determination of phenol using an enhanced chemiluminescent assay

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Received 22 August 2001; revised 2 May 2002; accepted 22 May 2002

ABSTRACT: Enhanced chemiluminescence (ECL) describes the phenomenon of increased light output in the luminol oxidationreaction catalysed by horseradish peroxidase (HRP) in the presence of certain compounds, such as para-iodophenol. In this work, theeffects of phenol on the para-iodophenol-enhanced HRP-catalysed chemiluninescent reaction intensity in an aqueous buffer (Tris–HCl buffer, pH 8.5) and in a surfactant–water–octane mixture were compared. Preincubation of HRP at low phenol concentrationsstimulated the chemiluminescent intensity in the assay performed in an aqueous buffer, but did not have significant effect in thesodium bis(2-ethylhexyl)sulphosuccinate) (Aerosol OT, AOT) applied system. It was also observed that HRP preincubation withphenol concentration higher than 0.003 mg/mL produced an inhibitory effect on the enzyme activity for both assay systems. Only aninhibitory effect of phenol on the chemiluminescent intensity in the surfactant system in octane (as organic solvent) was observed.Three assays were developed to determine phenol concentration in water and in an organic solvent mixture. The detection limits were0.006, 0.003 and 0.0005 mg/mL, respectively, for the buffer-containing system, the AOT-applied system with phenol standardsolutions in water and for the AOT-applied system with phenol standard solutions in octane. Copyright � 2002 John Wiley & Sons,Ltd.

KEYWORDS: horseradish peroxidase; luminol; phenol; surfactant–water–octane system

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Phenols are the major organic pollutants present inaqueous effluents from coal-conversion processes, aswell as from other industrial processes (1, 2). Since mostphenols are toxic, their presence in natural water presentsan environmental threat (1, 2). The presence andconcentration of these pollutants must be detected andmonitored to maintain environmentally acceptable con-ditions.

Current laboratory methods for phenol detection inwastewaters include pretreatment of the sample, coupledwith spectrophotometric detection of the compounds thatare formed by the reaction between the phenol and theassay reagents (3). The time taken to complete thesetypes of assays and the variations in sensitivity makethem unsuitable for on-site applications (3).

A more rapid enzymatic assay using different peroxi-dases and o-dianisidine has been earlier reported (4).However, the sensitivity of this assay for phenol was 10times less than current analytical methods (3, 4).

The enhanced chemiluminescent oxidation of luminolcatalysed by horseradish peroxidase (EC 1.11.1.7) offersanalytical advantages in the development of techniquesthat allow detection of environmental pollutants (5, 6).The high selectivity, simplicity and extreme sensitivity ofthe chemiluminescence methods underlies the success ofthis method in a variety of applications, includingdetection of enzymatically generated H2O2, HRP labelsin enzyme immunoassay and DNA probe assays, andcontrol of bioactive compounds and detection ofenvironmental pollution (5–10).

We have developed a chemiluminescence assay for thedetermination of phenol in aqueous and organic mediausing a surfactant–water–octane system. The response tophenol in the different assays was compared andcalibration plots were obtained. It was demonstrated thatthe enhanced chemiluminescent (ECL) reaction may beuseful for monitoring phenol concentration in aqueous aswell as in organic media.

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HRP (type IVA, 1100 U/mg), Trizma, hydrochloric acid5-amino-2,3-dihydri-1,4-phtalazinedione (luminol),para-iodophenol, EDTA, hydrogen peroxide, NaOH,

Luminescence 2003;18:31–36Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bio.698

Copyright 2002 John Wiley & Sons, Ltd.

ORIGINAL RESEARCH

*Correspondence to: A. D. Ilyina, Blvd. V. Carranza and Ing. J.Cardenas V., Col. Republica, A.P. 935, C.P. 25000, Saltillo, Mexico.Email: [email protected]

Contract/grant sponsor: CONACyT-SIREYES; contract/grant number:970406030.

sodium bis(2-ethylhexyl)sulphosuccinate [also known asaerosol OT (AOT)], octane and phenol were purchasedfrom Sigma Chemical Co. Chemiluminescence intensitywas measured by an EMILITE EL 1003 portableluminometer (Russia).

