [81]. Other Cyt cbased biosensors for determination of H2O2 in biological systems have been obtained by deposition of Cyt onto different nanosized TiO2 structures [8284]. Myoglobin, hemoglobin, and ferredoxin can be also utilized for the construction of H2O2 biosensors [85]. Nanomaterial-based biosensors are very attractive sensing tools due to the synergistic effects obtained by combining the outstanding features of nanomaterials (small size and increased active surface) with substrate specificity that enzymes and proteins offer. The nanobiosensor designed by means of HRP immobilization onto AuNPs [86] and ZnONPs [87] has demonstrated electrocatalytic activity toward H2O2 reduction. Also a 3D biomimetic electrochemical biosensor was obtained by immobilization of MnO2 within the pores of PPY and successfully employed to detect H2O2 released from cells [88].

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4.3 ELECTROCHEMICAL BIOSENSORS FOR THE ASSESSMENT OF TOTAL ANTIOXIDANT CAPACITY

ROS are normally present in living aerobic organisms and are involved in various biochemical reactions corresponding either to physiological or pathological states. The in vivo and/or in vitro monitoring of ROS are valuable tools for the assessment of human health and allow more accurate diagnostics and effective therapies of diseases that improve the quality of people’s lives. Electrochemical biosensors demonstrate to be remarkable analytical tools for selective and sensitive determination of different ROS, being successfully applied to track the oxidative stress and related pathologies even at the level of a single cell.

4.3 ELECTROCHEMICAL BIOSENSORS FOR THE ASSESSMENT OF TOTAL ANTIOXIDANT CAPACITY OF PLANTS AND FOODS Plants are a rich and natural source of antioxidant compounds. Especially medicinal plants containing a tremendous variety of natural antioxidants such as flavonoids, phenolic acids, and tannins, which possess more powerful antioxidant activity than common dietary plants. Owing to their antioxidant and freeradical scavenging activity they are used in adjuvant therapy for the treatment of antiinflammatory, antitumor, antiallergic, antiviral, and antibacterial processes. The presence of polyphenolic and phenolic compounds in plants assures protection against pathogen attacks or ultraviolet radiation. Several studies have demonstrated that the consumption of some cereal products is of importance for the prevention of diabetes, cancer, and cardiovascular diseases. The phenolic compounds prevent oxidative damage to cellular organelles, proteins, lipids, DNA, and RNA [89]. For example, oat contains different kinds of phytochemicals with antioxidant activities as flavonoids, phenolic acids, and tocols [90].

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Also, this kind of cereal includes antioxidants with low-molecular weight, and substituted N-cinnamoylanthranilic acids that are named avenanthramides (AVs) [91]. Emmons and Peterson [92] have shown that these acids have a significant antioxidant capacity, with 1030times higher radical scavenging activities than caffeic acid, ferulic acid, and vanillin. Nie and Wise [93] discovered that AVs have an antiproliferative influence on vascular smooth muscle cells, limiting the development of restenosis and atherosclerosis after angioplasty. These phytochemicals in plants exhibit health protection against several biomolecules damages. Daily, at the cell level around 2 3 104 DNA damaging events occurs leading to diseases such as arteriosclerosis, hypertension, diabetes, and neurodegenerative disorders [94,95]. The damage could be assigned to ROS
as HO
, H2O2, and superoxide radical (O2 2 ) [96]. Thus the monitoring of ROS has attracted a great deal of interest over the past few years. For instance, it was demonstrated that a DNAbased biosensor against ROS is a useful approach in total antioxidants capacity assessment because the principle of measurement is closer to the antioxidant activity in biological systems [97]. The radical attack on DNAmodified electrodes is similar to the process that occurs within the cell, which may generate replication errors and subsequent misleading protein synthesis that produce cell ageing. Consequently, the development of analytical methodologies for the quantification of the antioxidants or evaluation of the antioxidant capacity in plants, food, and beverages is of paramount importance. The analytical methods could be classified as hydrogen atom transfer (HAT) and single electron transfer (SET) methods based on the reaction mechanisms involved. Both mechanisms almost always occur together according to their antioxidant structure and pH [98]. HAT-based methods used in the literature are total radical-trapping antioxidant parameter (TRAP), total oxidant scavenging capacity (TOSC), oxygen radical

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absorbance capacity (ORAC), low-density lipoprotein (LDL) oxidation, and β-carotene bleaching by ROO
. SET-based techniques used in antioxidant capacity determination are total phenolic assays by Folin-Ciocalteu (FC), ferric reducing antioxidant power (FRAP), trolox equivalent antioxidant capacity assay (TEAC), and DPPH-based assay (2,2-diphenyl-1-picrylhydrazyl) [99]. In the Introduction, Fig. 4.1 illustrates most studied antioxidants classified according to their exogenous or endogenous characters. Due to the utilization of herbal extracts in medicine and food technology, it is important to evaluate their antioxidant capacity and this issue has been performed by a range of analytical methodologies including liquid or gas chromatography, capillary electrophoresis, spectrophotometry, fluorescence, and mass spectrometry [100]. In spectrophotometric assay, antioxidant activity of plant extracts is often determined against different freeradical species, such as the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the bleaching rate is monitored at a characteristic wavelength in the presence of the plant extract. These methods provide precise quantification of several antioxidants in various samples, but they are time consuming and sometimes sample pretreatment is required. The use of electrochemical methods like cyclic voltammetry and differential pulse voltammetry in the quantification of various antioxidants has several benefits like simplicity, high sensitivity, rapidity of analytical measurements, and analysis of colored samples without pretreatment, compared to traditional analytical methodologies [101]. The electrochemical parameters of importance in antioxidants determination via electrochemical methods are: (1) the anodic (oxidation) peak potential, Epa; (2) the anodic (oxidation) peak current, ipa; and (3) the electric anodic charge, which is related to the area under the anodic oxidation wave, Qa. The Epa value is related to

the electron donor capability of the measured antioxidant, while the ipa parameter refers to the concentration of the antioxidant. The electric anodic charge has been successfully used to quantify the antioxidant capacity usually expressed as total polyphenolic content. The electrochemical methods are typically applied in connection with metals (Pt, Au) or glassy carbon electrodes and semiconductors, as well as electrodes modified with various nanomaterials and organic polymers to improve the selectivity and the sensitivity of the analytical measurements. The judicious modification of conventional electrode substrates with a range of inorganic, organic, and composite materials represents the most important achievement and development in the electrochemical science. The modification of the electrode surface revealed the possibility to achieve new properties like selectivity, sensitivity, and polarity that greatly expand the final applications of obtained modified electrodes. Chemically modified electrodes (CMEs) are usually obtained by deposition of chemical modifiers, that is, inorganic, organic, and polymeric compounds, in forms of monomolecular, multimolecular, and polymeric layers. The CMEs are actually functioning as electrochemical sensors and have found several applications in the electroanalysis of antioxidants. Another approach consists in the use of electrochemical biosensors based on oxidase enzymes such as tyrosinase, laccase, and HRP for the quantification of various antioxidants. Electrochemical biosensors represent a subclass of electrochemical sensors and have the advantages of both the sensitivity of the electrochemical transducers (electrode substrates) and the selectivity of the biological recognition element, namely, the enzymes. The immobilization of the enzymes onto electrode surfaces is of paramount importance for the proper functioning of the electrochemical biosensors. The main enzyme immobilization procedures are adsorption, entrapment into a matrix,

