Flow injection analysis coupled with carbon electrodes as the tool for analysis of naphthoquinones with respect to their content and functions in biological samples

Senso s 2006, 6, 1466-1482 senso s ISSN 1424-8220 © 2006 by MDPI h p://www.mdpi.o g/senso s Full Pape Flow Injec ion Analysis Coupled wi h Ca bon Elec odes as he Tool o Analysis o Naph hoquinones wi h Respec o Thei Con en and Func ions in Biological Samples Pe Babula 1 , Dalibo Huska 2,4 , Pa el Hanus iak 2 , Ji i Baloun 2,4 , Sona K izko a 2 , Voj ech Adam 2 , Ja omi Hubalek 3 , Ladisla Ha el 4 , Milan Zemlicka 1 , Ales Ho na 5 , Mi osla a Beklo a 6 and Rene Kizek 2,* 1 Depa men o Na u al D ugs, Uni e si y o Ve e ina y and Pha maceu ical Sciences, Palackeho 1-3, CZ-612 42 B no, Czech Republic 2 Depa men o Chemis y and Biochemis y, and 4 Depa men o Plan Biology, Facul y o Ag onomy, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic 3 Depa men o Mic oelec onics, Technical Uni e si y o B no, Udolni 53, CZ-602 00 B no, Czech Republic 5 Depa men o Food Enginee ing and Chemis y, Facul y o Technology, Tomas Ba a Uni e si y, CZ- 762 72 Zlin, Czech Republic 6 Depa men o Ve e ina y Ecology and En i onmen al P o ec ion, Uni e si y o Ve e ina y and Pha maceu ical Sciences, Palackeho 1-3, CZ-612 42 B no, Czech Republic * Au ho o whom co espondence should be add essed; E-mail: [email p o ec ed] Recei ed: 16 Oc obe 2006 / Accep ed: 27 Oc obe 2006 / Published: 6 No embe 2006 Abs ac : Naph hoquinones a e one o he g oups o seconda y me aboli es widesp ead in na u e, whe e hey mos ly appea as ch oma ic pigmen s. They embody b oad- ange o biological ac ions om phy o oxic o ungicidal. An an icance e ec o naph hoquinones s imula es an in e es in de e mina ion and cha ac e iza ion o single de i a i es o 1,2- and 1,4-quinones in biological samples. The main aim o his wo k was o sugges a echnique sui able o de e mine lawsone, juglone and/o plumbagin in biological samples and o s udy o hei in luence on BY-2 obacco cells. The BY-2 obacco cells we e cul i a ed in he p esence o he naph hoquinones o in e es (500 µg.l -1 ) o 24 h and hen he mo phological changes we e obse ed. We ound ou ha naph hoquinones igge ed he p og ammed cell dea h a BY-2 cells, which can be con i med by he apop o ic bodies in nucleus. A e ha we sugges ed and op imized di e en elec ochemical echniques such di e en ial pulse Senso s 2006, 6 1467 ol amme y (DPV) coupled wi h hanging me cu y d op (HMDE) and ca bon pas e elec ode, mic o low de ice coupled wi h ca bon sc een p in ed elec odes and low injec ion analysis coupled wi h Coulochem III de ec o o de e mine hem. The de ec ion limi s o naph hoquinones o in e es we e exp essed as 3S/N and a ied om uni s o hund eds o ng pe millili es acco ding o me hods used. Mo eo e , we u ilized DPV coupled wi h HMDE and mic o low de ice o de e mine con en o juglone in lea es Pe sian walnu (Juglans egia). We de e mined ha he lea es con ained juglone en hs o g pe 100 g o esh weigh . The esul s ob ained show he con incing possibili ies o using o hese me hods in analysis o plan seconda y me aboli es. Keywo ds: Naph hoquinones; Plumbagin; Lawsone; Juglone; BY-2 