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Copyright © 2006 John Wiley & Sons, Ltd. Luminescence (In press)DOI: 10.1002/bio

Kinetic and mechanistic aspects of PVP hydrogel-supported luminol CL ORIGINAL RESEARCH 1 ORIGINAL RESEARCHLuminescence (In press)Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bio.934 ORIGINAL RESEARCH

Studies on PVP hydrogel-supported luminolchemiluminescence: 1. Kinetic and mechanistic aspectsusing multivariate factorial analysis

Erick Leite Bastos, Luiz Francisco Monteiro Leite Ciscato, Fernando Heering Bartoloni,Luiz Henrique Catalani and Wilhelm Josef Baader*

Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil

Received 14 March 2006; revised 16 May 2006; accepted 17 May 2006

ABSTRACT: The chemiluminescent oxidation of luminol by hydrogen peroxide in the presence of hemin is revisited in an UV-Ccross-linked PVP hydrogel. Chemiluminescence properties such as initial light intensity (I0), area of emission (S) and observed rateconstants (kobs) are studied, varying the concentration of all reactants using a multivariate factorial approach. Copyright © 2006John Wiley & Sons, Ltd.

KEYWORDS: luminol; chemiluminescence; hemin; hydrogel; PVP

Copyright © 2006 John Wiley & Sons, Ltd.

*Correspondence to: W.J. Baader, Instituto de Química, Universidadede São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP,Brazil.E-mail: [email protected]

Contract/grant sponsor: FAPESP, Brazil; Contract/grant number:98/05445-9; 00/06652-0; 01/07477-0.Contract/grant sponsor: CNPq, Brazil.Contract/grant sponsor: CAPES, Brazil.Contract/grant sponsor: PADCT, Brazil.

We describe a convenient method for the preparationof a cross-linked PVP hydrogel containing luminol andhemin and the use of multivariate factorial analysis torationalize the influence of reactant concentrations onthe CL parameters.

MATERIALS AND METHODS

Reagents

Luminol (5-amino-2,3-dihydro-phthalazine-1,4-dione)was obtained from Merck (Germany) and a stocksolution (10 mmol/L) was prepared in 1 mol/L NaOH,kept at 4°C and used within 10 days. The workingstock solution was prepared by dilution in phosphatebuffer (0.1 mol/L, Na3PO4/Na2HPO4), pH 11.6. The finalluminol concentration was determined spectrophoto-metrically at 347 nm (ε = 7600 mol/L/cm). Hydrogenperoxide (Peróxidos do Brasil, Brazil) was obtainedas a 60% w/w unstabilized aqueous solution. The finalconcentration after dilution with demineralized water(18 MΩ, Milli-Q, Millipore) was determined spectro-photometrically as described by Cotton and Dunford(17). Hemin (ferriprotoporphyrin IX chloride) waspurchased from Sigma (St. Louis, USA). A stock solu-tion was prepared by dissolving 2.5 mg hemin in 5 mL1 mol/L aqueous NaOH. The working solution is a1:100 dilution with 1 mol/L NaOH (8 µmol/L). Theconcentration was determined spectrophotometricallyusing ε = 58 400 mol/L/cm at 382 nm (18). Poly(N-vinyl-2-pyrrolidone), known as Plasdone K-90 (MW = 1.2 × 106;Mn = 3.6 × 105), was obtained from GAF Chem. Co.(USA).

INTRODUCTION

Luminol and its derivatives can be oxidized, in thepresence of catalysts and enhancers, resulting in chemi-luminescence (CL) (1). The main applications of thisreaction are the quantification of hydrogen peroxide,analysis of trace metals, as Fe(III), Cu(II) and Cr(III),detection of reductants and antioxidants, and deter-mination of labelled antibodies in immunoassays (1, 2).Hemin is widely used as catalyst for the oxidationof luminol by hydrogen peroxide (3, 4). It constitutes astable and cheap mimetic of metalloporphyrins such ashorseradish peroxidase (HRP), providing reproducibleresults in several analytical methods (5).

Various polymeric sorbent coatings and hydrogelshave been utilized in the development of chemical andenzymatic sensors based on luminol CL (6–9). However,the use of poly(N-vinyl-2-pyrrolidone) (PVP) for theseapplications is scarcely described (10–15). Lopérgoloet al. have developed a method to produce PVP hy-drogels by UV-mediated polymer cross-linking (16).Thus, PVP hydrogel-supported luminol and hemincan be easily prepared directly in a well microplate andthis system can be utilized in a variety of analyticalapplications.

ORIGINAL RESEARCH E. L. Bastos et al.


Scheme 1. Representation of a well microplate with the full set of experimentscombining hemin, luminol and hydrogen peroxide in 27 runs. The line segmentsindicate addition of reactant in random order.

Table 1. Levels selected for concentration of luminol (factor A), hemin (factor B) and H2O2 (factor C)

Levels

Factors Abbr. Low (1) Intermediate (2) High (3)

A. Luminol concentration (mmol/L) LC 0.10 1.00 10.0B. Hemin concentration (nmol/L) HC 0.77 7.70 77.0C. Hydrogen peroxide concentration (mmol/L) PC 0.01 0.10 1.00

Instruments

The CL emission experiments were performed on aBerthold EG&G 96V Lumimat microplate lumino-meter. Absorption spectra were recorded using aShimadzu Multispec 1500 UV-visible spectrophoto-meter. Chemiluminescence spectra were recorded in aVarian Eclipse spectrofluorimeter.

Irradiation of PVP in the presence of luminoland hemin

200 µL of a solution containing 80 mg/mL PVP andadequate concentrations of luminol and hemin inphosphate buffer (pH 11.6, µ = 0.1 mol/L) were placed ina 96-well microplate, which was positioned 20 mm dis-tant from the irradiation source. Irradiations were car-ried out using a pen-type Heraeus (Hanau, Germany)20 W low-pressure Hg lamp (λem = 254 nm; 200 mmlong). This setting produces 11.5 MW/cm2 of radiant fluxat the plate position or 4.89 J/s on a flat specimen (16).

CL from the luminol–hemin–hydrogen peroxidesystem

The resulting microplate, containing in every well200 µL aqueous hydrogel solution, was put into thecavity of a luminometer and thermostated to 25.0 ±0.5°C. The reaction was initiated by automatic jet injec-tion of 100 µL aqueous hydrogen peroxide solution inadequate concentration. Final reagent concentrationsare reported for a final volume of 300 µL. Emissiondecay was monitored for 1 h in cycles of 60 measure-ments/well/hour.