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4.3 ELECTROCHEMICAL BIOSENSORS FOR THE ASSESSMENT OF TOTAL ANTIOXIDANT CAPACITY

microencapsulation, cross-linking, and covalent bonding. The incorporation of enzymes within conducting polymer-based matrices has attracted great interest because of the simplicity, versatility, and reproducibility of this procedure. PPY, PANI, PEDOT, and their derivatives have been successfully and extensively used in the fabrication of enzymatic electrochemical biosensors [102104]. The use of tyrosinase [105107] and laccase [108,109] for the detection of antioxidants like polyphenols is very convenient since the enzymatically generated quinone derivative may be reduced at low potentials, thus the electrochemical interferences are considerably minimized. The reduction current of the generated quinone derivative is measured and is related to the concentration of the investigated polyphenols. The functioning principle of the electrochemical biosensors based on oxidase enzymes for polyphenols detection is schematically depicted in Fig. 4.2. Tyrosinase (polyphenol oxidase, E.C. 1.14.18.1), in the presence of oxygen, catalyzes the hydroxylation of monophenols to o-diphenols (Reaction 1), and the oxidation of o-diphenols to o-quinones (Reaction 2) [110]. The resulted quinone derivatives can be easily detected by electrochemical reduction at low potential values (Reaction 3). This is the basic principle of tyrosinase-based amperometric biosensors and can be described by these reaction schemes:

117

mono-phenol1TyrosinaseðO2 Þ-o-diphenols (4.1) o-diphenols1TyrosinaseðO2 Þ-o-quinones1H2 O 2

1

o-quinones12e 12H -o-diphenols

(4.2) (4.3)

Laccase (EC 1.10.3.2) catalyzes the oxidation of phenol, diphenols, and various polyphenols to quinone derivatives and does not require the H2O2 as a cosubstrate [111,112]. Similarly to tyrosinase-based amperometric biosensors, the reduction of the enzymatically generated quinone derivatives provides the electrochemical signal in laccase-based biosensors. Actually, the antioxidant capacity is measured using a standard compound like caffeic acid, catechin, chlorogenic acid, or catechol, and this compound displays good electrochemical behavior at the electrode surface. The analytical performance of tyrosinase and laccase-based amperometric biosensors depends mainly on the enzyme immobilization method, the enzyme loading and activity, and pH of the sample solution. The immobilization method is the most important parameter in the development of enzyme-based electrochemical biosensors. The main achievements in this topic will be discussed taking into account the enzyme immobilization procedure. For instance, the adsorption of enzymes is a simple and versatile approach in biosensors construction [105]. In this study, the beneficial role of FIGURE 4.2 The functioning mechanism of electrochemical biosensors based on oxidase enzymes.

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the immobilization matrix alongside to the enzyme adsorption procedure by covering the adsorbed tyrosinase enzyme with polyethylene glycol and/or the ion-exchanger Nafion was demonstrated. The use of Nafion coating ensured the highest analytical performance in synthetic samples (low detection limit of 0.064 μM, sensitivity of 8 3 103 nA/μmol L cm2, Michaelis-Menten constant of 67.1 μmol/L for catechol), as well as in real samples. In another study [113], a tyrosinase-based biosensor was constructed by the immobilization of the enzyme onto screen-printed electrodes using various methods such as crosslinking with glutaraldehyde, entrapment with polyvinyl alcohol, and cross-linking with glutaraldehyde and human serum albumin. The best analytical performance in terms of the lowest detection and quantification limits (1.5 μM and 5.1 μM catechol, respectively) were obtained for the biosensor prepared via cross-linking of tyrosinase with glutaraldehyde. This biosensor was successfully applied in the determination of trolox equivalent antioxidant capacity of infusions prepared with various medicinal plants and the results were compared with a well-established method, namely, the DPPH spectrophotometric method. The incorporation of the enzyme within carbon-paste electrodes is a versatile and lowcost approach applied successfully in the construction of biosensors. The use of ionic liquids in conjunction with carbon paste demonstrated higher stability and sensitivity of the obtained electrodes. As an example, a laccase-biosensor for rosmarinic acid determination was fabricated using carbon paste and 1-N-butyl3-methylimidazolium hexafluorophosphate (BMIPF6) ionic liquid [108]. The quinone derivative produced in the enzymatic reaction was electrochemically reduced at 10.2 V versus Ag/AgCl and detected by using square-wave voltammetry. The optimized biosensor displayed a linear response toward rosmarinic

acid in the concentration range of 0.9965.4 μM, a very low detection limit of 0.188 μM, and very good sensitivity and stability. The laccase-biosensor was used in the determination of rosmarinic acid in plant extracts with very good recovery values ranging from 96.1% to 105.0% and good agreement with the data obtained via capillary electrophoresis method at the 95% confidence level was observed. Besides these analytical performances, the preparation procedure based on the incorporation of the enzyme within a carbon paste with ionic liquid has several advantages like low cost, simplicity, rapidity, and renewability of the obtained laccase-biosensor. Another biosensor construction approach consists in the entrapment of the enzyme within a polymer membrane aiming to improve the stability of the biosensor. For instance, a laccase-based biosensor was built by immobilization of the enzyme within a polyvinyl alcohol polymer membrane onto graphite screenprinted electrodes [109]. The immobilization procedure ensured very good stability and excellent analytical performance in terms of detection limit of 0.5 μM caffeic acid and sensitivity of 24.9 nA/μM. The laccase biosensor was successfully applied in the determination of phenolic content of tea infusions with minimal sample preparation by using chronoamperometric detection mode and standard addition analytical protocol. The results obtained with the laccase biosensor were compared with those gathered by the Folin-Ciocalteu spectrophotometric method. The proposed laccase biosensor has several advantages like simplicity, rapidity, sensitivity, and direct analysis of real samples. Alternatively, the incorporation of enzymes within conducting polymers matrix during the electrochemical polymerization of the corresponding monomers onto various electrode substrates represents an efficient and simple biosensor construction procedure [103,114116].

