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Sensors and Actuators B: Chemical
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ioactive paper platform for colorimetric phenols detection
arcella Arciuli a, Gerardo Palazzob, Anna Gallonea,∗, Antonia Mallardi c,∗∗
Dipartimento di Scienze Mediche di Base, Neuroscienze ed Organi di Senso, Università di Bari, ItalyCSGI and Dipartimento di Chimica, Università di Bari, ItalyCNR-IPCF, Istituto per i Processi Chimico-Fisici, UOS di Bari, Italy
a r t i c l e i n f o
rticle history:eceived 10 March 2013eceived in revised form 7 June 2013ccepted 16 June 2013vailable online 24 June 2013
In this study the development of a low cost, portable and disposable paper-based bioassay for phenoliccompounds is described. The colorimetric detection of the analyte is based on an enzymatic assay. Thetyrosinase enzyme has been immobilized on a filter paper by simple over-spotting with 3-methyl-2-benzothiazolinone hydrazone (MBTH), that allows the detection of phenols by forming stable colouredadducts with their enzymatic oxidation products. The colour intensity of the adduct (developed after5 min of reaction) was found to increase proportionally with the increase of the phenolic substrate con-centrations. Analyte detection can be achieved by eye and quantification can be simply obtained by usinga camera phone and an image analysis software.
The response characteristics of the sensor were determined using l-3,4-dihydroxyphenyl-alanine (l-DOPA), an archetype substrate of tyrosinase, as the analyte. The sensor gave a LOD of 5 �M and a linear
ine analysis response up to 0.5 mM. The polyphenol content in wine was determined by the biosensor and resultswere compared with that obtained for the phenolic-pool determined by tyrosinase enzymatic assay insolution and for the antioxidant-pool probed by the Folin–Ciocalteau assay.
This paper-based platform holds potential for the detection of different types of phenolic compounds.The proposed assay has the advantage of rapidity and simplicity over other detection methods, withoutneed of sophisticated instrumentation and trained personnel.
. Introduction
Phenolic compounds are widely used chemicals which findpplications in several fields. Phenolic derivatives may be pollutingndustrial products released into the environment and adsorbedy animals and humans with detrimental effects on health [1].olyphenols, as food antioxidants, are of great interest due to theirealth benefits as they decrease the risks of cancer and coro-ary cardiopathy [2]. Moreover they influence the quality andrganoleptic characteristics of foods [3]. Lastly, some neurotrans-itters are phenolic compounds. On this basis, a need has arisen
or fast, sensitive and inexpensive detection methods to monitor
hese compounds over a wide interval of concentrations depend-ng on the application/matrix. The example of the neurotransmitter-DOPA clarify this last point: values of l-DOPA in body fluids are
∗ Corresponding author at: Dipartimento di Scienze Mediche di Base, Neuro-cienze ed Organi di Senso, Facoltà di Medicina e Chirurgia, Universita’ degli Studii Bari, Palazzina del Nuovo Complesso di Scienze Biomediche – Policlinico, Piazzaiulio Cesare, Bari I-70124, Italy. Tel.: +39 080 5448555; fax: +39 080 5448538.
∗∗ Corresponding author at: Istituto per i Processi Chimico Fisici – CNR, Viarabona, 4, Bari I-70126, Italy. Tel.: +39 080 5442028; fax: +39 080 5442128.
below few �g/L, but its concentration can reach the mg/L level inpatient treated with exogenous l-DOPA [4] and in pharmaceuticalformulations it can reach the 80% by weight (e.g. in Madopar pillsthe composition is l-DOPA 200 mg/benserazide 50 mg).
In recent years, there has been a growing interest in the devel-opment of cost-effective and practical analytical tools in the food,medicine, and environmental sectors. Paper has been used as aplatform in analytical and clinical chemistry due to the fact thatit is abundant, inexpensive, sustainable, disposable and easy touse, store and transport [5–8]. Paper-based bio-assays have beendeveloped which can function without laboratories, significantinstrumentation or trained personnel.
In these colorimetric platforms the sample can be delivered bysimple spotting [9,10], or by both patterned microfluidic [11–13]and lateral flow [14,15].
