sensors

Letter Use of an Artificial Miniaturized in Detection by

Gerardo Zambrano , Flavia Nastri , Vincenzo Pavone , Angela Lombardi and Marco Chino * Department of Chemical Sciences, University of Naples “Federico II”. Via Cintia, 80126 Napoli, Italy; [email protected] (G.Z.); fl[email protected] (F.N.); [email protected] (V.P.); [email protected] (A.L.) * Correspondence: [email protected]; Tel.: +39-081-674421

 Received: 25 May 2020; Accepted: 3 July 2020; Published: 6 July 2020 

Abstract: Advanced oxidation processes represent a viable alternative in water reclamation for potable reuse. Sensing methods of hydrogen peroxide are, therefore, needed to test both process progress and final quality of the produced water. Several bio-based assays have been developed so far, mainly relying on peroxidase , which have the advantage of being fast, efficient, reusable, and environmentally safe. However, their production/purification and, most of all, batch-to-batch consistency may inherently prevent their standardization. Here, we provide evidence that a synthetic de novo miniaturized designed heme-enzyme, namely Mimochrome VI*a, can be proficiently used in hydrogen peroxide assays. Furthermore, a fast and automated assay has been developed by using a lab-bench microplate reader. Under the best working conditions, the assay showed a linear response in the 10.0–120 µM range, together with a second linearity range between 120 and 500 µM for higher hydrogen peroxide concentrations. The detection limit was 4.6 µM and quantitation limits for the two datasets were 15.5 and 186 µM, respectively. In perspective, Mimochrome VI*a could be used as an active biological sensing unit in different sensor configurations.

Keywords: ; hydrogen peroxide; heme ; artificial metalloenzymes;

1. Introduction According to the United Nations (UN) and World Health Organization (WHO) reports about the consequences of climate change, extreme weather events and variable climates are affecting food and water supplies [1,2]. Moreover, free access to drinking water is considered a fundamental and universal human right [3]. In this context, water reclamation for potable reuse is nowadays considered a necessary approach to face near-future water scarcity. Among the chemical, physical, and biological treatments to which reclaimed water must be subjected, advanced oxidation processes (AOPs), coupling either UV irradiation or ozonation in the presence of hydrogen peroxide, are effective both in microbial sterilization and organic pollutant oxidative degradation [4–6]. Hydrogen peroxide determination is, therefore, crucial in: (i) Assessing the undesired residual peroxide concentration of the final treated water; (ii) monitoring, hopefully on a real-time basis, the process performance; (iii) tuning the reagent amount in order to get the best results in terms of its ecological and economic costs. The determination of hydrogen peroxide is a historically fervent research field, as its concentration in solution is directly or indirectly related to the activity of several enzymes [7,8]. Many bio-based assays have been developed with potential food, clinical, and biotechnological applications [9–33]. Different techniques may be coupled to these assays, such as spectrophotometry [9], fluorimetry [10,11], electrochemistry [12–17], and chemiluminescence (CL) [18–33], giving rise to a wide spectrum of sensitivities and variable ranges of detection. CL offers several advantages, such as the use of very

Sensors 2020, 20, 3793; doi:10.3390/s20133793 www.mdpi.com/journal/sensors SensorsSensors2020 2020,,20 20,, 3793x FOR PEER REVIEW 22 ofof 1414

a wide spectrum of sensitivities and variable ranges of detection. CL offers several advantages, such sensitiveas the use and of very miniaturized sensitive and detectors, miniaturized from lab dete benchctors, PMT from detectorslab bench to PMT smartphone detectors CCDto smartphone cameras, andCCD the cameras, almost and complete the almost absence complete of background absence of signal background [8]. To thissignal aim, [8]. luminol To this hasaim, beenluminol widely has preferredbeen widely over preferred other CL over reagents other thanks CL reagents to its high thanks quantum to its yield, high enabling quantum the yield, detection enabling of trace the amountsdetection of of materials trace amounts [7,23–28 of,34 materials]. Upon oxidation, [7,23–28,34]. luminol Upon (LH oxidation,2) develops luminol (LH with2) develops an emission blue maximumlight with an at 425emission nm. Severalmaximum oxidation at 425 nm. conditions Several haveoxidation been conditions reported in have the literature,been reported involving in the eitherliterature, catalyzed involving activation either ofcatalyzed hydrogen activation peroxide of in hydrogen alkaline medium,peroxide orin inalkaline situ formation medium, of or singlet in situ 1 1 dioxygenformationspecies of singlet ( O 2dioxygen)[26,35–38 species]. Biomolecule-based ( O2) [26,35–38]. activation Biomolecule-based of hydrogen activation peroxide of has hydrogen several advantagesperoxide has over several other advantages methods, it over is generally other methods, highly selective it is generally and effi highlycient, the selective catalyst and can efficient, be recycled, the andcatalyst the usecan ofbe toxic recycled, reagents and is the avoided use of [ 22toxic–25 ].reag Horseradishents is avoided peroxidase [22–25]. (HRP) Horseradish catalyzes peroxidase hydrogen peroxide(HRP) catalyzes activation hydrogen and further peroxide oxidation activation of luminol, and further thus generating oxidation a of moderately luminol, thus high generating and durable a luminescencemoderately high signal and (LS). durable The mechanismluminescence of luminolsignal (LS). oxidation The mechanism by HRP has of beenluminol studied oxidation in detail by andHRP involves has been several studied steps in detail before and light involves emission several [37,39 steps–44]. before First, hydrogenlight emission peroxide [37,39–44]. induces First, the formationhydrogen ofperoxide the high induces oxidation the state formation compound of the I (C-I)high intermediateoxidation state (Scheme compound1; step I 1),(C-I) which, intermediate in turn, oxidizes(Scheme two I; step equivalents 1), which, of luminol,in turn, inoxidizes the deprotonated two equivalents form LH of— luminol,under alkaline in the conditions,deprotonated through form theLH— formation under alkaline of the compoundconditions, IIthrough (C-II) intermediate the formation (Scheme of the1 ;compound steps 2–3). II Subsequently, (C-II) intermediate rapid dismutation(Scheme I; steps of the 2–3). early Subsequently produced radical, rapid species dismutation yields the of fullythe early oxidized produced diazaquinone radical species (L; Scheme yields1; stepthe fully 4). Finally, oxidized an uncatalyzed diazaquinone coupling (L; scheme between I; step a second 4). Finally, equivalent an uncatalyzed of H2O2 and coupling L takes place between in the a rate-limitingsecond equivalent step, whichof H2O gives2 and rise L takes to dinitrogen place in the release rate-limiting and formation step, which of 3-aminophtalate gives rise to dinitrogen (3-AP) in anrelease excited and triplet formation state (Schemeof 3-aminophtalate1; step 5). Intersystem (3-AP) in crossingan excited then triplet leads state to the (scheme decay toward I; step the 5). emissiveIntersystem singlet crossing state. then leads to the decay toward the emissive singlet state.

HRP + H2O2  C-I + H2O (step 1)

C-I + LH—  C-II + LH•— (step 2)

C-II + LH—  HRP + LH•— (step 3)

2 LH•—  L + LH2 (step 4)

L + H2O2  3-AP + N2 + hν (step 5)

SchemeScheme 1.I. Mechanism of -catalyzed luminolluminol oxidation by hydrogen peroxide, and subsequent luminescence. and subsequent luminescence.