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In this case, the immobilization of the enzyme occurs mainly via electrostatic interactions between the positively charged backbone of the conducting polymer and the negative charges carried out by the enzyme at a given pH value. Even if there is some diffusion limitation within the polymer matrix of the reactive species, the rapidity and simplicity of this preparation method ensured its wide use in the development of enzyme-based electrochemical biosensors. Selected applications of tyrosinase,

horseradish peroxidase, and laccase-based biosensors in the determination of various antioxidants and mainly polyphenols in plants, foods, and beverages are presented in Table 4.2. The data displayed in Table 4.2 point out to the peculiar analytical performances of the enzyme-based electrochemical biosensors, namely, the very low detection limit values and the wide linear response ranges that can be achieved. The possibility to directly measure real samples with no (or minor)

TABLE 4.2 Enzymatic-Based Biosensors Used in the Determination of Various Antioxidants Linear Limit of Antioxidant Range (µM) Detection (µM) References

Enzyme

Immobilization Procedure

Sample

Tyrosinase

Nafion coated sonogel-carbon electrode

Beers and industrial wastewaters

Catechol



0.064

[105]

Tyrosinase

Multiwalled nanotube ionic liquid-chitosan coated on ITO electrode

Red wines

Phenol

1080



[106]

Tyrosinase

On diazonium-functionalized Tea screen-printed gold electrodes

Catechol

0.122

0.1

[107]

Tyrosinase

Cross-linking with glutaraldehyde

Medicinal plants

Catechol

Up to 136

1.5

[113]

Tyrosinase

Cross-linking onto polypyrrole

Environmental water

Phenol



1.71

[117]

Tyrosinase

Entrapment within PEDOT

Synthetic sample Catechol

20300

12.9

[116]

Horseradish peroxidase

Immobilization on selfassembled monolayers

Wine and tea

Catechin

Up to 25

2

[118]

Laccase

Incorporation with ionic liquid into carbon paste

Plant extracts

Rosmarinic acid

0.9965.4

0.188

[108]

Laccase

Entrapment in polymer membrane onto graphite screenprinted electrodes

Tea

Caffeic acid

0.5130

0.5

[109]

Laccase

Physical adsorption

Plant extract

Caffeic acid

Up to 10

0.56

[119]

Laccase

Covalent immobilization on dendrimers

Tea

Catechin

0.110

0.05

[120]

Laccase

Entrapment in nanocomposite Salvia officinalis multiwall carbon nanotubesand Mentha chitosan piperita extracts

Rosmarinic acid

0.912.1

0.23

[121]

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pretreatment of the sample constitutes the main appealing feature of electrochemical biosensors. Furthermore, the low-cost instrumentation, possibility of miniaturization, and rapidity and simplicity of the measurements represent other advantages of electrochemical biosensors compared to conventional analytical methodologies.

4.4 BIOSENSORS FOR THE ANALYSIS OF POLYPHENOLS IN BEVERAGES As it has been previously mentioned, polyphenols possess an important role in the antioxidant capacity. This antioxidant capacity has a strict relationship with several health benefits. Thus the determination of polyphenols, not only in plants but in the very common products made by them, is a critical factor. This section tries to show several approaches which can be used to solve this topic out. Some of the principles used have been briefly mentioned in the previous section; however, the approach will be slightly different, being mainly focused on beverages such as beers, wines, teas, juices, and others.

4.4.1 Beverages: Role of Antioxidant Capacity for Healthcare Purposes In the last decades the interest for relating the health benefits of beverages and the prevention of diseases is increasing. Numerous research studies show that moderate consumption of a certain beverages (e.g., wine, beer, coffee, juice, teas) produces positive benefits in the human body. However, these drinks are highly consumed, as shown in Fig. 4.3. Tea and beer seem to be the most-consumed beverages in the world. One of the most representative studies of this topic is the “French paradox.” In 1992 Renaud and De Lorgeril [122] pointed out the relation between wine consumption and health from the study of the mortality statistics of seventeen countries, such as Japan, France, Switzerland, Spain, Italy and Ireland, among others. In some of these countries (i.e., France) the intake of lactic fats was very high, although the mortality caused by heart disease was much lower than could be expected. Wine was considered as being responsible for longevity due to its high antioxidant capacity and its medium-high consumption. Grapes and, consequently wines, are wealthy sources of antioxidants. It has been demonstrated that the antioxidant presence in

FIGURE 4.3 Worldwide consumption of the most popular beverages in 2016.

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4.4 BIOSENSORS FOR THE ANALYSIS OF POLYPHENOLS IN BEVERAGES

this fruit has protective properties against several diseases [123]. They also participate in the slowing of thrombosis procedures due to the platelet aggregation inhibition, lipids peroxidation, oxidation of proteins of low density, and others. Moreover, their activity against oxidative diseases such as cellular aging, mutations and even cancer, is well-known [124]. Other beverages, such as teas, present anticarcinogenic effects, participating in the scavenging of reactive oxygen species (ROS) [125]. In addition, it has been demonstrated that they have an important role in the prevention of many diseases such as heart diseases and obesity [126]. Several procedures have been applied in the determination of the antioxidant capacity of beverages. The usual approach is the imitation of the radical scavenging, which can be found in the sample, between the native antioxidants and the ROS species. A chromophore reacts with a strong oxidant, obtaining a colored radical compound. The scavenging phenomenon leads to a decay in the concentration of the colored radical attributed to the antioxidant capacity. By spectrophotometric assays, a change of the signal in the UV-vis spectrum is found. Different radicals species can be used such as (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) ABTS [127], (dimethyl-4-phenylenediamine) (DMPD) [128], (2,2-diphenyl-1-picrylhydrazyl) (DPPH) [129], ferric reducing ability of plasma (FRAP) [130], and cupric reducing antioxidant capacity (CUPRAC) [131], among others. Fluorescence properties can be also used for this purpose, for example the oxygen radical absorbance capacity (ORAC) assay [132]. Some of these methods which have been applied to plants and food samples, have been previously commented in Section 4.3. A similar approach is the employment of DNA biosensors. These biosensors monitor the damage to DNA chains caused by radical species, analogous to the scavenging capacity of the compounds used in other methods. DNA biosensors will be explained deeply in Section 4.4.2.2.