obacco cells (Nico iana abaccum); Pe sian walnu (Juglans egia); Senso s; Flow analysis; Di e en ial pulse ol amme y; Elec ochemical de ec ion; Ca bon elec odes. 1. In oduc ion Naph hoquinones belong o g oup o allelopa hic me aboli es widesp ead in na u e. They ha e been ound in highe plan s such as Plumbaginaceae, Juglandaceae, e c. [1-4], ungi (Ma asmius g amium and Ve icillium dahliae) [5] and mic oo ganisms (S ep omyces and Fusa ium) [6]. Juglone, lawsone and plumbagin a e ones o hei mos ly in es iga ed ep esen a i es. Juglone (5-hyd oxy-1,4- naph hoquinone) is he cha ac e is ic compound o Juglans spp., which is epo ed o occu in esh walnu lea es [7,8]. Ne e heless, because o polyme iza ion phenomena, juglone is epo ed o occu in he d ug (d y lea es) only in es igial amoun s, which means ha he compound is no sui able o use in he quali y con ol o he d y plan [8]. Plumbagin (5-hyd oxy-2-me hyl-1,4-naph hoquinone) is a na u ally occu ing yellow pigmen de i a i e om he he b Plumbago zeylanica L., a semi-climbing subsh ub dis ibu ed in hicke s o g assland. The whole plan and i s oo ha e been used as a olk medicine o he ea men o heuma ic pain, menos asis, ca buncle, and inju y due o bumping [9]. Plumbagin has been shown o ha e an icance , an i-leishmanial, an i-bac e ial and an i- ungal p ope ies [10-12], as well as a con acep i e e ec [9]. Mo eo e plumbagin supp esses NF-kappa B ac i a ion and NF-kappa B- egula ed gene p oduc s h ough modula ion o p65 and I kappa B alpha kinase ac i a ion, leading o po en ia ion o apop osis induced by cy okine and chemo he apeu ic agen s [13]. The ac i e dye ing edien , lawsone (2-hyd oxy-1,4-naph hoquinone), which cons i u es abou 1% o he d y weigh o he lea es o Lawsonia alba, has been implica ed in he causa ion o henna-induced hemoly ic anemia because o i s s uc u al simila i y o o he o ho-subs i u ed 1,4-naph hoquinones, such as menadione (2-me hyl-1,4-naph hoquinone), ha a e known o induce oxida i e inju y wi hin ed cells [14]. 1,4-Naph hoquinones a e hough o induce oxida i e damage as a consequence o hei abili y o unde go edox cycling [15], wi h he gene a ion o eac i e oxygen species. High pe o mance liquid ch oma og aphy wi h spec ome ic de ec ion and elec ochemical echniques a e he mos commonly used me hod o hese pu poses [9,16-21]. On he o he , elec ochemical senso has no been sugges ed ye o de e mina ion o he lawsone, juglone and/o Senso s 2006, 6 1468 plumbagin. The main aim o his wo k was o sugges a echnique sui able o de e mine lawsone, juglone and/o plumbagin in biological samples. Fo hese pu poses, we op imized bo h s a iona y and low elec ochemical echniques using ca bon elec odes as senso s. Mos o all, we aimed on using o ca bon sc een p in ed elec odes. Besides ha we in es iga ed he in luence o he compounds o in e es on BY-2 obacco cells and de e mined hei con en in Pe sian walnu (Juglans egia). 