Senso s 2015, 15, 2232-2243; doi:10.3390/s150202232
senso s
ISSN 1424-8220
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A icle
A Ch onoampe ome ic Sc een P in ed Ca bon Biosenso
Based on Alkaline Phospha ase Inhibi ion o W(VI)
De e mina ion in Wa e , Using 2-Phospho-L-Asco bic Acid
T isodium Sal as a Subs a e
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 School o Chemis y & CELEQ, Uni e si y o Cos a Rica, San Ped o de Mon es de Oca,
11500-2060 San José, 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,
09001 Bu gos, Spain; E-Mails: [email p o ec ed] (M.A.A.-L.); [email p o ec ed] (O.D.-R.);
[email p o ec ed] (M.J.A.-M.)
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +1-506-2511-2442; Fax: +1-506-2511-2446.
Academic Edi o : Robe o Pillo on
Recei ed: 10 No embe 2014 / Accep ed: 13 Janua y 2015 / Published: 22 Janua y 2015
Abs ac : This pape p esen s a ch onoampe ome ic me hod o de e mine ungs en in wa e
using sc een-p in ed ca bon elec odes modi ied wi h gold nanopa icles and c oss linked
alkaline phospha ase immobilized in he wo king elec ode. Enzyma ic ac i i y o e
2-phospho-L-asco bic acid isodium sal , used as subs a e, was a ec ed by ungs en ions,
which esul ed in a dec ease o ch onoampe ome ic cu en , when a po en ial o
200 mV was applied on 10 mM o subs a e in a T is HCl bu e pH 8.00 and 0.36 M o KCl.
Calib a ion cu es o he elec ochemical me hod alida ion, gi e a ep oducibili y o 5.2%
(n = 3), a epea abili y o 9.4% (n = 3) and a de ec ion limi o 0.29 ± 0.01 µM. En iched ap
wa e , pu i ied labo a o y wa e and bo led d inking wa e , wi h a ce i ied ungs en
e e ence solu ion aceable o NIST, ga e a eco e y o 97.1%, 99.1% and 99.1%
espec i ely (n = 4 in each case) and a dynamic ange om 0.6 o 30 µM. This s udy was
pe o med by means o a Linewea e –Bu k plo , showing a mixed kine ic inhibi ion.
OPEN ACCESS
Senso s 2015, 15 2233
Keywo ds: alkaline phospha ase; ch onoampe ome y; 2-phospho-L-asco bic acid
isodium sal ; sc een p in ed ca bon elec ode; ungs en
1. In oduc ion
Tungs en is a me al which occu s na u ally in soils and sedimen s, usually in small concen a ions
anging be ween 0.2 and 2.4 mg·kg−1 in he li hosphe e [1–3]. This me al is also p esen in oceanic
wa e s in ace amoun s, o ins ance i s con en s o he No he n A lan ic and Paci ic Oceans epo ed
in he li e a u e a e 100 ng·L−1 and 8 ng·L−1 espec i ely [1]. In aqueous media, WO42− is he dominan
species, occu ing as monome in a pH ange o 6.9 o 9.3, while poly ungs a e species o m a lowe pH
and highe W concen a ion. Ions such as NH4+ shi he pH a which polycondensa ion occu s, hus
poly ungs a e species in ammoniacal solu ion a e s able a pH alues whe e no mally only
mono ungs a e ions would exis . On he o he hand, W has been ound wi h a concen a ion be ween 0.27
and 742 µM in g oundwa e in Ca son Dese (Ne ada, USA) [4].
Na u al p ocesses o ungs en isola ion include wea he ing o W- ich ocks and soils, dissolu ions,
hyd o he mal and olcanic ac i i y, a mosphe ic p ecipi a ion (we and d y) and exc e ion o
me aboli es. Tungs en occu s in he oxida ion s a es III, IV, V and VI, howe e , oxida ion s a e VI
ep esen s he mos s able o ungs en species. I has ensile s eng h a high empe a u e, a densi y o
19.1 g·cm−1 and he highes mel ing/boiling poin s among elemen s. These p ope ies make ungs en
sui able o a wide a ie y o uses. Tungs en-based p oduc s ha e been in use in a wide ange o
applica ions, s e ching om daily household supplies o highly specialized componen s o mode n
science and echnology [1].