These simple and portable diagnostic tests can be a good alter-native for point-of-need testing and could be extremely useful inremote locations or developing countries which do not have readyaccess to laboratory facilities and where simple, sensitive and lowcost bioassays are essentials.
The purpose of this work is to develop a bioactive paper-basedstrip test, which allows the portable sensing for colorimetric detec-tion of phenolic compounds. The bioassay is based on the tyrosinaseenzyme which catalyses the conversion of phenolic substrates to
Fig. 1. Picture of the filter paper with the bioactive spots. In the middle part of thefilter are shown the spots of the calibration (concentrations of l-DOPA ranging from0.01 to 10 mM). Spots marked with “C” are blanks. In the upper part of the filter five
58 M. Arciuli et al. / Sensors and
atechol and then to quinone [16]. The enzyme was immobilizedn paper by simple adsorption (spotting) without any further stabi-ization treatment. The determination of phenolic compounds waserformed by simple visualization of quinone product, by usinghe reagent 3-methyl-2-benzothiazolinone hydrazone hydrochlo-ide hydrate (MBTH) which interacts with quinone in the enzymaticeaction to produce a stable coloured adduct [17]. The analyte col-rimetric detection is very compelling, because it provides resultshich can be observed by the naked eye, recorded by a simple cam-
ra phone and transmitted to a computer for further quantificationf the digital results. The assay was successfully applied for theetermination of polyphenols in a real matrix (wine).
. Experimental
.1. Reagents
Lyophilized mushroom tyrosinase from Agaricus bisporusMT) (EC.1.14.18.1; 4187 U/mg), 3-methyl-2-benzothiazolinoneydrazone hydrochloride hydrate (MBTH), l-3,4-dihydroxyphenyl-lanine (l-DOPA) and Folin–Ciocalteau reagent were purchasedrom Sigma–Aldrich. All other chemicals were of analytical gradend were used without further purification. All aqueous solutionsere prepared with bidistilled water from Carlo Erba Reagents.oth enzyme and l-DOPA solutions were prepared in a 50 mMa–phosphate buffer, pH 6.8. Wines of different types werecquired from a local supermarket. The only sample treatmenterformed consisted of a 1/1 vol:vol dilution with water beforenalysis.
.2. Procedures
.2.1. Fabrication of bioactive paper sensorPrior to constructing an efficient paper-based sensor, all reagent
oncentrations were optimized initially in solution and later onaper in single-factor experiments (varying one parameter at aime).
The bioactive paper sensor was prepared on round-shapedhatman no. 4 filter papers. The sensing spot was obtained by
equentially depositing on the very same position 1 �L each of00 mM Na–phosphate buffer at pH 6.8, 10 mg/mL tyrosinase solu-ion, and finally 6 mM MBTH (in 100 mM Na–phosphate buffer, pH.8) solution via a micropipette; this results in a bioactive spot of
mm of diameter. After deposition of each component the paperas allowed to dry in air for 1 min at room temperature. The same
ound-shaped filter accommodates several spots (centre-to-centreistance of about 15 mm) that will be used for the calibration curve,or the blanks and for the unknown samples. The prespotted paperlters were stored at 4 ◦C, covered by an aluminium foil withoutxcluding air.
.2.2. Colorimetric assayThe presence of phenols was assayed on the bioactive paper strip
y a tyrosinase-based assay. A sample drop of 1 �L was placed onhe bioactive spot. When phenols are present, they are oxidized byhe tyrosinase enzyme forming a stable pink adduct with the MBTH.oloured spots appear on the paper within few minutes. The filter isllowed to dry in air (5 min are enough) and a digital image is taken.n the same paper filter can be deposited (on different bioactive
pots) drops of: solutions at different substrate concentrations foralibration, buffer without substrate for the blank (5 replicates), andhe samples to be assayed. A typical arrangement of the bioactive
pots on the filter is shown in Fig. 1. The reference analyte used inhis study for the calibration was always the l-DOPA.
For stability studies, bioactive spots were prepared simulta-eously on different filters that have been stored under the same
wine samples have been loaded (in triplicate). Wines are Franciacorta (Fc), Pinot (P),Sauvignon (S), Table wine (T), Frascati (F).
conditions. The colour intensity developed by bioactive spots uponloading with the same l-DOPA concentration was then measuredat different times.
