A Disposable Alkaline Phosphatase-Based Biosensor for Vanadium Chronoamperometric Determination

Senso s 2014, 14, 3756-3767; doi:10.3390/s140203756 senso s ISSN 1424-8220 www.mdpi.com/jou nal/senso s A icle A Disposable Alkaline Phospha ase-Based Biosenso o Vanadium Ch onoampe ome ic De e mina ion Ana Lo ena Al a ado-Gámez 1,*, Ma ía Asunción Alonso-Lomillo 2, Olga Domínguez-Renedo 2 and Ma ía Julia A cos-Ma ínez 2 1 CELEQ and School o Chemis y, Uni e si y o Cos a Rica, San Ped o de Mon es de Oca, San José P.O. Box 11500-2060, Cos a Rica 2 Depa men o Chemis y, Facul y o Sciences, Uni e si y o Bu gos, Plaza Misael Bañuelos s/n, Bu gos 09001, Spain; E-Mails: [email protected] (M.A.A.-L.); [email protected] (O.D.-R.); ja [email protected] (M.J.A.-M.) * Au ho o whom co espondence should be add essed; E-Mail: ana.al a adogamez@uc .ac.c ; Tel.: +506-2511-2442; Fax: +506-2511-3217. Recei ed: 10 Decembe 2013; in e ised o m: 24 Janua y 2014 / Accep ed: 8 Feb ua y 2014 / Published: 24 Feb ua y 2014 Abs ac : A ch onoampe ome ic me hod o anadium ion de e mina ion, based on he inhibi ion o he enzyme alkaline phospha ase, is epo ed. Sc een-p in ed ca bon elec odes modi ied wi h gold nanopa icles we e used as ansduce s o he immobiliza ion o he enzyme. The enzyma ic ac i i y o e 4-ni ophenyl phospha e sodium sal is a ec ed by anadium ions, which esul s in a dec ease in he ch onoampe ome ic cu en egis e ed. The de eloped me hod has a de ec ion limi o 0.39 ± 0.06 µM, a epea abili y o 7.7% (n = 4) and a ep oducibili y o 8% (n = 3). A s udy o he possible in e e ences shows ha he p esence o Mo(VI), C (III), Ca(II) and W(VI), may a ec anadium de e mina ion a concen a ion highe han 1.0 mM. The me hod was success ully applied o he de e mina ion o anadium in spiked ap wa e . Keywo ds: alkaline phospha ase; biosenso ; gold nanopa icles; 4-ni ophenyl phospha e; sc een p in ed elec ode; anadium; wa e analysis OPEN ACCESS Senso s 2014, 14 3757 1. In oduc ion Being conside ed as one o he impo an ansi ion elemen s in biological sys ems, anadium is an ul a- ace me al ha can be ound in some ma ine o ganisms, in he p os he ic g oup o b omope oxidases in ce ain ma ine algae [1–3], as pa o he ni ogenase sys em o some bac e ia and plan s [4,5], as well as in plasma and inside cells o mammals [6]. I pa icipa es in he syn hesis o chlo ophyll in pho osyn he ic o ganisms and is a mic onu ien o ma ine and e es ial species [7]. In he pas , anadium compounds we e used as a he apeu ic agen o diabe es, anemia, chlo osis, and e en o ube culosis. I is also a onic, an isep ic and as a spi oche icide. Ne e heless anadium, especially as anadium pen oxide, has a b oad spec um o known oxic e ec s on he espi a o y, ci cula o y and cen al ne ous sys ems, diges i e o gans, kidneys and skin in humans. Howe e con i ma i e mu agenici y and ca cinogenici y s udies a e no consis en , hough hey should be gi en p io i y in long exposu e s udies [8,9]. In ecen yea s, anadium has been used in he de elopmen o no el ma e ials in biochemis y and indus ial p ocesses [10–12]. I s me allic o m is used as a ca bide s abilize in making s eels. Vanadium pen oxide is used in ce amics, as a ca alys , and in he p oduc ion o supe conduc i e magne s, and anadyl sul a e and sodium me a anada e ha e been used in die a y supplemen s [8]. Indus ies using ossil uels like pe oleum, coal and oil, cause mos o he discha ges o anadium in o he en i onmen . Mining a eas a e o he sou ces o his con amina ion, while dis illa ion and pu i ica ion o c ude oils con ibu e less