2. Ma e ials and me hods 2.1. Chemicals Naph hoquinones (lawsone, juglone and plumbagin) we e pu chased om Sigma Ald ich Chemical Co p. (S . Louis, USA). Me hanol o HPLC and o he analy ical eagen s o ACS pu i y we e also pu chased om Sigma Ald ich. Solu ions we e p epa ed using deionised ACS wa e (Sigma). The s ock s anda d solu ions o naph hoquinones a (100 µg.ml -1 ) we e p epa ed in ACS me hanol and s o ed in he da k a 4°C. The wo king s anda d solu ions we e p epa ed daily by dilu ion o he s ock solu ions. All solu ions we e il e ed h ough a 0.45 µm Te lon memb ane il e (Me aChem, To ance, USA) p io o FIA analysis. The pH alue was measu ed using WTW inoLab Le el 3 (Mul iLab Pilo ; Weilheim, Ge many) con olled by a pe sonal compu e p og am (Mul iLab Pilo ). 2.2. Elec ochemical measu emen s Di e en ial pulse ol amme y (DPV). The elec ochemical analysis o naph hoquinones we e pe o med wi h AUTOLAB Analyse (EcoChemie, Ne he lands) connec ed o VA-S and 663 (Me ohm, Swi ze land), using a s anda d cell wi h h ee elec odes. The wo king elec odes we e a hanging me cu y d op elec ode (HMDE) wi h a d op a ea o 0.4 mm 2 and/o ca bon pas e elec ode. The e e ence elec ode was an Ag/AgCl/3M KCl elec ode and he auxilia y elec ode was a g aphi e elec ode. Fo smoo hing and baseline co ec ion he so wa e GPES 4.4 supplied by EcoChemie was employed. Phospha e bu e (0.2 M) was used as suppo ing elec oly e. The measu emen was ca ied ou in po en ial ange om –0.6 o 0 V wi h po en ial s ep 5 mV.s -1 and pulse ampli ude o 25 mV. The phospha e bu e (0.2 M) was deoxygena ed p io he measu emen . P io o analysis o naph hoquinones on ca bon pas e elec ode DPV pa ame e s we e used as ollows: po en ial ange om 0 o 1.2 V wi h po en ial s ep 5 mV.s -1 and pulse ampli ude 25 mV. All measu emen s we e pe o med a oom empe a u e. P epa a ion o ca bon-pas e elec ode. The ca bon pas e (abou 0.5 g) was made o g aphi e powde (Ald ich) and mine al oil (Sigma; ee o DNase, RNase, and p o ease). The a io o he g aphi e powde and mine al oil was 70/30 (w/w). This pas e was housed in a Te lon body ha ing a 2.5-mm-diame e disk su ace. P io o measu emen s, he elec ode su ace was enewed by polishing wi h a so il e pape . Then, he su ace was eady o measu emen o a sample olume o 5 µl [22,23]. Mic o Flow sys em. The one channel pe is al ic pump PP10 consis s o minia u e mo o wi h plane a y gea and s ainless body o pe is al ic pump (BVT echnologies, B no). The low was egula ed by PC so wa e (BVT echnologies, B no). The measu emen was ca ied ou in low cells FC2 (BVT Technologies, B no). The measu ing elec odes we e home made a Technical Uni e si y o Senso s 2006, 6 1469 B no, pa icula ly ca bon wo king elec ode, sil e elec ode as auxilia y and Ag/AgCl as e e ence ones. The low sys em was connec ed wi h AUTOLAB Analyse (EcoChemie). All measu emen s we e pe o med a oom empe a u e. 2.3. Flow injec ion analysis wi h ampe ome ic de ec ion An FIA-ampe ome ic sys em consis ed o sol en deli e y pump ope a ing in ange o 0.001-9.999 ml.min -1 (Model 583 ESA Inc., Chelms o d, MA, USA), a gua d cell (Model 5020 ESA, USA), a eac ion coil (1 m) and an elec ochemical de ec o . The elec ochemical de ec o (ED) includes one low olume low- h ough analy ical cells (Model 5040, ESA, USA), which is consis ed o glassy ca bon wo king elec ode, palladium elec ode as e e ence elec