2.2.3. Quantification methodThe digital image of the whole filter paper (containing several
spots) can be taken by using different devices with different res-olutions such as professional digital cameras, scanners or cameraphone. Since our goal is a simple, fast and low-cost assay, the colourintensity of the sensing areas was quantified by means of a cam-era phone. Camera phones have several advantages: are portable,lightweight, with high digital resolution and have a widespread dif-fusion also in less developed countries. When used as a detector forpaper-based assays, camera phones easily transmit the image to anoff-site laboratory, where the data can be analyzed.
The images of filter papers with bioactive spots have been regis-tered using the camera of a smartphone (Samsung GT-I9100 GalaxyS) and directly sent to a personal computer by e-mail. The photoshave been taken holding the smartphone by hands (without anycamera support systems) and using the customary illuminationpresent in the lab (no flash or ad hoc illumination set-up).
The ImageJ free software [18] has been used to analyze thejpeg images. This software furnishes a numerical value of the spotcolour intensity (in arbitrary units). Colour intensity has beenassessed leaving out the edges of spots. To account for variationsin colour intensity owing to differences in environmental illumi-nation, a background subtraction (colour intensity of the papersurface closest to the sensing area) was done for each data point.The colour intensity of the blank spots was averaged giving themean intensity of the blank I0. The sensor response was finallyevaluated as the fractional change in colour intensity, i.e. as �I/I0where �I represents the difference between the colour intensityof the spot in presence of analyte (calibration or unknown) and themean blank intensity I0. Also the data from blank spots have beentreated in such a way giving a null mean blank response and a
standard deviation of the blank SB. The SB value was profitably usedto estimate either the limit of detection (LOD), as the concentrationcorresponding to a response that is three times the SB, either the
Actuators B 186 (2013) 557– 562 559
lt
2
omtTlaMct6a
2
i1wtowTs
w
3
3
napobu(ppat
sAoaA
sa
aoowcoacgT
0 2 4 6 8 100
2
4
6
8
10
12
Δ I/I
0
L-DOPA (mM)
0
1
2
3
4
10
2× V
0 (
Ab
s/s
)
Fig. 2. Calibration curve for l-DOPA. Full circles and left ordinate: fractional inten-sity of colour developed on the spots of the bioactive paper (quantified by usingImage J software upon digital image acquisition). The points refer to the mean ofeight calibrations, error bars = ± standard deviation. Solid line represents the non-
M. Arciuli et al. / Sensors and
imit of quantification (LOQ), as the concentration correspondingo a response that is ten times the SB [19,20].
.2.4. Enzymatic assay in solutionFor comparison purposes, phenol determination in wines was
btained in solution by Winder and Harris method, with minorodification [21]. The method is based on the estimation of the ini-
ial rate of the l-DOPA oxidation catalyzed by tyrosinase enzyme.he reaction was spectrophotometrically followed (for 5 min) using-DOPA or wine as substrates and recording the rate of increase inbsorbance at 505 nm due to the formation of adducts betweenBTH and l-DOPA oxidation. Briefly, 100 �L of l-DOPA at different
oncentrations (for calibration) or wine (as unknown) were addedo 890 �L of 100 mM Na–phosphate buffer at pH 6.8, containing
mM MBTH. The reaction was started with the addition of 10 �L of 1 mg/mL tyrosinase solution.
.2.5. Folin–Ciocalteau assayWines were also analyzed by the spectrophotometric method
nvolving the use of Folin–Ciocalteau reagent [22]. In this method,.58 mL of deionized water and 0.10 mL of Folin–Ciocalteau reagentere added to 0.02 mL of wine (appropriately diluted with bidis-
illed water). The mixture was stirred for about 10 min, then 0.3 mLf a 20% Na–carbonate solution was added. The resulting solutionas allowed to stand for 2 h at room temperature in darkness.
he absorbance was then read at 765 nm by using a JASCO V-530pectrophotometer.
The same procedure was used to construct a calibration plotith standard solutions of l-DOPA.