anadium in o he a mosphe e [13]. Vanada e in aqueous solu ion in luences nume ous enzyme-ca alyzed eac ions. I s e ec s on li ing sys ems and he di e en esponses o he in luence o anadium a e well documen ed [14]. As i can assume many s able anionic o ms in aqueous solu ion, depending on acidi y and concen a ion [15], i has been desc ibed as an inhibi o o di e en enzymes. Lindquis in 1973 [16] desc ibed he inhibi ion o ibonuclease by anada e in he p esence o u idine, explaining in some way he o igin o he biological in luences o anadium compounds. A yea la e , in 1974, Van E en and cowo ke s [17], demons a ed he in luence o anada e, molybda e and ungs a e on phosphohyd olases such as acid phospha ases which a e ela i ely nonspeci ic enzymes ha ca alyze he hyd olysis o se e al alkyl and a yl phospha e es e s a a pH be ween 4 and 6. Lopez e al. showed ha alkaline phospha ase, which is a me allop o einase, ca alyzes he hyd olysis o a numbe o phospha e es e s, and he e a e a ew compe i i e inhibi o s o alkaline phospha ase aside om ino ganic phospha e and a sena e, such as oxo anadium (IV) VO2+. I is also possible ha anadium (V) migh adop a igonal bipy amidal s uc u e since c ys alline hyd a ed me a anada es (VO3−·H2O) a e i e-coo dina e wi h oxygen a oms, and he geome y is app oxima ely igonal bipy amidal like phospha e, which is one o he easons why anada e is a known inhibi o (and some imes s imula o ) o many phospha e-me abolizing enzymes [18]. This includes he inhibi ion o a egula o y p o ein phospha ase, which is likely o lead o ac i a ion o a p o ein kinase, he ac i i y o which is key o he insulin-mime ic ac ion o anada e [17,18]. I also can inhibi hexokinase, adenyla e kinase and phospho uc okinase [15]. Vanada e-dependen halope oxidases ha e been shown o a ain phospha ase ac i i y, and his inding may ha e some impac on medical applica ions. Ano he impo an impe us o anadium coo dina ion chemis y has a isen om he obse a ion ha anada e, pe oxo anada e, anadyl and se e al anadium complexes exe an insulin-mime ic e ec [6]. Senso s 2014, 14 3758 Elec ochemical biosenso s based on he p inciple o enzyme inhibi ion ha e been applied o a wide ange o oxic analy es such as pes icides, de i a i es o insec icides, hea y me als and glycoalkaloids [19]. Because o hei excellen pe o mance capabili ies, such as apid esponse, high speci ici y and sensi i i y, ela i ely compac size, low cos and easy ope a ion, hese biosenso s can be a good al e na i e o he de ec ion o anadium [20]. Alkaline phospha ases (ALPs), which ca alyze he hyd olysis o phospha e es e s, a e widely dis ibu ed in mammalian issues, and a e p esen in high concen a ions in bones, in es ines, kidneys, placen a, and li e [21]. ALP is p obably he mos commonly used conjuga ed enzyme o immunoassays due o i s high u no e numbe , b oad subs a e specifici y and possibili y o applica ion. The de e mina ion o i s ac i i y has been ca ied ou using a ious spec opho ome ic and elec ochemical me hods [21–25]. In he de elopmen o sensi i e elec ochemical ALP-based assays s able subs a es such as phenyl phospha e [26–28], naph hyl phospha e [28,29], asco bic acid 2-phospha e [28,30], p-ni ophenyl phospha e [28,31] and i o la in-5-monophospha e [20] ha e been used. Among hem, p-ni ophenyl phospha e is p obably one o he mos widely used subs a e o ALP, since he enzyma ically p oduced p-ni ophenol can be de ec ed elec ochemically [31]. Re e sible inhibi ion o ALP by anadium has been p e iously epo ed [18,20,25], al hough his in e ac ion has been sca cely