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Other alternative approaches focused on electrochemical devices have emerged. They can be classified according to two different principles. The first is based on the direct relation between an electrochemical signal and the antioxidant capacity of the sample [133] and this relation generates a new parameter, namely, the electrochemical index (EI). However, the main drawback of this procedure resides in the high amount of interferents found in complex real samples. The second principle is based on the important role of the polyphenolic content in the antioxidant capacity of beverages. A polyphenol index (Ip), usually calculated using certain standard polyphenol, is correlated with the antioxidant capacity of the sample. Wine, coffee, beer, and juice constitute a rich source of polyphenols. For instance, wine contains a large number of different polyphenols, which can be split in two groups, nonflavanoids and flavonoids. Nonflavanoids include polyphenols like gallic, caffeic, and ferulic acid. On the other hand, flavonoids contain tannins, anthocyanins, or flavanols such as catechins. All these polyphenols have been widely studied for different reasons. In the majority of cases, the antioxidant capacity of the beverages can be attributed to o-diphenols. Gallic acid, caffeic acid, and catechins, among others, have been widely studied [134139]. Inside this group, some authors indicated the higher antioxidant capacity of gallic acid and caffeic acid over the others [140]. This study corroborates the use of these polyphenols as standard polyphenols to recalculate the Ip in electrochemical assays. Wine has the highest polyphenols content, although beer, tea, coffee, and juice also possess a significant amount of these polyphenols. Consequently, the study of these beverages is a very prolific field [97,141147]. Table 4.3 shows the most relevant polyphenols and the beverages in which they mainly appear.

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TABLE 4.3 The Most Relevant Polyphenols in Beverages Polyphenol

Chemical Structure

Beverage

Quercitin

Juice and wine

Chlorogenic acid

Coffee

Caffeic acid

Coffee, wine, and beer

Catechin

Juice, wine, tea, and beer

Epicatechin

Juice, wine, and tea

(Continued)

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TABLE 4.3 (Continued) Polyphenol

Chemical Structure

Beverage

Epicatechingallate

Tea

Epigallocatechin- gallate

Tea

Gallic acid

Wine

(Continued)

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TABLE 4.3 (Continued) Polyphenol

Chemical Structure

Beverage

Salycilc acid

Beer

Theaflavin

Tea

Theaflavin-3-gallate

Tea

Theaflavin-3,3’-digallate

Tea

(Continued)

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4.4 BIOSENSORS FOR THE ANALYSIS OF POLYPHENOLS IN BEVERAGES

TABLE 4.3 (Continued) Polyphenol

Chemical Structure

Beverage

Vanillic acid

Beer and wine

4.4.2 Electrochemical Biosensing of Polyphenols in Beverages There are two main trends in polyphenols determination in these kinds of samples. The first is focused on the individual determination of these polyphenols. For this purpose, chromatography techniques [136,148] have an important role due to their power and good sensitivity. Other techniques such as electrophoresis [149] or mass spectrometry [150] can be used for their determination as well. However, all these procedures are very expensive and time-consuming. Besides, the individual determination of all polyphenols in beverages is, at best, a hard task. Some authors indicate that the contribution in the antioxidant capacity of some groups of polyphenols is higher than others, so the total polyphenolic content is not so important as the polyphenolic content of certain groups [151]. The second technique to determine polyphenols is based on the collective determination. Spectrophotometric methods stood out for their simplicity. One of the most representative methods in this category is the FolinCiocalteau (FC) method [152]. It has been wellknown for decades as a reference method for the determination of polyphenols content in

food samples. FC reagent is a mixture that contains sodium molibdate, sodium tungstate, and phosphates in basic media. The reaction of this mixture and polyphenols evolves into a chromophore. Thus this compound can be measured by using spectrophotometric assays. However, this reagent can interact with other reducers in the samples, as sugars, giving false information [153]. Therefore the main drawback of this method is the lack of selectivity [154]. Researchers are making great efforts in the development of different techniques to monitor polyphenols in real samples. The ideal technique should be low cost, selective, and sensitive. In addition, for the benefits of the population, the perfect technique should be easily applied in situ. Real-time control of antioxidant capacity of food samples would have a direct and positive impact in people health. These requirements can be found in electrochemical biosensors. First, the antioxidant capacity of polyphenols is related with their capacity to donate electrons, so these substances are good candidates for electrochemical-sensing purposes. Second, biosensors contain a biological recognition element, providing selectivity, good sensitivity, and very quick responses. Third, these kinds of devices can be easily

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miniaturized and used in online analyses. They can also be considered low cost in comparison with other techniques like HPLC-MS, among others. Different biological recognition elements have been successfully employed in biosensors such as enzymes, antibodies, DNA chains, microorganisms, and others. The most-used biosensors are based on enzymatic compounds. 4.4.2.1 Enzymatic Biosensors These devices have been commented in Section 4.3. because of their utility in the plant extracts analysis. However, there is also a great number of studies related to the application of these kinds of biosensors in beverages [101,137,143,144,153,155161]. Enzymatic biosensors owe their popularity in this sector to several factors. First, the biological recognition element, enzymes, provides selectivity toward the analyte, in this case polyphenols. This selectivity plays an important role in the analytical determinations of complex real samples such as wine, juice, or beer. Second, the enzymatic catalysis usually improves the signal provided by the biosensor. Third, the immobilization of the enzyme in the transducer can be easily carried out by using several physical and chemical methods as reported in the literature. The most common methods are physical adsorption [162], entrapment [163], covalent

binding [164], and affinity immobilization [165], among others. Most enzymes used as biological recognition elements in enzymatic biosensors belong to the family of oxidases. Their activity has been briefly commented on Section 4.3, although here we will clarify some additional issues. These enzymes are mainly based on the oxidation of several substrates. Molecular oxygen is used as electron donor and hydrogen as electron receptor. Peroxidase is also frequently applied in biosensing with the use of H2O2 as receptor, whereas another different molecule has the role of electron donor. A large number of substances can act as donor, even the target analyte. These enzymes, together with laccase and tyrosinase, constitute the biological receptor in biosensor devices for beverage assays. Laccase is an oxidase enzyme which possesses four copper atoms to perform its enzymatic reactions. This is mainly focused on the oxidation of orthodiphenols and paradiphenols to their respective quinones. In Fig. 4.4 the usual oxidation process performed by laccase is illustrated. The substrate, a p-polyphenol, is oxidized to its quinone form which can be reduced again to the original polyphenol by the application of a certain potential, resulting in an electrochemical reduction response. This signal can be correlated with the concentration of the polyphenol. Besides, many FIGURE 4.4 Scheme of the reaction catalyzed by laccase enzymes.