Fo a long ime, ungs en was conside ed an insoluble me al ha did no exhibi se ious oxicological
o en i onmen al e ec s, and i was placed among less oxic elemen s. Ne e heless ungs en e ec s on
en i onmen al sys ems ha e no been in es iga ed ex ensi ely in ega ds o i s eco oxicological e ec s,
so published da a a e agmen a y. In ac , an h opogenic sou ces include a a ie y o indus ial,
comme cial and mili a y ac i i ies along wi h non-sus ainable disposal p ac ices o municipal,
ag icul u al and indus ial was es. Howe e , in spi e o i s ex ensi e uses, biological and biochemical
e ec s o ungs en and ungs en compounds a e no well known [1,5].
Fo ins ance, i has been epo ed ecen ly ha ungs en as a ace elemen is oxic o people and
animals, such as 5 μg·kg−1 o ungs en led o he dea h o animal emb yos [2,6,7]. O he s udies ha e
shown ha dissolu ion o me allic ungs en pa icles may cause ad e se en i onmen al e ec s such as
soil acidi ica ion as well as di ec and indi ec oxic e ec s in plan s, soil mic oo ganisms and
in e eb a es. The e o e, i has been ound necessa y o e-e alua e he en i onmen al egula ions o
ungs en, o con i m he “non oxic” and “en i onmen ally ine ” o he me al [6,8].
The e a e many echniques o analyse ungs en in en i onmen al samples; mos ly spec opho ome ic
me hods such as A omic Abso p ion and ICP-OES, as well as ol amme ic and pola og aphic me hods
ha e been used [2,3,9–11]. The applica ion o enzyme biosenso s o de e mining oxic compounds is a
dynamic p omising esea ch end, because he associa ed analy ical sys ems a e simple, apid and
selec i e o ampe ome ic, po en iome ic o conduc ime ic echniques. They unc ion by combining
Senso s 2015, 15 2234
an elec ochemical p ocess wi h he ac i i y o an immobilized enzyme. The selec ed enzyme mus
p o ide selec i i y h ough i s biological a ini y o a pa icula subs a e, usually o biological o igin.
These ype o biosenso s cons i u e one o he mos widesp ead, nume ous and success ully
comme cialized de ices in biomolecula elec onics since hei incep ion, which implied he
de elopmen o a no el ield in analy ical bio echnology [12,13].
The enzyme immobiliza ion on he elec ode su ace is a c i ical s ep ha de ines an enzyme
elec ode’s e ec i eness. Di e en chemical, biochemical and physical ac o s such as he chemical
c oss linking wi h glu a aldehyde, o he unc ional an igen–an ibody agen in e ac ions, as well as he
magne ic in e ac ions, en apmen o encapsula ion wi hin polyme s and he o ma ion o pas e
ma e ials, a e gene ally used o immobilize he enzyme on he elec ode su ace [9,13].
Alkaline phospha ase has been used o analyse some hea y me als, such as anadium and
molybdenum h ough hese echniques. Me als such as anadium and ungs en exe inhibi o y e ec s on
he enzyme alkaline phospha ase (ALP). Fo ins ance, in 1974, Van E en [14] 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 pH alues be ween 4 and 6. Ou esea ch g oup ecen ly epo ed p omising esul s using wo di e en
biosenso s wi h his enzyme o anadium de ec ion a ace le els in wa e [15,16], s udying
modi ica ion o elec ode su aces wi h gold nanopa icles in o de o p o ide a mic o-en i onmen
simila o na i e sys ems o edox p o eins and allowing hese molecules mo e eedom in o ien a ion,
he eby educing he insula ing e ec o he p o ein shell owa ds di ec elec on ans e h ough gold
nanoc ys al conduc ing unnels [17–19].
The eby, we epo in his pape he de elopmen o a new me hodology o de e mine ungs en in
wa e a low µmol·L−1 concen a ions using a biosenso wi h a disposable sc een p in ed ca bon
elec ode (SPCE) modi ied wi h gold nanopa icles, using he enzyme alkaline phospha ase immobilized
o e he wo king elec ode and 2-phospho-L-asco bic acid isodium sal as subs a e.