Factorial analysis

The effect luminol, hemin and hydrogen peroxideconcentrations on emission properties was evaluatedthrough a 33 factorial design. Factors were defined inthree levels and the design is shown in Table 1.

The factors and levels were combined to obtain 27experimental conditions and the experiments carried outin a well microplate as depicted in Scheme 1. Hemin andluminol concentrations were varied horizontally, whilehydrogen peroxide concentration was varied vertically inthe microplate.

Response curves were calculated by two-way analysis(linear, quadratic) of the 33 factorial planning, usingequation 1:

z = y0 + ax + by + cxy + dx2 + ey2 (1)+ fxy2 + gx2y + hx2y2

Statistical and mathematical analysis

All values were expressed as mean ± standard deviation(SD) of at least three independent experiments. Theobserved rate constants (kobs) and initial intensities (I0)were obtained by non-linear fitting of the kinetic emis-sion curves (intensity vs. time). Emission areas were de-termined by numerical integration of the emission curvesextrapolated to intensity zero. Statistical data analysiswas achieved by one or two-way analysis of variance(ANOVA). The level of statistical significance was takenat p < 0.05. All calculations and 2D fittings were per-formed using Origin 6.1 (OriginLab Corp., 2000). Three-dimensional plots and fittings were obtained usingSigmaplot 6.1 (SPPS, Inc., 2000). Multivariate factorial


Kinetic and mechanistic aspects of PVP hydrogel-supported luminol CL ORIGINAL RESEARCH 3 ORIGINAL RESEARCH

analysis was calculated with the Statistica 6.0 (Statsoft,Inc., 2001).

RESULTS

Luminol CL in PVP hydrogel

Poly(N-vinyl-2-pyrrolidone) was submitted to directphotocross-linking in aqueous solution containingluminol and hemin using a low pressure Hg lamp(λem = 254 nm) (16). Microplate wells were filled with200 µL of a solution containing adequate concentra-tion of luminol and hemin and PVP K-90 (80 mg/mL)in phosphate buffer, pH 11.6, and submitted to UV-Cirradiation for 30 min under a nitrogen atmosphere.After this period, a hydrogel film ca. 3 mm thickwas formed. Oxygen enhances gel formation in PVPphotocross-linking probably due to hydroperoxideformation (16). The use of a nitrogen flux through theirradiation chamber drops the gel fraction by about15–20% but proved to be necessary as it avoids luminolconsumption. After irradiation, the microplate wasflushed with nitrogen, protected with a PVC plasticfilm and kept at 20°C. The microplates can be utilizedafter a storage time of at least 1 month without any lossof activity.

The chemiluminescent reaction was initiated byaddition of 100 µL of an aqueous solution of hydrogenperoxide (10 µmol/L) to the well containing thehydrogel. The obtained results were compared with twocontrol systems: a standard aqueous system, in theabsence of PVP, and a system containing 80 mg/mL PVPwithout irradiation (Fig. 1). As can be seen, photocross-linking proved to be adequate to obtain the PVPhydrogel containing luminol and hemin, as irradiationshows no significant effect on the CL emission intensityof the system after cross-linking, despite the fact that thepresence of PVP reduces CL intensity (Fig. 1).

The effect of PVP hydrogel on the CL spectra wasalso investigated. The presence of cross-linked PVP(80 mg/mL) shows no effect on the maximum wave-length of the CL emission (λmax = 421 nm; Fig. 2).

Effect of reagents concentrations on CLproperties

Multivariate factorial approach. Factorial experi-mental design provides mathematical ways to identifyand quantify the more important variables in a givensystem, as well as any possible interaction between them(19). This approach requires that factors and levels aredefined. Factors are the independent variables of in-terest and levels are experimental conditions relatedto a specific factor. In order to investigate the effectof reagents on CL properties of the luminol-hydrogel

Figure 1. Comparison of emission kinetics in the presenceand absence of cross-linked PVP. [Luminol], 0.1 mmol/L;[Hemin], 77 nmol/L; [H2O2], 0.1 mmol/L; [PVP], 80 mg/mL.

system, the experiment was designed as follows: factorA, luminol concentration 0.1, 1.0 and 10 mmol/L; factorB, hemin concentration 0.77, 7.70 and 77.0 nmol/L; andFactor C, hydrogen peroxide concentration 0.01, 0.10and 1.00 mmol/L. The concentration of PVP was keptconstant, as the optimized conditions for gel formationhave already been described (16). The different formu-lations of the factorial design consist of systematicalcombinations of all factors at all levels resulting, in thiscase, in 27 experiments (33). Experiments were per-formed in a fully randomized order and the dependentvariables initial emission intensity (I0), area under emis-sion curve (S) and observed rate constant (kobs) moni-tored. The results obtained are shown in Table 2.

Figure 2. Chemiluminescence spectra of the luminol–hemin–H2O2 system in the presence and the absence of cross-linkedPVP. [Luminol], 0.1 mmol/L; [hemin], 77 nmol/L; [H2O2],0.1 mmol/L; [PVP], 80 mg/mL.



As a general tendency, I0 and S show the same pro-file at the three different peroxide concentrations; bothvariables showing an increase with increasing heminconcentrations, whereas the luminol dependence showsmaximum values for both I0 and S at the intermediate(1 mmol/L) luminol concentration (Table 2). The behav-iour of the kobs values is clearly distinct from that of thetwo other dependent variables, showing a relativelysmall variation with the luminol and hemin concentra-tion for the three different peroxide concentrations,mainly at low H2O2 concentrations. For high peroxideconcentrations (1 mmol/L), there is a tendency towardshigher kobs values with the lower luminol concentrationused, whereas no clear-cut tendency is observed withrespect to variation of the hemin concentration.