ode and auxilia y ca bon elec ode, and Coulochem III as a con ol module. The sample (5 µl) was injec ed manually. The ob ained da a we e ea ed by CSW 32 so wa e. The expe imen s we e ca ied ou a oom empe a u e. Gua d cell po en ial was 0 V. A glassy ca bon elec ode was polished mechanically by 0.1 µm o alumina (ESA Inc., USA) and sonica ed a he oom empe a u e o 5 min using a Sono ex Digi al 10 P Sonica o (Bandelin, Be lin, Ge many) a 40 W. 2.4. Plan ma e ials BY-2 obacco cell suspension. A suspension cul u e o Nico iana abacum BY-2 line was g own in liquid Mu ashige and Skoog medium supplemen ed wi h suc ose (30 g.l -1 ), KH 2 PO 4 (0.2 g.l -1 ), hiamine (1 mg. l -1 ) and 2,4-dichlo ophenoxyae ic acid (0.2 mg.l -1 ), acco ding o Naga a [24]. The suspension cul u es (20 ml) we e g own in 50 ml E lenmeye lasks a 27°C wi h shaking a 135 pm (Kühne Shake , ype: LT-W, Adol Kühne AG, Swi ze land). Subcul i a ion was pe o med a e 3 o 4 days by ans e ing 2 o 1 ml, espec i ely, o suspension cul u e in o a esh medium ( o al olume 20 ml). Juglans egia. The lea es o Pe sian walnu (Juglans egia) we e ha es ed in Bo anic ga den o Mendel Uni e si y o Ag icul u e and Fo es y in B no, Oc obe 2005 and July 2006. The ha es ed lea es (0.10 g ± 5 mg), we e lyophilized a –51°C o 48 h (CHRIST-Alpha 1-2). Then he lyophilized samples we e homogenized by an Ika A11 basic g inde (IKA We ke GmbH and Co., S au en, KG, Ge many). The homogenized samples (0.02 – 0.10 g) we e dissol ed in 5 ml o 99.999% me hanol and sonica ed a he labo a o y empe a u e o 30 min using a K5 Sonica o (Czech Republic) a 150 W, 38 kHz. The samples we e il e ed h ough a 0.45 µm Te lon memb ane il e (Me aChem) p io o injec ion in o he FIA sys em and 100 imes dilu ed wi h ACS wa e . 2.5. Cy ological analysis The sample o obacco cell suspension was mixed wi h PEM bu e (100 mM PIPES, 10 mM EGTA, 10 mM MgCl 2 , pH 6.9) con aining 4 % o o maldehyde in a io 1:1. A e he incuba ion (30 min., 25°C) he ixed cells we e h ee imes washed wi h PEM bu e and esuspended in PEM bu e wi h 0.1 % T i on X100 and 1 µg.ml -1 Hoechs 22385. The nucleus mo phology was obse ed by he luo escen mic oscope (Olympus AX 70) using he b oad band UV exci a ion (cube U-MWU). Senso s 2006, 6 1470 2.6. S a is ical analysis STATGRAPHICS® (S a is ical G aphics Co p®, USA) was used o s a is ical analyses. Resul s a e exp essed as mean ± S.D. unless no ed o he wise. Value o p < 0.05 was conside ed signi ican . 3. Resul s and discussion Al hough naph hoquinones ha e been in ensi ely s udying o mo e hen o y yea s [25], hei biochemical mechanism o ac ion is s ill unclea . I is a common knowledge ha hey can en e and in luence a cell cycle [10,26-32]. Due o his hei e ec s on umou cell lines like such as P 338, BG-1 and HL-60 ha e been in es iga ed [33]. He e we aimed on sugges ing o a senso o de e mina ion o ce ain naph hoquinones (lawsone, plumbagin and juglone, see in Fig. 1A) and on in es iga ing o hei e ec s on BY-2 obacco cells. The e ec s o hese subs ances on whole plan s ha e been desc ibed bu hei in luence on plan cell suspension is no clea ye , whe eas BY-2 obacco cells a e e y sui able o hese pu poses. 