senso s
Le e
Combined SPRi Senso o Simul aneous De ec ion o Ni a e
and Ammonium in Was ewa e
Ma ina V áblo á1,* , I an Kou ník1,2, Ka eˇ ina Smu ná1, Dominika Ma ko á1,2 and Nikola Ve e ko á1,3


Ci a ion: V áblo á, M.; Kou ník, I.;
Smu ná, K.; Ma ko á, D.; Ve e ko á,
N. Combined SPRi Senso o
Simul aneous De ec ion o Ni a e
and Ammonium in Was ewa e .
Senso s 2021,21, 725. h ps://
doi.o g/10.3390/s21030725
Academic Edi o : Venka
R. Bhe hanabo la
Recei ed: 10 Decembe 2020
Accep ed: 16 Janua y 2021
Published: 21 Janua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Ins i u e o En i onmen al Technology, CEET, VSB-Technical Uni e si y o Os a a, 17. lis opadu 15,
708 00 Os a a, Czech Republic; [email p o ec ed] (I.K.); [email p o ec ed] (K.S.);
[email p o ec ed] (D.M.); [email p o ec ed] (N.V.)
2Facul y o Ma e ials Science and Technology, VSB-Technical Uni e si y o Os a a, 17. lis opadu 15,
708 00 Os a a, Czech Republic
3Facul y o Mining and Geology, VSB-Technical Uni e si y o Os a a, 17. lis opadu 15,
708 00 Os a a, Czech Republic
*Co espondence: [email p o ec ed]
Abs ac :
Wa e pollu ion is a se ious p oblem in mode n socie y. Ag icul u e, being esponsible
o he discha ge o ag ochemicals, o ganic ma e , o d ug esidues, p oduces a huge amoun
o was ewa e . Aquaponics has he po en ial o educe bo h wa e consump ion and he impac
o wa e pollu ion on ish a ming and plan p oduc ion. In he aqua ic en i onmen , ino ganic
ni ogen is mos ly p esen in he o m o ni a e and ammonium ions. Ni a e, as a inal p oduc
o ammonia mine aliza ion, is he mos common chemical con aminan in aqui e s a ound he
wo ld. Fo con inuous moni o ing o ni ogen compounds in was ewa e , we p opose a senso
o he simul aneous de ec ion o ni a e and ammonium. A su ace plasmon esonance imaging
me hod wi h enzyme-media ed de ec ion was used. Ac i e laye s o ni a e educ ase and glu amine
syn he ase we e c ea ed on he gold su ace o a biochip and es ed o he sensing o ni a e and
ammonium in wa e om an aquaponic sys em. The p oposed senso was applied in wa e samples
wi h a concen a ion o NO
3−
and NH
4+
in a ange be ween 24–780 mg
·
L
−1
and 0.26–120 mg
·
L
−1
,
espec i ely, wi h minimal p e ea men o a sample by i s dilu ion wi h a bu e p io o con ac on a
biochip su ace.
Keywo ds: ni a e; ammonium; su ace plasmon esonance; was ewa e ; aquaponics; senso
1. In oduc ion
Wa e consump ion is g owing wo ldwide, whe eas i s supplies a e dwindling. Mo e-
o e , wa e esou ces all o e he wo ld a e acing he p oblem o pollu ion. Ag icul u e
plays a majo ole in wa e pollu ion [
1
]. Chemical subs ances o igina ing om in en-
si ely a med landscape (ag ochemicals, o ganic ma e , d ug esidues, sedimen s, and
saline d ainage) each he g oundwa e and ha e a se e e impac on wa e quali y [
2
].
The o e use o misuse o e ilize s in ag icul u e leads o a la ge numbe o nu ien s
(ni ogen and phospho us) leaching om soils [
3
]. An o e abundance o nu ien s in wa e
associa ed wi h he excessi e use o e ilize s inc eases he p obabili y o algae g ow h,
which induces he eu ophica ion o wa e bodies [
4
]. Toxins c ea ed by some species o
algal blooms can be ha m ul o e en deadly o humans and biodi e si y.
In he aqua ic en i onmen , ino ganic ni ogen is mos ly p esen in he o m o ni a e
(NO
3−
) and ammonium (NH
4+
) ions. Ammonium ends o be oxidized o ni a e in he ae -
obic p ocess o ni i ica ion. Ni a e (NO
3−
), as a inal p oduc o ammonia mine aliza ion,
is he mos common chemical con aminan in aqui e s a ound he wo ld [
5
]. On a global
scale, ni a e concen a ions may exceed alues as high as 110 mg
·
L
−1
in su ace wa e s
and 440 mg
·
L
−1
in g oundwa e [
6
]. Ele a ed ni a e concen a ion in d inking wa e poses
a se ious h ea o human heal h, especially o bo le- ed in an s unde six mon hs o age,
Senso s 2021,21, 725. h ps://doi.o g/10.3390/s21030725 h ps://www.mdpi.com/jou nal/senso s
Senso s 2021,21, 725 2 o 11
p egnan women, and people wi h low s omach acid [
7
–
9
]. The Wo ld Heal h O ganiza ion
(WHO) hus ecommends limi ing ni a es in d inking wa e o 50 mg·L−1[10].
