New Ultralow-Cost Telemetric System for a RapidElectrochemical Detection of Vitamin C in FreshOrange Juice

Antonio Barberis,† Gianfranco Bazzu,‡ Giammario Calia,‡ Giulia M. G. Puggioni,‡

Gaia G. Rocchitta,‡ Rossana Migheli,‡ Mario Schirra,† Maria Speranza Desole,‡ andPier Andrea Serra*,‡

Institute of Food Sciences, National Research Council, Traversa La Crucca, 3 Regione Baldinca,07040 Li Punti, Sassari, Italy, and Department of Neuroscience, Medical School, University of Sassari,v.le S. Pietro 43/b-07100, Sassari, Italy

Ascorbic acid (AA), one of the principal micronutrientsin horticultural crops, plays a key role in the humanmetabolism, and its determination in food products hasa great significance. Citrus fruits are rich in AA, but itscontent is highly susceptible to change during postharvestprocessing and storage. We present a new ultralow-costsystem, constituted of an amperometric microsensorcomposed of three rod carbon electrodes connected to atelemetric device, for online detection of AA in orangejuice, as an alternative to conventional analytical methods.The in vitro calibration, ranged from 0 to 5 mM, and AAjuice content was calculated by adding low volumes ofsample into an acetate buffer solution at a constantpotential of +120 mV vs carbon pseudoreference. Thisnew approach, which is simple, expandable, and inex-pensive, seems appropriate for large scale commercialuse.

Ascorbic acid (AA, vitamin C), a water-soluble antioxidant, isone of the principal micronutrients in horticultural crops. It ispresent in variable amounts in fresh fruit and vegetables, and itis often added to foodstuffs as an antioxidant and a stabilizer ofcolor and aroma. It plays an important role in the humanmetabolism, helping to prevent many diseases, like cancer,diabetes mellitus, stroke,1 and Parkinson’s disease,2 most of themlinked to oxidative stress. Epidemiological studies underlined AAfunction as a free-radical scavenger and demonstrated that dietsrich in AA are often associated with lower risk of cardiovasculardiseases.3 Most mammals are able to synthesize ascorbic acidfrom glucose; but AA is not produced by human beings, and itmust be introduced with diet.4 AA is involved in several biological

reactions such as collagen, carnitine and catecholamine synthesis,peptide amidation, and tyrosine metabolism. In the brain, AA isthe most important component of the antioxidant pool and actsas a radical scavenger against reactive oxygen species (ROS) andreactive nitrogen species (RNS) and it is also implicated inprotection against glutamate excitotoxicity. Recently, vitamin Cis also proposed as a modulator in the neuronal energeticmetabolism.5

Many horticultural crops and citrus fruits, in particular,represent an important source of AA, depending on species,variety, cultural practice, fruit age, climate, and postharvesthandling; its content usually declines during fruit storage orprocessing,6 thus influencing the quality of fresh products,7 theirderived juices,8 or other byproducts.

Due to the importance of this analyte, the determination ofAA in food products, natural or transformed, has a great signifi-cance. A large number of methods of analysis have been applied tofruits, vegetables, biological fluids, and pharmaceuticals. They areusually based on spectrophotometric9 and nonspectrophotometric10,11

techniques among which the titrimetric12-14 and chromatographic15

ones are the most commonly used. All these procedures can providea precise and accurate assessment of AA when applied in thelaboratory. However, they are time-consuming and currently tooexpensive to be adopted into most routine quality control operations.

* Corresponding author. E-mail: paserra@uniss.it. Tel: +39079228558. Fax:+39079228525.

† National Research Council.‡ University of Sassari.

(1) Yokoyama, T.; Date, C.; Kokubo, Y.; Yoshiike, N.; Matsumura, Y.; Tanaka,H. Stroke 2000, 31, 2287–2294.

(2) Serra, P. A.; Sciola, L.; Delogu, M. R.; Spano, A.; Monaco, G.; Miele, E.;Rocchitta, G.; Miele, M.; Migheli, R.; Desole, M. S. J. Biol. Chem. 2002,277, 34451–33461.

(3) Civit, L.; Nassef, H. M.; Fragoso, A.; O’Sullivan, C. K. J. Agric. Food Chem.2008, 56, 10452–40455.

(4) Hediger, M. A. Nat. Med 2002, 8, 445–446.

(5) Castro, M. A.; Beltran, F. A.; Brauchi, S.; Concha, I. I. J. Neurochem. 2009,110 (2), 423–440.