2. Expe imen al Sec ion
2.1. Chemical Reagen s
Se e al inks we e used in he ab ica ion o he sc een p in ed ca bon elec odes (SPCEs),
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 u he 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), alkaline phospha ase (ALP), bo ine
se um albumine (BSA) and glu a aldehyde (GA) we e ob ained om Sigma Chemical Co. (S . Louis,
MO, USA).
2-Phospho-L-asco bic acid isodium sal was acqui ed om Sigma Ald ich (S einheim, Ge many).
Ammonium ungs a e Ce iPUR aceable o SRM om NIST (Me ck, Da ms ad , Ge many) was used as
s ock solu ion o ungs en. Tungs en me al 99.99% pu i y, 2% / HNO3 ce i ied alue 1000 ± 4 mg·L−1
(CRM aceable o NIST con i med agains SRM 3163, High-Pu i y S anda ds, Cha les on, SC, USA)
Senso s 2015, 15 2235
was used o spike samples o eco e y. 28 mM T is (hyd oxyme hyl) aminome hane (Ald ich Chemical
Co., Buchs, Swi ze land) bu e 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 0.1 mM (Me ck) was used o adjus he pH alue.
2.2. Appa a us
Sc een P in ed Ca bon Elec odes (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).
2.3. So wa e
Da a analysis was p ocessed wi h a S a g aphics Plus (S a Poin Technologies, Inc. Wa en on, VA,
USA, 1994–1999) so wa e package o he expe imen al design p ocess.
2.4. Manu ac u ing o Sc een P in ed Ca bon Elec odes
Home-made SPCEs we e used in he de e mina ion o ungs en. Fo he cons uc ion o he SPCEs,
successi e laye s o di e en inks we e p in ed on o a polyes e s ip subs a e ollowing he p in ing
p ocedu e desc ibed in p e ious wo ks [19].
2.5. P epa a ion o Modi ied SCPEs Biosenso
The wo king elec ode was elec ochemically modi ied by gold nanopa icles (AuNPs), using a
0.1 mM solu ion o HAuCl4 in 0.5 mM 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 [17,19–21]. The biologically sensi i e
laye o he biosenso was o med by c oss-linking he enzyme alkaline phospha ase on he su ace o he
AuNPs-modi ied SPCEs, by d opping 10 µL o a 2:1:2 mix u e o 0.6% 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. Volume and concen a ion o ALP, GA and BSA we e op imized o ob ain he maximum
analy ical esponse o he inhibi ion o he enzyme wi h W(VI) [15,22]. Finally, he mix u e was le o
eac a 4 °C du ing one hou and he ALP-AuNPs-SPCEs we e s o ed a 4 °C. Unde hese s o age
condi ions he de eloped biosenso showed good s abili y o a leas one week.
2.6. Op imiza ion o Expe imen al Condi ions
In o de o ob ain a sensi i e analy ical signal, a 23 cen al composi e design was ca ied ou conside ing
h ee impo an ac o s: pH, Cl− and subs a e concen a ions [23]. A g oup o 17 expe imen s we e
pe o med a di e en pH, Cl− and subs a e concen a ions acco ding o he design, and he bes
condi ions ob ained om an es ima ed su ace. Ano he expe imen was ca ied ou o op imize he
wo king po en ial. Conside ing ha highe po en ials a e no selec i e and lead o mo e oxidized species,
we ied o apply a po en ial as low as possible, a ying i om 1.0 V o 0.1 V.
Senso s 2015, 15 2236
2.7. Tungs en Ch onoampe ome ic De e mina ion P ocedu e
The ALP/SPCEs biosenso s we e placed in he elec ochemical cell con aining 5 mL o T is-HCl
bu e solu ion, pH 8. 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 2-phospho-L-asco bic acid isodium sal 10 mM s ock solu ions was added o he
measu ing cell. A la ge anodic cu en was obse ed due o he addi ion o 2-phospho-L-asco bic acid
isodium sal . Then, once a pla eau co esponding o he s eady-s a e esponse was eached again,
50 µL aliquo s o he ungs en s ock solu ion we e added consecu i ely. The addi ion o each aliquo
esul ed in a cu en dec ease p opo ional o he amoun o he me al added. Enzyme elec odes we e
condi ioned in a bu e solu ion o 5 min be ween each calib a ion se ing.