Results in Table 2 were submitted to a multivariatefactorial analysis (two-way linear/quadratic model) andthe predicted values and experimental data are shown inTable 3. From the observed correlation coefficients(0.92 < R2 < 0.97) one can see that this model is ableto describe the complex effect of luminol, hemin andhydrogen peroxide concentration on I0, S and kobs

adequately.The effects of the three factors (I0, S and kobs) and

some of their combinations were obtained by centred

Table 2. Calculated values of the dependent variables I0, S and kobs from five sets of 27 experiments

Level I0 × 10−5 (a.u./s) S × 10−8 (a.u.) kobs × 104 (s−1)

Factor A Factor B Factor C Mean SD Mean SD Mean SDEntry (LC) (HC) (PC)

1 1 (0.1)a 1 (0.77)b 1 (0.01)c 0.015 0.001 0.023 0.001 8.155 0.6872 1 2 (7.70)b 1 0.037 0.002 0.056 0.002 8.162 0.6913 1 3 (77.0)b 1 0.166 0.012 0.280 0.018 7.729 0.5874 2 (1.0)a 1 1 0.027 0.003 0.045 0.003 7.453 0.7555 2 2 1 0.157 0.011 0.295 0.024 6.287 0.5216 2 3 1 0.203 0.018 0.333 0.026 7.851 0.5587 3 (10)a 1 1 0.014 0.001 0.026 0.002 7.733 0.6688 3 2 1 0.087 0.003 0.171 0.014 6.906 0.7019 3 3 1 0.062 0.003 0.106 0.008 7.955 0.69810 1 1 2 (0.10)c 0.124 0.010 0.195 0.012 5.830 0.45811 1 2 2 0.225 0.018 0.333 0.021 6.090 0.35112 1 3 2 1.528 0.098 3.029 0.024 4.678 0.36513 2 1 2 0.150 0.011 0.247 0.018 5.723 0.55914 2 2 2 0.909 0.082 1.618 0.124 4.852 0.39715 2 3 2 3.583 0.185 8.923 0.962 2.837 0.19816 3 1 2 0.155 0.012 0.279 0.016 5.447 0.24517 3 2 2 0.657 0.042 1.402 0.128 5.193 0.45818 3 3 2 1.420 0.128 4.069 0.385 1.164 0.27419 1 1 3 (1.00)c 0.036 0.001 0.059 0.003 4.450 0.36820 1 2 3 0.071 0.003 0.125 0.011 4.956 0.44221 1 3 3 0.204 0.019 0.429 0.033 3.744 0.28422 2 1 3 0.029 0.002 0.058 0.004 4.368 0.97723 2 2 3 0.147 0.009 0.349 0.031 2.546 0.22124 2 3 3 0.321 0.028 0.769 0.064 3.122 0.20125 3 1 3 0.020 0.001 0.046 0.002 2.370 0.24426 3 2 3 0.055 0.002 0.137 0.012 2.213 0.26827 3 3 3 0.083 0.002 0.191 0.012 2.121 0.200

a[luminol] in mmol/L; b[hemin] in nmol/L; c[H2O2] in mmol/L.

and scaled polynomial regression of data consideringtwo-way interactions (linear, quadratic) (equation 2;Table 4).

z = y0 + ax1 + bx2 + cx3 + dx1x2 + ex1x3 + fx2x3 (2)+ gx2

1 + hx22 + ix2

3 + jx21x2 + kx2

1x3

+ lx22x3 + mx2

1x22 + nx2

1x23 + ox2

2x23

The concentration of hemin (linear) and peroxide(quadratic) and their combination (linear, quadratic)causes the highest effects on both I0 and S. In the caseof kobs, significant negative effects are observed byluminol concentration (linear), peroxide (linear andquadratic) and the combination of hemin and peroxideconcentrations (linear, quadratic).

The effect of luminol concentration on I0 wasfurther investigated, using a conventional non-factorialapproach, in order to confirm the decrease of I0 asthe concentration of luminol increases above 1 mmol/L.The concentration of luminol was varied at three dif-ferent hydrogen peroxide concentrations (0.01, 0.10and 1.00 mmol/L) using a hemin concentration of7.7 nmol/L (Fig. 3).

The increase of luminol concentration up to avalue of 1 mmol/L results in the rise of I0 values in all



Table 3. Comparison between predicted and experimental values for maximum intensity, area and observed rate constant

Factor A Factor B Factor C I0 × 10−5 (a.u./s) S × 10−8 (a.u.) kobs × 104 (s−1)

(LC)a (HC)b (PC)c Expt. Pred. Q Expt. Pred. Q Expt. Pred. Q

0.1 0.77 0.01 0.015 0.122 −0.107 0.023 0.357 −0.334 8.155 7.840 0.3150.1 7.7 0.01 0.037 0.052 −0.015 0.056 0.189 −0.133 8.162 7.916 0.2460.1 77 0.01 0.166 0.044 0.122 0.280 −0.187 0.467 7.729 8.291 −0.5621 0.77 0.01 0.027 −0.158 0.185 0.045 −0.415 0.460 7.453 7.697 −0.2441 7.7 0.01 0.157 0.055 0.102 0.295 −0.025 0.320 6.287 6.229 0.0581 77 0.01 0.203 0.490 −0.287 0.333 1.113 −0.780 7.851 7.665 0.18610 0.77 0.01 0.014 0.092 −0.078 0.026 0.152 −0.126 7.733 7.804 −0.07110 7.7 0.01 0.087 0.173 −0.086 0.171 0.358 −0.187 6.906 7.210 −0.30410 77 0.01 0.062 −0.102 0.164 0.106 −0.207 0.313 7.955 7.580 0.3750.1 0.77 0.1 0.124 −0.088 0.212 0.195 −0.453 0.648 5.830 6.179 −0.3490.1 7.7 0.1 0.225 0.222 0.003 0.333 0.114 0.219 6.090 6.628 −0.5380.1 77 0.1 1.528 1.743 −0.215 3.029 3.895 −0.866 4.678 3.791 0.8871 0.77 0.1 0.150 0.498 −0.348 0.247 1.081 −0.834 5.723 5.792 −0.0691 7.7 0.1 0.909 1.090 −0.181 1.618 2.206 −0.588 4.852 4.698 0.1541 77 0.1 3.583 3.054 0.529 8.923 7.501 1.422 2.837 2.922 −0.08510 0.77 0.1 0.155 0.019 0.136 0.279 0.092 0.187 5.447 5.029 0.41810 7.7 0.1 0.657 0.479 0.178 1.402 1.033 0.369 5.193 4.809 0.38410 77 0.1 1.420 1.734 −0.314 4.069 4.625 −0.556 1.164 1.966 −0.8020.1 0.77 1 0.036 0.141 −0.105 0.059 0.373 −0.314 4.450 4.417 0.0330.1 7.7 1 0.071 0.059 0.012 0.125 0.211 −0.086 4.956 4.664 0.2920.1 77 1 0.204 0.112 0.092 0.429 0.029 0.400 3.744 4.069 −0.3251 0.77 1 0.029 −0.134 0.163 0.058 −0.316 0.374 4.368 4.055 0.3131 7.7 1 0.147 0.067 0.080 0.349 0.081 0.268 2.546 2.758 −0.2121 77 1 0.321 0.563 −0.242 0.769 1.412 −0.643 3.122 3.224 −0.10210 0.77 1 0.020 0.078 −0.058 0.046 0.107 −0.061 2.370 2.717 −0.34710 7.7 1 0.055 0.147 −0.092 0.137 0.319 −0.182 2.213 2.293 −0.08010 77 1 0.083 −0.067 0.150 0.191 −0.052 0.243 2.121 1.694 0.427

R2 0.9314 0.9213 0.9692

a[luminol] in mmol/L; b[hemin] in nmol/L; c[H2O2] in mmol/L.