(6) Lee, S. K.; Kader, A. A. Postharvest Biol. Technol. 2000, 20, 207–220.(7) Schirra, M.; Palma, A.; D’Aquino, S.; Angioni, A.; Minello, E. V.; Melis, M.;

Cabras, P. J. Agric. Food Chem. 2008, 56, 455–460.(8) Lee, H. S.; Coates, G. A. J. Agric. Food Chem. 1997, 45, 2550–2555.(9) Rahman Khan, M. M.; Rahman, M. M.; Islam, M. S.; Begum, S. A. J. Biol.

Sci. 2006, 6 (2), 388–392.(10) Sauberlich, H. E.; Green, M. D.; Omaye, S. T. Determination of ascorbic

acid and dehydroascorbic acid. In Ascorbic acid: Chemistry, Metabolism, andUses; American Chemical Society: 1982, Vol. 200 (9); pp 199-221.

(11) Arya, S. P.; Mahajan, M.; Jain, P. Anal. Chim. Acta 2000, 417, 1–14.(12) Horwitz, W. Official Methods of the Association of Official Analytical Chemists,

Association of Official Analytical Chemists: Washington, DC, 1980, 13thed.; p 43061.

(13) Ting, S. V.; Rouseff, R. L. Vitamins. In Citrus fruits and their products, Analysisand Technology; Ting, S. V.; Rouseff, R. L., Eds.; Marcel Dekker, Inc.: NewYork, 1986; pp 121-136.

(14) AOAC Official methods of analysis of the Association of Official AnalyticalChemists, 15th ed.; Association of Official Analytical Chemists: Arlington,VA, 1990; pp 1058-1059.

(15) Lee, H. S.; Coates, G. A. J. Micronutr. Anal. 1987, 3, 199–209.

Anal. Chem. 2010, 82, 5134–5140

10.1021/ac100303p 2010 American Chemical Society5134 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010Published on Web 05/26/2010

The lack of an appropriate analytical system at low cost forlarge scale commercial application redirected the attention of thescientific community to electrochemical sensors and telemetry,well-known systems in medicine but still a pioneer in the foodand agricultural industry.16

The application of sensor and biosensor technology to the foodindustry, as a powerful and low cost alternative to conventionalanalytical techniques, has been widely discussed by severalauthors.17,18 Amperometric devices are the most commonlyreported class of sensors since they are cheap and based uponthe use of disposable electrodes; they are highly sensitive and donot need extensive sample pretreatment or large sample volumes.Amperometric biosensors for detection of AA in food have beendeveloped since the 80s,19 with the determination of L-ascorbicacid by immobilizing ascorbate oxidase on a Clark oxygenelectrode. Later, Volotovsky and Kim20 used an ion-sensitive field-effect transistor (ISFET) biosensor containing horseradish per-oxidise immobilized into bovine serum albumin (BSA) gel for anindirect detection of ascorbic acid in fruit juices and beverages.O’Connell et al.21 proposed a sensor based on electropolymerizedaniline (PANI) to selectively catalyze the oxidation of AA at lowpotential (+100 mV) in order to minimize the effects of the mostcommon interfering agents in juices and pharmaceuticals. A glassycarbon electrode modified with (PVP/Mo(CN)8

4-) was used forL-ascorbic acid estimation directly in orange fruit juice,22 anda good correlation was attained with the official titrimetricmethod. In the same year, an electrochemically etched platinummicroelectrode was developed to evaluate the spatial distribu-tion of AA concentration in oranges by in situ measurements.23

More recently a modified carbon paste electrode with SiO2/SnO2/phosphate/Meldola’s blue was used to study the elec-trocatalytic oxidation of AA in commercial fruit juices.24 Thevoltammetric behavior and the amperometric determination ofAA was also studied by a cadmium pentacyanonitrosylferratefilm modified glassy carbon electrode in fruit juices25 and bya polyaniline-based microelectrode in wine and other bever-ages.26 All the above-mentioned systems implicate a certaindegree of complexity in the phase of sample preparation and/or in the electrode modification. For this reason, a correctapplication is possible only when performed by specializedpersonnel in well-equipped laboratories.

Telemetry is a well-established technique for real-time monitor-ing of several biological molecule levels. Sophisticated telemetrydevices have been recently developed to analyze neurochemical

data and successfully used in conjunction with microbiosensorsfor the in vivo measurement of brain AA, O2, glucose, andlactate.27,28

In this paper, we present a telemetric wireless device connectedto a really simple and low cost microsensor for on line detectionof AA in orange fruit juice.