. Results and discussion
.1. Bioactive paper assay sensing performances
With the aim to test the effective catalytic properties of tyrosi-ase in a dry form on paper and in view of potential applications sensor, experiments are performed using the o-diphenolic com-ound l-DOPA, an archetype substrate of tyrosinase, as the analyte.-Diphenols are usually unsuited for end-point enzymatic assaysased on tyrosinase because the resulting o-quinones are verynstable and undergo to a sequence of non-enzymatic reactionspolymerizations included) that preclude any isolation of an endroduct. To use the end-point approach, the addition of a nucleo-hilic compound which is able to produce a stable stoichiometricdduct with the o-quinones is required [17]. In this case, we addedhe MBTH reagent.
Both tyrosinase and MBTH have been deposited by simpleequential over spotting on a filter paper made of pure cellulose.fter the l-DOPA addition, its enzymatic oxidation produces an-quinone which reacts with MBTH to produce a pink coloureddduct, visualized as a spot on the paper after 5 min of exposure.fter this time no further colour change was observed.
The middle part of the filter paper shown in Fig. 1 shows thepots of the calibration obtained using l-DOPA as the referencenalyte.
The resulting spot colour changes from almost colourless to pinkt increasing of the substrate concentration. After colour devel-ping the sensing results can be first determined by naked eyebservation and then, as already reported by Whitesides and co-orkers [12], it is possible obtain quantitative analysis by using
olour image managing tools and software. We captured the resultsf the colorimetric assay by a camera phone (a digital camera or
portable scanner can be also used), and transmitted electroni-ally the image to a personal computer. The “Image J” softwareave the colour intensity of the pictures obtained from the assay.he obtained data allowed the assembly of the calibration curve,
linear best fit of the data to Eq. (1). Hollow stars and right ordinate: initial rate (V0)of l-DOPA oxidation obtained in an experiment performed in solution.
relating the response (�I/I0 i.e. the fractional increment in colourintensity) to the substrate concentration, reported in Fig. 2.
An hyperbolic growth and saturation upon increasing the sub-strate (l-DOPA) concentration is evident. Accordingly, the data havebeen fitted by nonlinear regression to the following equation
�I
I0= ImaxC
K ′m + C
(1)
Eq. (1) has the same form of the Michaelis and Mentenrelationship being c the substrate concentration and Imax themaximum response attainable; K ′
m represents the apparentMichaelis–Menten constant. The best fit values obtained are: K ′
m =(0.84 ± 0.04) mM and Imax = (11.4 ± 0.02) a.u.; the trend is fully con-sistent with the Michaelis–Menten dependence of reaction initialrate on the l-DOPA concentration observed in independent exper-iments performed in solution (stars in Fig. 2). Accordingly, the K ′
mvalue is also consistent with the literature results for the tyrosi-nase activity in solution assayed with MBTH that reports a trueMichaelis–Menten constant of 0.8 mM [23]. The points (error bars)in Fig. 2 refer to the mean (standard deviation) of eight calibra-tions performed on different filter papers in different days. Fig. 3Ashows the data of Fig. 2 in the low concentration range (0–0.5 mM).Here the data points obtained from calibrations performed on dif-ferent filter papers are indicated by different symbols (for the sakeof readability only four calibrations are shown). The scattering ofthe points at each concentration is a measure of the reproducibilityof the assay; the spread of the response is usually below 5% butreaches 15% for some points. In this concentration range the sensorresponse to l-DOPA loading is approximately linear. Accordinglythe low concentration data (<0.5 mM) have been fitted to a straightline and the analytical performances of the bioactive paper havebeen examined. The analytical sensitivity (defined as the slope ofthe response vs the concentration) is 9.5 mM−1. The standard devi-ation of blank response is SB = 0.06 and from this value the LOD,estimated as the concentration corresponding to a response that
is 3 SB, was estimated to be 5 �M while the LOQ, estimated as theconcentration corresponding to a response that is 10 SB, is 50 �M.