Table 4. Effect of variables on PVP-supported luminol oxidation. Results are expressed as two-way interaction [linear (L)/quad-ratic (Q)] estimate effects and p values (ANOVA)

I0 S kobs

Effect NEa P Effect NEa P Effect NEa P

Factor A (L) (luminol) −0.087 −0.082 0.743 0.043 0.016 0.951 −1.415 −0.300 0.020Factor A (Q) 0.342 0.324 0.202 1.000 0.371 0.165 −0.165 −0.035 0.734Factor B (L) (hemin) 0.608 0.575 0.018 1.668 0.619 0.015 −0.880 −0.186 0.053Factor B (Q) 0.161 0.152 0.434 0.290 0.108 0.592 −0.469 −0.099 0.243Factor C (L) (peroxide) 0.033 0.031 0.901 0.117 0.043 0.868 −4.722 −1.000 <0.01Factor C (Q) 1.057 1.000 0.003 2.693 1.000 0.003 −3.371 −0.714 <0.01A (L) × B (L) −0.146 −0.139 0.535 −0.104 −0.039 0.867 −0.003 −0.001 0.995A (L) × B (Q) 0.089 0.084 0.693 0.196 0.073 0.742 −0.335 −0.071 0.440A (Q) × B (L) 0.257 0.243 0.269 0.842 0.312 0.182 0.584 0.124 0.194A (Q) × B (Q) 0.111 0.105 0.606 0.186 0.069 0.745 −0.795 −0.168 0.079A (L) × C (L) −0.016 −0.015 0.939 −0.031 −0.011 0.957 −0.832 −0.176 0.066A (L) × C (Q) 0.070 0.066 0.732 0.378 0.140 0.491 −0.481 −0.102 0.234A (Q) × C (L) 0.004 0.004 0.983 0.044 0.016 0.935 −0.034 −0.007 0.930A (Q) × C (Q) 0.426 0.403 0.054 1.115 0.414 0.057 −0.068 −0.014 0.854B (L) × C (L) 0.034 0.032 0.884 0.107 0.040 0.864 −0.533 −0.113 0.250B (L) × C (Q) 0.838 0.793 0.005 2.277 0.845 0.004 −1.718 −0.364 0.003B (Q) × C (L) −0.009 −0.009 0.967 −0.006 −0.002 0.991 0.134 0.028 0.753B (Q) × C (Q) 0.114 0.108 0.598 0.160 0.059 0.779 0.335 0.071 0.420

R2 0.9315 0.9211 0.9692

aNormalized effect.



irradiation, reducing the formation of oxidant(s) (mostprobably hydroperoxides) (16) which could decrease theluminol concentration. Furthermore, different CL emis-sion spectra have been reported in the literature duringluminol oxidation in different reaction media (24).However, such an effect is not observed on changing themedium from aqueous to PVP hydrogel and the coin-cident emission band at 421 nm in water and PVPhydrogel indicates that the polymer does not stronglyinteract with the emitting species (Fig. 2).

The role of luminol, hemin and hydrogen peroxideconcentrations is important to modulate the amount ofproduced light and to access specific kinetic condi-tions (e.g. for the determination of antiradical capacityor monitoring reactive oxygen and nitrogen species gen-eration) (25). The use of a factorial approach for the op-timization of experimental conditions requires a reducednumber of experiments, when compared to a conven-tional kinetic approach, and provides the opportunityto find the more adequate experimental condition fordifferent applications.

Reaction mechanism and kinetics

There is no consensus about the mechanism ofluminol CL in alkaline aqueous media (26). Neverthe-less, it is certainly known that the emitting species is anexcited singlet 3-aminophthalate, which is formed in amultifaceted reaction, including multiple oxidation steps(Scheme 2) (1).

The mechanism of catalysis by haem-containingperoxidases such as catalases, horseradish peroxidase(HRP) and haem mono-oxygenases (cytochrome P-450)in the presence of hydrogen peroxide involves theformation of oxidizing intermediates that are formally‘Fe(V)’ species (27, 28). In most cases the structure is anFe(IV)=O (ferryl) center, combined with a porphyrinπ-cation radical. In the oxidation of protoferrihaem,the ‘Fe(V)’ intermediate is not observed because it isquickly reduced by unoxidized protoferrihaem to a for-mal Fe(IV) species (Scheme 3, steps 2 and 3), supposedto be mainly responsible for luminol mono-anion (1)oxidation and the initiation of the reaction sequence(Scheme 3, step 5) (4). In the main pathway of the CLreaction, the oxidized luminol species (2) is supposed toundergo disproportionation to luminol and the corre-sponding diazaquinone derivative (3, Scheme 3, step 7),

Figure 3. Dependence of the chemiluminescence intensity onluminol concentration at three hydrogen peroxide concentra-tions. [Hemin], 7.7 nmol/L; [PVP], 80 mg/mL.

conditions. However, as luminol concentration increasesbeyond this value a slight decrease of the I0 values isobserved. Additionally, the I0 values are significantlyhigher at 0.1 mmol/L H2O2 (Fig. 3). These results pro-vide a detailed complement to those obtained in thefactorial approach and confirm the tendencies observed.