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4.4 BIOSENSORS FOR THE ANALYSIS OF POLYPHENOLS IN BEVERAGES

FIGURE 4.5 Tyrosinase reaction scheme. (A) monophenolase activity.

interferences are avoided since a reducing potential is used instead of an oxidative one. It is important to clarify that beverages possess a number of substances that can be easily oxidized, such as sugars among many others. On the contrary, interferents susceptible to reduction are less abundant. Tyrosinase is another oxidase frequently used in biosensors applied to beverages. This oxidase has two copper atoms to carry out its reactions. First, tyrosinase may o-hydroxilate monophenols to o-diphenols (monophenolase activity). This enzyme can also oxidize orthodiphenols to quinones (cresolase activity). A typical reaction of tyrosinase can be found in Fig. 4.5. It should be noted that o-diphenols obtained in the first step could be oxidized in the second step, as shown in Fig. 4.5. Thus the signal obtained will include the component of the native monophenols which are transformed afterwards into o-diphenols, as well as the component from the orthodiphenols contained in the sample. Therefore this enzyme has two different substrates and for this reason it is possible to assume that selectivity is lower than that obtained with laccase enzyme.

activity;

and

(B)

cresolase

FIGURE 4.6 Peroxidase enzyme reaction scheme.

Third, the basis of peroxidase enzymatic catalysis is the electron transport from several electron donors (polyphenols, in this case) to H2O2. The core of this enzyme is a heme group. These biosensors can be used to determine H2O2 as well as polyphenols. The most used is HRP. Fig. 4.6 shows a typical reaction of peroxidase enzymes. As seen in Fig. 4.6, the enzyme as electron donor has the same product as the previous enzymes, although H2O2 is required. This is the main difference between this enzyme and the others. Thus biosensors based on peroxidase will need a previous addition of H2O2 to work properly. On the other hand, the principle is similar for the other enzymes; the quinone is reduced onto the surface of the electrode under a specific potential value.

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The control of the current will serve to monitor the concentration of the polyphenol. Despite of the selectivity of the recognition element, it is well-known that the real substrate of enzymes entails a family of compounds. Therefore these biosensors are usually focused on the collective determination of polyphenols in a real sample. There are several authors who have pointed out the relationship between the antioxidant capacity and the collective polyphenol amount determined with these kinds of biosensors [138,151]. Thus they can be considered as a feasible alternative to the classical methods in order to determine antioxidant capacity in beverages. Table 4.4 lists some of the most relevant enzymatic biosensors reported in the literature over the past few years. There are several interesting aspects concerning the information collected in Table 4.4. The first thing to point out is the predominant use of laccase enzymes, although tyrosinase is also frequently used. This fact can be justified by the higher selectivity provided by these enzymes in comparison with peroxidase-based biosensors. In contrast to peroxidase, a cosubstrate like H2O2 is not necessary to perform the enzymatic activity in laccase and tyrosinase-based devices, providing simplicity. Regarding selectivity, laccase only reacts with o- and p-diphenols, considered as the most relevant polyphenols due to their great antioxidant properties. Therefore laccase has been used in very different samples such as tea, wine, beer, and others, demonstrating its high versatility and potential. Tyrosinase is also widely used as biological recognition elements, obtaining good results in spite of their lack of selectivity. In order to highlight the results obtained with the relevance of these devices, some papers are focused in the correlation of tyrosinase biosensors and antioxidant capacity determined by employing a reference method [129].

Last, very good results have been obtained by using peroxidases. However, the use of an additional compound such as hydrogen peroxide constitutes the main drawback for online studies. Regarding the analytical technique employed, the most used is amperometry, which is based on the application of a fixed potential and the recording of the current obtained. The main advantage of this technique is the enhancement of the sensitivity of the analytical measurement compared with cyclic voltammetry. In contrast, by using this technique it is not possible to discriminate the analytical signal. However, the purpose of these biosensors is not the individual determination of a specific analyte, but the collective determination of a group of substances with similar properties. Voltammetric techniques, such as DPV and CV, are more selective for analytes, but they are less reported than amperometric techniques for polyphenol determination. There is a large number of electroactive compounds in real matrices, so several peaks can be overlapped. Despite this disadvantage, very good results in terms of sensitivity and limits of detection can be found when using voltammetric techniques [144,166,169,173]. The most-used standard phenolic compounds for the reported assays are catechol, caffeic acid, and gallic acid. The selection of the standard phenolic compound can also depend on the type of sample, although a general trend has not been observed. Gallic and caffeic acid are commonly chosen as representative of polyphenols in samples of wine and beer, but catechol is usually focused on tea samples. Different polyphenols, such as catechin and hydroquinone, are gaining importance as well. Hydroquinone is an isomer of catechol and it is widely employed in electrochemical sensing. Concerning other analytes, catechin and epicatechin (catechin isomer) are polyphenols which can be found in very high

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

TABLE 4.4 Features of the Most Relevant Enzymatic Amperometric Biosensors Used for the Determination of Antioxidant Capacity in Beverages Biological Recognition Element

Transducer

Technique Analyte

Limit of Detection (M) Linear Range (M)




29







28

25

Tyrosinase

GCE PEDOT RGO Fe2O3

DPV

CAT

7 10

4 10 6.2 10

Laccase

GCE SPCE

ChA

CAT

1 1026




5 10286 1025

Tyrosinase/ Laccase

GCE ERGOMWCNTs/GO

ChA

CAT

5 1027/ 3 1027

1 10263.4 1024/ 1 10263 1024

Laccase

GE AgNPs/ ZnO NPs

CV

Guayacol



5
10

Tyrosinase

CPE

ChA

HQ

1.6 1026

28






Tyrosinase

SPE CoAlSO4 LDH/ AuSPE

ChA

Mixture of polyphenols

Brasica Napus hairy roots

CPE Fc-MWCNTsMO

ChA

CA/Resveratrol 1.1 1027/ 1.1 1027

Laccase/ Tyrosinase

ITO/CS-MWCNTs

ChA

Rosmarinic acid/CA/GA









1
10



5
10

25




24




2 10251.2 1024




26

02.4 10





3.3 10273.8 1024/ 2.2 10273.3 1024




4 10276.4 1026/ 4 10277.4 1026/ 1.6 1026/8.1 1026

2.5 1027/ 2.88 1027/ 1.55 1027








27



























Pretreatment Employed



Extraction, filtration

Green tea [166]

13.4

Dilution, filtration

Tea, black [167] tea

310

Dilution, filtration

Fruit juice [168]

5.57

Dilution

Red wine

[169]

42.6

Dilution

Red wine

[161]

Sample

References



Green tea [160]



Wine, tea

[170]

148/152/33 Extraction, filtration, dilution

Plant extracts

[171]

Laccase

US PES

ChA

CA

8.8 10

5 10 3.5 1025

0.102

Dilution

Wine

[172]

Tyrosinase

CPE/MWCNTSs/ Naffion

ChA

Trolox







Dilution

Wine

[129]