The LOD value is very close to the polyphenol detection limitachieved by tyrosinase-based biosensors using electrochemical
560 M. Arciuli et al. / Sensors and Actuators B 186 (2013) 557– 562
0.00 0.02 0.04 0.06 0.08 0.100
1
2
3
4
10
3×V
0 (
Abs/s
)
L-DOPA (mM)
0.0 0.1 0.2 0.3 0.4 0.50
1
2
3
4
5
LOD l evel
DI/I0
L-DOPA] (mM)
LOQ le vel
A
B
Fig. 3. Linear low concentration range calibrations. (A) The results obtained fromfour separate calibrations performed on different filter papers are shown with dif-ferent symbols. Solid line is the linear best fit. LOD and LOQ levels are evidenced. (B)The results obtained from a tyrosinase-based enzymatic assay performed in solutionare shown; the ordinate represents the initial rate (V0) of absorbance increase. Notetr
[hb
ots
fiptDbfcMctrslf(twp
0.1 1 10
0
2
4
6
8
10
12
t= 3 weeks
t= 4 weeks
t= 2 weeks
ΔI/I
0
L-DOP A ( mM)
t=0
t= 1week
LOQ level
0 1 2 3 4 5
2
4
6
8
10
12(ΔI/I )@8mM
time (weeks)
half-life= 1.4 weeks
Fig. 4. Time stability of the tyrosinase activity on the bioactive paper. Calibrations
hat since the assay in solution imply a 1/10 dilution of the wine the concentrationange is limited to 0–0.11 mM. Solid line is the linear best fit.
24] or optical [25,26] techniques, while is one order of magnitudeigher of that obtained by Alkasir et al. [9] using a paper sensorased on a complex enzyme polyelectrolyte architecture.
More important, the LOD value we found is well below the rangef polyphenol concentrations commonly found in wines [27–29],hen our assay is applicable to polyphenol determination in realample matrices (see below).
The long-term stability of the tyrosinase immobilized in thelter paper has been also investigated. Bioactive spots were pre-ared simultaneously on several filters that have been stored inhe dark at 4 ◦C. Weekly one of the filter was loaded with l-OPA and buffer solutions to give the calibration curve and thelanks. The calibration curves obtained from active filters after dif-erent times from preparation are shown in Fig. 4, plotting theoncentration on a logarithmic scale. In this representation theichaelis–Menten behaviour appears sigmoidal and allows an easy
omparison with the response corresponding to the LOQ (horizon-al dashed line); the concentration at which each calibration curveeach this LOQ-level is the limit of quantification. The decay of theensor response follows a first-order kinetics with an apparent half-ife time of about 1.4 weeks (inset) and the LOQ increases passingrom 80 �M after one week to 0.16 mM (two weeks) and 0.3 mM
three weeks). It should be emphasized that either the LOQ eitherhe residual 37% of activity still present in the biosensor after twoeeks from its preparation are enough to allow its use for sensingurposes.
were performed weekly on bioactive papers prepared in the same conditions andstored at 4 ◦C. The response corresponding to the LOQ is evidenced. Inset shows�I/I0 values obtained in each calibration at 8 mM l-DOPA concentration.
3.2. Polyphenol content in wine
The practical usefulness of the assay was evaluated on real sam-ples, using commercial white wines. Wines contain a variety ofphenolic compounds (tannins) which cannot be determined singlyso they are measured collectively as so-called total “polyphenolindex” [30]. The amount of polyphenols is important given theirantioxidant properties and their impact in wine sensorial features.In addition they have gained increasing attention owing to theirpossible health benefits.
Since white wine is barely coloured it can be assayed directlywithout any pre-treatment beside a 1/1 dilution with distilledwater. The samples were analyzed by direct addition of an aliquot(1 �L) of diluted wine onto the paper strip and using l-DOPA asthe phenol standard. The polyphenol content of the sample wasdetermined as l-DOPA equivalent, expressed as mmol L−1. At leastthree determinations have been taken for each wine (see picture inFig. 1), and the results (mean and standard deviation) for the fivewines examined are listed in Table 1.
The same wines were also analyzed according to two differentsolution assays: the Winder–Harris enzymatic assay [21] and theFolin–Ciocalteau assay for antioxidant [22] commonly used in wineindustries.
It should be stressed that the two assays probe different chemi-cal properties. The Winder–Harris enzymatic assay can be thoughtas the solution counterpart of the paper-based assay we propose.It is based on the estimation of the enzymatic rate (a representa-tive calibration in the linear low concentration range is shown inFig. 3B) and it probes the substrates of tyrosinase activity i.e. thetrue phenolic pool. Compared to the paper-based assay, the enzy-matic solution assay requires larger amounts of reagents/sample,a relatively sophisticated instrumental setup (spectrophotometer)to collect the kinetics of absorbance change and it is time consum-ing because kinetics have to be measured over five minutes for eachcalibration/unknown point.