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Enzyme solution (20 �L) (1 L U/ml) was preincubatedwith 0.02 mL phenol solution for 1 min at 25°C.Measurements of chemiluminescent intensity wereperformed in 0.1 mol/L Tris–HCl buffer containing2 mmol/L EDTA, pH 8.5. Aliquots of 0.1 mL0.2 mmol/L luminol, 0.1 mL 0.3 mmol/L para-iodophe-nol and 0.01 mL 0.44 mol/L H2O2 were added to acuvette containing Tris–HCl buffer to a final volume was1.01 mL. The reaction was initiated by addition of0.02 mL enzyme–phenol solution. The same procedureswere performed using distilled water (as control) insteadof the phenol solution. The ECL intensity profiles wererecorded and the maximum intensity was used to plot thecalibration graphs. Each measurement was in duplicateand the assays were repeated in triplicate.

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The ECL reaction for samples in water and an organicphase was performed using AOT as surfactant and octaneas organic solvent. The conditions permitted for detectionof the ECL intensity were as defined previously (11). Thetechnique applied for this is similar to that used in studiesof the reversed micelle system (11) but with a lowconcentration of surfactant (0.001 mol/L). In this case,the optimum value of W0 was 2700. W0 is the hydrationratio (W0 = [H2O]/[surfactant]) commonly used forcharacterization of reversed micelle systems. Thiselevated value of W0 demonstrated the non-existence ofreversed micelles, which are commonly characterized inthe range W0 10–40 (12, 13).

�� 0�� 3�� Phenol standards were prepared in distilled waterand the AOT (0.001 mol/L) was dissolved in octane. Inthe assay, 0.02 mL enzyme solution (22 U/mL) werepreincubated with 0.02 mL pollutant solution for 30 s at25°C. To perform the ECL reaction, 1 mL surfactantsolution was placed in a luminometer cuvette, followedby 0.02 mL enzyme–phenol mixture and 0.01 mL H2O2

(0.022 mol/L). This mixture was shaken for 30 s at250 rpm until it became uniform. Finally, a solution of0.2 mmol/L luminol and 0.3 mmol/L para-iodophenol(0.02 mL) was added to initiate the ECL reaction. The

value of the light intensity emitted during the ECLreaction was determined rapidly after shaking thereaction mixture for 7–10 s. The maximum intensityvalue was used to plot the calibration graph.

�� Phenolstandard solutions were prepared in octane. The assaymethod was similar to the above method, except that only0.95 mL AOT solution was used, then 0.05 mL phenolsolution in octane was added. The enzyme concentrationin this case was 11 U/mL and there was no preincubation.The maximum intensity of the light emission was used toplot a calibration graph.

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Three samples of wastewater were used to compare theECL-based phenol assay in an aqueous buffer system, theECL-based phenol in the surfactant–water–octane sys-tem, and the current spectophotometric analytical method(3).

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Peroxidases from different sources (fungal anionic per-oxidase and cationic peroxidase from alfalfa) showedsimilar types of inhibition of activity in the presence ofphenol. The decrease of the reaction rate of o-dianisidineoxidation was used to plot a calibration graph for phenoldetermination (4). Horseradish peroxidase can not beused to determine phenol because the inhibition effectwas only observed over a narrow range of phenolconcentrations (47–94 mg/mL) (4).

However, it was reported previously (5–9) that a widerange of chemicals, including phenol, inhibited theenhanced chemiluminescent oxidation of luminol cata-lysed by horseradish peroxidase. We have adapted thisassay for phenol detection, using commercial horseradishperoxidase. For an aqueous phenol standard (1.25 mg/mL), we observed that the presence of phenol in thereaction mixture in Tris–HCl buffer at this concentrationdecreased light intensity (Fig. 1, Top). Further, the curvesare characterized by rapid light decay during the ECLreaction, which makes estimation of the Imax difficultfrom data obtained without plotting the light-emissionkinetics.