MATERIALS AND METHODSReagents. All chemicals were of analytical grade and used as

received without any further purification. Solutions were preparedwith Milli-Q water (Millipore, Inc.; 18 MΩ/cm). L-Ascorbic acid(99%) was purchased from Merck (Germany); stock solutions ofAA (5 mM) were prepared daily in acetate buffer (AB) (Merck-Germany) at pH 3.75. K3Fe(CN)6 was purchased from Sigma(Milano, Italy). For titrimetric measurements, oxalic aciddehydrate and 2,6-dichlorophenol-indophenol sodium salt di-hydrate, sodium hydrogen carbonate (NaHCO3, powder g99.5%)were purchased from Merck (Germany). N2-saturated (0% O2)and oxygen-saturated (100% O2) solutions, used for in vitrocalibrations, were prepared by dissolving (bubbling) the ap-propriate gas in 10 mL of AB for 1 h. The air-saturated solutionof AB (21% O2) was prepared by bubbling filtered air in 10 mLof AB for 1 h, using a diaphragm air pump.27 All in vitrocalibrations were performed in freshly prepared N2 and O2

solutions under standard conditions of pressure and temperature.Fruit Samples and Juice Preparation. Commercially mature

oranges [Citrus sinensis (L.) Osbek], cultivars Salustiana, Hamlin,and Ovale calabrese, were hand-harvested in an experimentalorchard of the Institute of Sciences of Food Production in central-western Sardinia, Italy, managed under standard horticulturalpractices. Fruits were placed in plastic trays and delivered to thelaboratory immediately after harvest. Then, medium size fruits,free from rind defects, were randomly selected, placed in plastictrays, and left overnight at 17 °C. The juice used for in vitrocalibrations and for AA determination was squeezed and filteredwith a kitchen strainer.

Sensor Description and Preparation. The sensor assemblywas composed of three rod carbon (length ) 30 mm; Ø ) 300µm, 2H Staedler graphite pencil leads) electrodes (Figure 1). Theworking electrode (WE) was coated with an insulating thin layerof epoxy resin, and its active surface (0.158 mm2) was ellipse-shaped using a high speed drill equipped with an aluminumoxide grinding wheel. The epoxy resin solutions were preparedas follows: solution (A), 1 g of Araldite-M (Sigma-Aldrich, Milan,Italy) was dissolved in 500 µL of acetone; solution (B), 1 g ofharderer was dissolved in 500 µL of acetone. The insulatingsolution was obtained by mixing 200 µL of solution (A) to 50µL of acetone and 100 µL of solution (B). The pencil lead thatconstituted the WE was insulated by a single dip in the epoxyresin mixture and then dried in oven at 70 °C overnight. Threemillimeters of not insulated carbon rod guaranteed goodelectrical contact when inserted in a gold-plated socket. Theinsulation of the WE was measured before and after theinsulating process using a digital multimeter (Fluke mod. 28

(16) Nakamura, H.; Karube, I. Anal. Bioanal. Chem. 2003, 377, 446–468.(17) Velasco-Garcia, M. N.; Mottram, T. Biosyst. Eng. 2003, 84 (1), 1–12.(18) Terry, L. A.; White, S. F.; Tigwell, L. J. J. Agric. Food Chem. 2005, 53,

1309–1316.(19) Matsumoto, K.; Yamada, K.; Osajima, Y. Anal. Chem. 1981, 53, 1974–

1979.(20) Volotovsky, V.; Kim, N. Sens. Actuators, B 1998, 49, 253–257.(21) O’Connell, P. J.; Gormally, C.; Pravda, M.; Guilbault, G. G. Anal. Chim.

Acta 2001, 431, 239–247.(22) Thangamuthu, R.; Senthil Kumar, S. M.; Chandrasekara Pillai, K. Sens.

Actuators, B 2007, 120, 745–753.(23) Paixao, T. R. L. C.; Lowinsohn, D.; Bertotti, M. J. Agric. Food Chem. 2006,

54, 3072–3077.(24) Castilho, R. F.; Souza, E. B. R.; Alfaya, R. V. S.; Alfaya, A. A. S. Electroanalysis

2008, 20 (2), 157–162.(25) Razmi, H.; Harasi, M. Int. J. Electrochem. Sci. 2008, 3, 82–95.(26) Kilmartin, P. A.; Martinez, A.; Bartlett, P. N. Curr. Appl. Phys. 2008, 8,

320–323.

(27) Bazzu, G.; Puggioni, G. M. G.; Dedola, S.; Calia, G.; Rocchitta, G.; Migheli,R.; Desole, M. S.; Lowry, J. P.; O’Neill, R. D.; Serra, P. A. Anal. Chem. 2009,81, 2235–2241.