Folin–Ciocalteau assay is based on the chemical reduction ofthe reagent, a mixture of tungsten and molybdenum oxides. Thisreagent, by reacting with the phenol OH group, slowly developed ablue coloured complex whose absorbance is read, after two hoursof incubation, at 765 nm. The resulting solutions, being made byheavy metals, must be treated as hazardous waste. It should be
noticed that, being based on a chemical reduction, such an assayis positive to a several reductants other than phenolics, so thatit strictly probes the antioxidant-pool instead of the polyphenol-pool. As a consequence the Folin–Ciocalteau assay overestimates
M. Arciuli et al. / Sensors and Actuators B 186 (2013) 557– 562 561
Table 1Estimation of the content of phenolic compounds in wines by using the tyrosinase bioactive filter-paper, the Winder–Harris assay and the Folin–Ciocalteau assay. The phenolindex is expressed as l-DOPA equivalent (mM). Values represent the mean (standard deviation) of at least three replicates.
Wine type Bioactive filter-paper Winder–Harris assay Folin–Ciocalteau assay
he phenolics content when other reductant molecules are present31].
The values obtained by these standard assays on the very sameines tested by the paper-based assay are listed in Table 1. The val-es obtained for the phenolic pool by the assay based on bioactivelters and by the enzymatic assay in solution are mutually close forll the wines. Indeed the statistical comparison of the mean valuesbtained by the two methods indicates that there are not significantifference between the values (differences are not significant at the% level applying t-test for two sample means [32]). On the con-rary, the antioxidant-index evaluated using the Folin–Ciocalteaussay is strongly larger than the phenolic-index in all the wine sam-les but not in the Sauvignon. This is in agreement with the wide
iterature demonstrating how the Folin–Ciocalteau usually highlyverestimates the phenolic pool because of interference with sul-hites, reducing sugars and non-phenolic antioxidants [28–31].
. Conclusions
In this study, we have reported a method to prepare a veryimple, portable bioactive paper sensor which allows for rapidfew minutes) and sensitive colorimetric detection of phenolicompounds. The determination is based on an enzymatic assay.yrosinase is coimmobilized on Whatman filter paper with theolouring agent MBTH that allows detection of oxidation productsf phenols by forming stable pink colour adducts. Detection can bechieved by eye and in-field quantification can be simply achievedy using a camera phone and image analysis software running on
portable computer, avoiding the need for ad-hoc instrumentationnd trained personnel.
This bioactive paper sensor presents many advantages listedn the following. (1) Entrapment of the sensing components ontohe paper platform is obtained by the use of simple over spot-ing deposition without any further stabilization treatment. (2) Thessay does not require addition of external reagents, because allhe sensing components are deposited onto the paper substrate.he only step needed to perform the analysis is the addition ofhe analyte. (3) Rapid response time: “spot and read” colorimetricetection without need of additional sophisticated instrumenta-ion. This should be compared with classical enzymatic assay inolution that requires the collecting of kinetic traces over severalinutes for each calibration/unknown point. (4) The simplicity of
potting the sample followed by visual readout is attractive foroint-of-need applications. (5) Our method is less expensive inomparison to other discussed ones, because of the very small vol-me of chemicals used, the absence of hazardous waste and of themployment of a single filter paper instead of several disposablelastic cuvettes for each determination.
The sensors presented in our paper exhibits a good analyticalerformance associated with a simple and fast preparation, reliabil-
ty and low cost. Otherwise it represents a good and easy methodor monitoring polyphenols in real samples and the accuracy of the
easurements is comparable with that obtained with the referencepectrophotometric method.
[
0.15 ± 0.10 0.22 ± 0.050.21 ± 0.09 1.0 ± 0.12
References
[1] D. Puig, D. Barceló, Determination of phenolic compounds in water and wastewater, Trends in Analytical Chemistry 15 (1996) 362–375.