3.1. Mo phological changes o BY-2 obacco cells caused by naph hoquinones The BY-2 obacco cells we e cul i a ed in he p esence o he naph hoquinones o in e es (500 µg.l -1 ) o 24 h and hen he mo phological changes we e obse ed. The cell nuclei o non- ea ed BY-2 obacco cells we e well obse able and hei cy oplasm luo esced weakly g eenly (Fig. 1B). On he o he hand he ea ed BY-2 cells embodied a numbe o mo phological changes. The cell cy oplasm luo esced in ensi ely in compa ison wi h non- ea ed ones (Fig. 1C-G), which is p obably associa ed o he inc ease o ac i i y o in acellula es e ases [34-36]. The ma ked changes o ch oma in o ea ed BY-2 cells we e obse ed. As we obse ed in he mic opho og aphy, he nucleus p e-apop o ic changes, pa icula ly, he s ong ini ia ion condensa ion o ch oma in on nuclea pe iphe y was obse ed (Fig. 1D, G). In addi ion, we ound ou ha naph hoquinones igge ed he p og ammed cell dea h a BY-2 cells, which can be con i med by he apop o ic bodies in nucleus (Fig. 1E,F) [37,38]. This e ec could be explained by he abili y o naph hoquinones o gene a e he eac i e oxygen species, he po en ial deple ion on mi ochond ial memb anes and he inhibi ion o opoisome ases I and II [5,10,39]. Mo eo e , plumbagin is a po en inhibi o o he NF-kappa B ac i a ion pa hway ha leads o supp ession o NF-kappa B- egula ed gene p oduc s [13]. Thanks o p ope ies o he naph hoquinones, he in e es sugges ing and op imizing o easy- o-use and sensi i e de e mina ion o hem inc eases. Senso s 2006, 6 1471 Figu e 1. (A) Chemical s uc u es o lawsone, juglone and plumbagin. The mo phological changes o BY-2 cells induced by he naph hoquinones juglone and plumbagin. The obacco cells we e ixed and dyed wi h luo escen dye Hoechs 33258 and obse ed by epi luo escen mic oscopy. (B) Con ol, non- ea ed BY-2 cells. (C-G) The obacco cells ea ed by plumbagin and juglone (500 µg.l-1) o 24 hou s; (C) o e all insigh ; (D) beginning ch oma in condensa ion on he nuclea pe iphe y – p e-apop o ic nucleus; (E) apop o ic nucleus – plumbagin; (F) apop o ic nucleus – juglone; (G) – nucleus wi h s ong ch oma in condensa ion. The a ows show on desc ibed phenomenon. Magni ica ion o he igu es: (B-C) × 400 and (D-F) × 1000. B E C D F G Lawson Juglon Plumbagin A OOH O O OOH OCH 3 OOH Senso s 2006, 6 1472 0.0 1.0 2.0 3.0 4.0 00.20.40.6 Lawsone Concen a ion (µg/ml) Peak heigh (nA) 0 4 8 12 0.0 1.0 2.0 Concen a ion (µg/ml) Peak heigh (nA) Plumbagin 0 4 8 0.0 0.2 0.4 0.6 0.8 1.0 Concen a ion (µg/ml) Peak heigh (nA) 0 20 40 60 80 100 5.56.06.57.07.5 lawsone plumbagin juglone pH o he phospha e bu e Peak heigh (%) 0 20 40 60 80 100 0 50 100 150 200 250 lawsone plumbagin juglone Time o accumula ion (s) Peak heigh (%) Plumbagin Plumbagin Lawsone Lawsone Juglone Juglone 2.5 nA -0.4 -0.2 0.0 Po en ial (V) scan Juglone A B CD Figu e 2. Naph hoquinone analysis by s a iona y elec ochemical me hod. (A) The DP ol ammog am o lawsone, juglone and plubagin (1 µg.ml-1) simul aneously de e mined on he su ace o HMDE. The change o signal heigh o he naph hoquinones wi h inc easing (B) ime o accumula ion and (C) pH o phospha e bu