(28) Calia, G.; Rocchitta, G.; Migheli, R.; Puggioni, G.; Spissu, Y.; Bazzu, G.;Mazzarello, V.; Lowry, J. P.; O’Neill, R. D.; Desole, M. S.; Serra, P. A. Sensors2009, 9, 2511–2523.

5135Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

II). In Figure 2, a SEM image of the WE at four differentmagnifications is shown. The pseudoreference (ref) and auxiliary(Aux) electrodes were not insulated. All the in vitro characteriza-tions were carried out at room temperature 24 h after sensormanufacturing (day 0) while a calibration was performed im-mediately before every juice analysis.

Electrochemical Characterization, in Vitro Calibration,and Juice Analysis. All electrochemical characterizations andcalibrations were performed using a four channel system (eDAQQuadStat, e-Corder 410 and Echem software, eDAQ Europe,Poland) placing the microsensors in a cell, a glass beakercontaining 10 mL of acetate buffer at pH 3.75.

A preliminary electrochemical characterization of WE wascarried out using cyclic voltammetry starting from -300 mV to+700 mV (vs carbon pseudoreference electrode) at a scan rate of50 mV/s. AA oxidation was studied on the microsensor surfacein AB containing variable O2 concentrations: 0%, 21%, and 100%.

In order for all changes in the measured electrode potentialto be reliably attributed solely to the WE, the potential of thereference electrode must not change during the measurement.With this aim in view, according to Ruch et al.,29 we previouslytested the suitability of carbon as a pseudoreference electrodeperforming CV in a 0.1 M KNO3 solution with the addition ofK3Fe(CN)6 10 mM, first using our graphite pencil lead as areference electrode and then substituting it with an Ag/AgClwire.

All AA calibrations were made on day 0 (the day after thesensor preparation) in air-bubbled AB applying a positive potentialof +120 mV (vs carbon pseudoreference electrode) after a stablebaseline was achieved. A five-point calibration was performed byadding known amounts of AA (40 µL of a 0.5 mM stock solution)in order to have concentrations comprised between 0 and 100 µM

in the cell. A second calibration was made adding 5 × 40 µL of a5 mM AA solution for obtaining concentrations 10 times higher(Figure 3C). The effect of the sample matrix on AA currents wasinvestigated by spiking juice with known amounts of AA. Six 10mL aliquots of Salustiana orange juice were spiked with increasingquantities of AA in a range comprised between 0 and 2.5 mM(standard addition method). As a result, we obtained a spiked juicecovering the entire range of AA concentrations detected in thethree studied varieties. Spiked samples were injected in triplicate,and a linear regression analysis was performed on baseline-subtracted data (Figure 4B).

A different group of pencil leads was studied in order todetermine the aging of the sensors. A five point AA calibration(up to 100 µM in cell) was performed at day 0 and repeated atdays 7, 14, and 21 and, in the meantime, sensors were used dailyfor AA determination in orange juice.

The quantification of AA in orange juice was achieved by thetelemetry system described in the next paragraph. AA determi-nation was made, into 10 mL of AB solution, by exposing thesensors (n ) 4) to triplicate aliquots (3 × 80 µL) of each orangejuice variety and interpolating the resulting currents in thecalibration plot obtained immediately before juice injections.

Telemetry System. The telemetric miniaturized device, weigh-ing less than 15 g, consists of a single-supply sensor driver,a current-to-voltage (I/V) converter, a microcontroller, and aminiaturized data transmitter (Figure 1). It is coupled with amicrosensor that generates electrical signals related to electro-chemical processes. The telemetric device was derived fromprevious designs28,30 and is capable of working in oxidation mode.A biological oxidable molecule such as AA can be directly detectedon the surface of the amperometric sensor connected to apotentiostat.30 The amperometric section of the telemetric device,capable of transducing currents up to 45 nA, was built around aquad single-supply operational amplifier (MCP6044, ArizonaMicrochip, Chandler, AZ) and a ZXRE4001 Zener diode (Zetex,Manchester, UK) using a 50 MΩ feedback resistor in the I/Vcircuit. The ADC was an integral part of the microcontroller(PIC12F683, Arizona Microchip) used in this system. The AAoxidation currents were digitized by means of an analog-to-digitalconverter integrated in a peripheral interface controller (PIC) andsent to a personal computer by means of a miniaturized AMtransmitter. The 433.92 MHz AM transmitter was a RT4-433.92(Telecontrolli, Casoria, Italy) while the RR3-433.92 module(Telecontrolli) was selected as the AM receiver. Both TC moduleswere equipped with external antennas. The serial-to-USB converterwas a FTDI-FT232R module with an internal E2PROM andintegrated clock synthesizer. The components were solderedon single side printed circuit board (PCB) boards producedas previously described.28 All electronic parts used in thisproject were Pb free and compliant to restriction of hazardoussubstances (RoHS) directives. The electronics were calibratedand tested in vitro under different experimental conditions andexhibited high stability, low power consumption, and goodlinear response in the nanoampere current range.28,30