[2] D. Del Rio, L.G. Costa, M.E. Lean, A. Croizer, Polyphenols and health: what com-pounds are involved? Nutrition, Metabolism, and Cardiovascular Diseases 20(2010) 1–6.
[3] I. Ignat, I. Volf, V.I. Popa, A critical review of methods for characterisation ofpolyphenolic compounds in fruits and vegetables, Food Chemistry 126 (2011)1821–1835.
[4] J. Dutton, L.G. Copeland, J.R. Playfer, N.B. Roberts, Measuring l-dopa in plasmaand urine to monitor therapy of elderly patients with Parkinson disease treatedwith l-dopa and a dopa decarboxylase inhibitor, Clinical Chemistry 39 (1993)629–634.
[6] C. Parolo, A. Merkoc i, Paper-based nanobiosensors for diagnostics, ChemicalSociety Reviews 42 (2013) 450–457.
[7] R. Pelton, Bioactive paper provides a low-cost platform for diagnostics, Trendsin Analytical Chemistry 28 (2009) 925–942.
[8] W. Zhao, A. Ven den Berg, Lab on paper, Lab on a Chip 8 (2008) 1988–1991.[9] R.S.J. Alkasir, M. Ornatska, S. Andreescu, Colorimetric paper bioassay for the
detection of phenolic compounds, Analytical Chemistry 84 (2012) 9729–9737.10] H.A. Oktem, O. Senyurt, F.I. Eyidogan, C. Bayrac, R. Yilmaz, Development of a
laccase based paper biosensor for the detection of phenolic compounds, Journalof Food, Agriculture and Environment 10 (2012) 1030–1034.
11] X. Chen, J. Chen, F. Wang, X. Xiang, M. Luo, X. Ji, Z. He, Determination of glucoseand uric acid with bienzyme colorimetry on microfluidic paper-based analysisdevices, Biosensors and Bioelectronics 35 (2012) 363–368.
12] A.W. Martinez, S.T. Phillips, E. Carrilho, S.W. Thomas III, H. Sindi, G.M.Whitesides, Simple telemedicine for developing regions: camera phones andpaper-based microfluidic devices for real time, off-site diagnosis, AnalyticalChemistry 80 (2008) 3699–3707.
13] J. Yu, S. Wang, L. Ge, S. Ge, A novel chemiluminescence paper microfluidicbiosensor based on enzymatic reaction for uric acid determination, Biosensorsand Bioelectronics 26 (2011) 3284–3289.
14] S.M.Z. Hossain, R.E. Luckham, M.J. Mc Fadden, J.D. Brennan, Reagentless bidi-rectional lateral flow bioactive paper sensors for detection of pesticides inbeverage and food samples, Analytical Chemistry 81 (2009) 9055–9064.
15] S.M.Z. Hossain, J.D. Brennan, �-galactosidase-based colorimetric paper sensorfor determination of heavy metals, Analytical Chemistry 83 (2011) 8772–8778.
16] J.D. Simon, D. Peles, K. Wakamatsu, S. Ito, Current challenges in understand-ing melanogenesis: bridging chemistry, biological control, morphology, andfunction, Pigment Cell Melanoma Research 22 (2009) 563–579.
17] F. García-Molina, J.L. Munoz, R. Varón, J.N. Rodríguez-López, F. García-Cánovas,J. Tudela, A review on spectrophotometric methods for measuring themonophenolase and diphenolase activities of tyrosinase, Journal of AgriculturalFood Chemistry 55 (2007) 9739–9749.
18] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years ofimage analysis, Nature Methods 9 (2012) 671–675.
19] M. Thompson, S.L.R. Ellison, R. Wood, Harmonized guidelines for single-laboratory validation of methods of analysis, Pure and Applied Chemistry 74(2002) 835–855.
20] ISO 11843, Capability of detection (several parts), International StandardsOrganisation, Geneva.
21] A.J. Winder, H. Harris, New assays for the tyrosine hydroxylase and dopa oxi-dase activities of tyrosinase, Journal of Biochemistry 198 (1991) 317–326.
22] O. Folin, W. Denis, On phosphotungstic–phosphomolybdic compounds ascolour reagents, Journal of Biological Chemistry 12 (1912) 239–243.