SuitabilitySuitability ofof thethe HRPHRP/luminol/luminol reactionreaction systemsystem inin thethe determinationdetermination ofof hydrogenhydrogen peroxideperoxide hashas beenbeen recognized many many years years ago ago [18]. [18 Both]. Both batch batch and andflow flowassays assays have been have developed, been developed, even though even thoughonly a limited only a limitedresponse, response, in terms in of terms linearity of linearityrange and range limit and of detection limit of detection (LOD), could (LOD), be couldachieved be achieved[9,21–25]. [ 9A,21 –substantial25]. A substantial upgrade upgrade of the of sensin the sensingg capacity capacity was was accomplished accomplished both both by by enzymeenzyme immobilizationimmobilization onto onto di differentfferent matrices matrices and and by by LS LS enhancers enhancers [9, 22[9,22–25,45,46].–25,45,46]. LS LS could could be modulatedbe modulated by theby the presence presence of di offf differenterent organic organic molecules molecules (generally (generally aromatic), aromatic), detergents, detergents, and and metal metal ions, ions, both both in thein the enhancement enhancement and and in the in suppressionthe suppression of the of emitted the emitted light, allowinglight, allowing the determination the determination of several of analytesseveral analytes [23–25]. [23–25]. Finally, bi-enzymaticFinally, bi-enzymatic sensors could sensors be could developed be developed by coupling by coupling HRP/luminol HRP/luminol LS either toLS H either2O2-dependent to H2O2-dependent enzymes, asenzymes, glucose as oxidase glucose (GOx) oxidase enzyme (GOx) for enzyme glucose determination,for glucose determination, or to highly selectiveor to highly antibodies selective for antibodies antigen detection for antigen [34 ,detection47]. [34,47]. ArtificialArtificial metalloenzymes metalloenzymes (ArMs) (ArMs) have have shown shown their their versatility versatility in modeling in modeling natural natural functions functions as well as aswell in engineeringas in engineering catalytic catalytic sites able sites to able perform to pe uncommonrform uncommon or non-natural or non-natu substrateral substrate conversion conversion [48–52]. In[48–52]. this respect, In this we respect, developed we deve differentloped metal-binding different metal-binding sites in the context sites in of the either context four-helix of either bundle four-helix [53,54] orbundle peptide-porphyrin [53,54] or peptide-porphy conjugates [55rin–57 conjugates]. In the former [55–57]. scaffold, In the we former afforded scaffold, the design we ofafforded non-heme the di-irondesign oxidasesof non-heme and oxygenases, di- oxidases as well asand the oxygenases, stabilization as of anwell unprecedented as the stabilization tetra-zinc of metal an cofactorunprecedented [58–61]. Intetra-zinc the latter, metal we miniaturized cofactor [58–61] and then. In reengineered the latter, the we heme-binding miniaturized site ofand globins, then obtainingreengineered a tailor-made the heme-binding set of catalysts site namedof globins, Mimochromes obtaining (MC)a tailor-made [62–68]. The set MC of scaffoldcatalysts recovers named manyMimochromes natural hemeprotein(MC) [62–68]. prerogatives, The MC scaffold by mimicking recovers many thefirst natural and hemeprotein second coordination prerogatives, sphere by

Sensors 2020, 20, x FOR PEER REVIEW 3 of 14

mimicking the first and second coordination sphere interactions around the metal cofactor. MCs comprise two small peptide chains covalently linked to deuteroporphyrin and arranged to fold into a helix–heme–helix sandwich (Figure 1a). Over the years, we rationally evolved the construct from a coordinatively saturated non-catalytic complex to penta-coordinated catalytic analogs [62–64]. An

Sensorsiterative2020 process, 20, 3793 of design and redesign allowed us to engineer and optimize the peroxidase activity3 of 14 into an MC scaffold, thus affording MC6*a (Figure 1a) [65]. MC6*a comprises: A tetradecapeptide (TD) chain bearing a His residue acting as the heme axial proximal ligand; a decapeptide (D) chain, interactionslacking the aroundmetal coordinating the metal cofactor. residue, MCs thus comprise resembling two the small distal peptide site chainsof heme-peroxidase covalently linked for toaccommodating deuteroporphyrin hydrogen and arranged peroxide. to The fold helix–heme– into a helix–heme–helixhelix sandwich sandwich structure (Figure is stabilized1a). Over by the an years,inter-chain we rationally salt bridge evolved between the the construct Glu2 residue from a in coordinatively the (D) chain saturatedand the Arg10 non-catalytic residue complexin the (TD) to penta-coordinatedchain. Further, two catalytic 2-aminoisobutyric analogs [62– acid64]. An(Aib), iterative at positions process 3 ofand design 7 of the and (D) redesign chains, allowed stabilize us the to engineerhelical conformation and optimize theand peroxidase drive the activity peptide into chain an MC to scaffold,face the thus metalloporphyrin affording MC6*a ring, (Figure thanks1a) [ 65 to]. MC6*ahydrophobic comprises: interactions A tetradecapeptide (Figure 1b). (TD) chain bearing a His residue acting as the heme axial proximalFeMC6*a ligand; overcomes a decapeptide the catalytic (D) chain, activity lacking of the HR metalP in coordinatingABTS oxidation residue, [65] thus and resemblingin thioanisole the distaloxygenation site of heme-peroxidase [66]. This minimal for accommodatingscaffold showed hydrogen higher resistance peroxide. against The helix–heme–helix oxidative damage, sandwich thus structurehighlighting is stabilized the protective by an roleinter exerted-chain salt by bridgethe pept betweenide matrix the on Glu2 the residue metal cofactor, in the (D) and chain it was and able the Arg10to tune residue the reactivity in the (TD) of the chain. metalloporphyrin. Further, two 2-aminoisobutyric Indeed, MC6*a manganese acid (Aib), atcomplexes positions were 3 and found 7 of the to (D)be a chains, competent stabilize catalyst the helical in peroxygenase conformation activities and drive [66], the whereas peptide the chain cobalt to face derivative the metalloporphyrin was active in ring,hydrogen thanks evolution to hydrophobic interactions [67,68]. (Figure1b).

(a) (b)

Figure 1. (a) FeMC6*a designed model. Key functional residues and the deuteroporphyrin IX are Figure 1. (a) FeMC6*a designed model. Key functional residues and the deuteroporphyrin IX are highlighted as sticks. Iron is represented as an orange sphere. Aib residues face the porphyrin ring at highlighted as sticks. Iron is represented as an orange sphere. Aib residues face the porphyrin ring at the distal site. The designed inter-chain ion pair interaction is depicted, together with the hydrogen the distal site. The designed inter-chain ion pair interaction is depicted, together with the hydrogen bond network presumably involved in hydrogen peroxide activation. (b) FeMC6*a peptide sequences. bond network presumably involved in hydrogen peroxide activation. (b) FeMC6*a peptide Proximal His and distal axial residues are indicated in bold. The Glu and Arg residues involved in the sequences. Proximal His and distal axial residues are indicated in bold. The Glu and Arg residues inter-chain ion pair interaction are depicted in red and blue, respectively. involved in the inter-chain ion pair interaction are depicted in red and blue, respectively. FeMC6*a overcomes the catalytic activity of HRP in ABTS oxidation [65] and in thioanisole oxygenationSubstituting [66]. natural This minimal enzymes sca wiffoldth properly showed higherdesigned resistance metalloen againstzymes oxidative would be damage, valuable, thus as highlightingfine-tuning the the enzyme protective performances role exerted by by design the peptide should matrix open onthe the way metal to the cofactor, construction and it was of tailor able tomade tune catalysts. the reactivity The ofinteresting the metalloporphyrin. results obtained Indeed, on the MC6*a MC6*a manganese artificial complexesenzyme prompted were found us toto beexploit a competent its practical catalyst application. in peroxygenase To this end, activities we have [66 recently], whereas reported the cobalt the feasibility derivative of was FeMC6*a active inas hydrogena clickable evolution artificial catalysisperoxidase, [67,68 which]. easily reacts with azide-functionalized molecules and/or nanomaterialsSubstituting to afford natural functional enzymes bioconjugates with properly [69]. designed Further, metalloenzymes the ability of FeMC6*a would beto overcome valuable, asHRP fine-tuning catalytic thebehavior enzyme suggested performances its application by design shouldas a substitute open the of way natura to thel enzymes construction in biosensor of tailor madetechnology. catalysts. The interesting results obtained on the MC6*a artificial enzyme prompted us to exploit its practical application. To this end, we have recently reported the feasibility of FeMC6*a as a clickable artificial peroxidase, which easily reacts with azide-functionalized molecules and/or nanomaterials to afford functional bioconjugates [69]. Further, the ability of FeMC6*a to overcome HRP catalytic behavior suggested its application as a substitute of natural enzymes in biosensor technology. Sensors 2020, 20, 3793 4 of 14

In this study, we describe the FeMC6*a proficiency in catalyzing the luminol oxidation and further light emission. First, we compared the activities of FeMC6*a to commercial HRP. Then, we developed a batch assay for H2O2 determination, which can be performed with a simple microplate reader, by screening the best reaction conditions in terms of pH, enzyme, and luminol concentrations. Given the complex kinetic interplay of the reaction steps, the outcome of these experiments is not trivial, and they could be interpreted in terms of the mechanism reported for the natural peroxidase. Finally, we showed that FeMC6*a induces H2O2-dependent LS, which is linear over a wider range of concentrations, compared to HRP, and we tested this in the simulated AOP of a reducing aromatic compound.