Ammonia (NH
3
) in he en i onmen o igina es om me abolic, ag icul u al, and
indus ial p ocesses, and disin ec ion wi h chlo amine. I s occu ence in wa e indica es
possible bac e ial, sewage, and animal-was e pollu ion. Na u al le els in g oundwa e
and su ace wa e a e usually below 0.2 mg
·
L
−1
, whe eas anae obic g oundwa e may
con ain up o 3 mg
·
L
−1
. In ensi e li es ock a ming can gi e ise o much highe le els in
su ace wa e . The p esence o ammonia in an aqua ic en i onmen has a nega i e e ec
on ish g ow h, gill condi ion, o gan weigh s, and hema oc i [
11
]. Ammonia le els ha e
no been es ablished in d inking wa e , since i occu s a concen a ions well below hose
o heal h conce n. Toxicological e ec s o humans a e obse ed only a exposu es abo e
abou 200 mg/kg body weigh [10].
Cu en ly, he e is a b oad ange o analy ical me hods o ni a e and/o ammo-
nia concen a ion assessmen , including spec opho ome ic and luo ome ic me hods,
elec ochemical analysis, ch oma og aphic me hods, and elec opho e ic me hods [
12
,
13
].
Flow-injec ion analysis (FIA) echniques can be applied o enhance he e iciency o he
analysis. The choice o a sui able me hod depends on he concen a ion ange and he
p esence o in e e ences. Al hough he chemical educ ion o ni a e (NO
3−
) o he mo e
eac i e ni i e (NO
2−
) is o en he only way in which he ela i ely ine ni a e ion can be
de ec ed, he use o bioca aly ic educ ion (using he ni a e educ ase enzyme) is possible
and o e s an ad an age o e he mo e oxic chemical- educ ion a ian , a oiding he use
o oxic subs ances [14,15].
The mos widely used me hods o he de e mina ion o ni a es a e spec opho o-
me ic. The mos common me hod is he colo ime ic es o he analysis o ni i es and
ni a es, o example, he well-known G iess assay [
14
,
16
]. In he de e mina ion o am-
monium, he indophenol blue (IPB) me hod based on he classic Be helo eac ion is he
mos widely used spec opho ome ic me hod [
17
]. Recen ly, a modi ied IPB me hod,
eplacing oxic and odo ous phenol wi h o-phenylphenol (OPP), was epo ed [
18
–
21
]. The
analyze de eloped by Li e al. [
19
] was used o he online moni o ing o ammonium. In
he gas-di usion-based me hods, an acid–base indica o (e.g., b omo hymol blue, ni azine
yellow) is used, and he colo change is measu ed spec opho ome ically.
On he o he hand, he usage o a biological ca alys o elec ochemical de ec ion
seems o be a mo e ingenious way o de e mine ni a e le els, conside ing he quali y o bio-
ca alys s in imp o ing he sensi i i y and selec i i y o an elec ode. In ecen yea s, ni a e
biosenso s wi h ni a e educ ase as he biological ecogni ion elemen ha e gained pa ic-
ula in e es as hey enable online and con inuous moni o ing and a e non oxic [22–25].
The su ace plasmon esonance (SPR) o me allic s uc u es pe mi s he iden i ica ion
o pollu an molecules in he en i onmen . A simple colo ime ic me hod o ni a e
de ec ion using gold nano ods (AuNRs) was epo ed by Akba i e al. [
26
]. Miao e al. [
27
]
ha e come up wi h an SPR-based ni i e nanosenso combining su ace-modi ied gold
nanopa icles (AuNPs) and he adi ional colo ime ic de ec ion wi h a de ec ion limi
o 3.0
µ
g.L
−1
. The use o AuNPs unc ionalized wi h G iess eac ion eagen s was also
epo ed by Daniel e al. [
28
]. The ad an ages o a simple and compac p obe design, low
cos , and sui abili y o in si u and
in i o
measu emen s a e o e ed by ibe -op ic su ace
plasmon esonance (FOSPR) senso s [
29
]. A FOSPR-based p obe u ilizing a nanocomposi e
o ca bon nano ubes/Cu nanopa icles was p oposed o he ul a ace sensing o ni a e
by Pa een e al. [
30
]. Mo eo e , an FOSPR-based senso p oposed by Zhang e al. [
31
]
enables he simul aneous measu emen o ni a e concen a ion and empe a u e. Fo he
con inuous moni o ing o ni a e in an aqua ic en i onmen , many in si u senso s ha e been
epo ed [
32
,
33
]. Howe e , hese echnologies ha e no been adop ed on a la ge scale due
o p ohibi i e cos s [
34
]. Thus, simple low-cos op ical de ec o s a e being de eloped, such
as a senso employing a UV LED sou ce ecen ly designed by [
35
] o he abo e-men ioned
SPR senso s.