23] J.C. Espin, R. Varon, L.G. Fenoll, M.A. Gilabert, P.A. Garcia-Ruiz, J. Tudela, F.Garcia-Canovas, Kinetic characterization of the substrate specificity and mech-anism of mushroom tyrosinase, European Journal of Biochemistry 267 (2000)1270–1279.
24] C. Apetrei, P. Alessio, C.J.L. Constantino, J.A. de Saja, M.L. Rodriguez-Mendez,F.J. Pavinatto, E. Giuliani Ramos Fernandes, V. Zucolotto, O.N. Oliveira Jr.,Biomimetic biosensor based on lipidic layers containing tyrosinase and
lutetium bisphthalocyanine for the detection of antioxidants, Biosensors Bio-electronics 26 (2011) 2513–2519.
25] J. Abdullah, M. Ahmad, N. Karuppiah, L.Y. Henga, H. Sidek, Immobilization oftyrosinase in chitosan film for an optical detection of phenol, Sensors andActuators B 114 (2006) 604–609.
Antonia Mallardi is Researcher at the Institute for Chemical and Physical Processesof the National Council of Research (CNR) of Italy. Her main research activitiesdeal with the biophysical chemistry of membrane proteins and deposition ofbiomolecules and biosensors design.
62 M. Arciuli et al. / Sensors and
26] D. Fiorentino, A. Gallone, D. Fiocco, G. Palazzo, A. Mallardi, Mushroom tyrosi-nase in polyelectrolyte multilayers as an optical biosensor for o-diphenols,Biosensors and Bioelectronics 25 (2010) 2033–2037.
27] L. Campanella, A. Bonanni, E. Finotti, M. Tomassetti, Biosensors for deter-mination of total and natural antioxidant capacity of red and white wines:comparison with other spectrophotometric and fluorimetric methods, Biosen-sors and Bioelectronics 19 (2004) 641–651.
28] V. Carralero Sanz, M.L. Mena, A. Gonzalez-Cortés, P. Yanez-Sedeno, J.M.Pingarron, Development of a tyrosinase biosensor based on gold nanoparticles-modified glassy carbon electrodes. Application to the measurement of abioelectrochemical polyphenols index in wines, Analytica Chimica Acta 528(2005) 1–8.
29] M. Di Fusco, C. Tortolini, D. Deriu, F. Mazzei, Laccase-based biosensor for thedetermination of polyphenol index in wine, Talanta 81 (2010) 235–240.
30] A.J. Blasco, M.C. Rogerio, M.C. Gonzalez, A. Escarpa, “Electrochemical index” asa screening method to determine “total polyphenolics” in foods: a proposal,Analytica Chimica Acta 539 (2005) 237–244.
31] A.L. Waterhouse, Determination of total phenolics, in: Current Protocols in FoodAnalytical Chemistry, John Wiley & Sons, Inc., 2001.
32] J.N. Miller, J.C. Miller, Statistics and Chemometrics for Analytical Chemistry, 4thed., Prentice Hall, Harlow, England, 2000.
iographies
arcella Arciuli has a PhD in “Biochemistry and Medical Biology” and workst the Department of Basic Medical Sciences, Neurosciences and Sense Organs,
tors B 186 (2013) 557– 562
Faculty of Medicine of Bari. Her research activity focuses on the study of tyrosi-nase of pigmented cell of cold-blooded Vertebrate and Mammals. She performschemical, enzymatic and structural characterization of tyrosinase by mean spec-trophotometric and radiometric analysis. She also assesses regulation aspects of themelanogenic system by using molecular studies of gene expression.
Gerardo Palazzo is an Associate Professor in Physical-Chemistry at the University ofBari. The main research activities of Gerardo Palazzo deal with biophysical chemistryof proteins and the characterization of membrane and complex fluids by means ofphysicochemical techniques (NMR, SAXS, SANS, etc.)
Anna Gallone is an Associate Professor in Applied Biology at the University of Bariand lecturer of Biology in Courses of Faculty of Medicine. Her research activitiesconcern with the following scientific topics: cytological, biochemical and physicalstudies on pigment cells; molecular studies on regulation of the melanogenic sys-tem; development of tyrosinase-based biosensors and biotechnological applicationsof the melanogenic systems; molecular systems for the detection of food-deliveredpathogens.