e – suppo ing elec oly e. Signal heigh s o 7 nA o lawsone, o 4 nA o plumbagin and o 8 nA o juglone co espond o he 100 %. (D) The dependence o naph hoquinone peak heigh on i s concen a ion. The naph hoquinones we e measu ed in he p esence phospha e bu e (0.2 M) in po en ial ange om –0.6 o 0 V wi h po en ial s ep 5 mV.s-1 and pulse ampli ude 25 mV. Fo o he de ails see “Ma e ials and Me hods” sec ion. 3.2. Elec ochemical de e mina ion o naph hoquinones The highe a en ion was gi en o ch oma og aphic me hods o naph hoquinones de e mina ion [18,40-45]. The elec ochemical echniques ha e been u ilizing mainly o s udy o naph hoquinones chemical in e ac ion [46-48], bu hei u ilizing o naph hoquinone de e mina ion in biological samples has no been published ye . He e, we ocused p ima ily o s udy o lawsone, plumbagin and juglone beha iou on me cu y d op elec ode measu ed in he p esence o phospha e bu e (pH 6.0). We ob ained he cha ac e is ic ol ammog ams wi h elec ochemical signals co esponds o elec ochemical changing o compounds o in e es . Due o elec oanaly ical pu poses, he cha ac e is ic signals a po en ial o -0.37 V o lawsone, o -0.23 V o plumbagin and o -0.04 V o juglone we e chosen. Mo eo e , we we e able o de e mine he naph hoquinones o in e es Senso s 2006, 6 1473 simul aneously (Fig. 2A). Du ing simul aneous de e mina ion he sligh po en ial shi o single naph hoquinones signals was obse ed (± 5%). I clea ly ollows om he esul s ob ained ha he naph hoquinones can in e ac s ongly wi h he su ace o HMDE. Pa icula ly, we obse ed inc ease in he heigh o juglone and plumbagin signal wi h inc easing ime o accumula ion up o A o 120 s and hen he signal dec eased. As o lawsone, i s signal inc eased mo e han 60 % a A o 300 s in compa ison wi h he signal measu ed a A o 0 s and hen he signal inc eased slowly (Fig. 2B). I clea ly ollows om he esul s ha he ime o accumula ion o 120 s was he mos sui able o simul aneous de e mina ion o he naph hoquinones (Fig. 2B). I is a common knowledge ha he pH o he suppo ing elec oly e in luences ma kedly he heigh o a signal. I clea ly ollows om he esul s ob ained ha he highes signals o he naph hoquinones o in e es we e obse ed a pH 5.5. The elec ochemical signals dec eased a highe pH alues, mos ly a he plumbagin and a he leas a lawsone (Fig. 2C). A e he op imizing s ep, measu ing he dependence o he naph hoquinone elec ochemical signal on i s concen a ion ollowed (Fig. 2D). The calib a ion cu es ob ained we e linea wi hin he es ed concen a ion ange om 0.1 o 5 µg.ml -1 . The de ec ion limi s we e exp essed as 3S/N and a ied om 5 o 100 ng.ml -1 wi h ela i e s anda d de ia ions abou 3% (Table 1). Consequen ly, he sugges ed me hod was u ilized o s udy he naph hoquinones con en di ec ly in plan ma e ial. The lea es o Pe sian walnu (Juglans egia) was chosen as plan ma e ial analysed [16]. This ee was chosen no only because i ’s mul ila e al usage, bu also o he high naph hoquinones le el, especially juglone and plumbagin. We de e mined ha he lea es con ained plumbagin a 320 µg.ml -1 and juglone a 290 µg.ml -1 , which co esponds o he naph hoquinone con en o 0.3 and 0.2 g pe 