DISCUSSION

The incorporation of CL reagents within hydrogelsand polymeric thin films has been investigated mainlyin the detection of toxic inorganic and organic chemi-cals (20). The cross-linking of PVP can be performedphotochemically using an UV-C irradiation source andthe properties of the resulting hydrogel are discussedelsewhere (16). This cross-linked polymer network isused as support for the luminol–hemin–hydrogen per-oxide CL reaction and it is shown that the CL proper-ties of this system are maintained (Fig. 1). The emissionintensity is still high enough to be easily observed in acommercial luminometer, although the area under theCL emission curve resulting from hydrogel-supportedreaction is ca. four times lower than that obtained for thestandard aqueous system (Fig. 1). As PVP has noabsorption band higher than 300 nm, reabsorption ofemission by PVP cannot be responsible for this effect.However, it could be explained in terms of the occur-rence of a physical quenching event, the formation ofPVP–hemin and PVP–H2O2 complexes, or less effectivecontact of the reactants in the heterogeneous condition(21–23). On the other hand, irradiation has no effecton the emission profile (Fig. 1), due to the use ofN2-saturated solutions and nitrogen purging during

Scheme 2. CL oxidation of luminol in alkaline aqueousmedia.



Scheme 3. Reaction mechanism postulated for the CL oxidation of luminol in alkaline media.

which reacts with the hydrogen peroxide anion toa hydroperoxy species (4), responsible for excited 3-aminophthalate formation, probably with the interme-diacy of a ‘diaza-endoperoxide’ intermediate (Scheme 3,step 8). Fluorescence emission from singlet excited 3-aminophthalate is finally the origin of the observed CLin this reaction (Scheme 3, step 9) (1).

This minimum reaction scheme indicates that: (a) thereaction is initiated by hemin oxidized with hydrogenperoxide; (b) a second-order term in luminol concentra-tion may be involved in the rate equation, in conditionswhere the disproportionation step is rate-limiting;(c) hydrogen peroxide plays a dual role in this reaction,as it is responsible for the formation of the active



form of the catalyst and also involved as a nucleophilewhich adds to one of the carbonyls of the diazaquinoneintermediate.

The kinetics of a CL reaction can be followed by thetime course of the emission intensity. At any given timeof the reaction, the emission intensity corresponds to thevelocity of excited state formation and is therefore ameasure for the rate constant of the rate-limiting step inthe reaction sequence. The variation of I0 in differentreaction conditions is proportional to the variation of thevelocity of the limiting step, supposing that the emissionquantum yield is not altered. If we limit ourselves to theminimum mechanistic scheme proposed (Scheme 3), wecan predict possible candidates for rate-limiting steps.

Formation of the formal Fe(V) hemin species and theformation of the active form of the catalyst by its inter-action with native hemin (Scheme 3, steps 2 and 3)should not be rate-limiting, as the rate constants forthese reaction are in the order of 107 mol/L/s for HRP(29, 30). Furthermore, it is known that in the case ofHRP the formation of the enzymes active form by hy-drogen peroxide is rate limiting only at very low enzymeand peroxide concentrations (31). Proton transfer forluminol anion formation (step 4, Scheme 3) shouldcertainly be fast and not rate-limiting. The interactionbetween two luminol radicals (Scheme 3, step 7) has anestimated rate constant of 5 × 108 mol/L/s and should notbe rate-limiting in normal conditions (26). Moreover, theintramolecular reaction steps involved in the transforma-tion of 4 to excited 3-aminophthalate (5*) and theradiative decay of 5* are very fast and not rate-limitingunder any experimental condition (1).

The most likely candidates for rate limiting steps are:(a) oxidation of luminol anion by Hem-Fe(IV), whichdepends on the concentration of all reagents (Scheme 3,step 6, k (HRP) = 2.3 × 104 mol/L/s) (32) and should beconsiderably slower than step 5 in analogy with the re-activity of HRP compounds I and II (4, 32). Therefore,under ‘normal’ conditions, step 4 should be rate-limitingand variation in the emission intensity is supposed toreflect changes in the velocity of this step; (b) additionof H2O2 to diazaquinone 3 should only be rate-limitingat low peroxide concentrations (Scheme 3, step 8,k = 5 × 107 mol/L/s) (33).

Effect of reagent concentrations on initialintensities and emission areas

From the results shown in Table 2, response surfacescan be plotted for the concentration dependence of theemission intensity and areas which show similar behav-iour (Figs 4, 5). When hydrogen peroxide is the thirdvariable, the emission intensity is low and reasonablyconstant, independent of the concentrations of the otherreagents, except at intermediate hydrogen peroxide con-centrations (Fig. 4, row A). At low hydrogen peroxide

concentration, steps 5 and 8, should always be slow, in-dependent of the concentration of the other reagents(Scheme 3); therefore, an increase in both hemin andluminol concentrations will not lead to a significant over-all rate increase (Fig. 4A1). The I0 values are consider-able higher at the intermediate [H2O2], increasing withthe [hemin] and showing maximum curves with the[luminol] (Fig. 4A2). An increase in the peroxide andhemin concentrations will increase the turn-over of thehemin catalyst and the overall reaction rate will increasein consequence (Scheme 3, steps 1–3, 5, 6). The nearlyconstant and much lower I0 values at high [H2O2] (Fig.4A3) indicate that the high peroxide concentrations leadto hemin destruction (34, 35) and therefore the reactionrate will always be low, due to the lack of active catalystfor luminol oxidation.

The reaction rate rises with increasing hemin concen-trations (Fig. 4, row B), which is more pronounced atintermediate luminol and peroxide concentrations (Fig.4A2, C2). This fact can be explained by considering twofactors: (a) higher active catalyst concentrations resultsin rate increase of step 5 (Scheme 3); and (b) high per-oxide concentrations can lead to hemin destruction andtherefore the maximum effect is observed at intermedi-ate H2O2

concentrations.The highest emission intensities are obtained at inter-

mediate luminol concentrations (Fig. 4, row C), alsoillustrated by the maximum curves observed with respectto [luminol] in the plots A2 and B3 (Fig. 4). In thesecases, it appears that at higher luminol concentrationsthe hemin is rapidly recycled by fast reaction withluminol (Scheme 3, steps 1, 5 and 6), resulting in hydro-gen peroxide consumption. With peroxide as the limit-ing reagent, the nucleophilic attack of HOO− to thediazaquinone intermediate 3 (step 8, Scheme 3) mightbecome the rate-limiting step and the overall reactionrate decreases with increasing luminol concentrations.Moreover, the diazaquinone 3 may suffer attack byother nucleophiles present in the reaction medium (e.g.OH−), a reaction that does not contribute to excited stateformation (26–33). The surprising effect of high luminolconcentrations on I0 has been confirmed by our addi-tional experiments with [luminol] variation at three[H2O2] and intermediate [hemin], which show an in-crease in I0 with increasing concentrations only for low[luminol] (<1 mmol/L) followed by a decrease of I0 forhigher concentrations (Fig. 3).