used o anadium de e mina ion [20]. The p esence o anadium p oduces a dec ease o he ch onoampe ome ic educ ion signal egis e ed ha can be ela ed o he concen a ion o his species. Thus, he aim o his wo k has been he de elopmen o a sc een-p in ed based ampe ome ic biosenso , easily usable in any analy ical labo a o y, o he de ec ion o anadium. ALP has been c oss-linked o he wo king elec ode o sc een-p in ed ca bon elec odes (SPCEs) p e iously modi ied by gold nanopa icles (ALP-AuNPs-SPCEs). In o de o ob ain a biosenso wi h imp o ed conduc i i y and pe o mance o anadium de ec ion, AuNPs we e deposi ed on o he wo king elec ode p e ious o he enzyme immobiliza ion [32]. The use o AuNPs ha e been epo ed in o de o enhance he ch onoampe ome ic cu en esponse, yielding a senso wi h an excellen elec oca aly ic esponse, as esponse ime, long e m s abili y and ep oducibili y [32–38]. The ALP-based biosenso has been cha ac e ized o he de ec ion o anadium in wa e samples. Figu es o me i , such as p ecision o limi o de ec ion, ha e been e alua ed. 2. Resul s and Discussion In a p e ious pape 5- ibo la in monophospha e was used as a subs a e o an alkaline phospha ase biosenso because he e was no epo o such a subs a e being used o a biosenso . P eechawo apun [28] p esen ed a lis o he subs a es o his ype o enzymes, and Fanjul [31] s udied he de ec ion o p-ni ophenol in alkaline phospha ase assays. In ou case we p o ed se e al subs a es ecommended by P eechawo apun such as 3-indoxyphenyl phospha e, 1-na yl phospha e and p-ni ophenyl phospha e, bu he las one p esen ed highe cu en s, and also ALP inhibi ion cu en s dec eased signi ican ly wi h anadium addi ions. As men ioned abo e, p-ni ophenyl phospha e is hyd olyzed by ALPs, unde alkaline condi ions, o 4-ni ophenol, which is elec ochemically oxidized, o igina ing a well-de ined oxida ion cu en [31]. Taking in o accoun ha he enzyma ic ac i i y o ALPs is inhibi ed by anadium [18,20,25], he Senso s 2014, 14 3759 p esence o his me al in o he elec ochemical cell esul s in a cu en dec ease. In his way, he di e ence be ween he s eady-s a e cu en in he absence o anadium (I0) and he s eady-s a e cu en in he p esence o anadium (I) (I (I0–I)) can be quan i a i ely ela ed o concen a ion o anadium added. In o de o quan i y his kind o elec ochemical cu en , an ALP-based biosenso was buil acco ding o he p ocedu e desc ibed in Sec ion 3.3. This ch onoampe ome ic cu en depends on expe imen al ac o s, such as pH o suppo ing elec oly e, subs a e concen a ion, wo king po en ial o ionic s eng h o he medium (concen a ion o Cl− ions in o he elec ochemical cell). In o de o maximize he egis e ed inhibi ion cu en , he e ec o hese a iables and hei in e ac ions in he ch onoampe ome ic esponse was a i s e alua ed by he expe imen al design me hodology [39–41]. The expe imen al domain was de ined by he alues shown in Table 1, co esponding o he high (+) and low (–) le els o each ac o . Then, he 17 expe imen s co esponding o all hose possible combina ions, bea ing in mind he h ee eplica es in he cen al poin necessa y o es ima e he esidual alue, we e ca ied ou . Once he oxida ion cu en egis e ed due o he enzyma ically p oduced p-ni ophenol was s able, anadium was added, quan i ying he ch onoampe ome ic cu en o a 1.8 μM solu ion as esponse a iable o he analysis. Table 1. Values co esponding o he high (+) and low (–) le els o each ac o used in he op imiza ion o he expe imen al condi ions o anadium de ec ion. Low Le el High Le el Suppo ing elec oly e pH 7.0 9.6 Wo king po en ial + 0.5 V