2. Materials and Methods

2.1. Reagents All reagents have been purchased from Sigma-Merck, if not differently specified. HRP Type VI-A (batch SLBH1737V) lyophilized powder was at > 950 U/mg (using ABTS) and directly renatured under the assayed buffer conditions (ε(403) = 100 mM 1 cm 1). Hydrogen peroxide and FeMC6*a stock − × − solutions were prepared using 39.4 M 1 cm 1 (at 240 nm) and 117 mM 1 cm 1 (at 387 nm) as the − × − − × − molar absorptivities, respectively.

2.2. Enzyme Synthesis and Purification MC6*a free base has been synthesized as previously reported [65]. Iron insertion has been accomplished by a slightly modified acetate method, as reported elsewhere [63]. FeMC6*a purity has been ascertained by LC–MS analysis (LC-20 Prominence coupled to an ESI IT-TOF high-resolution mass spectrometer, Shimadzu Corporation, Japan) to be higher than 95%.

2.3. Luminescence Standard Assay The standard assay was prepared in a 135 µL final volume reaction mixture containing 0.3 µM FeMC6*a and 0.100 mM luminol. After 2 min of incubation at 25 ◦C, aliquots of H2O2 stock solutions (15 µL) were added to the assay solution. Chemiluminescence measurements were carried out in a TECAN Spark plate reader (Tecan Trading AG, Switzerland) using a 96-well plate (Greiner PS Microplate, 96 Well, solid F-bottom (flat), chimney well with black sides). The rate of luminol oxidation 1 was determined by monitoring the increase in luminescence at 425 nm. Molar absorptivity of 7630 M− cm 1 at 347 nm was used for luminol standardization [40]. All measures were carried out at least × − three times, arbitrary units (a.u.) have been reported by dividing the instrumental response (counts per second) by 106, and the data were analyzed by the software OriginPro 8 (Copyright 1991–2007 OriginLab Corporation, Northampton, MA, USA).

2.4. Steady-State Luminescence Kinetics Steady-state luminescence kinetic traces were recorded on a FluoroMax 4 (Horiba, Minami-ku Kyoto, Japan) instrument in the absence of any excitation (5 nm emission slit width). Temperature control was kept by an external Peltier unit. A reduced volume four-windowed cell (10/3.3 mm path lengths) was used and agitation was granted by a stirring bar. The assays were carried out in a dark room by injecting 10 µL of H2O2 to a final concentration of 3 mM with a Hamilton syringe into a 2 mL solution of 0.1 M Tris/HCl pH 8.5 buffer containing 0.5 mM luminol and 2 nM catalyst.

2.5. Simulated Advanced Oxidation Process of Thioanisole Two four-windowed quartz cells (10 mm path lengths) were filled with 2.5 mL of either water or 81 µM thioanisole. H2O2 was then added to both cuvettes to a final concentration of 8.1 mM. Then, the samples were exposed to a UV lamp photopolymerization apparatus (type Zp lamp UV-A/B/C at 500 W, HELIOS ITALQUARTZ, Milan, Italy) for two hours. Further, 200 µL aliquots were collected at time zero and after each hour and stored at 4 ◦C in dark. Thereafter, all the collected aliquots were Sensors 2020, 20, 3793 5 of 14 Sensors 2020, 20, x FOR PEER REVIEW 5 of 14 processed to the luminol assay according to the procedureprocedure reported in Section 2.32.3,, with the exception that aliquots taken at time zerozero werewere primarilyprimarily diluteddiluted 1:41:4 withwith water.water.

3. Results and Discussion

3.1. Assessment of the Artificial Artificial Peroxi Peroxidasedase ProficiencyProficiency in Luminol OxidationOxidation In order to to test test whether whether FeMC6*a FeMC6*a could could proficiently proficiently catalyze catalyze luminol luminol oxidation oxidation by by activation activation of ofhydrogen hydrogen peroxide, peroxide, we we performed performed a apreliminary preliminary steady-state steady-state kinetic kinetic study study of of LS LS production by FeMC6*a, and compared it to that obtained by HRP in thethe absenceabsence ofof anyany enhancer.enhancer. When 3 mM hydrogen peroxideperoxide was was added added to to a solutiona solution containing containing 0.5 0.5 mM mM of luminolof luminol and and 2 nM 2 ofnM FeMC6*a, of FeMC6*a, a very a intensevery intense LS at 425LS nmat 425 was nm developed was developed that lasted that more la thansted 10more min, than corresponding 10 min, corresponding to the relaxation to time the ofrelaxation the 3-AP time [35] of (Figure the 3-AP2). [35] (Figure 2).

Figure 2.2. Chemiluminescence kineti kineticc trace trace of luminol/H luminol/H2OO22 (0.5(0.5 mM/3 mM/3 mM) mM) system when 2 nM FeMC6*a (black trace) or HRP (red(red trace) isis usedused asas aa catalystcatalyst (100(100 mMmM TrisTris bubufferffer pHpH 8.5).8.5).

A very brilliant LS peak approached its maximummaximum after approximately 1 min and then rapidly quenched, following a kinetic trace that has been previouslypreviously observed in the literature when a very high concentration of peroxidase was adopted [19,37, [19,37,44],44], or when luminol was in high excess with respect to hydrogen peroxide [[41–45].41–45]. Instead, in thethe presence of HRP as a catalyst, only a modest LS was observedobserved inin the the first first 600 600 s, unders, under these these conditions, conditions, as previously as previously reported reported for this for catalytic this catalytic system insystem the absence in the absence of enhancers of enhancers [46]. [46]. These encouraging results prompted us to elaborateelaborate a CL-based hydrogen peroxide assay that could be be easily easily monitored monitored by by a abe benchnch microplate microplate reader. reader. Given Given the di thefferent different sensitivity sensitivity of these of thesekind kindof instrument, of instrument, and andthe thedifferent different geometry geometry of ofthe the experimental experimental setup, setup, we we first first tested tested the the best conditions inin terms terms of samplingof sampling time time and optimaland op FeMC6*atimal FeMC6*a concentration concentration under very under harsh very conditions harsh ofconditions H2O2/luminol of H2 (0.5O2/luminol/10 mM). (0.5/10 Kinetic mM). traces Kinetic of LS as traces taken byof theLS plateas taken reader by underthe plate different reader FeMC6*a under concentrationsdifferent FeMC6*a show concentrations a strong signal show after 1a min,strong as previouslysignal after shown 1 min, for as the previously steady-state shown kinetic for trace. the However,steady-state we kinetic preferred trace. to However, take our measurements we preferred to at take 2 min our to measurements decrease the extent at 2 min of variability to decrease in the LSextent value of (Figurevariability3a). Thein the signal LS value intensity (Figure (herein 3a). expressed The signal in intensity a.u., see Materials(herein expressed and Methods), in a.u., read see afterMaterials 2 min, and increased Methods), almost read exponentially after 2 withmin, FeMC6*aincreased concentration almost exponentially up to 1 µM, slowlywith FeMC6*a decaying thereafterconcentration to very up lowto 1 valuesμM, slowly at 10 µ decayingM concentration thereafter (Figure to very3b). low values at 10 μM concentration (Figure 3b). The observed trend has been previously documented, and it is expected when considering that at very high amounts of catalyst, hydrogen peroxide is rapidly and entirely consumed to produce

Sensors 2020, 20, x FOR PEER REVIEW 6 of 14

Sensors 2020the ,luminol20, 3793 radical, and no H2O2 is then available to react with the diazaquinone (formed upon 6 of 14 radical dismutation) to give the 3-AP (Scheme I) [41–45].

