Combined SPRi sensor for simultaneous detection of nitrate and ammonium in wastewater

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]. 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