In contrast, in the surfactant (AOT)–water–octanesystem, the kinetics of light emission (Fig. 1, bottom) ismore stable than in the buffer system (Fig. 1, top). This isprobably related to a greater stability of peroxidase in thissystem, indicating that enzyme inactivation is the mainreason for light decay in the course of the reaction (14).The higher signal stability facilitated detection of the

Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2003;18:31–36

32 ORIGINAL RESEARCH A. D. Ilyina et al.

maximum light intensity, which was used to obtain thecalibration curves for phenol quantification.

To detect the light intensity in the system containingAOT and octane, the phenol concentration was reducedsignificantly (Fig. 1, bottom). The ECL reaction wasinhibited completely at phenol concentrations �0.4 mg/mL (phenol concentrations of 0.0005–0.128 mg/mL weretested in this study).

It was observed that the response of the ECL reactionat low phenol concentrations was different in the buffer-

containing system as compared to the AOT–water–octane system. In the buffer system an increase inchemiluminescent intensity was observed over the phenolconcentration range 0.0005–0.003 mg/mL (Fig. 2). Therelationship between light intensity and phenol concen-tration is linear: y = 2.90x � 75.23 (r2 = 0.986). Theintensity increase is not significant at low phenolconcentration (Fig. 2), but the linear relationshippermitted the determination of concentration with highprecision.

In the buffer system, the inhibitory effect of phenolwas observed for concentrations higher than 0.006 mg/mL (n = 3, p = 0.95), which is the minimum concentra-tion of the calibration linear graph [y = 75.87 � 0.131x(r2 = �0.986)] (Fig. 4).

As the enhancement or inhibition of chemiluminescentintensity in the buffer system is dependent on the phenolconcentration (Figs 2, 4), the interpretation of results ismore difficult (15).

In contrast to the buffer system, the AOT–water–octane system does not present these difficulties in theinterpretation of results (Fig. 3). The aqueous phenolsolution over the same low concentration range did notaffect chemiluminescence intensity significantly (Fig. 3,top).

The linear function of the calibration graph obtainedwith aqueous phenol solutions and demonstrated in Fig. 5is y = 101.73 � 0.25x (r2 = �0.976). The minimumphenol concentration detectable was 0.003 mg/mL(n = 3, p = 0.95).

Using the standard phenol solution prepared in octaneand the AOT-applied system, we demonstrated that inthis case the phenol inhibited the chemilumininescentintensity at all concentrations tested (Figs 3, bottom and

Figure 1. ECL intensity–time profiles. Top: in the absence ofphenol in the buffer-containing system and in the presence ofphenol (1.25 mg/mL), thick and thin lines, respectively. Bottom:in the absence of phenol and in the presence of phenol(0.128 mg/mL) in the AOT-containing system, thick and thinlines, respectively. The peroxidase concentrations were11 U/mL and 22 U/mL respectively.

Figure 2. Maximum ECL intensity as a function of lowphenol concentrations a in buffer-containing system (11 U/mLperoxidase).


Phenol detection using an enhanced chemiluminescence ORIGINAL RESEARCH 33

6). At low phenol concentrations (Fig. 3, bottom) theinhibition is described by the linear functiony = 32.62 � 2.48x (r2 = �0.988). Using this calibrationgraph, concentrations up to 0.0005 mg/mL can bedetected (n = 3, p = 0.95).

The linear function of the calibration plot, obtainedusing phenol standard solution in octane at concentra-tions higher than 0.006 mg/mL (presented in Fig. 6) isy = 27.06 � 0.14x (r2 = �0.990). This technique may beuseful to determine phenol in a hydrophobic medium, e.g.in cosmetics, chemicals and oils as well as in industrialwastes containing organic solvents.

In the present study the phenol concentration inwastewater samples was also determined using differenttechniques (Table 1): (a) ECL reaction in buffer; (b) ECLreaction in the surfactant–water–octane system; and (c)

Figure 3. Maximum ECL intensity as a function of lowphenol concentrations in the AOT–water–octane system. Top,in the presence of aqueous phenol standard solution (22 U/mLperoxidase). Bottom, in the presence of phenol standardsolution prepared in octane (11 U/mL peroxidase).