2.8. Valida ion
Se e al calib a ion cu es wi h he same and di e en elec odes we e used o e alua e he igu es o
me i . To es ablish he accu acy, eliabili y, and ep oducibili y o he collec ed da a, all es s we e
eco ded in iplica e and only a e age alues a e epo ed. Blank es s we e un in pa allel. All he lab
wa e used in he s udy was p e iously soaked in Alconox, insed wi h dis illed wa e , and inally wi h
ul apu e wa e om TKA and allowed o d y a oom empe a u e.
3. Resul s and Discussion
To de elop he alkaline phospha ase biosenso , ini ially wo subs a es we e used,
p-ni ophenylphospha e, and 2-phospho-L-asco bic acid isodium sal , because in p e ious pape s we
epo ed i s use o de e mine V(V) and As(V) [15,24]. Based on di e en expe imen s esul s, we
decided o wo k wi h 2-phospho-L-asco bic acid isodium sal , which had been es ed only wi h acid
phospha ase, bu no wi h he alkaline enzyme. To he bes o au ho s’ knowledge, he de e mina ion o
ungs en using biosenso s based on he inhibi o y e ec o his me al o e ALP ha e no been desc ibed
up o now. The alkaline phospha ase biosenso is based on he ollowing eac ion:
𝑎𝑙𝑘𝑎𝑙𝑖𝑛𝑒 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑎𝑠𝑒
𝑅 − 𝑃𝑂3
2− + 𝐻2𝑂 ↔ 𝑅 − 𝐻 + 𝐻𝑃𝑂4
2−
whe e R-PO32− is he o ganic phospha e subs a e and HPO42− he monohyd ophospha e. When
p-ni ophenyl phospha e is used as subs a e, he p oduc RH is p-ni ophenol and when
2-phospho-L-asco bic acid is used as subs a e, he p oduc R-H is L-asco bic acid. The e o e, in he
p esence o alkaline phospha ase, he eac ion induces o a change in he pH and in conduc i i y [25].
I is well-known ha ch onoampe ome ic measu emen s a e in luenced by di e en ac o s such as
pH o he medium, he applied po en ial, he subs a e concen a ion, and also ionic s eng h among
o he s, depending on he s udied sys em. Based on he op imiza ion p ocess o ob ain he bes cu en
signals, he op imized pa ame e s a e summa ized in Table 1, which we e used in ungs en de e mina ion:
suppo ing elec oly e pH 8.00, wo king po en ial o +0.20 V s. Ag/AgCl SPE, a 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 ungs en.

Senso s 2015, 15 2237
Table 1. Values co esponding o high (+) and low (–) le els o each ac o , used o
op imize expe imen al condi ions o ungs en de ec ion.
Low Le el
High Le el
Op imum
pH o suppo ing elec oly e
6.30
9.70
8.00
Subs a e concen a ion
0.036 mM
0.38 mM
0.32 mM
Ionic s eng h
0.10 M
0.50 M
0.36 M
Wo king po en ial
+0.1 V s. Ag/AgCl
+1.0 V s. Ag/AgCl
0.20 V
Table 2 show highe calib a ion cu e slopes a lowe applied po en ials, hus, 0.20 V was he bes
po en ial o ou expe imen s. To analyse he signal wi h he immobilized enzyme, 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 he
enzyme. No analy ical signal was ob ained; hence he inhibi ion esponse egis e ed a e he addi ion o
he subs a e is only ela ed o ungs en concen a ion. Consequen ly, ungs en can be de e mined by i s
inhibi o y e ec on he esponse o ALP o 2-phospho-L-asco bic acid isodium sal , by calib a ion
cu es as i is shown in Table 2.
Table 2. Slope a ia ion wi h applied po en ial, om di e en calib a ion cu es o W(VI).
Applied Po en ial/V
Slope, A/M
0.20
0.2531
0.30
0.2122
0.40
0.0913
0.50
0.0437
Figu e 1 ep esen s a ch onoampe og am ob ained o alkaline phospha ase biosenso , a base line
ep esen s he cu en o he bu e signal, he i s cu en s ep is due o subs a e addi ion, and he es ,
1 o 12, esembles consecu i e addi ions o aliquo s o W(VI) s anda d. The inse igu e is he calib a ion
cu e o his ch onoampe og am, expe imen al condi ions a e indica ed in he g aph.