Senso s 2021,21, 725 3 o 11
The con inuous moni o ing o ni ogen nu ien s is o pa icula in e es , o example,
in aquaponic sys ems. Aquaponics is a combina ion o aquacul u e and hyd oponics,
whe ein aquacul u e is de ined as he a ming o aqua ic o ganisms including ish, mol-
lusks, c us aceans, and aqua ic plan s [
36
], and hyd oponics is de ined as he p oduc ion o
plan s in a soilless medium whe eby all o he nu ien s supplied o he c op a e dissol ed
in wa e [
37
]. In aquaponics, he majo i y o nu ien s equi ed o plan g ow h in he
hyd oponic sys em a ise om was e o igina ing om aquacul u e [
38
,
39
]. The aquacul u e
e luen con ains ammonia, which is ans o med o ni a e ia ni i ying bac e ia. Dis-
sol ed ni a e is u he exploi ed as a nu ien sou ce o plan s in he hyd oponic pa . The
wa e emedia ed o cumula ed nu ien s is hen ecycled back o he aquacul u e ank [
40
].
Fo he smoo h ope a ion o aquaponics, i is necessa y o keep he concen a ions o all
ni ogenous compounds wi hin accep able limi s and o a oid an undesi able accumula ion
o ni ogenous subs ances in he sys em [
41
,
42
]. A e age alues in aquaponics come o
60 mg·L−1 o ni a e and 15 mg·L−1 o o al ammonia ni ogen [43].
The aim o his wo k was o design and es a senso based on he enzyme-media ed
de ec ion o ni a es and ammonium by su ace plasmon esonance imaging (SPRi). SPRi
wi h a cha ge-coupled de ice (CCD) de ec o combines a as de ec ion wi h a spa ial
esolu ion on a biochip su ace. The e o e, se e al enzymes can be simul aneously immobi-
lized on he biochip su ace and used o he online de ec ion o wo o mo e compounds
dissol ed in wa e . Ou esea ch was ocused on he de ec ion o ni a e and ammonium
ions p esen in was ewa e om aquaponics due o he need o he con inuous moni o ing
o wa e quali y in hese sys ems.
2. Ma e ials and Me hods
2.1. Wa e Samples
A ba e SPRi-Biochip
™
wi h a gold su ace (HORIBA F ance SAS, Longjumeau, F ance)
was es ed o sodium, po assium, calcium ni a e, and ni ic acid dissol ed in wa e .
Senso s (biochips wi h enzymes immobilized on hei su aces) we e es ed on model wa e
samples p epa ed om ammonium sul a e o po assium ni a e dissol ed in a mobile phase
(T is-HCl o PBS bu e , espec i ely). Knop’s solu ion was p epa ed by dissol ing 1.44 g
Ca(NO
3
)
·
4H
2
O, 0.25 g KH
2
PO
4
, 0.125 g KCl, 0.51 g MgSO
4·
7H
2
O, and 2.0 g FeCl
3·
6H
2
O
in ul apu e wa e . All used chemicals we e o p.a. quali y. Samples o was ewa e we e
collec ed om (i) he hyd oponic pa o a small-scale aquaponic sys em (50 L), (ii) he
aquacul u e pa o a small-scale aquaponic sys em (50 L), and (iii) hyd oponics ha was
no connec ed wi h aquacul u e. The aquaponic sys em was popula ed by c ucian ca ps
(Ca assius ca assius) and Mexican min (Coleus amboinicus).
2.2. Su ace Plasmon Resonance Imaging (SPRi) and Biochips P epa a ion
SPRi de ec ion is based on changes in su ace p ope ies due o he in e ac ion o an
analy e wi h an enzyme bound o he gold biochip su ace. Model wa e samples we e
es ed on SPRi-Biochips
™
(HORIBA F ance SAS, Longjumeau, F ance) wi h a monolaye
o an enzyme. Fo he de ec ion o ni a e and ammonium, monolaye s o ni a e educ ase
(NR) and glu amine syn he ase (GS), espec i ely, we e p epa ed. Real wa e samples we e
es ed on a combined biochip wi h spo s o bo h NR and GS. The p inciple o enzyma ic
de ec ion was based on enzyma ic assays (Sigma-Ald ich, S . Louis, MO, USA):
Ni a e +β-NADH Ni a e Reduc ase
→Ni i e +β-NAD +H2O
Glu ama e +NH+
4+ATP Glu amine Syn he ase
→L-Glu amine +ADP +Pi
whe e
β
-NADH and glu ama e a e co ac o s (chemical compounds ha a e equi ed o an
enzyme’s ac i i y as a ca alys ).