Firmware and Software. The firmware was realized in Clanguage. The program, which runs on the telemetry device,

(29) Ruch, P. W.; Cericola, D.; Hahn, M.; Kotz, R.; Wokaun, A. J. Electroanal.Chem. 2009, 636, 128–131.

(30) Serra, P. A.; Rocchitta, G.; Bazzu, G.; Manca, A.; Puggioni, G. M.; Lowry,J. P.; O’Neill, R. D. Sens. Actuators, B: Chem. 2007, 122 (1), 118–126.

Figure 1. Sensor assembly and telemetry system. 3D representationof the three electrodes (A). Pseudoreference (REF) and auxiliary(AUX) electrodes were not coated while the working electrode (WE)was completely insulated except for the active surface where the AAoxidation happened. L-Ascorbic acid is readily oxidized on the carbonelectrode surface at +120 mV vs carbon as follows: AA (+120 mV)f dehydroascorbate (DHAA) + 2e- + 2H+. The microsensor isconnected to a transmitter unit (B) which sends the electrochemicalsignal to a receiver (C) linked to a PC (via USB) where it is recordedand plotted in real-time.

5136 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

consists of two routines: the main procedure and a timer-interruptroutine called every second. The software, running on the PCunder Windows XP Professional or Vista, communicates throughthe USB by the use of the low-level driver. The graphic userinterface was developed using Profilab Expert (version 4.0 fromAbacom) while the dynamic link library (DLL) serial-data-parserwas programmed in C (Dev-C++ ver. 4.9.9.2). The application(capable of plotting, storing, and retrieving data) interfaces thesystem with a printer via USB and a Local Area Network (LAN)and Internet via TCP/IP. A software alarm was generated if batteryvoltage dropped down to 2.7 V or a data reception time-outoccurred.

Titrimetric and Chromatographic (HPLC) Measurements.Results attained with the sensor method were validated bycomparing the AA content achieved by the sensor with thoseobtained by both a standard titrimetric method13 and a chromato-graphic method.15 AA content was expressed as mg/100 mL.

Statistical Analysis. AA currents were expressed in nanoam-peres and given as mean ± standard error of the mean (SEM) ofabsolute oxidation currents (nA) or baseline-subtracted currents(∆nA). After in vitro calibrations, the AA currents were plottedvs the AA concentration and the linear regression was calculated.AA-spiked data were obtained by subtracting the baseline aftereach injection of juice and expressed as ∆nA. AA juice contentwas expressed as molarity and then converted to mg/100 mL tobe comparable with reference methods.

Statistical analysis was performed by Statgraphics software(Statgraphics Centurion, version XV, Herdon, Virginia). Analy-sis of variance (one-way ANOVA) was carried out using aunifactorial complete randomized block design. Mean com-

parisons were calculated by Fisher’s least significant differencetest at P e 0.05.

RESULTS AND DISCUSSIONSensor Characterization: Cyclic Voltammetry (CV), Sensor

Characteristics, and Calibration. Preliminary studies werecarried out in order to investigate the electrochemical behaviorof AA at the carbon working electrode surface vs a carbonpseudoreference electrode. The use of such a reference electrode,as an alternative to an Ag/AgCl one, has already been successfullyexperimented by Dilleen et al.31 and Nie et al.32 The results ofthe CV (data not shown), performed using the carbon pseudoref-erence electrode, evidenced a potential difference (∆Ep) betweenthe ferricyanide anodic (Epa) and cathodic (Epc) peaks of 68mV and an half-wave potential (E1/2 ) Epc + ∆Ep/2) corre-sponding to +374 mV. Such a characterization was repeatedweekly showing an E1/2 and a ∆Ep variation lower than a 10mV. Substituting the carbon pencil lead with an Ag/AgCl wire asquasi-reference electrode, we obtained a parallel shift ofK3Fe(CN)6 peaks (E1/2 ) +691 mV) with a ∆Ep equal to 73 mV.