100 g o esh weigh . E en despi e he numbe o in e e ing compounds like ca boxylic acids (linolic, linolenic and ca eic acid), e pens (ge mak en D), i amins (p o i amin A, some i amins B, i amin C and E) and no leas annins and hei de i a i es (galic and egalic acid) he naph hoquinones we e easily de ec able by he sugges ed echnique in he lea es o Pe sian walnu . Table 1. Di e en ial pulse ol amme ic cha ac e is ics o he naph hoquinones o in e es (n = 5). Naph hoquin one Equa iona R 2 LOD (nM)c LOQ (nM)d R.S.D. (%)e Lawsone y = 6.4353x + 0.1462b 0.9921b 5 16 3.5 Plumbagin y = 4.8977x - 0.3042b 0.9922b 100 333 2.8 Juglone y = 8.7097x + 0.2237b 0.9921b 18 60 2.9 a … The concen a ion ange was om 0.1 o 5 µM o he compound o in e es . b … The equa ion was de i ed om he dependence o he peak heigh on he naph hoquinone concen a ion. c … Limi o de ec ion is exp essed as 3 S/N. d … Limi o quan i ica ion is exp essed as 10 S/N. e … Rela i e s anda d de ia ion. Senso s 2006, 6 1474 Juglone Plumbagin Lawsone Posi ion = 0.69 V 0.5 0.7 0.9 Po en ial (V) 0.5 0.7 0.9 Po en ial (V) 30nA 0.7 0.9 1.1 Po en ial (V) 15nA 0.7 0.9 1.1 Po en ial (V) 0.7 0.9 1.1 Po en ial (V) Posi ion = 0.98 V Posi ion = 1.00 V 5nA 30nA 10nA 10nA scan 10nA 0.2 0.6 1.0 Po en ial (V) Full scan o lawsone Posi ion = 0.58 V Posi ion = 0.78 VPosi ion = 0.85 V Juglone Lawsone Plumbagin 0 4 8 12 16 01020 Plumbagin Lawsone Juglone Peak heigh (nA) Concen a ion (µg/ml) Posi ion = 0.7 V Juglans egia 0.5nA AB C DE Figu e 3. Ca bon pas e elec ode. (A) Baseline co ec ed DP ol ammog ams o lawsone, juglone and plumbagin (1 µg.ml-1) measu ed on he su ace o ca bon pas e elec ode. DPV pa ame e s we e used as ollows: po en ial ange om 0 o 1.2 V wi h po en ial s ep 5 mV.s-1 and pulse ampli ude 25 mV. Ca bon sc een p in ed elec ode. (B) Full scan o Lawsone measu ed on ca bon sc een p in ed elec ode using mic o low de ice. (C) Baseline co ec ed ol ammog ams o lawsone, juglone and plumbagin (1 µg.ml-1) on measu ed on he su ace o ca bon sc een p in ed elec odes. (D) Dependence o signal heigh on he concen a ion o single naph hoquinone. (E) Vol ammog am o he eal sample o walnu lea e 100 imes dilu ed. Pumping o he suppo ing elec oly e (0.2 M phospha e bu e , pH 5.5) by using he mic o pump (140 s, 5 000 pm), he samples we e injec ed each 30 s om s ock ube (5 000 pm). Fo o he de ails see “Ma e ials and Me hods” sec ion. 3.3. Elec ochemical analysis o naph hoquinones using a ca bon pas e elec ode The me cu y elec ode can no be u ilized o he analysis in low a angemen , hus we used he solid elec odes (ca bon, gold, e c.) o hese pu poses. The oxida ion signals o naph hoquinones o in e es can be obse ed on ol ammog ams ob ained. These signals a e baseline co ec ing a e shown in Fig. 3A [49,50]. The signals o lawsone we e obse ed a he po en ial o 0.68 ± 0.03 V (n = 5), o juglone a 1.00 ± 0.02 V (n = 5) and o plumbagin a 0.98 ± 0.02 V (n = 5). The peak heigh o Senso s 2006, 6 1481 49. B omba, M.U.A.; Ziegle , H. Applica ion Hin s o Sa i zky-Golay Digi al Smoo hing Fil e s. Anal. Chem. 1981, 53, 1583-1586. 50. B omba, M.U.A.; Ziegle , H. Digi al Smoo hing o Noisy Spec a. Anal. Chem. 1983, 55, 648-653. 