Senso s 2021,21, 725 4 o 11
2.2.1. Biochips wi h Monolaye s o Ni a e Reduc ase o Glu amine Syn he ase
Ni a e educ ase om Aspe gillus nige (Sigma-Ald ich, S . Louis, MO, USA, CAS
9029-27-0) was dissol ed in PBS bu e (10 mM, pH 7.3) in a concen a ion o 11.2 mg
·
mL
−1
.
L-glu amine syn he ase om Esche ichia coli (Sigma-Ald ich, S . Louis, MO, USA, CAS 9023-
70-5) was dissol ed in T is-HCl bu e (20 mM, pH 7.1) in a concen a ion o 0.672 mg
·
mL
−1
.
Each solu ion was applied o he su ace o one ba e SPRi-Biochip
™
(wi h a golden laye ,
HORIBA F ance SAS, Longjumeau, F ance) by a sequence o injec ions in o he measu ing
cell. The mobile phase in he monolaye applica ion and du ing expe imen s was he
same bu e used o enzyme dissolu ion. The con ac ime be ween he enzyme and gold
su ace was 30 min o each injec ion, and he injec ions we e epea ed un il he signal
s abilized. The e ec o binding be ween he ligand (enzyme) and he analy e (ion) in
p esence o a co ac o (a ca alys ) was de ec ed. As a co ac o , 0.2 mM solu ion o
β
-NADH
(Sigma-Ald ich, S . Louis, MO, USA) in PBS bu e o expe imen s wi h ni a e educ ase
and 2 mM solu ion o L-glu amic acid (Sigma-Ald ich, S . Louis, MO, USA) in T is-HCl
bu e o expe imen s wi h glu amine syn he ase was used.
2.2.2. Combined Biochip wi h Spo s o Ni a e Reduc ase and Glu amine Syn he ase
Solu ions o NR and GS in PBS bu e (20 mM, pH 7.2, enzyme concen a ion o
1 mg
·
mL
−1
) we e pu in d ops on he su ace o CS-LD SPRi-Biochip
™
(biochip wi h a
chemically modi ied gold su ace, HORIBA F ance SAS, Longjumeau, F ance). Con ac ime
was 2 h. Fu he , he biochip was insed wi h ul apu e wa e , blocked by e hanolamine
solu ion (pH 9) o 15 min, and insed wi h ul apu e wa e again. As a mobile phase, PBS
bu e (20 mM, pH 7.2) wi h dissol ed co ac o s (0.2 mM
β
-NADH and 2 mM L-glu amic
acid) was used.
2.2.3. Su ace Plasmon Resonance Imaging (SPRi)
The measu emen was pe o med on an OpenPlex SPRi ins umen (HORIBA F ance
SAS, Longjumeau, F ance). Op ical exci a ion o su ace plasmons was achie ed by he
me hod o a enua ed o al e lec ion (p ism coupling). The measu emen s we e pe o med
a a ixed angle, and he ampli ude was measu ed. The mobile phase (bu e ) was de-
gassed h ough a acuum degasse and pumped in o he appa a us wi h a cons an low
(50
µ
L.min
−1
) ia a pe is al ic pump. The measu emen o he p epa ed samples was
pe o med by injec ing he analy e h ough he low loop ( olume 200
µ
L). The samples
we e undilu ed (model wa e samples) o dilu ed en imes by mobile phase (samples om
hyd oponics and aquaponics, Knop’s solu ion). Repea abili y was calcula ed du ing ali-
da ion o he me hod om epea ed measu emen s o eal samples; he a e age s anda d
de ia ion was 1.4% o he mean when he maximum signal o he senso was ead, and
6.9% o he mean when a slope o he a ini y cu es was calcula ed.
2.3. Ion Ch oma og aphy
The ni a e con en o samples was de e mined by measu emen on an Eco IC Ion
ch oma og aph wi h a conduc i i y de ec o (Me ohm AG, He isau, Swi ze land). A Me -
osep A supp 17 (150/4.0) column and a mobile phase (sodium bica bona e: 0.2 mmol.L
−1
;
sodium ca bona e: 5.0 mmol.L
−1
) we e used. A mixed s anda d As asol (Analy ika, P ague,
Czech Republic) was used o calib a ion. Resul s (exp essed in mg
·
L
−1
) we e calcula ed
using MagIC Ne 3.2 so wa e (Me ohm AG, He isau, Swi ze land).