Laccase

SPCE/Pt Nps/ RGO

CV/ChA

CA

9 1028

2 10272 1026

2147.38

Dilution

Tea

[158]

Laccase

CPE

DPV

GA/CAT/RUT 





Dilution

Honey

[144]

Laccase

Pt-Ag

ChA

CA



Dilution

Wine

[135]

2.29/2.53/ 2.84

Extracted

Coffee

[159]

Dilution

Wine

[153]






26

Sensitivity (µA/mM)














27






26




25

Laccase

Au/Au-DTSP/Au- ChA MTPS

HQ

9.1 10 / 8.9 107/ 2.5 107

3 10 1.5 10 / 3 10261 1025/ 9 10272 1025

Laccase

SPEs-MWCNTs

GA

0.1/0.3 (ppm)

0.117/0.118 (ppm)

ChA





(Continued)

TABLE 4.4 (Continued) Biological Recognition Element

Transducer

Technique Analyte

Limit of Detection (M) Linear Range (M)

Sensitivity (µA/mM)

Pretreatment Employed

Sample

References

2.944 1026 (μA/ppb)

Filtered

Wine

[173]

3.64

No treatment Must and [174] wine

217

Diluted

130

No treatment Wine

[156]

Degassing, dilution

Beer

[175]

Degassing, dilution

Beer

99.45/ Degassing, 12.75/ dilution 11.00/ 89.06/28.13

Beer

Degassing, dilution

Beer

[139]

Dilution

Wine

[138]

0.009/0.007 (μA/ppm)




PPO

GCE

DPV

CATE

1.76 (ppb)

Peroxidase

SPE ChitosanMWCNTs

ChA

Gluconic acid

2.6 1026




4 10266.2 1024

Tyrosinase

SPE

ChA

CATE

3 1028

5 10281.5 1025




ITO-MWCNTs/ ChA ITO poly(GVPB)-gMWCNTSs/ITOpoly(HEMA)-gMWCNTs

CATE/GA



Tyrosinase

SNGC

CA/FA/GA/ CATE/EPI

1.43 1026/ /3.38 1026/ 3.30 1027/ 4.2 1027

Peroxidase

Laccase

SNGC

SNGC

ChA

ChA

CA/FA/GA/ CATE/EPI

CA/FA/GA/ CATE/EPI








5
10


3.5
10

25

Tyrosinase

ChA

40200 (ppb)

6 1028/ 1.6 1027/ 4.1 106/ 1 1026/ 1.6 1026









2
10 5.15
10 / 2
10 4
10 / 2
10 8.36
10 / 2
10 2
10 / 2
10 3.2
10 4
10 2
10 / 4
10 2
10 / 1
10 2.2
10 / 4
10 3
10 / 4
10 8
10












24

2 1027/ 3.2 1027/ 9.4 1025/ 1.4 1027/ 1.8 1027
















6 10272.45 1025/ 0.72/ /3.6 10265.8 1025/ /0.08/ 1.57/1.23 6 10271.03 1025/ 6 10272 1025 27 27

26

26

25 27

24

26

27

26

28

26

28

26

7

26

28

26

28

26

8.55/4.32/ 0.024/ 15.925/ 11.192

Laccase

SNGC

ChA

GA







Laccase

SNGC

ChA

GA

0.11 (mg/L)

50200 (mg/L)

3.65 1027 (A L/mg)




Tea

[137]

CA, caffeic acid; CAT, catechol; CATE, catechin; ChA, chronoamperometry; CPE, carbon paste electrode; CS, chitosan; CV, cyclic voltammetry; DPV, differential pulse voltammetry; DTSP, 3,3’-Dithiodipropionic acid di(N- succinimidyl) ester; EPI, epicatechin; ERGO, electrochemical reduced graphene oxide; FA, ferulic acid; Fc, ferrocene; g, glucose; GA, gallic acid; GCE, glassy carbon electrode; GE, gold electrode; GO, oxide; GVPB, 4-vinylphenylboronate; HEMA, 2-hydroxyethylmethacrylate; HQ, hydroquinone; ITO, indium tin oxide; LDH, layer double hydroxide; MO, mineral oil; MTPS, (3-mercaptopropyl)-trimethoxysilane; MWCNTs, multiwalled carbon nanotubes; Nps, nanoparticles; PEDOT, poly(3,4ethylenedioxythiophene); PES, polyethersulfone membranes; PPO, polyphenol oxidase; RGO, reduced graphene oxide; RUT, rutin; SCE, screen printed electrode; SCPE, screen printed carbon electrode; SNGC, Sonogel-carbon.; US, universal sensor.

4.4 BIOSENSORS FOR THE ANALYSIS OF POLYPHENOLS IN BEVERAGES

concentration in samples such as wine and beer. Last, other polyphenols such as resveratrol can be used as well. The importance of resveratrol in human health has been previously demonstrated [149]. As reported in Table 4.4, wines, beers and teas are the most commonly analyzed samples. This trend is similar to the one found in the consumption graph, (Fig. 4.1). Beer and tea are highly consumed due to cultural factors or the health benefits attributed to these beverages, as widely exposed until now. Wine is also highly consumed, but not as much as beer and tea. However, there has been a great amount of research regarding this sample. The lower worldwide consumption level cannot be correlated with a smaller economic impact. In 2018 the International Organisation of Vine and Wine reported that 2017 wine global market generated benefits of about 34.2 billions of US dollars, which can be considered a very significant amount since it supposes a rise of 4.8% compared with 2016. In terms of antioxidant capacity, wines have the highest index. Therefore higher benefits of health could be expected under responsible and advised consumption of wines by health authorities. The usual pretreatments applied to the real sample are dilution, filtration, extraction, and degassing. Extraction is only required in tea samples due to the different solubility of tea compounds. Dilution is almost always needed to improve the sensitivity of the biosensors. Filtration is not common, but in some cases with juice or tea it can be performed to avoid some suspended material. Degassing is required in beer in order to remove the CO2, which hinders the correct application of the electrochemical procedure. It should be noted that the best biosensor would involve the least treatment possible to easily perform in situ and online analyses. The large number of approaches dealing with the transducer can be also noticed. Transducers convert chemical information into an electrical signal. Ceramic, carbon, and