Optimum conditions for high intensity emission areobtained for high hemin concentrations ([hemin] =77 nmol/L), intermediate peroxide ([H2O2] = 0.1 mmol/L) and luminol ([luminol] = 1.0 mmol/L) concentrations.Furthermore, several conditions can be identified inwhich large reagent concentration variations lead to onlysmall variations in I0 (Fig. 4), a situation which might beof interest for several analytical applications where con-stant emission intensity is desired.



As previously mentioned, S and I0 plots show similarprofiles and optimum total emission conditions are simi-lar to those for maximum intensity: high hemin, interme-diate peroxide and luminol concentrations. However,whereas I0 is related to the overall reaction rate, S indi-cates the total number of generated photons and shouldbe determined by the limiting reagent concentration.The quantum yields for the luminol reaction are knownto be constant for luminol concentrations up to 2 mmol/L (36, 37); therefore, in our experiments, constant quan-tum yields are expected for low and intermediateluminol concentrations.

At low and high peroxide concentrations the areasshow little variation with the other two reagent con-centrations (Fig. 5A1, A3), confirming that at lowH2O2 concentration, the peroxide is always the limit-ing reagent and at the high concentration it causes

hemin destruction, which is the reason for the lowemission areas. At intermediate peroxide concentra-tion, the areas show an increase with the hemin con-centration also confirming that hemin destructionlimits the emission efficiency (Fig. 5A2). At low andintermediate hemin concentrations, the areas showonly small variations with the luminol as well as per-oxide concentration, indicating hemin as the limitingreagent (Fig. 5, row B). However, for the highhemin concentration the areas show considerable de-pendence on both other variables, the maximum valuebeing observed for luminol and H2O2 concentrationsof 1.0 mmol/L and 0.10 mmol/L, respectively (Fig. 5B3).The reasons for this are: (a) hemin destruction athigh peroxide concentration; (b) lack of peroxide fornucleophilic attack at high luminol concentration, asdiscussed before.

Figure 4. Response curves for the dependence of the maximum emission intensity (I0) as a function of two factorsat three different concentrations of the third factor. (A) Luminol and hemin as independent variables at hydrogenperoxide concentrations of 0.01, 0.1 and 1 mmol/L. (B) Luminol and hydrogen peroxide as independent variables athemin concentrations of 0.77, 7.7 and 77 nmol/L. (C) H2O2 and hemin as independent variables at luminol concentra-tions of 0.1, 1.0 and 10 mmol/L.



The plots obtained with luminol as the third variable(Fig. 5, row C) show all maximum curves for peroxideconcentration and increasing areas with increasinghemin concentrations, showing [hemin] to be limitingand the importance of hemin destruction at high [H2O2].The effects are more pronounced at the intermediateluminol concentration, which is explained by the lack ofhydrogen peroxide for nucleophilic attack to thediazaquinone intermediate 3 at high [luminol] (Scheme3, step 8), which may be destroyed by addition of othernucleophiles, not leading to light emission and, conse-quently, lower quantum yields (26, 33).

Effect of reagent concentrations on observedrate constants

Whereas the emission intensities are directly relatedto the global reaction rate, as discussed above, the

observed rate constants reflect the consumption of thelimiting species and may correspond to the depletion ofany component of the system (luminol, hydrogen perox-ide and hemin), since it can become the limiting reagent,depending on the concentration ratio of the reagents. Itshould be kept in mind that the factorial design em-ployed screens 27 conditions where reagents concentra-tions are varied in three orders of magnitude. In the firstinstance, the concentration of the catalyst hemin shouldnever be limiting, as it is not consumed in the main re-action pathway (Scheme 3). However, it is known thathemin inactivation can occur, especially at high perox-ide concentrations, causing hemin to become the limit-ing reagent and thus the observed rate constant berelated to its destruction (4, 34, 35).

The observed rate constants show a clearly dis-tinct behaviour from the other two dependent vari-ables (Fig. 6). At the peroxide concentrations studied,

Figure 5. Response curves for the dependence of the area of emission as a function of two factors at three differentconcentrations of the third factor. (A) Luminol and hemin as independent variables at hydrogen peroxide concentrationsof 0.01, 0.1 and 1 mmol/L. (B) Luminol and hydrogen peroxide as independent variables at hemin concentrations of 0.77,7.7 and 77 nmol/L. (C) H2O2 and hemin as independent variables at luminol concentrations of 0.1, 1.0 and 10 mmol/L.



variation of the other independent factors leads to onlysmall changes in kobs values, which decrease as the con-centration of peroxide increases (Fig. 6, row A). Thisindicates that H2O2 might be limiting at the low concen-tration and that the other reagents will become limitingas the peroxide concentration increases. The decrease ofthe kobs values with increasing luminol concentration forthe high peroxide concentration (Fig. 6A3) indicates thatat low luminol concentration the rate constant corre-sponds to luminol consumption and, when luminol con-centration is increased, hemin might become the limitingreagent as [H2O2] are high.

For low and intermediate hemin concentrations therate constants values are similar and nearly independentof the luminol and H2O2 concentration, whereas at highhemin concentrations the kobs values show considerablevariation (Fig. 6, row B). This behaviour indicates

changes in the rate-limiting reagent from hemin (at thelow concentration) to peroxide and luminol (at the highconcentration).

The plots in which luminol concentration is used asthe third independent variable (Fig. 6, row C) indicatemore pronounced variation in the rate constants withthe other two reagent concentrations, showing that thehemin and peroxide concentrations are the most deter-minant factors for this dependent variable. However,the central regions of the surface are very similar atall [luminol], indicating kobs values independent of theluminol concentration. Interestingly, these plots showsignificant differences in the kobs values for high andlow peroxide to hemin concentration ratios, more pro-nounced at the higher [luminol] (Fig. 6C3). In theseconditions, a change in the limiting reagent from hemin(at high peroxide:hemin ratios) to peroxide (at low

Figure 6. Response curves for the dependence of the observed rate constants (kobs) as a function of two factors at threedifferent concentrations of the third factor. (A) Luminol and hemin as independent variables at hydrogen peroxideconcentrations of 0.01, 0.1 and 1 mmol/L. (B) Luminol and hydrogen peroxide as independent variables at heminconcentrations of 0.77, 7.7 and 77 nmol/L. (C) H2O2 and hemin as independent variables at luminol concentrations of0.1, 1.0 and 10 mmol/L.



peroxide:hemin ratios) appears to occur and in theintermediate region both reagents are limiting in vary-ing degree.