2.4. UV-VIS Spec opho ome y
The ammonium con en o samples was de e mined by S anda d es NANOCOLOR
Ammonium (Ammonium-Indophenol me hod; o concen a ion ange 0.1–2.5 mg
·
L
−1
)
(Mache ey-Nagel GmbH & Co, Dü en, Ge many). Measu emen s we e pe o med using a
Speco d 250 Plus UV-VIS spec opho ome e (Jena Analy ik AG, Jena, Ge many). Signals
we e de ec ed in a wa eleng h o 690 nm.
Senso s 2021,21, 725 5 o 11
3. Resul s and Discussion
Expe imen s based on sensing ni a es in model wa e samples on he biochip wi h
ba e gold su ace e ealed di e en peak shapes and a eas depending on he ca ion p esen
in he calib a ion solu ion (Figu e 1). The e was no speci ic in e ac ion be ween he gold
su ace and NO
3−
when HNO
3
was dissol ed in ul apu e wa e . In ha case, he signal
was dependen on he concen a ion o he solu ion due o he di e en e ac i e indices.
On he o he hand, K
+
, Na
+
, and Ca
2+
ions in e ac ed wi h he gold and caused ailing o
he peak. The e o e, he sensed signal did no co ela e wi h he concen a ion o NO
3−
ions and was a ec ed by he ype o ca ion.
Senso s 2021, 21, x FOR PEER REVIEW 5 o 12
3. Resul s and Discussion
Expe imen s based on sensing ni a es in model wa e samples on he biochip wi h
ba e gold su ace e ealed di e en peak shapes and a eas depending on he ca ion p e-
sen in he calib a ion solu ion (Figu e 1). The e was no speci ic in e ac ion be ween he
gold su ace and NO3− when HNO3 was dissol ed in ul apu e wa e . In ha case, he
signal was dependen on he concen a ion o he solu ion due o he di e en e ac i e
indices. On he o he hand, K+, Na+, and Ca2+ ions in e ac ed wi h he gold and caused
ailing o he peak. The e o e, he sensed signal did no co ela e wi h he concen a ion
o NO3− ions and was a ec ed by he ype o ca ion.
Figu e 1. Su ace plasmon esonance imaging (SPRi) signals o di e en o ms o ni a es (HNO3
(A), KNO3 (B), NaNO3 (C), Ca(NO3)2 (D)) in wa e sensed by a biochip wi h unmodi ied gold su -
ace.
A monolaye o he enzyme ni a e educ ase (NR) on he gold su ace p e en ed he
nonspeci ic in e ac ion be ween an analy e and he senso su ace. In his case, NR was a
speci ic ligand o he analy e (NO3−) dissol ed in an aqueous sample [24,44]. Expe imen s
wi h KNO3 dissol ed in PBS bu e p o ed a good co ela ion be ween he concen a ion
o NO3− ions and he heigh o he peak (Figu e 2).
Figu e 1.
Su ace plasmon esonance imaging (SPRi) signals o di e en o ms o ni a es (HNO
3
(
A
),
KNO3(B), NaNO3(C), Ca(NO3)2(D)) in wa e sensed by a biochip wi h unmodi ied gold su ace.
A monolaye o he enzyme ni a e educ ase (NR) on he gold su ace p e en ed he
nonspeci ic in e ac ion be ween an analy e and he senso su ace. In his case, NR was a
speci ic ligand o he analy e (NO
3−
) dissol ed in an aqueous sample [
24
,
44
]. Expe imen s
wi h KNO
3
dissol ed in PBS bu e p o ed a good co ela ion be ween he concen a ion
o NO3−ions and he heigh o he peak (Figu e 2).
Senso s 2021, 21, x FOR PEER REVIEW 6 o 12
Figu e 2. (A) SPRi signals o KNO3 dissol ed in PBS bu e (pH 7.3) sensed on a monolaye o ni-
a e educ ase and (B) SPRi signal o (NH₄)₂SO₄ dissol ed in T is bu e (pH 7.1) sensed on a mon-
olaye o glu amine syn he ase.
A simila sys em o speci ic ligand and analy e can be ound o ammonium ions dis-
sol ed in T is bu e , whe e sensing on a monolaye o he enzyme glu amine syn he ase
(GS) led o symme ic peaks wi h a heigh dependen on he concen a ion o NH4+ ions
(Figu e 2B). Bu e s we e used wi h espec o he chemical p ope ies o he enzymes
[45,46], and ensu ed a s able pH because pH can in luence he enzyma ic ac i i y and hus
he SPR signal [44,47]. In me hods based on su ace plasmon esonance, he signal depends
no only on he speci ic in e ac ion be ween an analy e and a ligand bu also on he e ac i e
index o he solu ion [48]. The e o e, he heigh o a ea o he peak can only be used as a
concen a ion indica o o one-componen solu ions. In eal samples, a mix u e o com-
pounds is p esen and ma ix e ec s appea [49]. To sol e his p oblem, he slope o he
signal in a pe iod o peak “pla eau” (e.g., 50–150 min in ou case) can be e alua ed. To e i y
his app oach, we measu ed samples o Knop’s solu ion and a s anda d solu ion o KNO3
dilu ed by PBS bu e o ob ain di e en concen a ions o NO3− and ound a good co ela-
ion be ween he slope o he peak and he concen a ion o NO3− ions (Figu e 3).