s. Ag/AgCl SPE + 1.0 V s. Ag/AgCl SPE Subs a e concen a ion 0.13 mM 0.47 mM Ionic s eng h 0.26 M 0.46 M F om his op imiza ion p ocess, he ollowing op imum alues o he expe imen al a iables in he anadium de e mina ion we e used: suppo ing elec oly e pH 8.7, wo king po en ial o + 0.8 V s. Ag/AgCl SPE, subs a e concen a ion o 0.32 mM and Cl− concen a ion o 0.36 M. Easily quan i iable ch onoampe ome ic signals a e egis e ed unde hese op imized condi ions o anadium (Figu e 1). Figu e 1. Con ou s o es ima ed esponse su ace o alkaline phospha ase and p-ni ophenyl phospha e a [Cl−] = 0.40 M. The cen al g een poin in he cen e o he ci cle is conside ed he op imum alues o expe imen al a iables men ioned abo e. Senso s 2014, 14 3760 Figu e 2. Ch onoampe og am egis e ed using an ALP-based biosenso unde he op imum condi ions (applied po en ial, + 0.80 V s. Ag/AgCl SPE; suppo ing elec oly e pH 8.7 (T is bu e , 0.36 M o al Cl− ) and p-ni ophenyl phospha e, 0.32 mM) in he anadium concen a ion ange om 3.0 μM o 30.0 μM. Inse igu e, a calib a ion cu e o anadium addi ions a he op imum condi ions. Con ol expe imen s we e ca ied ou unde he op imum condi ions using ba e SPCEs and AuNPs-SPCEs bu wi hou enzyme as epo ed in Sec ion 3.3. No analy ical signal was ob ained, ha is o say, he inhibi ion esponse egis e ed a e he addi ion o he subs a e is only ela ed o anadium concen a ion. The e o e, anadium can be de e mined by i s inhibi o y e ec on he esponse o ALP o p-ni ophenyl phospha e. Figu e 2 shows he ampe ome ic signals o he subs a e addi ion and anadium addi ions 1 o 10 unde op imal condi ions. An inse igu e shows a calib a ion cu e o anadium V. The inhibi o y e ec o his me al in he enzyma ic ac i i y, when using p-ni ophenyl phospha e as subs a e, was also s udied by he kine ic pa ame e s o he Linewea e -Bu k plo (Vmax and Km), in absence and p esence o anadium. In absence o he me al, Vmax and Km we e 1.1 × 10−6 and 2.8 × 10−4, espec i ely. In p esence o anadium, i was obse ed ha Vmax and Km inc eased: wi h 3.8 μM o anadium, Vmax = 2.1 × 10−6, Km = 6.5 × 10−4, and wi h 11 μM o anadium Vmax = 2.5 × 10−6 M and Km = 9.9 × 10−4 which sugges a mixed inhibi ion [42]. Thus, he inhibi o y e ec o anadium on he ALP/p-ni ophenyl phospha e eac ion has been con i med o he highe a ini y o ALP o p-ni ophenyl phospha e in he absence o his me al. Senso s 2014, 14 3761 The de ec ion o anadium h ough he inhibi ion o ALP/p-ni ophenyl eac ion (Calib a ion ange om 0.8 μM o 30.0 μM) has esul ed mo e sensi i e han he epo ed one based on he inhibi ion o ALP/ ibo la in-5-monophospha ase (calib a ion ange om 1.8 μM o 15.0 μM) [15]. In his way, he limi o de ec ion based on he s anda d de ia ion (Sy/x) o he esponses o he blank injec ion in iplica e and he slope o he calib a ion cu e was 0.39 ± 0.07 μM, one o de lowe han he p e iously one epo ed [20]. P ecision o he de eloped p ocedu e was s udied in e ms o epea abili y (in a-biosenso ) and ep oducibili y (in e -biosenso s). Bo h igu es o me i ha e been de e mined as he ela i e s anda d de ia ion (RSD) o he slopes o ou calib a ion cu es buil unde he op imum condi ions o he expe imen al a iables. Values o 7.7% and 8% (n = 4) we e ob ained o epea abili y and ep oducibili y, espec i ely. The pe o mance o he de eloped p ocedu e was checked by i s accu acy and ueness. The accu acy o he p oposed me hod was e alua ed by means o he analysis o a anadium ce i ied sample (High Pu i y S anda ds(R) Vanadium S anda d solu ion wi h a Ce i ica e o Analysis con i med agains SRM 3165, lo 992706, ce i ied