(a) (b)

FigureFigure 3. (a) Luminescence3. (a) Luminescence signal (LS)signal kinetic (LS) traceskinetic as traces registered as registered at different at FeMC6*adifferent concentrationsFeMC6*a (0.07 µconcentrationsM, black; 0.1 µ(0.07M, red;μM, 0.3black;µM 0.1 blue). μM, Insertred; 0.3 shows μM blue). the enlargementInsert shows the of theenlargement LS traces. of Each the LS point is the meantraces. of threeEach point measurements, is the mean withof three error measurem bars representingents, with error the standardbars representing deviations. the standard (b) FeMC6*a concentrationdeviations. dependence (b) FeMC6*a of concentration chemiluminescence dependence as measured of chemiluminescence at 2 min after as measured H2O2 addition at 2 min in after the plate readerH (0.52O2 mMaddition H2O in2 ,the 10 plate mM luminol,reader (0.5 100 mM mM H2O Tris2, 10 bumMffer luminol, pH 8.5). 100 Dashed mM Tris line buffer corresponds pH 8.5). Dashed to the best biexponentialline corresponds fit of the toexperimental the best biexponential data. fit of the experimental data. μ The observedBased on trendthe FeMC6*a has been concentration previously dependence documented, of LS and (see it isFigure expected 3b), we when selected considering 0.3 M as that at the FeMC6*a concentration to be used for the optimization of other experimental conditions. This very high amounts of catalyst, hydrogen peroxide is rapidly and entirely consumed to produce the concentration appeared to us as a good compromise to obtain a strong signal without detector luminolsaturation. radical, and no H2O2 is then available to react with the diazaquinone (formed upon radical dismutation) to give the 3-AP (Scheme1)[41–45]. Based3.2. Effect on of the pH FeMC6*a and Luminol concentration Concentration dependence of LS (see Figure3b), we selected 0.3 µM as the FeMC6*aOur preliminary concentration results to beprompted used forus thein optimizationscreening for ofthe other best experimentalconditions for conditions. the This concentrationperoxide-dependent appeared CL assay. to usThe as optimal a good pH compromise for the peroxidase to obtain activity a strong of FeMC6*a signal was without detectorpreviously saturation. found to be 6.5 [65] in other oxidation reactions; however, the LS efficiency of luminol was maximum around pH 10 [18,22,25] where a luminol deprotonated form is abundant. The 3.2. Effmaximumect of pH andLS is Luminol therefore Concentration expected in the range of 6.5–9.5. The LS was measured at 2 min after the hydrogen peroxide addition at various pH in the range of 6.5–9.5. We found optimal LS at pH of 8.5 Our preliminary results prompted us in screening for the best conditions for the (Figure 4a), quite similar to that reported for HRP [18,22,25]. MC6*a and HRP catalytic activities are peroxide-dependentpractically indistinguishable CL assay. The in optimalterms of pHpH fordependence, the peroxidase making activity the artificial of FeMC6*a metalloenzyme was previously a foundviable to be 6.5alternative [65] in to other the natural oxidation enzyme reactions; in this specific however, application. the LS e fficiency of luminol was maximum around pHGiven 10 [18 that,22, 25luminol] where is involved a luminol in deprotonatedseveral reaction form steps, is luminol abundant. concentration The maximum is an important LS is therefore expectedvariable in the to rangeoptimize. of 6.5–9.5.In this respect, The LS we was decrease measuredd the luminol at 2 min concentration after the hydrogen from 10 mM peroxide down to addition 3 μ at variousM under pH in the the optimized range of FeMC6*a 6.5–9.5. concentration We found optimal and pH LSand at kept pH the of 8.5H2O (Figure2 concentration4a), quite constant similar to that reportedat 0.5 mM. for The HRP observed [18,22,25 LS]. vertically MC6*a and increased HRP catalyticwith the increase activities in areluminol practically initial concentration indistinguishable and reached its maximum at 0.1 mM (Figure 4b). in terms of pH dependence, making the artificial metalloenzyme a viable alternative to the natural enzyme in this specific application. Given that luminol is involved in several reaction steps, luminol concentration is an important variable to optimize. In this respect, we decreased the luminol concentration from 10 mM down to 3 µM under the optimized FeMC6*a concentration and pH and kept the H2O2 concentration constant at 0.5 mM. The observed LS vertically increased with the increase in luminol initial concentration and reached its maximum at 0.1 mM (Figure4b). Sensors 2020, 20, x FOR PEER REVIEW 7 of 14

Sensors 2020, 20, 3793 7 of 14 Sensors 2020, 20, x FOR PEER REVIEW 7 of 14

(a) (b) (a) (b) Figure 4. (a) Effect of the pH on LS, as measured at 2 min after hydrogen peroxide addition. (0.3 μM FigureFigure 4. (a)E 4. ff(aect) Effect of the of the pH pH on on LS, LS, as as measured measuredat at 22 minmin after hydrogen hydrogen peroxide peroxide addition. addition. (0.3 μ (0.3M µM FeMC6*a, 0.5 mM H2O2, 10 mM luminol, 100 mM Tris buffer). Dashed line corresponds to the best FeMC6*a,FeMC6*a, 0.5 mM 0.5 mM H2O H22,O 102, 10 mM mM luminol, luminol, 100 100 mMmM Tris buffer). buffer). Dashed Dashed line line corresponds corresponds to the to best the best Gaussian fit of the experimental data. (b) Effect of luminol concentration on LS (0.3 μM FeMC6*a, 0.5 GaussianGaussian fit of fit the of the experimental experimental data. data. ( b))E Effectffect of of luminol luminol concentration concentration on LS on (0.3 LS μM (0.3 FeMC6*a,µM FeMC6*a, 0.5 mM H2O2, 100 mM Tris buffer pH 8.5). Dashed line corresponds to the best biexponential fit of the 0.5 mMmM H 2HO2O2,2, 100 100 mM mM TrisTris bubufferffer pH 8.5). Dashed Dashed line line corre correspondssponds to the to thebest best biexponential biexponential fit of fitthe of the experimental data. experimentalexperimental data. data.

The furtherfurtherThe further increase increase increase in luminolin inluminol luminol concentration concentratio concentratio causedn causedcaused an exponential anan exponential exponential drop in drop the drop signal, in thein approaching signal,the signal, 4approaching a.u.approaching at luminol 4 a.u. concentrations 4 a.u.at luminolat luminol higher concentrations concentrations than 10 mM. higherhigher As previously thanthan 1010 mM. discussed,mM. As As previously previously the multi-step discussed, discussed, mechanism the the multi-step mechanism leading to CL involves the consumption of two equivalents of H2O2 in two leadingmulti-step to CLmechanism involves theleading consumption to CL involves of two equivalents the consumption of H O ofin two two equivalents steps as shown of H in2O Scheme2 in two1 steps as shown in Scheme I and in Figure 5a. 2 2 andsteps in as Figure shown5a. in Scheme I and in Figure 5a.

(a) (a)

(b) (b)

Figure 5. (a) Proposed reaction scheme of luminol oxidation catalyzed by FeMC6*a. (b) Step-by-step hydrogen peroxide assay developed in this work. Figure 5. (a) Proposed reaction scheme of luminol oxidation catalyzed by FeMC6*a. (b) Step-by-step FigureTherefore, 5. (a) Proposed one would reaction expect scheme that of LS lumino wouldl oxidation be maximized catalyzed when by theFeMC6*a. hydrogen (b) Step-by-stepperoxide to hydrogenluminol ratio peroxide is 2:1. assayassay However, developeddeveloped luminol inin thisthis radical work.work. formation (LH•–) is generally considered faster than the subsequent diazaquinone (L) oxidation [42]. Given that LS depends on the latter (Figure 5a), a Therefore, one would expect that LS would be maximized when the hydrogen peroxide to Therefore,higher amount one of would H2O2 is expectactually that needed LS towould maximize be themaximized signal, for whentwo reasons: the hydrogen (i) To increase peroxide the to – luminol ratio is 2:1. However, luminol radical formation (LH )•– is generally considered faster than the luminolrate ratio for the is uncatalyzed2:1. However, second-order luminol reactionradical betweenformation H2 O(LH2• and )L; is (ii) generally to prevent considered total consumption faster than subsequent diazaquinone (L) oxidation [42]. Given that LS depends on the latter (Figure5a), a higher the subsequent diazaquinone (L) oxidation [42]. Given that LS depends on the latter (Figure 5a), a amounthigher amount of H2O of2 is H actually2O2 is actually needed needed to maximize to maximize the signal, the signal, for two for reasons: two reasons: (i) To (i) increase To increase the rate the forrate the for uncatalyzedthe uncatalyzed second-order second-order reaction reaction between between H2O H2 2andO2 and L; (ii)L; (ii) to preventto prevent total total consumption consumption of H2O2 in the first oxidation step. Our results indeed show that maximum LS is reached at 0.1 mM

Sensors 2020, 20, x FOR PEER REVIEW 8 of 14

Sensors 2020, 20, 3793 8 of 14 of H2O2 in the first oxidation step. Our results indeed show that maximum LS is reached at 0.1 mM luminol, when 0.5 mM hydrogen peroxide was used (5:1 hydrogen peroxide:luminol ratio). luminol,Therefore, when we 0.5 adopted mM hydrogen this luminol peroxide concentrat was usedion (5:1 hydrogenfor quantitative peroxide:luminol hydrogen ratio).peroxide Therefore, assay wedevelopment. adopted this luminol concentration for quantitative hydrogen peroxide assay development.