Figu e 3. De ec ion o ni a e in Knop’s solu ion (whi e ci cles) used in hyd oponics and a
s anda d solu ion o KNO3 (black ci cles) on a monolaye o ni a e educ ase (NR). KNO3 was
dissol ed in PBS bu e (pH 7.3), and he Knop’s solu ion was dilu ed by his PBS bu e en imes.
A slope o he SPRi signal e lec ing he in e ac ion be ween ni a e and NR was calcula ed om
eco ded peaks o e lec i i y. Linea eg ession was calcula ed om all poin s.
Figu e 2.
(
A
) SPRi signals o KNO
3
dissol ed in PBS bu e (pH 7.3) sensed on a monolaye o ni a e
educ ase and (
B
) SPRi signal o (NH
4
)
2
SO
4
dissol ed in T is bu e (pH 7.1) sensed on a monolaye
o glu amine syn he ase.

Senso s 2021,21, 725 6 o 11
A simila sys em o speci ic ligand and analy e can be ound o ammonium ions
dissol ed in T is bu e , whe e sensing on a monolaye o he enzyme glu amine syn he ase
(GS) led o symme ic peaks wi h a heigh dependen on he concen a ion o NH
4+
ions
(Figu e 2B). Bu e s we e used wi h espec o he chemical p ope ies o he enzymes [
45
,
46
],
and ensu ed a s able pH because pH can in luence he enzyma ic ac i i y and hus he SPR
signal [
44
,
47
]. In me hods based on su ace plasmon esonance, he signal depends no
only on he speci ic in e ac ion be ween an analy e and a ligand bu also on he e ac i e
index o he solu ion [
48
]. The e o e, he heigh o a ea o he peak can only be used
as a concen a ion indica o o one-componen solu ions. In eal samples, a mix u e o
compounds is p esen and ma ix e ec s appea [
49
]. To sol e his p oblem, he slope o
he signal in a pe iod o peak “pla eau” (e.g., 50–150 min in ou case) can be e alua ed. To
e i y his app oach, we measu ed samples o Knop’s solu ion and a s anda d solu ion o
KNO
3
dilu ed by PBS bu e o ob ain di e en concen a ions o NO
3−
and ound a good
co ela ion be ween he slope o he peak and he concen a ion o NO3−ions (Figu e 3).
Senso s 2021, 21, x FOR PEER REVIEW 6 o 12
Figu e 2. (A) SPRi signals o KNO3 dissol ed in PBS bu e (pH 7.3) sensed on a monolaye o ni-
a e educ ase and (B) SPRi signal o (NH₄)₂SO₄ dissol ed in T is bu e (pH 7.1) sensed on a mon-
olaye o glu amine syn he ase.
A simila sys em o speci ic ligand and analy e can be ound o ammonium ions dis-
sol ed in T is bu e , whe e sensing on a monolaye o he enzyme glu amine syn he ase
(GS) led o symme ic peaks wi h a heigh dependen on he concen a ion o NH4+ ions
(Figu e 2B). Bu e s we e used wi h espec o he chemical p ope ies o he enzymes
[45,46], and ensu ed a s able pH because pH can in luence he enzyma ic ac i i y and hus
he SPR signal [44,47]. In me hods based on su ace plasmon esonance, he signal depends
no only on he speci ic in e ac ion be ween an analy e and a ligand bu also on he e ac i e
index o he solu ion [48]. The e o e, he heigh o a ea o he peak can only be used as a
concen a ion indica o o one-componen solu ions. In eal samples, a mix u e o com-
pounds is p esen and ma ix e ec s appea [49]. To sol e his p oblem, he slope o he
signal in a pe iod o peak “pla eau” (e.g., 50–150 min in ou case) can be e alua ed. To e i y
his app oach, we measu ed samples o Knop’s solu ion and a s anda d solu ion o KNO3
dilu ed by PBS bu e o ob ain di e en concen a ions o NO3− and ound a good co ela-
ion be ween he slope o he peak and he concen a ion o NO3− ions (Figu e 3).
Figu e 3. De ec ion o ni a e in Knop’s solu ion (whi e ci cles) used in hyd oponics and a
s anda d solu ion o KNO3 (black ci cles) on a monolaye o ni a e educ ase (NR). KNO3 was
dissol ed in PBS bu e (pH 7.3), and he Knop’s solu ion was dilu ed by his PBS bu e en imes.
A slope o he SPRi signal e lec ing he in e ac ion be ween ni a e and NR was calcula ed om
eco ded peaks o e lec i i y. Linea eg ession was calcula ed om all poin s.