51. Rolka, D.; Poghossian, A.; Schoning, M.J. In eg a ion o a capaci i e EIS senso in o a FIA sys em o pH and penicillin de e mina ion. Senso s 2004, 4, 84-94. 52. De Oli ei a, M.F.; Saczk, A.A.; Ne o, J.A.G.; Roldan, P.S.; S adio o, N.R. Flow injec ion ampe ome ic de e mina ion o pe sul a e in cosme ic p oduc s using a P ussian Blue ilm- modi ied elec ode. Senso s 2003, 3, 371-380. 53. P asek, J.; Adamek, M.; Hubalek, J.; Adam, V.; T nko a, L.; Kizek, R. New Hyd odynamic Elec ochemical A angemen o Hea y Me als De ec ion Using Thick-Film Chemical Senso Elec odes. Senso s 2006, 6, 1498-1512. 54. Supalko a, V.; Pe ek, J.; Ha el, L.; K izko a, S.; Pe lo a, J.; Adam, V.; Po esil, D.; Babula, P.; Beklo a, M.; Ho na, A.; Kizek, R. Elec ochemical Senso s o De ec ion o Ace ylsalicylic Acid. Senso s 2006, 6, 1483-1497. 55. Po esil, D.; Mikelo a, R.; Adam, V.; Kizek, R.; P usa, R. Change o he p o ein p53 elec ochemical signal acco ding o i s s uc u al o m - Quick and sensi i e dis inguishing o na i e, dena u ed, and agg ega ed o m o he "gua dian o he genome". P o ein J. 2006, 25, 23- 32. 56. 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De e mina ion o iso la ones in soybean ood anal human u ine using liquid ch oma og aphy wi h elec ochemical de ec ion. J. Ch oma og . B 2004, 806, 101-111. 60. Vi ecek, J.; Pe lo a, J.; Pe ek, J.; Adam, V.; Po esil, D.; Ha el, J.; Mikelo a, R.; T nko a, L.; Kizek, R. Elec ochemical s udy o S ni osoglu a hione and ni ic oxide by ca bon ib e NO senso and cyclic ol amme y - possible way o moni o ing o ni ic oxide. Elec ochim. Ac a 2006, 51, 5087-5094. 61. T nko a, L.; Kizek, R.; Vacek, J. Squa e wa e and elimina ion ol amme ic analysis o azido hymidine in he p esence o oligonucleo ides and ch omosomal DNA. Bioelec ochemis y 2004, 63, 31-36. 62. Billo a, S.; Kizek, R.; Jelen, F.; No o na, P. Squa e-wa e ol amme ic de e mina ion o ce ope azone in a bac e ial cul u e, pha maceu ical d ug, milk, and u ine. Anal. Bioanal. Chem. 2003, 377, 362-369. Senso s 2006, 6 1482 63. T nko a, L.; Jelen, F.; Pe lo a, J.; Adam, V.; Po esil, D.; Kizek, R. Elimina ion ol amme y wi h linea scan as a new de ec ion me hod o DNA senso s. Senso s 2005, 5, 448-464. 64. Adam, V.; Pe lo a, J.; Po esil, D.; Zehnalek, J.; Su es, B.; T nko a, L.; Jelen, F.; Kizek, R. S udy o me allo hionein modi ied elec ode su ace beha io in he p esence o hea y me al ions- biosenso . Elec oanalysis 2005, 17, 1649-1657. 65. Adam, V.; Pe lo a, J.; Po esil, D.; Lubal, P.; Zehnalek, J.; Su es, B.; Kizek, R. New elec ochemical biosenso o de e mine pla inum cy os a ics o DNA s uc u e. Chem. Lis y 2005, 99, 353-393. 66. Adam, V.; Zehnalek, J.; Pe lo a, J.; Po esil, D.; Su es, B.; T nko a, L.; Jelen, F.; Vi ecek, J.; Kizek, R. Phy ochela in modi ied elec ode su ace as a sensi i e hea y-me al ion biosenso . Senso s 2005, 5, 70-84. 67. Pe lo a, J.; Po esil, D.; Zehnalek, J.; Su es, B.; Adam, V.; T nko a, L.; Kizek, R. Cispla in elec ochemical biosenso . Elec ochim. Ac a 2006, 51, 5169-5173. © 2006 by MDPI (h p://www.mdpi.o g). 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