3.3.3.3. HydrogenHydrogen Peroxideperoxide Determinationdetermination UnderUnder the the optimized optimized conditions conditions (0.3 (0.3µM FeMC6*a,μM FeMC6*a, 0.1 mM 0.1 luminol, mM luminol, pH 8.5, pH 2 min), 8.5, the2 min), LS response the LS toresponse H2O2 concentration to H2O2 concentration was followed was accordingfollowed according to the developed to the developed procedure procedure (Figure5b). (Figure A bimodal 5b). A linearbimodal trend linear was trend found was (Figure found6). (Figure LS increased 6). LS increased slowly and slowly linearly and atlinearly H 2O2 atconcentrations H2O2 concentrations lower thanlower 120 thanµM, 120 whilst μM, LS whilst increased LS moreincreased linearly more and linearly steeply and at higher steeply concentrations. at higher concentrations. Nonetheless, theNonetheless, observed lag-phase the observed gave uslag-phase the opportunity gave us to the extrapolate opportunity a linear to intervalextrapolate of response a linear in interval the range of ofresponse 10.0–120 inµ theM (Figurerange of6a). 10.0–120 μM (Figure 6a).

(a) (b)

Figure 6. (a) LS response to H2O2 concentration, as measured at 2 min after hydrogen peroxide addition Figure 6. (a) LS response to H2O2 concentration, as measured at 2 min after hydrogen peroxide in the 10.0–120 µM range. (0.3 µM FeMC6*a, 0.1 mM luminol, 100 mM Tris buffer pH 8.5) (b) LS addition in the 10.0 – 120 μM range. (0.3 μM FeMC6*a, 0.1 mM luminol, 100 mM Tris buffer pH 8.5) observed at different H2O2 concentrations in the 120–500 µM range. Short- and long-dashed lines (b) LS observed at different H2O2 concentrations in the 120 – 500 μM range. Short- and long-dashed correspond to the best linear fits of the two datasets, respectively. lines correspond to the best linear fits of the two datasets, respectively. 3 The sensitivity (slope of the linear fit) was found to be 10.1 10− a.u./µM, the agreement was very The sensitivity (slope of the linear fit) was found to be 10.1 10–3 a.u./μM, the agreement was very good, and a detection limit (LOD) of 4.6 µM and quantitation limit (LOQ) of 15.5 µM were calculated good, and a detection limit (LOD) of 4.6 μM and quantitation limit (LOQ) of 15.5 μM were calculated from the y-residuals (R2 = 0.9997, n = 24). At peroxide concentrations higher than 120 µM (Figure6b), from the y-residuals (R2 = 0.9997, n = 24). At peroxide concentrations higher than 120 μM (Figure 6b), more intense signals are observed, and a linear fit of the data was obtained (R2 = 0.991, n = 15), with more intense signals are observed, and a linear fit of the data was obtained (R2 = 0.991, n = 15), with the LOD far below the observed linearity range (55.8 µM) and the LOQ being 186 µM (0.12–0.50 mM; μ μ the LOD3 far below the observed linearity range (55.8 M) and the LOQ being 186 M (0.12–0.50 mM; 25.0 10− a.u./µM). 25.0 10–3 a.u./μM). By contrast, HRP showed a typical Michaelis–Menten dependence of the LS response toward By contrast, HRP showed a typical Michaelis–Menten dependence of the LS response toward H2O2 concentration [41,42]. Nonetheless, a nonlinear response against H2O2 concentration in the H2O2 concentration [41,42]. Nonetheless, a nonlinear response against H2O2 concentration in the pre-saturation phase was previously reported by many authors in the case of HRP and lactoperoxidase pre-saturation phase was previously reported by many authors in the case of HRP and in batch assays, when a high concentration of the catalyst was used [22,42,44]. Although a more lactoperoxidase in batch assays, when a high concentration of the catalyst was used [22,42,44]. detailed study of the kinetic mechanism of FeMC6*a is required, on the basis of the HRP mechanism Although a more detailed study of the kinetic mechanism of FeMC6*a is required, on the basis of the proposed by Arnold and co-workers [42], it appeared reasonable to us to hypothesize that: (i) When the HRP mechanism proposed by Arnold and co-workers [42], it appeared reasonable to us to [H2O2]/[luminol] ratio is lower than 1, H2O2 activation (Scheme1; step 1) and diazaquinone oxidation hypothesize that: (i) When the [H2O2]/[luminol] ratio is lower than 1, H2O2 activation (Scheme I; step are both rate-limiting, affecting LS changes; (ii) when the ratio is higher than 1, diazaquinone is rapidly 1) and diazaquinone oxidation are both rate-limiting, affecting LS changes; (ii) when the ratio is formed (within 2 min) and LS rapidly increases with exceeding H2O2. higher than 1, diazaquinone is rapidly formed (within 2 min) and LS rapidly increases with Interestingly, the increased CL efficiency of FeMC6*a enables H2O2 detection in the µM range, exceeding H2O2. as compared to the mM range when HRP is used as a catalyst [22]. In order to circumvent such

Sensors 2020, 20, x FOR PEER REVIEW 9 of 14

SensorsInterestingly,2020, 20, 3793 the increased CL efficiency of FeMC6*a enables H2O2 detection in the μM range,9 of 14 as compared to the mM range when HRP is used as a catalyst [22]. In order to circumvent such limitation, HRP has been either coupled to an CL enhancer [23,24], or immobilized [25], increasing thelimitation, signal-to-noise HRP has ratio been and either response coupled linearity. to an CLGiven enhancer the observed [23,24], analogies or immobilized between [25 FeMC6*a], increasing and HRP,the signal-to-noise we expect that ratio such and approaches response linearity.could lead Given to further the observed enhancement analogies of FeMC6*a between performances FeMC6*a and inHRP, luminol we expect oxidation. that such approaches could lead to further enhancement of FeMC6*a performances in luminol oxidation. 3.4. Sample analysis and organic contaminant interference study 3.4. Sample Analysis and Organic Contaminant Interference Study The utility of the assay developed here was checked by simulating a real-case scenario of AOP. The utility of the assay developed here was checked by simulating a real-case scenario of AOP. Two Two samples were prepared either with or without 10 ppm thioanisole as organic contaminant, in samples were prepared either with or without 10 ppm thioanisole as organic contaminant, in which which H2O2 was added to a final concentration of 8.1 mM (1:100 thioanisole:H2O2 ratio). Both H O was added to a final concentration of 8.1 mM (1:100 thioanisole:H O ratio). Both samples samples2 2 were then exposed to UV irradiation for two hours to induce2 hydroxyl2 radical (OH•) were then exposed to UV irradiation for two hours to induce hydroxyl radical (OH ) formation and formation and oxidation of the organic matter. Subsequently, various aliquots• were taken at oxidation of the organic matter. Subsequently, various aliquots were taken at different times to measure different times to measure the H2O2 content by the proposed FeMC6*a/luminol assay (Figure 7). the H2O2 content by the proposed FeMC6*a/luminol assay (Figure7).