Figu e 3.
De ec ion o ni a e in Knop’s solu ion (whi e ci cles) used in hyd oponics and a s anda d
solu ion o KNO
3
(black ci cles) on a monolaye o ni a e educ ase (NR). KNO
3
was dissol ed in
PBS bu e (pH 7.3), and he Knop’s solu ion was dilu ed by his PBS bu e en imes. A slope o he
SPRi signal e lec ing he in e ac ion be ween ni a e and NR was calcula ed om eco ded peaks o
e lec i i y. Linea eg ession was calcula ed om all poin s.
Ano he app oach is o use a wo-channel SPR [
50
]. This was no possible in ou case
due o he one-channel cons uc ion o ou sys em. Ne e heless, he SPR imaging allows
using di e en a eas on he biochip su ace o sub ac signals acqui ed by speci ic and
nonspeci ic in e ac ions. The e o e, we p epa ed a biochip wi h h ee measu ing zones:
enzymes (NR and GS) o med wo zones, and he hi d zone was a e e ence o med by a
blocked su ace o he CS-LD biochip ha could no in e ac wi h ions o ou in e es (NO
3−
and NH
4+
). SPR imaging allowed scanning o he whole a ea o he measu ing zone o he
biochip a one ime due o he CCD de ec o [
51
,
52
]. We sensed he signal om enzymes
and sub ac ed he signal om he e e ence a ea. This led o he educ ion o ma ix
e ec s. Then, we eco ded he maximum ela i e e lec i i y o each sample ob ained on
bo h NR and GS su aces. The de ec ion limi o he senso was 17.8 mg
·
L
−1
o ni a e
and 0.115 mg
·
L
−1
o ammonium; he e o e, only samples wi h a highe concen a ion
o ions we e e alua ed. Selec ed wa e samples om aquaponics and hyd oponics had
concen a ions o NO
3−
and NH
4+
be ween 24 and 773 mg
·
L
−1
and 0.26 and 219 mg
·
L
−1
,
espec i ely (Table 1). Samples we e dilu ed en imes in a bu e o con ol pH and o lowe
ma ix e ec s. Indi idual measu emen s on enzymes e ealed a nonlinea ela ionship
be ween concen a ion measu ed by an independen me hod (ion ch oma og aphy o
spec opho ome y) and he signal om he senso (Figu e 4).
Senso s 2021,21, 725 7 o 11
Table 1.
Concen a ions o ni a e and ammonium in wa e samples om aquaponics and hyd opon-
ics measu ed by ion ch oma og aphy (NO3−) and UV-VIS spec opho ome y (NH4+).
Sample No. c (NO3−) mg·L−1c (NH4+) mg·L−1
1 24.0 0.45
2 25.1 0.40
3 25.2 0.52
4 88.7 11.34
5 251.0 122.29
6 772.4 218.14
7 32.2 0.26
8 108.3 0.57
9 136.3 1.27
10 307.0 9.31
Figu e 4
Figu e 5
Figu e 4.
The ela ionship be ween SPRi signals measu ed by combined biosenso s o he simul ane-
ous de ec ion o ni a e (
A
) and ammonia (
B
) and he concen a ion o ions in wa e samples om
aquaponics measu ed by con en ional me hods. The senso su ace was modi ied by ni a e educ-
ase (NO
3−
de ec ion) and glu amine syn he ase (NH
4+
de ec ion). The signal om he unmodi ied
pa o he senso was used as a e e ence.
Six samples (1–6) we e used o es he senso , and ano he ou samples (7–10)
we e used o e i y esul s. We plo ed ion concen a ions (Figu e 5) ob ained om ion
ch oma og aphy (NO
3−
) and spec opho ome y (NH
4+
) on a loga i hmic scale. The i s
g oup o samples was i ed wi h he linea unc ion, and he con idence and p edic ion
in e als we e calcula ed. F om he second g oup o samples (Figu e 5A, open symbols),
h ee we e ou side he p edic ion in e al wi h a ela i ely highe concen a ion o ni a e
han ammonium ions compa ed o he es o he samples (8–10).
The nex s ep was o measu e he same en samples on he combined biochip by SPRi
and co ela e he e lec i i y sensed on NR (belonging o NO
3−
ions) and GS (belonging
o NH
4+
ions) (Figu e 5B). When applying he same p o ocol o da a i ing, he ou lie s
we e iden i ied as samples 8–10. The ag eemen be ween he ela ionship o concen a ions
measu ed by con enien me hods and signals om he senso sugges s he possibili y o
using he senso o de ec de ia ions in ni ogen-compound concen a ions when mon-
i o ing he s a e o he aquaponic sys em. The simul aneous assessmen o ni a e and
ammonia allows e alua ion o he balance be ween aquacul u e (a dono o ni ogen) and
hyd oponics (an accep o o ni ogen) as well as he p ope unc ion o ni i ying bac e ia.