Figu e 1. Ch onoampe og am egis e ed using an ALP-based biosenso unde he op imum
condi ions (applied po en ial, +0.20 V s. Ag/AgCl SPE; suppo ing elec oly e pH 8.00
(T is HCl bu e , 0.36 M o al Cl−) and 2-phospho-L-asco bic acid isodium sal 0.32 mM,
in he W(VI) concen a ion ange om 3.0 µM o 30.0 µM . Inse igu e, a calib a ion cu e
o wel e aliquo W(VI) addi ions unde op imum condi ions.
Senso s 2015, 15 2238
3.1. Inhibi o y E ec o W(VI) O e he ALP Enzyme
The inhibi o y e ec o W in he enzyma ic ac i i y, when using 2-phospho-L-asco bic acid isodium
sal as subs a e, was s udied by means o kine ic pa ame e s o he Linewea e -Bu k plo . The y-in e cep
o his g aph is equi alen o he in e se o Vmax, he x-in e cep o he g aph ep esen s −1/Km (Km is he
Michaelis–Men en cons an and Vmax is he maximum eac ion eloci y). I also gi es a quick, isual
imp ession o he di e en o ms o enzyme inhibi ion, bo h in absence and p esence o ungs en.
This s udy was pe o med unde di e en ionic s eng h, acco ding o alues o Km and slopes,
sugges ing a mixed inhibi ion [26,27]. In ac , he inhibi o y e ec o ungs en on he
ALP/2-phospho-L-asco bic acid isodium sal eac ion was con i med h ough he highe a ini y o
ALP o he subs a e in he absence o his me al.
As i can be seen om Table 3 and Figu e 2, slopes and Km appa en inc eases di ec ly wi h he
concen a ion o W(VI) as inhibi o . In addi ion, Table 4 shows ha Km appa en inc eases di ec ly wi h
ionic s eng h, so we decided o use an ionic s eng h o 0.36 M KCl, because he change in cu en when
adding he subs a e was highe a his concen a ion.
Table 3. Michaelis-Men en appa en cons an alues a di e en W(VI) concen a ion.
W(VI) µg·L−1
Km
0
8.14 × 10−4
291
1.78 × 10−3
740
1.96 × 10−3
Figu e 2. Linewea e -Bu k double ecip ocal plo in p esence and absence o W(VI) as inhibi o .
Table 4. Michaelis-Men en appa en cons an alues a di e en KCl concen a ion.
Condi ions
Km
0.10 M KCl wi hou W(VI)
4.32 × 10−4
0.10 M KCl wi h W(VI)
7.49 × 10−4
0.25 M KCl wi h W(VI)
1.67 × 10−3
0.50 M KCl wi h W(VI)
3.54 × 10−3
Senso s 2015, 15 2239
3.2. Cha ac e iza ion o ALP Biosenso Pe o mance
To cha ac e ize an analy ical me hod, i is impo an o es ablish i s p ecision in e ms o
ep oducibili y and epea abili y. The i s one was calcula ed aking in o accoun calib a ion cu es
egis e ed using di e en biosenso s (in e -biosenso s), and he second one wi h one single biosenso
(in a-biosenso ). In his way, se e al calib a ion cu es we e pe o med in he ange o concen a ion
om 0.6 µM o 10 µM a op imum condi ions o he expe imen al a iables. Bo h igu es o me i we e
calcula ed by he ela i e s anda d de ia ion (RSD) o he slopes om di e en calib a ion cu es. The
ep oducibili y associa ed o slopes o hese calib a ion cu es in e ms o RSD was 4.2% (n = 3).
De e mina ion o he epea abili y was pe o med simila ly using a single ALP based SPCE, which kep
83% o i s ini ial sensibili y a e he hi d calib a ion cu e, ob aining a alue o 9.4% (n = 3) in e ms o
RSD. Table 5 shows he alida ed pa ame e s o he calib a ion cu es egis e ed using di e en
biosenso s and one biosenso espec i ely.
Table 5. P ecision pa ame e s ob ained h ough o dina y leas squa e (OLS) eg ession o
W(VI) using one o di e en ALP modi ied SPCEs unde op imum condi ions on Table 1.