Figure 7. Advanced oxidation processes (AOPs) as followed by the FeMC6*aFeMC6*a/luminol/luminol assay. Blue (without thioanisole) and magenta (81 µM thioanisole) columns report the evaluated H O concentration (without thioanisole) and magenta (81 μM thioanisole) columns report the 2evaluated2 H2O2 concentrationbefore and after before 1 and and 2 h of after UV-light 1 and irradiation. 2 h of UV-light Thin barsirradiation. report the Thin statistical bars re errorport the for datastatistical regression. error for data regression. The assay was able to recover the starting concentration of hydrogen peroxide, being only slightly overestimated in the sample without thioanisole. In fact, the measured concentrations were 8.2 0.2 The assay was able to recover the starting concentration of hydrogen peroxide, being ±only and 8.6 0.2 mM for samples with and without thioanisole, respectively (106% and 101% recovery). slightly ±overestimated in the sample without thioanisole. In fact, the measured concentrations were Virtually no difference could be observed within the experimental error between the two samples after 8.2 ± 0.2 and 8.6 ± 0.2 mM for samples with and without thioanisole, respectively (106% and 101% one and two hours. Very strong consumption of hydrogen peroxide was observed in the first hour recovery). Virtually no difference could be observed within the experimental error between the two (106 1 µM), while only a minimal decrease in H O concentration was observed after the second samples± after one and two hours. Very strong cons2 umption2 of hydrogen peroxide was observed in hour of UV-light exposureμ (105 1 µM). the first hour (106 ± 1 M), while± only a minimal decrease in H2O2 concentration was observed after μ the4. Conclusions second hour of UV-light exposure (105 ± 1 M).

4. ConclusionsIn the present work, the artificial enzyme, FeMC6*a, was applied in the development of a synthetic peroxidase-based batch-assay for H2O2 determination. Notably, FeMC6*a proved itself as an extremely In the present work, the artificial enzyme, FeMC6*a, was applied in the development of a proficient catalyst in luminol oxidation, despite its miniaturized scaffold. In comparison to HRP-based synthetic peroxidase-based batch-assay for H2O2 determination. Notably, FeMC6*a proved itself as CL sensors for H O , FeMC6*a-catalyzed oxidation of luminol and further light emission detection an extremely proficient2 2 catalyst in luminol oxidation, despite its miniaturized scaffold. In proceed within a short time range (~2 min) without the need of enhancers. An efficient detection of comparison to HRP-based CL sensors for H2O2, FeMC6*a-catalyzed oxidation of luminol and further H O was observed using low concentrations of enzyme (0.3 µM; 1.05 mg/L) with good linearity both light2 2 emission detection proceed within a short time range (∼2 min) without the need of enhancers. in the micromolar (R2 = 0.9997, n = 24) and in the low millimolar (R2 = 0.991, n = 15) ranges. LOD and An efficient detection of H2O2 was observed using low concentrations of enzyme (0.3 µM; 1.05 mg/L) LOQ were satisfactory, being as low as 4.6 and 15.5 µM, respectively. Moreover, the synthetic origin of with good linearity both in the micromolar (R2 = 0.9997, n = 24) and in the low millimolar (R2 = 0.991,

Sensors 2020, 20, 3793 10 of 14

FeMC6*a provides batch-to-batch consistency, paving the way to method standardization and reduced batch-to-batch discrepancies. This work has demonstrated that FeMC6*a can be adopted in luminescence-based sensors. Moreover, the results obtained from a real-case scenario not only show that the reported assay is able to satisfactorily recover the starting H2O2 concentration but also that H2O2 concentration could be actually monitored during an AOP test. We found that H2O2 was not completely consumed within two hours of UV-light irradiation, thus demonstrating the suitability of the FeMC6*a/luminol assay to assess undesired residual hydrogen peroxide content. Moreover, the presence of 10 ppm of a well-known reducing aromatic compound did not influence the assay outcome, demonstrating the usability of FeMC6*a for this kind of application in hydrogen peroxide sensing. Future efforts will be devoted to enhance the performances of FeMC6*a in luminol-based H2O2 determination either by using enhancers or immobilization onto solid supports [16,19,23,25,30,31,64], and to the construction of a standalone sensor device. Process control and quality assessment of reclaimed water may be further improved by developing efficient multi-purpose devices [4,28]. In particular, different oxidants, other than hydrogen peroxide, such as chlorite and chlorine dioxide [70], may be revealed through the methodology developed here, and their consumption rate may be eventually coupled to the presence of several aromatic and organophosphate pollutants [71–73].

Author Contributions: Conceptualization, M.C. and A.L.; Data curation, G.Z. and F.N.; Formal analysis, V.P. and M.C.; Funding acquisition, A.L.; Investigation, G.Z. and M.C.; Methodology, G.Z.; Supervision, A.L., V.P. and M.C.; Validation, M.C., V.P. and A.L.; Writing—original draft, M.C.; Writing—review and editing, F.N., V.P., A.L and M.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Campania Region, “Nuove strategie per la diagnostica medica e molecolare e per la tracciabilità ed il monitoraggio dei prodotti alimentari”—POR Campania FESR 2014/2020, Asse 1, [CUP B63D18000350007]. Acknowledgments: The authors wish to thank Ferdinando Febbraio and Ornella Maglio for helpful discussions. Conflicts of Interest: The authors declare no conflict of interest.

References

1. UN-Water Climate Change. UN-Water. Available online: https://www.unwater.org/water-facts/climate- change/ (accessed on 14 January 2020). 2. WHO|10 Facts on Climate Change and Health. Available online: https://www.who.int/features/factfiles/ climate_change/en/ (accessed on 14 January 2020). 3. OHCHR|Special Rapporteur on the Human Rights to Safe Drinking Water and Sanitation. Available online: https://www.ohchr.org/EN/Issues/WaterAndSanitation/SRWater/Pages/SRWaterIndex.aspx (accessed on 14 January 2020). 4. Sherchan, S.; Miles, S.; Ikner, L.; Yu, H.-W.; Snyder, S.A.; Pepper, I.L. Near Real-Time Detection of Ecoli in Reclaimed Water. Sensors 2018, 18, 2303. [CrossRef][PubMed] 5. Hijnen, W.A.M.; Beerendonk, E.F.; Medema, G.J. Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo) cysts in water: A review. Water Res. 2006, 40, 3–22. [CrossRef][PubMed] 6. Roszak, D.B.; Colwell, R.R. Survival strategies of bacteria in the natural environment. Microbiol. Rev. 1987, 51, 365–379. [CrossRef] 7. Marquette, C.A.; Blum, L.J. Applications of the luminol chemiluminescent reaction in analytical chemistry. Anal. Bioanal. Chem. 2006, 385, 546–554. [CrossRef] 8. Barni, F.; Lewis, S.W.; Berti, A.; Miskelly, G.M.; Lago, G. Forensic application of the luminol reaction as a presumptive test for latent detection. Talanta 2007, 72, 896–913. [CrossRef] 9. Fernandez-Romero, J.M.; Luque de Castro, M.D. Flow-through optical biosensor based on the permanent immobilization of an enzyme and transient retention of a reaction product. Anal. Chem. 1993, 65, 3048–3052. [CrossRef] 10. Wu, Y.; Gao, Y.; Du, J. Bifunctional gold nanoclusters enable ratiometric fluorescence nanosensing of hydrogen peroxide and glucose. Talanta 2019, 197, 599–604. [CrossRef] Sensors 2020, 20, 3793 11 of 14