Senso s 2021,21, 725 8 o 11
Figu e 4
Figu e 5
Figu e 5.
(
A
) The ela ionship be ween concen a ions o ni a e and ammonium in samples om
aquaponics and hyd oponics measu ed by ion ch oma og aphy (ni a e) and UV-VIS spec opho-
ome y (ammonium). (
B
) Signals om he SPRi senso o he simul aneous de ec ion o ni a e
and ammonium. A se ies o six samples we e used o linea i (closed symbols), and ano he ou
samples o alida ion o he senso (open symbols). Samples ha a e ou o he p edic ion band o
highe han he se limi (which is op ional and co esponds o he maximum equi ed concen a ion)
may p edic an unwan ed s a e o nu ien s in wa e .
The ad an ages and disad an ages o SPR-based senso s we e discussed by Pil-
ia ik e al. [
53
]. We p oposed a senso based on SPR imaging wi h ampli ude measu emen .
SPRi-based senso s a e o en used o he de ec ion o small molecules [
54
–
57
] and he
de ec ion o pollu an s in wa e [
58
–
60
]. An applica ion o SPRi in spa ially di e en ia ed
sensing (“elec onic ongue”) was epo ed by Genua e al. [61].
In aquaponics and hyd oponics, concen a ions o ni ogen compounds in wa e a e
ela i ely high in o de o ensu e he apid g ow h o plan s. Ru e al. [
62
] eached a
limi o up o 35 mg
·
L
−1
o ammonia in he aquaponic sys em. Gene ally, ni a e is a
non oxic compound which can be ound a le els exceeding 1000 mg
·
L
−1
in eshwa e
en i onmen s wi hou ad e se e ec s on aqua ic o ganisms [
63
]. In aquaponic sys ems,
ni a e has been epo ed o be ha mless a concen a ions o 150–300 mg
·
L
−1
[
41
,
64
].
The e o e, he low de ec ion limi o ni a es and ammonium is no a key ac o o used
senso s. The main ask is o sense changes in he balance o nu ien s in a imely manne .
Senso s o moni o ing wa e quali y should be use - iendly, as , and ully au oma ed,
simila o senso s o wa e empe a u e, wa e low a e, ligh in ensi y, pH le el, and plan
heigh [
65
,
66
]. Mo eo e , he simul aneous measu emen o mul iple pa ame e s o wa e
quali y is ad an ageous o lowe ope a ional cos s [
67
]. The e o e, he SPRi senso p oposed
in his wo k could be success ully used in aquacul u e and hyd oponic o aquaponic
sys ems o imp o e he moni o ing o wa e quali y, hus inc easing he e iciency o ish
and plan p oduc ion while educing he amoun o discha ged was ewa e .
4. Conclusions
A combined senso o he simul aneous de ec ion o ni a e and ammonium in
wa e was p oposed o es he biosenso echnology as an al e na i e o he chemical
de ec ion me hods o ni ogen-compound de e mina ion. Su ace plasmon esonance
imaging wi h he enzymes ni a e educ ase and glu amine syn he ase immobilized on
he su ace o a biochip was used as a de ec ion me hod. The p oposed senso was es ed
o s anda d solu ions o ni a e and ammonium as well as eal was ewa e samples om
aquaponics. The ad an age o he SPRi-based senso is he abili y o be used o online and
semicon inuous moni o ing o se e al compounds a he same ime wi h only minimal
sample p e ea men .
Senso s 2021,21, 725 9 o 11
Au ho Con ibu ions:
Concep ualiza ion, M.V.; me hodology: M.V., I.K., D.M., and N.V.; in es i-
ga ion, M.V., I.K., and N.V.; alida ion, M.V., I.K., and N.V.; da a cu a ion, M.V. and I.K.; w i ing—
o iginal d a , M.V., I.K., K.S., and D.M.; w i ing— e iew and edi ing, M.V., I.K., K.S., D.M., and N.V.;
supe ision, M.V. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
The esea ch was unded by EU s uc u al unding in Ope a ional P og amme Resea ch,
De elopmen and Educa ion [g an numbe CZ.02.1.01./0.0/0.0/17_049/0008419]. Expe imen al
esul s we e accomplished by using La ge Resea ch In as uc u e ENREGAT suppo ed by he
Minis y o Educa ion, You h and Spo s o he Czech Republic [g an numbe LM2018098]. S uden s
we e suppo ed by he p ojec o VSB-Technical Uni e si y o Os a a The mal was e ea men and
en i onmen al p o ec ion IV [g an numbe SP2020/14].
Da a A ailabili y S a emen :
The da a p esen ed in his s udy a e a ailable on eques om he
co esponding au ho .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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