131

screen-printed electrodes are widely used for these purposes, although indium and tin oxides popularity has risen over the past few years. All these transducers are commercially available. Other handmade electrodes can be excellent candidates as immobilization matrices. In this way, carbon-based ceramic electrodes obtained by using solgel process have emerged due to their excellent conductivity as well as a high surface area. A new type of ceramic electrode, namely, sonogel-carbon, emerged in the past decade as an excellent alternative for biosensor transducers. Several characteristics such as low residual current, renewable surface, and wide operational range can be attributed to this material [176]. All the materials mentioned above can be easily modified to improve their features, leading to an enhancement of electrical conductivity. However, higher conductivity is not the only aim for the modification of these materials. For instance, it has also demonstrated that metal nanoparticles, such as AuNps, improve the direct electron transfer between the biological recognition element and the transducer, increasing the sensitivity of the measurements [177]. On the other hand, conducting polymers can be used as the supporting element to improve the immobilization of the enzyme onto the electrode surface [116]. This immobilization process enhances the robustness of the biosensor obtained. Metal nanoparticles, carbon nanomaterials, and conducting polymers are frequently reported as modifiers. The modified transducers have better analytical performance such as limit of detection, limit of quantification, and sensitivity. In Table 4.4 very good sensitivities can be appreciated [158,168,175]. It is noteworthy to mention that correlation studies with antioxidant capacity methods are much more useful than having an excellent limit of detection in these applications. The procedure used should be as selective as possible. FC is not a good example for reasons previously stated. However, methods based in radical scavenging offer a more

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132

4. ELECTROCHEMICAL BIOSENSORS FOR ANTIOXIDANTS

suitable approach. In this line of reasoning, some assays have obtained very good relations [129,135,139,157] using methods such as DPPH, ABTS, and others. However, some authors keep using FC for the correlation in spite of the lack of selectivity this method shows. It is also possible to use several enzymatic biosensors simultaneously for sample analysis. According to this idea electronic tongues stood out. In general, an electronic tongue is a battery of amperometric sensors (biosensors, sensors, or a mixture) which are applied simultaneously to one sample. These devices provide a large number of different responses that require statistical treatment to extract useful conclusions [134,178]. The main disadvantage of electronic tongues is the timeconsuming treatment of the data, although it leads to a more complete characterization of the sample than that obtained with single biosensors. 4.4.2.2 DNA Biosensors In the previous sections, the relation between antioxidant compounds with biological harm has been mentioned. Biological damage is mainly caused due to ROS, obtained as by-products in breathing procedures. This group contains peroxides, superoxides, and hydroxyl radicals, among others, which affect tissues and even DNA and can lead to several diseases. Using this approach DNA biosensors are considered as an excellent alternative to determine the antioxidant capacity in beverages since they mimic the process and interactions in the human body under oxidative stress. DNA can be immobilized onto the surface of the transducer, using the genetic material as biological receptor. The nucleobases (adenine or guanine) are damaged by the presence of ROS, resulting in a decay of the electroanalytical signal. The addition of a polyphenolic sample removes ROS species, improving the response obtained. Consequently, this improvement can

FIGURE 4.7 Scheme of Fenton reaction in the human body.

be related to the antioxidant capacity of the sample [97]. Fig. 4.7 The most accepted reaction to study the degradation of DNA is the Fenton reaction, represented in Eq. (4.4). Fe21 1 H2 O2 -Fe31 1 OH2 1 OH


(4.4)

Fe (II) is used to promote the generation of hydroxyl species. Although it has been demonstrated that Cu (II) and also Cr (II) can also perform this generation, iron is the most considered due to its abundance in living beings and having the highest ratio of reactions. On the other hand, hydroxyl radical participates in reactions with other species as it can be noticed in Eqs. (4.5) and (4.6). OH
1 H2 O2 -H2 O 1 OH2


(4.5)

HO2
1 Fe31 -Fe21 1 O2 1 H1

(4.6)

In real conditions, Fenton reactions are not simple because of the existence of several cofactors and enzymes. For instance, the

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

4.4 BIOSENSORS FOR THE ANALYSIS OF POLYPHENOLS IN BEVERAGES

concentration of H2O2 is controlled by superoxide enzyme (SOD) and catalase. A scheme of a possible Fenton reaction adapted for living beings is presented in Fig. 4.5. Several oxidants and reducers contained in the environment participate in the formation of DNA
. The molecule resulting can be repaired in some cases, although it is possible to have hard damage in its structure. This damage can be due to the modification of more than 20 different nucleotides. Among these, 8-oxoguanine is the product mostly studied and it is used as a clinical biomarker for oxidative damage to DNA. A typical oxidation of guanine base to this biomarker is shown in Fig. 4.8 Within the biosensor, different strategies to detect DNA damage can be applied. The most common ones are based on the interaction between DNA and the transducer. • Studying the oxidation signal of DNA and its modifications. • Application of electrochemically active mediators. • Measuring possible changes in the properties of charge transfer of the DNA layer deposited onto the surface of the electrode. DNA biosensors in antioxidant fields are based on the abovementioned strategies. Their electrochemical signal can be excellently correlated with other reference analytical methods such as DPPH, ABTS, Trolox, and others. The antioxidants selected are added to the solution

133

at the same time as the Fenton reagent. The signal is compared with the signal in the absence of antioxidants. It is important to point out that damage in DNA is mostly irreversible, so these biosensors are, in general, for single use. These biosensors involve complex procedures to ensure their robustness, so they are not suitable for field work. On the other hand, good reproducibility can be achieved [142]. The most relevant DNA biosensors currently used are listed in Table 4.5. A comparison of the data displayed in Table 4.5 and Table 4.4 shows that DNA biosensors are less frequently applied to beverages than the enzymatic biosensors. This is due to factors previously mentioned such as less robustness or single use. However, there are several biosensors which provide very good results. In the first column the biological recognition elements are shown. Two trends are clearly identified. The first uses directly the nucleotide base, such as guanine, adenine, and others. The second trend is the use of some biological sample containing this nucleotide, for instance, salmon or herring sperm. In the latter, the DNA is directly dissolved in a buffer solution and used afterwards. It seems that the best results are obtained for the biosensors using the purified nucleotide base. One feasible reason to use another genetic compound such as herring and salmon sperm is their low cost. In contrast with enzymatic biosensors, transducers are not so much modified in this

FIGURE 4.8 Guanine oxidation to 8-oxoguanine.