alue (1,000 ± 4) mg L−1). The anadium mean concen a ion quan i ied, 1,055 ± 65 mg L−1 (n = 4; α = 0.05), ma ches he ce i ied alue o he sample. The me hod also showed a sa is ac o y alue o ueness, e alua ed by eco e y s udies, since he added anadium concen a ion alues (7.33 μM) we e in good ag eemen wi h he ound concen a ion alue o 7.56 ± 0.14 μM (n = 4, α = 0.05). The a e age eco e y o his analysis was 103.1 ± 3.6% wi h a RSD o 3.5%. The e o e, he p oposed me hod is bo h accu a e and sui able o he analysis o anadium. The possible in e e ence om o he me als, such as Ca(II), Sn(II), Al(III), Fe(III), C (III), As(V), Mo(VI) and W(VI), has been s udied. Thei e ec was analyzed by measu ing he inhibi ion cu en a e consecu i e addi ions o se e al solu ions o each me al. I was obse ed ha Al(III), As(V), Fe(III) and Sn(II) ha e a null in luence on he anadium ch onoampe ome ic esponse. Howe e , C (III), Mo(VI) and W(VI) p esen a highe inhibi ion cu en , so hese me als mus be aken in o accoun in he analysis o anadium. Finally, he de eloped p ocedu e was applied o he de e mina ion o anadium in spiked ap wa e samples (1.96 µM), by s anda d addi ion me hodology in quad uplica e. The concen a ion o anadium ound was 1.99 ± 0.23 µM (n = 4, α = 0.05, RSD = 6.8%), wi h an a e age eco e y o 101%. 3. Expe imen al Sec ion 3.1. Chemical Reagen s Se e al inks we e used in he ab ica ion o he sc een p in ed elec odes (SPEs), namely Elec odag PF-407 A (ca bon ink), Elec odag 6037 SS (sil e /sil e chlo ide ink) and Elec odag 452 SS (dielec ic ink) all supplied by Acheson Colloiden (Scheemda, The Ne he lands). Analy ical g ade chemicals wi h no addi ional pu i ica ion we e used. All solu ions we e p epa ed in ul apu e wa e , conduc i i y o 0.05 μS/cm (Gen-Pu e TKA, Niede elbe , Ge many). Hyd ogen e achlo oau a e (III) ihyd a e (HAuCl4), ALP, bo ine se um albumine (BSA) and glu a aldehyde (GA) we e ob ained om Sigma Chemical Co. (S . Louis, MO, USA). p-Ni ophenyl phospha e sodium sal was acqui ed om Fluka Analy ical (Buchs, Swi ze land). Ammonium Senso s 2014, 14 3762 me a anada e (Me ck, Da ms ad , Ge many) was used as s ock solu ion o anadium. 28 mM T is(hyd oxyme hyl)aminome hane bu e (Ald ich Chemical Co., Buchs, Swi ze land) was used oge he wi h 19 mM o MgCl2 (Me ck) and 0.36 M o al Cl−, (Me ck) as suppo ing elec oly e. HCl (Me ck) was used o adjus he pH alue. 3.2. Appa a us SPCEs we e p oduced on a DEK 248 p in ing machine (DEK, Weymou h, UK) using polyes e sc eens wi h app op ia e s encil designs. Elec ochemical measu emen s we e made wi h an Au olab 128N elec ochemical sys em wi h GPES so wa e (Echo Chemie, U ech , The Ne he lands). The pH measu emen s we e pe o med using a Me le -Toledo pHme e S47-K (Columbus, OH, USA). 3.3. Manu ac u ing o ALP-AuNPs-SCPEs SPCEs we e p oduce by sequen ial laye deposi ion o each componen , ha is conduc i e sil e acks, Ag/AgCl e e ence elec ode (Ag/AgCl SPE), ca bon coun e and wo king elec odes and inally, dielec ic ink, acco ding o he p ocedu e desc ibed anywhe e else [43]. The di e en inks we e cu e d acco ding o he manu ac u e ’s speci ica ions. Sc een-p in ed con igu a ions o h ee elec odes (wo king, e e ence and coun e elec ode) we e hus ob ained (Figu e 3). Figu e 3. ALP-AuNPs-SPCE used o anadium de e mina ion. Ca bon wo king elec ode a ea, 12.6 mm2. The wo king elec ode o hese de ices was elec ochemically modi ied by AuNPs, using a 0.1 mM solu ion o HAuCl4 in 0.5 M H2SO4. The deposi ion was pe o med by applying a po en ial o + 0.18 V ( s. Ag/AgCl SPE) du ing 15 s