11. Zhao, T.T.; Jiang, Z.W.; Zhen, S.J.; Huang, C.Z.; Li, Y.F. A (II)/cobalt (II) organic gel with enhanced peroxidase-like activity for fluorometric determination of hydrogen peroxide and glucose. Microchim. Acta 2019, 186, 168. [CrossRef] 12. Wang, J.; Lin, Y.; Chen, L. Organic-phase biosensors for monitoring phenol and hydrogen peroxide in pharmaceutical antibacterial products. Analyst 1993, 118, 277–280. [CrossRef] 13. Mulchandani, A.; Rudolph, D.C. Amperometric determination of lipid hydroperoxides. Anal. Biochem. 1995, 225, 277–282. [CrossRef] 14. Somasundrum, M.; Kirtikara, K.; Tanticharoen, M. Amperometric determination of hydrogen peroxide by direct and catalytic reduction at a copper electrode. Anal. Chim. Acta 1996, 319, 59–70. [CrossRef] 15. Astuti, Y.; Topoglidis, E.; Cass, A.G.; Durrant, J.R. Direct spectroelectrochemistry of peroxidases immobilised on mesoporous metal oxide electrodes: Towards reagentless hydrogen peroxide sensing. Anal. Chim. Acta 2009, 648, 2–6. [CrossRef] 16. Astuti, Y.; Topoglidis, E.; Durrant, J.R. Use of microperoxidase-11 to functionalize tin dioxide electrodes for the optical and electrochemical sensing of hydrogen peroxide. Anal. Chim. Acta 2011, 686, 126–132. [CrossRef] 17. Li, Q.; Zhang, Y.; Li, P.; Xue, H.; Jia, N. A nanocomposite prepared from hemin and reduced graphene oxide foam for voltammetric sensing of hydrogen peroxide. Microchim. Acta 2019, 187, 45. [CrossRef][PubMed] 18. Freeman, T.M.; Seitz, W. Rudolf Chemiluminescence fiber optic probe for hydrogen peroxide based on the luminol reaction. Anal. Chem. 1978, 50, 1242–1246. [CrossRef] 19. Olsson, B. Determination of hydrogen peroxide in a flow system with microperoxidase as catalyst for the luminol chemiluminescence reaction. Anal. Chim. Acta 1982, 136, 113–119. [CrossRef] 20. Blum, L.J.; Plaza, J.M.; Coulet, P.R. Chemiluminescent Analyte Microdetection Based on the Luminol-H2 O 2 Reaction Using Peroxidase Immobilized on New Synthetic Membranes. Anal. Lett. 1987, 20, 317–326. [CrossRef] 21. Hool, K.; Nieman, T.A. Immobilized luminol chemiluminescence reagent system for hydrogen peroxide determinations in flowing streams. Anal. Chem. 1988, 60, 834–837. [CrossRef] 22. Navas Díaz, A.; Ramos Peinado, M.C.; Torijas Minguez, M.C. Sol–gel horseradish peroxidase biosensor for hydrogen peroxide detection by chemiluminescence. Anal. Chim. Acta 1998, 363, 221–227. [CrossRef] 23. Ilyina, A.D.; Martínez Hernández, J.L.; López Luján, B.H.; Mauricio Benavides, J.E.; Romero García, J.; Rodríguez Martínez, J. Water quality monitoring using an enhanced chemiluminescent assay based on peroxidase-catalyzed peroxidation of luminol. Appl. Biochem. Biotechnol. 2000, 88, 45–58. [CrossRef] 24. Ramos, M.C.; Torijas, M.C.; Díaz, A.N. Enhanced chemiluminescence biosensor for the determination of phenolic compounds and hydrogen peroxide. Sens. Actuators B Chem. 2001, 73, 71–75. [CrossRef] 25. Li, B.; Zhang, Z.; Jin, Y. Chemiluminescence flow biosensor for hydrogen peroxide with immobilized reagents. Sens. Actuators B Chem. 2001, 72, 115–119. [CrossRef] 26. Yu, D.; Wang, P.; Zhao, Y.; Fan, A. Iodophenol blue-enhanced luminol chemiluminescence and its application to hydrogen peroxide and glucose detection. Talanta 2016, 146, 655–661. [CrossRef][PubMed] 27. Yamashoji, S. Determination of viable mammalian cells by luminol chemiluminescence using microperoxidase. Anal. Biochem. 2009, 386, 119–120. [CrossRef][PubMed] 28. Chai, J.; Yu, X.; Zhao, J.; Sun, A.; Shi, X.; Li, D. An Electrochemiluminescence Sensor Based on Nafion/Magnetic

Fe3O4 Nanocrystals Modified Electrode for the Determination of Bisphenol A in Environmental Water Samples. Sensors 2018, 18, 2537. [CrossRef][PubMed] 29. Niazov, A.; Freeman, R.; Girsh, J.; Willner, I. Following Glucose Oxidase Activity by Chemiluminescence and Chemiluminescence Resonance Energy Transfer (CRET) Processes Involving Enzyme-DNAzyme Conjugates. Sensors 2011, 11, 10388–10397. [CrossRef] 30. Lyu, Z.-M.; Zhou, X.-L.; Wang, X.-N.; Li, P.; Xu, L.; Liu, E.-H. Miniaturized electrochemiluminescent biochip prepared on gold nanoparticles-loaded mesoporous silica film for visual detection of hydrogen peroxide released from living cells. Sens. Actuators B Chem. 2019, 284, 437–443. [CrossRef] 31. Tian, H.; Tan, B.; Dang, X.; Zhao, H. Enhanced Electrochemiluminescence Detection for Hydrogen Peroxide Using Peroxidase-Mimetic Fe/N-Doped Porous Carbon. J. Electrochem. Soc. 2019, 166, B1594–B1601. [CrossRef] Sensors 2020, 20, 3793 12 of 14

32. Yu, J.; Cao, M.; Wang, H.; Li, Y. Novel manganese (II)-based metal-organic gels: Synthesis, characterization and application to chemiluminescent sensing of hydrogen peroxide and glucose. Microchim. Acta 2019, 186, 696. [CrossRef] 33. Wang, Z.; Dong, B.; Feng, G.; Shan, H.; Huan, Y.; Fei, Q. Water-soluble Hemin-mPEG-enhanced Luminol Chemiluminescence for Sensitive Detection of Hydrogen Peroxide and Glucose. Anal. Sci. 2019, 35, 1135–1140. [CrossRef] 34. Marks, R.S.; Bassis, E.; Bychenko, A.; Levine, M.M. Chemiluminescent optical fiber immunosensor for detecting cholera antitoxin. OptEn 1997, 36, 3258–3264. [CrossRef] 35. Huang, K.; Sun, Y.; Liu, L.; Hu, C. Chemiluminescence of 3-aminophthalic acid anion–hydrogen peroxide–cobalt (II). Luminescence 2020, 35, 400–405. [CrossRef][PubMed] 36. Yamazaki, T.; Kawai, C.; Yamauchi, A.; Kuribayashi, F. A highly sensitive chemiluminescence assay for superoxide detection and chronic granulomatous disease diagnosis. Trop. Med. Health 2011, 39, 41–45. [CrossRef][PubMed] 37. Kamidate, T.; Katayama, A.; Ichihashi, H.; Watanabe, H. Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine. J. Biolumin. Chemilumin. 1994, 9, 279–286. [CrossRef] 38. Yeh, H.-C.; Lin, W.-Y. Enhanced chemiluminescence for the oxidation of luminol with m-chloroperoxybenzoic acid catalyzed by microperoxidase 8. Anal. Bioanal. Chem. 2002, 372, 525–531. [CrossRef] 39. Gorsuch, J.D.; Hercules, D.M. Studies on the chemiluminescence of luminol in dimethylsulfoxide and dimethylsulfoxide-water mixtures. Photochem. Photobiol. 1972, 15, 567–583. [CrossRef] 40. Lee, J.; Seliger, H.H. Quantum Yields of the Luminol Chemiluminescence Reaction in Aqueous and Aprotic Solvents. Photochem. Photobiol. 1972, 15, 227–237. [CrossRef] 41. Cormier, M.J.; Prichard, P.M. An Investigation of the Mechanism of the Luminescent Peroxidation of Luminol by Stopped Flow Techniques. J. Biol. Chem. 1968, 243, 4706–4714. 42. Li, L.; Arnold, M.A.; Dordick, J.S. Mathematical model for the luminol chemiluminescence reaction catalyzed by peroxidase. Biotechnol. Bioeng. 1993, 41, 1112–1120. [CrossRef] 43. Cercek, B.; Cercek, B.; Roby, K.; Cercek, L. Effect of oxygen abstraction on the peroxidase–luminol–perborate system: Relevance to the HRP enhanced chemiluminescence mechanism. J. Biolumin. Chemilumin. 1994, 9, 273–277. [CrossRef] 44. Nakamura, M.; Nakamura, S. One- and Two-Electron Oxidations of Luminol by Peroxidase Systems. Free Radic. Biol. Med. 1998, 24, 537–544. [CrossRef] 45. Navas Díaz, A.; González García, J.A. Nonlinear Multicomponent Kinetic Analysis for the Simultaneous Stopped-Flow Determination of Chemiluminescence Enhancers. Anal. Chem. 1994, 66, 988–993. [CrossRef] 46. García Sanchez, F.; Navas Díaz, A.; González García, J.A. P-phenol derivatives as enhancers of the chemiluminescent luminol-horseradish peroxidase-H2O2 reaction: Substituent effects. J. Lumin. 1995, 65, 33–39. [CrossRef] 47. Li, F.; Ma, W.; Liu, J.; Wu, X.; Wang, Y.; He, J. Luminol, horseradish peroxidase, and glucose oxidase ternary functionalized graphene oxide for ultrasensitive glucose sensing. Anal. Bioanal. Chem. 2018, 410, 543–552. [CrossRef][PubMed] 48. Jeschek, M.; Reuter, R.; Heinisch, T.; Trindler, C.; Klehr, J.; Panke, S.; Ward, T.R. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 2016, 537, 661–665. [CrossRef] 49. Li, L.-L.; Yuan, H.; Liao, F.; He, B.; Gao, S.-Q.; Wen, G.-B.; Tan, X.; Lin, Y.-W. Rational design of artificial dye-decolorizing peroxidases using myoglobin by engineering Tyr/Trp in the heme center. Dalton Trans. 2017, 46, 11230–11238. [CrossRef] 50. Yin, L.; Yuan, H.; Liu, C.; He, B.; Gao, S.-Q.; Wen, G.-B.; Tan, X.; Lin, Y.-W. A Rationally Designed Myoglobin Exhibits a Catalytic Dehalogenation Efficiency More than 1000-Fold That of a Native Dehaloperoxidase. ACS Catal. 2018, 9619–9624. [CrossRef] 51. Hayashi, T.; Tinzl, M.; Mori, T.; Krengel, U.; Proppe, J.; Soetbeer, J.; Klose, D.; Jeschke, G.; Reiher, M.; Hilvert, D. Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme. Nat. Catal. 2018, 1, 578–584. [CrossRef] 52. Stenner, R.; Steventon, J.W.; Seddon, A.; Anderson, J.L.R. A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase. Proc. Natl. Acad. Sci. USA 2020, 117, 1419–1428. [CrossRef] Sensors 2020, 20, 3793 13 of 14