Briefly, from the data plotted in Fig. 6 we can affirmthat luminol concentration shows little influence on thedecay constants. This parameter is constant at interme-diate peroxide:hemin ratios, high kobs values are ob-tained for low peroxide:hemin ratios, whereas low kobs

values are obtained for high peroxide:hemin ratios.

Multivariate factorial analysis

The importance of any of the reagents concentrationfor the dependent variables (I0, S and kobs) can be quan-tified from the effects obtained by multivariate factorialanalysis (Table 4). The emission intensity as well asemission areas are strongly dependent on the quadraticperoxide concentration term [factor C (Q), NE = 1.0, forI0 and S]. Furthermore, both variables strongly dependon the linear term in hemin [factor B (L), NE = 0.575 forI0 and 0.619 for S] as well as the combination of bothterms [factor B (L) × factor C (Q), NE = 0.793 for I0 and0.845 for S]. The rate constants show to be determinedmainly by the linear and quadratic peroxide concentra-tion terms [factor C (L), NE = −1.0; factor C (Q), NE =−0.714]. The linear luminol term and the combina-tion hemin (linear) and hydrogen peroxide (quadratic)show smaller, although significant, negative contribu-tions [factor A (L), NE = −0.300; factor B (L) × factorC (Q), NE = −0.364]. The negative values of NE indi-cate a negative effect, indicating that increasing theindependent variable value leads to a decrease in thedependent variable.

The general results of this analysis appear to beconsistent with the mechanistic scheme proposed(Scheme 3). The dependent variable intensity and areaare strongly dependent on the square of hydrogen per-oxide concentration and its combination with the heminconcentration. Contrarily, the rate constant values showdependency on all reagent concentrations, although thesquare of the [H2O2] is most important. Especially, theimportance of the quadratic term of [H2O2] is in agree-ment with the fact that this reagent participates in tworeaction steps essential for excited states generation.

For special analytical applications, in which slow andnearly constant decay kinetics are of advantage, thismeans that the peroxide:hemin ratio should be in therange of 10−4 and luminol can be varied, if necessary,from 0.1 to 10 mmol/L. In combination with the resultsobtained on emission intensity (Fig. 4), we can find idealconditions for long-lasting high-intensity emission kinet-ics for intermediate luminol concentrations (1.0 mmol/L), intermediate peroxide concentrations (0.1 mmol/L)and intermediate to high hemin concentrations. On theother hand, if high intensity and fast emission kineticsare desired for a certain analytical application, the ideal

luminol and peroxide concentrations remain the same(1.0 mmol/L and 0.1 mmol/L, respectively) and thehemin concentration should be high; even higher valuesthan the one studied here might be used. Moreover,using concentration peroxide:hemin ratios of 103 oreven lower, both these concentrations can be increasedin order to obtain high-intensity flash emission kinetics.

CONCLUSIONS

In conclusion, photocross-linked poly(N-vinyl-2-pirrolidone) hydrogel is a suitable support for hemin-catalysed luminol oxidation by hydrogen peroxide, sincethe CL properties of this system are maintained. Thepresence of PVP seems to affect only the emission inten-sity, but the overall effect of reactants are in agreementwith previous kinetic studies in aqueous solution (3, 4).Using the factorial design, the effect of reagents con-centration on the dependent variables I0, S and kobs canbe determined with a reduced number of experiments.Multivariate factorial analysis of the data results in thequantification of the contribution (linear, quadratic andcombined) of each of the factors to the observed effecton the dependent variable. This approach, together withthe analysis of the obtained response surfaces, will beuseful to access adequate experimental conditions forspecific analytical applications, e.g. hydrogen peroxidedetermination and antiradical assays, which might beused to substitute, in a favourable way, for aqueousassay systems (38).

Acknowledgements

A donation of 60% hydrogen peroxide by Solvay–Peróxidos do Brasil LTDA Co. is gratefully acknow-ledged. This work was supported by Fundação deAmparo à Pesquisa do Estado de São Paulo (FAPESP;98/05445-9, 00/06652-0, 01/07477-0), Conselho Nacionalde Desenvolvimento Científico e Tecnológico (CNPq),Coordenação de Aperfeiçoamento de Pessoal deNível Superior (CAPES) and Programa de Apoio aoDesenvolvimento Científico e Tecnológico (PADCT).

REFERENCES

1. Baader WJ, Stevani CV, Bastos EL. Chemiluminescence oforganic peroxides. In The Chemistry of Peroxides, Rappoport Z(ed.). Wiley: Chichester, 2006; 1211–1278.

2. Garcia-Campana AM, Baeyens WRG, Cuadros-Rodriguez L,Barrero FA, Bosque-Sendra JM, Gamiz-Gracia L. Potential ofchemiluminescence and bioluminescence in organic analysis. Curr.Org. Chem. 2002; 6: 1–20.

3. Ewetz L, Thore A. Factors affecting specificity of luminol reactionwith hematin compounds. Anal. Biochem. 1976; 71: 564–570.

4. Jones P, Scowen NR. Kinetics and mechanism of catalysis byferrihaems in the chemiluminogenic oxidation of luminol byhydrogen peroxide. Photochem. Photobiol. 1987; 45: 283–289.



5. Zhang XR, Baeyens WRG, García Campaña AM. Recent devel-opments of luminol-based chemiluminescence analysis. Biomed.Chromatogr. 1999; 13: 169–170.

6. Zhang XR, Baeyens WRG, Garcia-Campana AM, Ouyang J.Recent developments in chemiluminescence sensors. Trends Anal.Chem. 1999; 18: 384–391.

7. Aboul-Enein HY, Stefan R-I, Van Staden JF.Chemiluminescence-based (bio)sensors—an overview. Crit. Rev.Anal. Chem. 1999; 29: 323–331.

8. Aboul-Enein HY, Stefan R-I, Van Staden JF, Zhang XR,Garcia-Campana AM, Baeyens WRG. Recent developments andapplications of chemiluminescence sensors. Crit. Rev. Anal. Chem.2000; 30: 271–289.

9. Collins GE, Rose-Pehrsson SL. Chemiluminescent chemicalsensors for oxygen and nitrogen dioxide. Anal. Chem. 1995; 67:2224–2230.