Figure 3A shows four cyclic voltammograms recorded at theWE vs carbon pseudoreference electrode, the first (baseline) inan air-bubbled AB solution in the absence of AA, the others inthe presence of 1 mM AA in AB at different O2 concentrations(0%, 21%, and 100%). The voltammograms demonstrated that

(31) Dilleen, J. W.; Birch, B. J.; Haggett, B. G. D. Anal. Commun. 1999, 36,363–365.

(32) Nie, Z.; Nijhuis, C.; Gong, J.; Chen, X.; Kumachev, A.; Martinez, A. W.;Narovlyansky, M.; Whitesides, G. M. Lab Chip 2009, 1-10, SupplementaryMaterial.

Figure 2. Scanning electron microscope (SEM) of the WE at four different magnifications: (A) ×100, (B) ×500, (C) ×1500, and (D) ×2000.Graphite rod (grf) and epoxy resin coating (epx) can be clearly distinguished.

5137Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

changes in solution oxygen levels did not affect the AAoxidation peak. At the potential of +120 mV, the AA oxidationoccurred and the relative lines split from the baseline. This iseven more evident in Figure 3B where baseline subtractedvoltammograms are reported, and then, the working potential wasfixed at that value to prevent the oxidation of interfering speciespresent in the orange juice.

The choice of the appropriate working potential is a crucialpoint discussed by several authors:3,21 carbon shows slow electrontransfer kinetics for the oxidation of AA by comparison to goldor platinum electrodes, but it is significantly less expensive andless susceptible to fouling by the oxidation products of AA.O’Connell et al.,21 using a glassy carbon electrode modified witha layer of polyaniline, found that the optimum potential fordetection of ascorbic acid determined from a linear sweepvoltammetry (LSV) scan was +300 mV, even though there wasonly a small decrease (9%) in response when +100 mV was used.More recently, Civit et al.,3 using a modified commercial screen-printed electrode, performed amperometric measurements at afixed potential of +200 mV (vs Ag/AgCl).

As previously described, the electrodes of the sensor are allin carbon. The hardness of pencil leads was chosen on the basisof the conclusions reported by Tavares and Barbeira33 whoshowed that harder graphite leads, regardless of their manufac-turer, are more appropriate as electrode material for voltammetricpurposes due to their higher peak currents, providing moresensitivity and reproducibility. The latter was also proved bycalibrating AA with pencil leads from different batches, but nosignificant differences were found among them (data not shown).The conductivity of the insulated WE was null along all the rodexcept for the active surface.

Since the presence of potential interfering agents can alter thesignal registered by the sensor, we evaluated the specificity ofour sensor investigating the effect of common potentially interfer-ing compounds present in orange juice on the stability of thebaseline. No alteration of the latter was registered in the presenceof citric and other organic acids, sugars, or antioxidants when

(33) Tavares, P. H. C. P.; Barbeira, P. J. S. J. Appl. Electrochem. 2008, 38, 827–832.

Figure 3. AA microsensor characterization in acetate buffer (pH 3.75).(A) Cyclic voltammetry with a scanned potential range (VApp) comprisedbetween -300 and +700 mV, in the absence (gray dotted line) and inthe presence of 1 mM AA at different O2 concentrations. (B) Baseline-subtracted voltammograms of AA oxidation: the arrow indicates thepotential of +120 mV which was used for all subsequent AA calibrationsand juice determinations. (C) Five point in vitro calibration and linearregression (inset) of AA starting from 0 up to 100 µM.

Figure 4. (A) Amperometric response obtained by 10 sequentialadditions of 40 µL of orange juice in acetate buffer. (B) AA calibrationwith the standard addition method: the addition of spiked juice to thebuffer determined a linear increase in the registered current (inset).(C) Aging of the sensors: the linear regression of the slopes of thefour calibrations indicates a loss of sensitivity of 50.4 pA/µM at theend of a 3 week period.

5138 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

working at a potential of +120 mV (data not shown). Similarresults were achieved by Akyilmaz and Dinckaya34 using abiosensor based on ascorbate oxidase. They detected the O2

consumption by a commercial dissolved oxygen probe and didnot register any electrode response for oxalic, L-aspartic,L-glutamic, succinic, citric and glycolic acids, D(+)-glucose, andD(-)-fructose. In their system, hydroquinone and catechol werethe only interfering agents oxidized by the ascorbate oxidase,likewise with AA. In our system, the absence of the enzymeand the low applied potential make the analysis free from theinterference of the above-mentioned molecules.