53. Chino, M.; Maglio, O.; Nastri, F.; Pavone, V.; DeGrado, W.F.; Lombardi, A. Artificial Diiron Enzymes with a De Novo Designed Four-Helix Bundle Structure. Eur. J. Inorg. Chem. 2015, 2015, 3371–3390. [CrossRef] 54. Lombardi, A.; Pirro, F.; Maglio, O.; Chino, M.; DeGrado, W.F. De Novo Design of Four-Helix Bundle Metalloproteins: One Scaffold, Diverse Reactivities. Acc. Chem. Res. 2019.[CrossRef][PubMed] 55. Chino, M.; Leone, L.; Zambrano, G.; Pirro, F.; D’Alonzo, D.; Firpo, V.; Aref, D.; Lista, L.; Maglio, O.; Nastri, F.; et al. Oxidation catalysis by iron and manganese porphyrins within enzyme-like cages. Biopolymers 2018, 109, e23107. [CrossRef][PubMed] 56. Perrella, F.; Raucci, U.; Chiariello, M.G.; Chino, M.; Maglio, O.; Lombardi, A.; Rega, N. Unveiling the structure of a novel artificial heme-enzyme with peroxidase-like activity: A theoretical investigation. Biopolymers 2018, 109, e23225. [CrossRef][PubMed] 57. Nastri, F.; D’Alonzo, D.; Leone, L.; Zambrano, G.; Pavone, V.; Lombardi, A. Engineering Metalloprotein Functions in Designed and Native Scaffolds. Trends Biochem. Sci. 2019.[CrossRef] 58. Chino, M.; Leone, L.; Maglio, O.; D’Alonzo, D.; Pirro, F.; Pavone, V.; Nastri, F.; Lombardi, A. A De Novo Heterodimeric Due Ferri Minimizes the Release of Reactive Intermediates in Dioxygen-Dependent Oxidation. Angew. Chem. Int. Ed. 2017, 56, 15580–15583. [CrossRef] 59. Chino, M.; Leone, L.; Maglio, O.; Lombardi, A. Designing Covalently Linked Heterodimeric Four-Helix Bundles. Methods Enzymol. 2016, 580, 471–499. [CrossRef] 60. Zhang, S.-Q.; Chino, M.; Liu, L.; Tang, Y.; Hu, X.; DeGrado, W.F.; Lombardi, A. De Novo Design of Tetranuclear Transition Metal Clusters Stabilized by Hydrogen-Bonded Networks in Helical Bundles. J. Am. Chem. Soc. 2018, 140, 1294–1304. [CrossRef] 61. Chino, M.; Zhang, S.-Q.; Pirro, F.; Leone, L.; Maglio, O.; Lombardi, A.; DeGrado, W.F. Spectroscopic and metal binding properties of a de novo metalloprotein binding a tetrazinc cluster. Biopolymers 2018, 109, e23339. [CrossRef] 62. Nastri, F.; Lista, L.; Ringhieri, P.; Vitale, R.; Faiella, M.; Andreozzi, C.; Travascio, P.; Maglio, O.; Lombardi, A.; Pavone, V. A Heme-Peptide Metalloenzyme Mimetic with Natural Peroxidase-Like Activity. Chem. Eur. J. 2011, 17, 4444–4453. [CrossRef] 63. Vitale, R.; Lista, L.; Cerrone, C.; Caserta, G.; Chino, M.; Maglio, O.; Nastri, F.; Pavone, V.; Lombardi, A. Artificial heme-enzyme with enhanced catalytic activity: Evolution, functional screening and structural characterization. Org. Biomol. Chem. 2015, 13, 4858–4868. [CrossRef] 64. Zambrano, G.; Ruggiero, E.; Malafronte, A.; Chino, M.; Maglio, O.; Pavone, V.; Nastri, F.; Lombardi, A. Artificial Heme Enzymes for the Construction of Gold-Based Biomaterials. Int. J. Mol. Sci. 2018, 19, 2896. [CrossRef][PubMed] 65. Caserta, G.; Chino, M.; Firpo, V.; Zambrano, G.; Leone, L.; D’Alonzo, D.; Nastri, F.; Maglio, O.; Pavone, V.; Lombardi, A. Enhancement of Peroxidase Activity in Artificial Mimochrome VI Catalysts through Rational Design. Chembiochem 2018, 19, 1823–1826. [CrossRef][PubMed] 66. Leone, L.; D’Alonzo, D.; Balland, V.; Zambrano, G.; Chino, M.; Nastri, F.; Maglio, O.; Pavone, V.; Lombardi, A. Mn-Mimochrome VI*a: An Artificial Metalloenzyme with Peroxygenase Activity. Front. Chem. 2018, 6. [CrossRef][PubMed] 67. Firpo, V.; Le, J.M.; Pavone, V.; Lombardi, A.; Bren, K.L. Hydrogen evolution from water catalyzed by cobalt-mimochrome VI*a, a synthetic mini-protein. Chem. Sci. 2018, 9, 8582–8589. [CrossRef][PubMed] 68. Le, J.M.; Alachouzos, G.; Chino, M.; Frontier, A.J.; Lombardi, A.; Bren, K.L. Tuning Mechanism through Buffer Dependence of Hydrogen Evolution Catalyzed by a Cobalt Mini-enzyme. Biochemistry 2020, 59, 1289–1297. [CrossRef][PubMed] 69. Zambrano, G.; Chino, M.; Renzi, E.; Di Girolamo, R.; Maglio, O.; Pavone, V.; Lombardi, A.; Nastri, F. Clickable artificial heme-peroxidases for the development of functional nanomaterials. Biotechnol. Appl. Biochem. 2020. [CrossRef] 70. Pepich, B.V.; Dattilio, T.A.; Fair, P.S.; Munch, D.J.; Gordon, G.; Körtvélyesi, Z. An improved colorimetric method for chlorine dioxide and chlorite ion in drinking water using lissamine green B and horseradish peroxidase. Anal. Chim. Acta 2007, 596, 37–45. [CrossRef] 71. Lin, Y.-W. Rational design of heme enzymes for biodegradation of pollutants toward a green future. Biotechnol. Appl. Biochem. 2019.[CrossRef] Sensors 2020, 20, 3793 14 of 14

72. Carullo, P.; Chino, M.; Cetrangolo, G.P.; Terreri, S.; Lombardi, A.; Manco, G.; Cimmino, A.; Febbraio, F. Direct detection of organophosphate compounds in water by a fluorescence-based biosensing device. Sens. Actuators B Chem. 2018, 255, 3257–3266. [CrossRef] 73. Cetrangolo, G.P.; Rusko, J.; Gori, C.; Carullo, P.; Manco, G.; Chino, M.; Febbraio, F. Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach. Sensors 2020, 20, 1365. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).