Rep oducibili y
Repea abili y
Elec ode
Slope
R2
Elec ode 3
R2
A/M
Slope A/M
1
0.0629
0.9974
0.0609
0.9902
2
0.0568
0.9966
0.0537
0.9932
3
0.0609
0.9906
0.0507
0.9930
Media
0.0595
0.0551
S d. De .
0.0025
0.0052
RSD%
4.2
9.4
The de ec ion o ungs en h ough he inhibi ion o ALP/2-PLAs eac ion was de e mined (calib a ion
ange om 0.60 µM o 10.0 µM). In his way, he limi o de ec ion based on he s anda d de ia ion
(3Sy/x/m) in iplica e o he calib a ion cu e was 0.29 ± 0.01 µM, and he limi o quan i ica ion was
0.58 ± 0.02 µM. The pe o mance o he de eloped p ocedu e was checked by i s accu acy and
eliabili y. The accu acy o he p oposed me hod was e alua ed by means o he analysis o a ungs en
ce i ied sample (High Pu i y S anda ds SRM, 1000 ± 4 mg·L−1 o W(VI) ), and hen he biosenso was
used o analyse en iched ap wa e wi h he same SRM, eco e ing 991 ± 68% (Table 5). Finally, he
de eloped p ocedu e was applied o he de e mina ion o ungs en in spiked ap wa e samples
(1.03 µM), by s anda d addi ion me hodology wi h High-Pu i y S anda ds aceable o NIST-SRM 3163
by iplica e. The mean concen a ion o W(VI) was (1.01 ± 0.03) µM (n = 3, α = 0.05 and RSD 2.9%,
wi h an a e age eco e y o (97.1 ± 2.9)%. O he samples en iched we e pu i ied labo a o y wa e om
a TKA Sys em and bo led d inking wa e , we e also en iched wi h he SRM s anda d, ob ained
eco e ies o 99.1% ± 2.9% and 99.1% ± 5.2% espec i ely (n = 4).
3.3. In e e ences
We also s udied he e ec o di e en ca ions a ou di e en concen a ions (1.0 µM, 10 µM,
0.1 mM and 1 mM) on he inhibi ion cu en o ALP unde biosenso -op imized condi ions. Thei e ec
Senso s 2015, 15 2240
was analysed by measu ing he inhibi ion cu en a e consecu i e addi ions o s anda d solu ions o
each me al. Figu e 3 shows ha a µM le el, W(VI) p oduces he highes inhibi ion cu en compa ed o
o he ca ions a he same concen a ion. Ne e heless, Figu e 4 p esen s he inhibi ion o possible
in e e ences could be p esen in na u al wa e s such as Ca(II), Al(III), Mg(II) and Fe(III), bu also o he
me als as Se(IV), As(V) and Sn(II). These elemen s and Fe(III) a e majo in e e ences a concen a ions
highe han 1.0 µM, howe e i hey a e p esen in wa e a highe concen a ions han W(VI), hey mus
be conside ed in he analysis o ungs en. Tha is a limi ing aspec o his me hod bu we can say ha in
some cases, many po en ial in e e ing ca ions end no o exis in many eal samples, only Fe(III) should
be a p oblem. In cases whe e he e could be ea ed wi h a sample p e iously p ecipi an , o example in
a basic medium, while o he ca ions a e as hyd oxides, he W would be as a ungs a e. Tha is o use
chemical means o p ecipi a ion and complexa ion o elimina e o dec ease he concen a ion o such
in e e ences. We can say ha in iew o he good esul s ob ained in spiked eal wa e samples, hese
in e e ences does no seem o be a p oblem.
Figu e 3. Pe cen age o inhibi ion cu en om se e al ca ions a 10−6 M, o ALP biosenso
T is HCl bu e pH 8.0, 0.36 M KCl; 0.20 V; 0.32 mM; 2-P-L-Asc as a subs a e.
Figu e 4. Pe cen age o inhibi ion cu en om se e al ca ions a di e en concen a ions, o
ALP biosenso , T is HCl bu e pH 8.0, 0.36 M KCl, 0.20 V, 0.32 mM 2-P-L-Asc as a subs a e.