The choice of placing the electrodes in acetate buffer (AB) atpH 3.75 derived from the fact that the orange juice pH of the threecultivars lies in that range (Hamlin, pH ) 3.8; Salustiana, pH )3.95; Ovale calabrese, pH ) 3.55). There is not agreement or adefinitive answer, among the authors, in order to establish whichbuffer solution and which pH of the buffer solution are the bestto calibrate sensors for AA. Our choice depended on the matrixwe were working on but, in some other cases, it depended onthe nature of the sensor. Ivanov et al.35 calibrated a platinumsensor, coated with a polyaniline (PANI) layer, for detection ofAA in orange juice, in phosphate buffer at pH 7. O’Connell et al.,21

working with a sensor made by a conductive PANI layer grownupon a carbon substrate, found that the optimum working bufferpH is near neutrality, between 6 and 7. This was an unexpectedresult because previous research had shown that polyaniline filmswere only efficient conductors under acidic conditions,36 andCasella and Guascito37 found that the optimum pH for AAoxidation on a PANI film was between 1 and 2.

Figure 3C shows a five point AA calibration. Plots of the currentresponse versus the concentration of AA over a dynamic rangeof 0-100 µM were obtained in triplicate. The linear response ofthe system was excellent (r2 ) 0.996, n ) 8, see inset) with asensitivity of 0.279 ± 0.021 nA/µM. A previous AA calibration(data not shown), performed over the range of 0-10 µM, gavean excellent response too (r2 ) 0.999, n ) 8, slope ) 0.283 ±0.002 nA/µM).

The limit of detection (LOD), which was calculated as thatconcentration that engendered a response 3 times the standarddeviation of the background noise of the sensor, was 0.61 ± 0.058µM. Quantification of AA in the juice was achieved by interpolatingthe response data in the calibration plot in a range from 0 to 5mM of AA after correction of the dilution factor. A typicalamperometric response, obtained by the sequential addition ofequal amounts (+40 µL per injection) of orange juice in acetatebuffer, is showed in Figure 4A.

The outcome of the calibration with the standard additionmethod was shown in Figure 4B. The addiction of spiked juicedetermined a linear increase in the baseline-subtracted currentvalues (r2 ) 0.997, n ) 12, slope ) 0.275 ± 0.071, see inset).These results, if compared with those of Figure 3C (slopes ofthe two linear regressions are quite similar, and there are not

any statistical differences between them) demonstrate that theexposition to juice did not affect the sensors performances.

The reproducibility of responses of the sensors was tested by10 subsequent measurements of standard solutions containingequal concentration of ascorbic acid (40 µM). The mean valueand the standard deviation were calculated as 13.6 nA/V and 0.12nA/V, respectively.

The operational stability of the electrode was assessed by itsstorage and operational efficiency in a 3 week time period. In orderto achieve the aging of the electrodes, each sensor, after apreliminary calibration, was used daily for AA determination inorange juice and then recalibrated again at days 7, 14, and 21.They were all linear with an r2 g 0.997. The slopes of thecalibrations made weekly were plotted on the same graph, andthe linear regression of the slopes were calculated as -0.0024± 0.00004 nA/µM/day (r2 ) 0.999; n ) 8) (Figure 4C). Theresults showed that after 7, 14, and 21 days the initial sensitivityof the sensors was reduced to 5.4%, 11.1%, and 17.4%, respectively,which means a linear loss of sensitivity of 2.4 pA/µM/day.

The results of the determination of the AA content in the juiceof fresh fruits of the three varieties were reported in Table 1. TheAA values obtained with the sensor significantly differed fromthose achieved with the titrimetric one for all varieties. Differently,the amperometric method, when compared with the HPLC one,showed significantly lower values of AA content only for theSalustiana variety. On average, AA amounts determined using ourmicrosensor were 7% and 4.5% smaller than those obtained,respectively, by titrimetric and chromatographic methods. Anerror within 20% between values registered by sensors and valuesobtained by redox titration is reported as tolerant in mostapplications.35 Moreover, an error of this magnitude is reallyacceptable since it is free from interferences. On the other hand,other studies carried out with constant potential amperometrictechniques showed an overestimate level of AA in comparisonwith the reference method.3 Such a result did not suggest thepresence of interfering agents but presumed a higher sensitivityof the sensor, especially in the function of the materials employedto build the working electrode. Our results, on the contrary,revealed a lower sensitivity when compared with the referencemethods, probably due to the lower applied potential.