unde s i ing condi ions [20,44]. The enzyme was hen immobilized by c oss-linking on he su ace o AuNPs modi ied SPCEs. To op imize an app op ia e mix u e o ALP enzyme, BSA and GA, se e al elec odes wi h di e en quan i ies o he enzyme om 20 μL o 80 μL, 10 μL–20 μL BSA and 20 μL–40 μL glu a aldehyde, we e p epa ed and he bes esul s we e ob ained wi h a mix u e made up o 40 μL o ALP 0.6%, 20 μL o BSA 1.75% (w/ ) and 40 μL o GA, 2.5% (w/ ) which ga e he bes cu en esponse (Figu e 4) by d opping 10 μL o a 2:1:2 mix u e o a 0.6% Senso s 2014, 14 3763 o enzyme solu ion, 1.75% (w/ ) o BSA solu ion and 2.5% (w/ ) o GA solu ion on o he su ace o a sc een-p in ed wo king elec ode [20]. Finally, he mix u e was le o eac a 4 °C du ing 1 h. ALP-AuNPs-SPCEs we e s o ed a 4 °C. Unde hese s o age condi ions he de eloped biosenso showed a good s abili y o a leas one week. Figu e 5 shows calib a ion cu es o anadium p epa ed he same day bu measu ed a di e en imes om elec ode p epa a ion. Figu e 4. Cu en s ob ained by p-NPP addi ions using elec odes wi h di e en ALP enzyme concen a ions. Figu e 5. Calib a ion cu es o ALP biosenso measu ed a di e en days om elec ode p epa a ion. Senso s 2014, 14 3764 3.4. Measu emen P ocedu e Ch onoampe ome ic measu emen s we e pe o med a oom empe a u e in a cell con aining 5 mL o suppo ing elec oly e solu ion, o he desi ed pH, unde cons an mechanical s i ing. An ALP- based biosenso was placed in he elec ochemical cell con aining 5 mL o suppo ing elec oly e solu ion. An adequa e po en ial was applied and, once a s eady-s a e cu en was se , a de ined amoun o p-ni ophenyl phospha e s ock solu ion was added o he measu ing cell. A la ge anodic cu en was obse ed due o he oxida ion o he enzyma ically p oduced p-ni ophenol. Then, once a pla eau co esponding o he s eady-s a e esponse was eached again, ixed po ions o he anadium s ock solu ion we e added consecu i ely. Enzyme elec odes we e condi ioned in a bu e solu ion o 5 min be ween each calib a ion se ing. 4. Conclusions The use o ALP based biosenso s using AuNPs/SPCEs allows he selec i e ch onoampe ome ic de e mina ion o anadium. This biosenso o e s be e igu es o me i compa ed wi h he p e ious wo k [20] using 5-monophospha e, lowe limi o de ec ion, wide linea ange, bu he same in e e ences, W(VI) and Mo(VI), which a e he mos signi ican a μM le els. The e ec o anadium in he ALP/p-ni ophenyl phospha e eac ion esul s in a mixed inhibi ion, which allows he quan i ica ion o anadium in ap wa e . The de eloped p ocedu e has shown a limi o de ec ion o 0.39 ± 0.06 µM, en imes lowe han p e iously epo ed. The ep oducibili y and epea abili y alues o RSD o he slopes o se e al calib a ions a e lowe han 10%. Acknowledgemen s Au ho s would like o acknowledge unding ia Resea ch Vice ec o y o Cos a Rica Uni e si y (P ojec 804-B0-058) and Spanish Minis y o Science and Inno a ion (TEC-2009/12029). This wo k was suppo ed by he Spanish Minis y o Science and Inno a ion (MICINN) 410 and he Eu opean Regional De elopmen Fund (FEDER) (INNPACTO SERIBIO 2011-411 2014) and TEC2009-12029, as well as h ough Jun a de Cas illa y León (BU212A12-2). Au ho Con ibu ions All au ho s collabo a ed o ca y ou he wo k p esen ed he e. M.J.A.-M. and M.A.A.-L. de ined he esea ch opic. A.L.A.-G. ca ied ou he expe imen s and in e p e ed he esul s. A.L.A.-G. and M.A.A.-L. w o e he pape . M.A.A.-L., M.J.A.-M. and O.D.-R. p epa ed he elec odes, e iewed and edi ed he manusc ip . All au ho s ead and app o ed he manusc ip . Con lic s o In e es The au ho s decla e no con lic o in e es .