Figure 4. Typical calibration curve obtained in assayperformed in a buffer-containing system, using a phenolconcentration of 0.006–0.128 mg/mL (11 U/mL peroxidase).

Figure 5. Typical calibration curve obtained in assayperformed in AOT–water–octane-containing system, usingaqueous standard phenol solution at a concentration range of0.006–0.128 mg/mL (22 U/mL peroxidase).

Table 1. Phenol detection in three samples of wastewatersby: (a) chemiluminescent enzymatic assay in the buffer-containing system; (b) the method carried out in thesurfactant–water–octane containing system; and (c) spec-trophotometric detection (3) using a common analyticaltechnique (n = 3, middle � standard deviation)

(a) Phenol, �g/mL (b) Phenol, �g/mL (c) Phenol, �g/mL

50.8 � 0.2 51.2 � 0.3 52.3 � 0.66.0 � 0.2 5.6 � 0.2 5.3 � 0.38.8 � 0.3 8.8 � 0.3 7.3 � 0.5



by the conventional analytical method (3). Both ECLmethods demonstrated good correlation with the currentanalytical method, which is based on the spectrophoto-metric detection of the product formed in the reactionbetween phenol and 4-aminoantipyrine (3).

The ECL assays have several advantages comparedwith the current analytical techniques for phenols. Thespectrophotometric assay is lengthy (more than 8 h)because it includes various distillation and extractionprocesses (3). It can not be used in situ, and its applicationfor determination on-line is difficult because of thelengthy analysis time. In contrast, the methods developedin the present study are rapid (a few minutes) and can beperformed using a portable luminometer that is suitablefor on-site determination of phenol concentration.

Moreover, in comparison with the enzymatic spectro-photometric assay (4), both methods described in thepresent study employ horseradish peroxidase, commonlyused in the laboratory, which has more storage stabilityand is economically affordable (10).

It is known that the maximum of chemiluminescentintensity is proportional to the reaction rate (16). Theintensity is directly detected on the luminometer, whereasthe reaction rate detected spectrophotometrically iscalculated from the kinetic curve of substrate oxidation(4). Hence, use of the ECL reaction of luminolperoxidation decreased the time of analysis.

The sensitivities of the determination of phenol in thepresent study assays are comparable to both spectro-photometric techniques (3, 4). Application of ECLreaction in the AOT–water–octane containing system(Fig. 6) further increased assay sensitivity by more thantwo-fold.

Rapid and simple assays based on ECL are useful toindicate general water contamination (5, 6). However, thenature of the peroxidase enzyme, as well as the mech-anism of enhancement (the radicals and oxygen speciesinvolved in the reaction) provide multiple points at whichthe light emission reaction can be modulated (5–9). Thispeculiarity of the ECL, reaction requires us to considerthat the presence of some substances (antioxidants, forexample) can influence the ECL phenol detectiondescribed in the present study. Considering the sourceof wastewater, a pretreatment of suitable samples, insome cases, or adequate choice of medium to a standardsolution preparation helped to overcome the problem ofassay selectivity (5–9).

Thus, in the present work it was demonstrated that theECL-reaction catalysed by HRP in surfactant–water–octane could be applied for the determination of phenolin both hydrophilic and a hydrophobic medium. Theassay was rapid, sensitive and the detection of thechemiluminescence signal simple to perform.

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The present research was supported by a project fromCONACyT-SIREYES, No. 970406030. We thank DrConsuelo de J. Cortes Penagos from the University ofMichoacan for her helpful discussion, and CorporacionMexicana de Investigacion en Materiales S.A. de C.V.(COMIMSA, Saltillo, Mexico) for their help in thedevelopment of this project.

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Figure 6. Typical calibration curve obtained in assayperformed in an AOT–water–octane-containing system usingstandard phenol solution prepared in octane, at a concentrationrange of 0.006–0.128 mg/mL (1 U/mL peroxidase).


Phenol detection using an enhanced chemiluminescence ORIGINAL RESEARCH 35

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determination of phenol using an enhanced chemiluminescent assay

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