Telemetry. The electronic circuit of the miniaturized telemetricdevice was composed of three different parts: the amperometricmodule, the microcontroller, and the transmitter. The ampero-metric module was made up of three “rail-to-rail” operationalamplifiers working as potentiostat, voltage follower, and current-to-voltage (I/V) converter. A dummy cell, based on a previously

(34) Akiylmaz, E.; Dinckaya, E. Talanta 1999, 50, 87–93.(35) Ivanov, S.; Tsakova, V.; Mirsky, V. M. Electrochem. Commun. 2006, 8,

643–646.(36) Shaolin, M.; Huaiguo, X.; Bidong, Q. J. Electroanal. Chem. 1991, 304, 7–

16.(37) Casella, I. G.; Guascito, M. R. Electroanalysis 1997, 8, 1381–1386.

Table 1. Ascorbic Acid Content (mg/100 mL) in theJuice of “Salustiana”, “Hamlin”, and “Ovale calabrese”Oranges As Determined by the Titrimetric, HPLC, andSensor (Electrochemical) Methodsa

orange cultivar

analytical method Salustiana Hamlin Ovale

titrimetric 41.13 ± 0.32 a 56.27 ± 0.64 a 49.47 ± 1.40 aHPLC 40.13 ± 0.35 b 54.73 ± 1.06 b 48.22 ± 1.05 absensor 38.13 ± 0.71 c 52.33 ± 0.83 b 46.29 ± 1.27 b

a Means in columns followed by unlike letters differ significantlyby Fisher’s least significant difference (LSD) procedure, P e 0.05.

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published design,30 was used to obtain a Thevenin current sourceable to reproduce the constant amperometric response of a trueelectrochemical cell, in order to test the amperometric modulebefore sensor calibration. The voltage applied to the dummy cellwas generated between the WE and the ref electrodes, and it isequal to the voltage difference between the above electrodes(+120 mV).

The electronics calibration was made indoors with a lineardistance between the transmitter and the receiver unit of about3 m. The average power consumption was 375 µW (125 µA)confirming that a 3 V lithium coin battery (Maxwell CR1216),having a capacity of 25 mA/h, can power the telemetric devicefor more than 1 week of continuous operation. The baseline wasrecorded for 7 days, and the electronic noise was on the order of20 pA according to previous observations.28,30 As previouslydiscussed,28,30 the use of the wireless connection dramaticallyreduces the background noise related to the residual ripple ofthe main power supply and the triboelectric effect expected if awired connection is used. The proposed system, characterizedby low noise, high stability, and good linear response, operatesonly in oxidation mode and it is particularly suited to work withdirect-oxidation sensors (AA) and in conjunction with oxidase-based AA biosensors containing ascorbate oxidase enzyme.

CONCLUSIONSIn this study, we presented an innovative telemetric system

for the real-time detection of AA in orange juice. The system, builtwith simple and inexpensive components, showed highly repro-ducible analytical performances and might be also easily proposedin environments, like developing countries, where the mostcommon used expensive materials (i.e., silver or platinum wires)cannot be easily found.

The transmitter and the receiver units can be used for accuratetransduction of the oxidation current generated on the surface ofthe AA carbon microsensor. This device consists of two compo-nents that, when assembled, polarize the sensor and send AA

oxidation data to a receiving unit connected to a notebook. Thesystem exhibited low power consumption, high stability, and agood linear response and offers the possibility of expanding thetelemetric network (NET) simply and quickly.

The results are in accordance with those obtained withtraditional reference methods, thus suggesting that the devicecould be used as a reliable model for the rapid monitoring of AA,on several biological substrates, during every step of the produc-tive chain.

The use of the telemetry in the postharvest sector of the foodand agricultural industry is a great novelty and goes over andabove the AA determination. It opens new perspectives giving thepossibility of realizing complicated or even sophisticated analysisin uncomfortable environments, like in the field for a directmeasure on fruits still on the plant, in storage and refrigeratingrooms where moving is difficult, or in particular situations like inmodified or controlled atmosphere conditions when displacingproducts out of the room is not possible.

At the moment, the fragility of the sensor, made by graphiterods, could limit the direct use of the device on fruits. On theother hand, rods are disposable, very easy to replace, and reallycheap. In conclusion, the system, based on a minimal samplepreparation (just a dilution), a miniaturized device, a notebook,and a wireless connection, can be considered like a portableworkstation, with high developing power and ultralow cost,especially if compared with traditional methods.

ACKNOWLEDGMENTThis research was partially funded by Fondazione Banco di

Sardegna. The authors acknowledge Domenico Mura for hiscontribution to laboratory analysis and Salvatore Marceddu forSEM analysis.

Received for review February 3, 2010. Accepted May 16,2010.

AC100303P

5140 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010