10. Lamarcq L, Lorimier P, Negoescu A, Labat-Moleur F, Durrant I,Brambilla E. Comparison of seven bio- and chemiluminescent rea-gents for in situ detection of antigens and nucleic acids. J.Biolumin. Chemilumin. 1995; 10: 247–256.

11. Emmons RE, Mauck JC, Heaney PJ, Freund DK, Latart DB,Chubet RG, Vizard DL. Dry elements, test devices, test kits andmethods for chemiluminescent detection of analytes usingperoxidase labelled reagents. Eur. Pat. Appl., EP 653637, 1995,CAN 123:5121.

12. Honzawa K, Kawaguchi S, Ishiguro T, Atsumi K, Shimomura F,Motojima H. Oxidizing composition in powder form and methodof measuring chemiluminescence using the same. Eur. Pat. Appl.,EP 653638, 1995, CAN 123:29057.

13. Collins GE, Rose-Pehrsson SL. Chemiluminescence chemicaldetection of vapors and device. U.S. Pat. Appl., US 500277, 1996,CAN 125:184484.

14. Hoeffkes H, Poppe E, Dolhaine H. Efficient oxidative hair dyecontaining radical scavengers for the protection of hair. Ger.Offen., DE 10315421, 2004, CAN 141:337254.

15. Edwards B, Voyta JC, Smith RM, Menchen SM. Arrays modifiedwith chemiluminescent enhancing materials for chemiluminescentassays, methods of making the arrays and methods of detectingchemiluminescent emissions on solid supports. U.S. Pat. Appl.Publ., US 2004259182, 2004, CAN 142:51794.

16. Lopérgolo LC, Lugão AB, Catalani LH. Direct UV photocross-linking of poly(N-vinyl-2-pyrrolidone) (PVP) to produce hydro-gels. Polymer 2003; 44: 6217–6222.

17. Cotton ML, Dunford HB. Studies on horseradish peroxidase. XI.On the nature of compounds I and II as determined from thekinetics of the oxidation of ferrocyanide. Can. J. Chem. 1973; 51:582–587.

18. Scolaro LM, Castriciano M, Romeo A, Patanè S, Cefali E,Allegrini M. Aggregation behaviour of protoporphyrin XI in aque-ous solution: clear evidence of vesicle formation. J. Phys. Chem.B 2002; 106: 2453–2459.

19. Keppel G, Wickens TD. Design and Analysis: A Researcher’sHandbook. Prentice Hall: New York, 2004.

20. Collins GE, Rose-Pehrsson SL. Chemiluminescent chemicalsensors for inorganic and organic vapors. Sens. Actuators B 1996;34: 317–322.

21. Inamura I, Isshiki M, Araki T. Solubilization of hemin in neutral

and acidic aqueous solutions by forming complexes with water-soluble macromolecules. Bull. Chem. Soc. Jpn 1989; 62: 2413–2415.

22. Inamura I, Uchida K. Association behaviour of protoporphyrin IXin water and aqueous poly(N-vinylpyrrolidone) solutions. Interac-tion between protoporphyrin IX and poly(N-vinylpyrrolidone).Bull. Chem. Soc. Jpn 1991; 64: 2005–2007.

23. Panarin EF, Kalninsh KK, Pestov DV. Complexation of hydrogenperoxide with polyvinylpyrrolidone: ab initio calculations. Eur.Polym. J. 2001; 37: 375–379.

24. Lind J, Merenyi G, Eriksen TE. Chemiluminescence mechanismof cyclic hydrazides such as luminol in aqueous solutions. J. Am.Chem. Soc. 1983; 105: 7655–7661.

25. Bastos EL, Romoff P, Eckert CR, Baader WJ. Evaluation ofantiradical capacity by H2O2-hemin-induced luminol chemilumine-scence. J. Agric. Food Chem. 2003; 51: 7481–7488.

26. Merényi G, Lind J, Eriksen TE. Luminol chemiluminescence—chemistry, excitation, emitter. J. Biolumin. Chemilumin. 1990; 5:53–56.

27. Dounce AL, Sichak SP. Hematin iron valence in catalase andperoxidase compound 1. Relationship to the free-radical reactionmechanism. Free Rad. Biol. Med. 1988; 5: 89–93.

28. Nakamura M, Nakamura S. One- and two-electron oxidationsof luminol by peroxidase systems. Free Rad. Biol. Med. 1998; 24:537–544.

29. Cormier MJ, Prichard PM. An investigation of the mechanism ofthe luminescent peroxidation of luminol by stopped flow tech-niques. J. Biol. Chem. 1968; 243: 4706–4714.

30. Davies DM, Jones P, Mantle D. The kinetics of formation ofhorseradish peroxidase compound I by reaction with peroxo-benzoic acids. Biochem. J. 1976; 157: 247–253.

31. Baader WJ, Bohne C, Cilento G, Dunford HB. Peroxidase cata-lysed formation of triplet acetone and chemiluminescence fromisobutyraldehyde and molecular oxygen. J. Biol. Chem. 1985; 260:217–225.

32. Candy TEG, Hodgson M, Jones P. Kinetics and mechanismof a chemiluminescent clock reaction based on the horseradishperoxidase catalysed oxidation of luminol by hydrogen peroxide.J. Chem. Soc. Perkin Trans. 1990; 2: 1385–1388.

33. Merényi G, Lind J, Eriksen TE. Nucleophilic addition todiazaquinones—formation and breakdown of tetrahedral inter-mediates in relation to luminol chemiluminescence. J. Am. Chem.Soc. 1986; 108: 7716–7726.

34. Brown SB, Jones P. Reactions between haemin and hydrogenperoxide. 3. Catalytic decomposition of hydrogen peroxide. Trans.Faraday Soc. 1968; 64: 999–1005.

35. Traylor TG, Kim C, Richards JL, Xu F, Perrin CL. Reactions ofiron(III) porphyrins with oxidants. Structure–reactivity studies.J. Am. Chem. Soc. 1995; 117: 3468–3474.

36. Lee J, Seliger HH. Quantum yields of luminol chemiluminescencereaction in aqueous and aprotic solvents. Photochem. Photobiol.1972; 15: 227–237.

37. Lind J, Merényi G. Determination of the chemiluminescencequantum yield of luminol in rapid chemical reactions. Chem. Phys.Lett. 1981; 82: 331–334.

38. Nguyen KT, West JL. Photopolymerizable hydrogels for tissueengineering applications. Biomaterials 2002; 23: 4307–4314.

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