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

TABLE 4.5 Features of the Most Relevant DNA-Based Biosensors Used for the Determination of Antioxidant Capacity in Beverages Biological Recognition Element

Transducer

Technique Employed

Guanine

GCE

SWV

AA/GA/ 0.29/0.09/0.06/ CA/COU/ 0.08/0.07 Resveratrol

0.52.5/0.10.5/ 0.40.8/0.310.73/ 0.100.5

Adenine

GCE

SWV

Deoxyadenylic acid

CPE

Thymus DNA

Sensitivity (µA/ppm)

Pretreatment Employed

2.82/9.33/ 8.76/9.20/ 11.8



AA/GA/ 0.99/0.08/0.07/ CA/COU/ 0.27/0.10 Resveratrol

26/0.110.44/ 0.4/7.38/ 0.10.5/0.11/0.10.5 11.9/3.81/ 8.78



SWV

AA/GA

2.7 (AA)

1030

CPE

DPV

GA/CA



Salmon sperm DNA

GCE/Polyvynil alcohol

CV

-

Salmon sperm DNA

SPCE Chitosan/Nafion CV, EIS



Deoxyadenylic acid

CPE

SWV

AA/GA/ 0.23 CA/p-COU

Herring sperm DNA

GCE/ Poly L-glutamic acid doped silver hybridized membrane

CV

AA



Guanine

GCE/ Graphene nanoribbon

SWV

AA

0.05

Analyte

Limit of Detection (ppm) Linear Range (ppm)

Sample

Reference

Flavored water

[179]

Filtered and diluted

Tea

[97]

2 10285 1028/ 167.53/ 7.5 10287.5 1027(M) 95.90 (μA/ mM)

Dried and extracted

Black [180] and green tea









White wine

[181]









Black tea, coffee

[182]

120

3.04

Filtered

Juice

[183]

1 10265 1025(M)



Filtered

Juice

[184]

0.14

4.16

Centrifuged and filtered

Juice

[185]










7.7 (AA)










AA, ascorbic acid; CA, caffeic acid; COU, coumaric acid; CPE, carbon paste electrode; CV, cyclic voltammetry; DPV, differential pulse voltammetry; EIS, electrochemical impedance spectroscopy; GA, gallic acid; GCE, Glassy carbon electrode; SPCE, screen printed carbon electrode; SWV, square wave voltammetry.

135

4.5 CONCLUSION

case. One possible reason is the difficulty to immobilize DNA onto the surface of the electrode. Nevertheless, some good results have been obtained using polymers and nanomaterials, such as polyvinyl alcohol [181] and silver nanoparticles [184]. Several membranes have been used in order to protect and provide robustness to these kinds of biosensors (chitosan, polyvinyl alcohol, etc.). Otherwise, metallic nanoparticles are used for the same reasons as for enzymatic biosensors, that is, enhanced conductivity and electrocatalytic effect. The lack of transducer modification reflects the difficulties to work with DNA instead of enzymes. In addition, the relevance of these kinds of biosensors relies on their ability to measure antioxidant capacity in an efficient way, very similar to real body conditions, hence higher sensitivities or limits of detection are not required. Voltammetric techniques such as SWV and CV are frequently used due to their ability to monitorize the DNA harming. Anodic SWV is applied in the detection of several compounds related to DNA damage. For instance, the analysis of thioridazine, an intercalator of these oxidative procedures, has been reported in the literature and as shown in Table 4.5 [182,186]. In addition, destruction of guanine and adenine nucleotide can be monitorized by using a similar procedure. Other compounds can be tested as electrochemical probes in order to evaluate the deterioration of DNA chain. Another approach based on the drop of the electrochemical response is the employment of EIS. Nyquist plots provide information about the resistance of the electrochemical system, which is related to the degradation phenomenon [187]. Regarding analytes used for electrochemical sensing, several similarities with Table 4.3 can be appreciated. The main compounds used are GA, CA, and resveratrol, among others. Ascorbic acid is also frequently used, since it is one of the most important benchmark analytes

reported in the literature. Beer and wine are much more commonly studied using enzymatic biosensors than DNA devices, since the application of these biosensors involves more simple matrices like teas and juices. Other complex samples contain more interfering species and protective membranes are necessary to avoid fouling processes. However, many difficulties arise due to the presence of these membranes. This is why, as shown in Table 4.4, pretreatments such as filtration, centrifugation, and dilution are frequently applied as well. To summarize this section, polyphenols have a critical role in the antioxidant capacity of highly consumed beverages. Consequently, there is a wide range of techniques to determine them individually and collectively. In this final Section, biosensors have been thoroughly discussed according to several advantages such as quick response, sensitivity, simplicity, robustness, limit of detection, low cost, and the possibility of in situ analyses. Two different approaches can be observed, using enzymes as biological recognition elements or the use of a DNA compound. In most cases enzymatic biosensors are more frequently used than the others. However DNA biosensors show a response that can be directly related with the antioxidant capacity. Hence the development of DNA-based biosensors is a very interesting and growing field that can expect a great raise in next years.

4.5 CONCLUSION As highlighted throughout this chapter, antioxidants and healthcare are intimately related to each other, since the former help us to prevent significant diseases affecting the population all around the world. This is why it is essential to possess a complete set of fast, simple, and versatile tools that are useful for the determination of these antioxidant

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136

4. ELECTROCHEMICAL BIOSENSORS FOR ANTIOXIDANTS

substances. Due to the advantages of electrochemical methods based on the use of biosensors, in terms of simplicity, rapidity, sample treatment, sensitivity, and limits of detection and quantification compared to classical analytical methodology, electrochemical biosensors are potentially improved alternatives for that purpose. In this chapter, the use of electrochemical biosensors in the assessment of various antioxidants in plants, food, and beverages was reported, paying special attention to the major achievements over the past decade. The preparation of oxidase enzymes-based electrochemical biosensors by various immobilization procedures has been thoroughly discussed from the point of view of the obtained analytical performances. The large number of selected applications commented on within this chapter clearly demonstrates the feasibility and utility of the electrochemical biosensors for the successful electroanalyses of antioxidants in real samples having complex matrix compositions. On the other hand, these results show that great attention has been devoted to the improvement of the analytical performance of biosensors in terms of limit of detection, linear response range, sensitivity, and stability. It was demonstrated that the enzyme immobilization procedure as well as the immobilization matrix play major roles in the improvement of the overall analytical performances. The reported limit of detection, linear response range, and sensitivity values underline the competitiveness of the electrochemical biosensors among other analytical methodologies. New directions in the development of biosensors for antioxidant determination may be related to the use of multienzymatic systems, more stable and size-reduced immobilization platforms, application of chemometrics tools in experimental data treatment, and the development of disposable biosensors.

4.6 Acknowledgments S. Lupu gratefully acknowledges the grant from the Romanian National Authority for Scientific Research, CNCSUEFISCDI, Project number PN-II-ID-PCE-2011-30271 (298/06.10.2011). J. J. Garcı´a-Guzma´n and D. Lo´pezIglesias acknowledge ESF funds, Sistema de Garantı´a Juvenil depending on Ministerio de Empleo y Seguridad Social of Spain and Junta de Andalucı´a for their employment contracts. The Spanish research group thanks Junta de Andalucı´a and the Institute of Research on Electron Microscopy and Materials (IMEYMAT) for their financial support.

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[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

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