Fresenius J Anal Chem (1999) 365 : 537–540
© Springer-Verlag 1999
O R I G I N A L PA P E R
A. Navas Díaz · F. G. Sanchez · M. C. Torijas · J. Lovillo
Chemiluminescent lipase determination based on the enhanced luminol/H2O2/horseradish peroxidase/fluorescein diacetate energy transfer system
Received: 1 March 1999 / Revised: 6 May 1999 / Accepted: 12 May 1999
Abstract A methodology for the determination of lipase, based on the coupled processes of energy transfer and enhancement of the chemiluminescence of the luminolH2O2-horseradish peroxidase (HRP) system has been developed. Fluorescein diacetate (FDA) was hydrolyzed to fluorescein by the action of the enzyme lipase, and this compound acted as an enhancer of the chemiluminescent process and acceptor of the chemiluminescent emission from the luminol-H2O2-HRP system. By measuring the transferred emission to fluorescein at 525 nm, lipase (range 0.2–1.5 U/mL, RSD 2.3%) was determined. This methodology permited the determination of every compound of the system, thus, H2O2 (range 0.5–2 mM, RSD 6.9%) and HRP (range 5.5–49.5 U/mL, RSD 3.6%) could also be determined. Lipase was determined in rabbit serum with 96.7 ± 3.3% and 102.9 ± 5.4% recoveries for two different lipase concentrations. Besides, H2O2 was determined in the disinfectant solution for contact lenses.
Introduction Chemiluminescence of the system luminol-H2O2-HRP is enhanced (up to 1000 times) by compounds such as benzothiazols [1–3], phenols [4–6], aromatic amines [7, 8] and arylboronic acids [9]. Enhanced chemiluminescent reactions have been used in immunoassays [10, 11] and in other analytical methods [12]. Fluorescein has also been described as an enhancer of this system [13]. It is a weak enhancer, but in addition to the enhancement, it acts as an acceptor of the chemiluminescent emission of luminol (425 nm) and re-emits the light at 525 nm by means of an efficient energy-transfer process [14–17]. This chemiluminescence enhanced energy transfer system combines the advantages of chemiluminescence techniques such as
A. Navas Díaz (쾷) · F. G. Sanchez · M. C. Torijas · J. Lovillo Department of Analytical Chemistry, Faculty of Sciences, University of Málaga, 29071-Málaga, Spain e-mail:
[email protected]
sensitivity and simplicity with the selectivity produced by measurements at the emission maximum of fluorescein. Compounds like 4-iodophenyl phosphate, 2-naphthyl phosphate, 2-naphthyl sulfate, 6-bromo-2-naphthyl-β-Dgalactopyranoside that can be converted into enhancers by a hydrolysis step, generally hydrolase enzyme mediated, are described as pro-enhancers of the luminol-H2O2-HRP system [18]. This type of coupled reactions have been used for the determination of the enzyme alkaline phosphatase, arylsulfatase, β-D-galactosidase, β-D-glucosidase [18] and cholinesterase [19]. Lipase plays an important role in human physiology and determination of its activity is important for the diagnosis of hyperlipidemia [20], acute pancreatitis [21] and other conditions too. This analysis needs to be carried out in many types of food and biotechnological industries [22]. Analytical methods to determine lipase are generally based on its enzymatic action on fatty compounds. Several assays for lipase activity are developed using different techniques [23–30]. The proposed method in this paper gives a new chemiluminescent technique. No bibliography antecedents have been found in the bibliographic search. In addition to this, several analytical methods to determine horseradish peroxidase and hydrogen peroxide involving enhanced chemiluminescent reactions have been described [31–33], most of these based on luminol chemiluminescence emission at 425 nm. In this method the emission is collected at 525 nm, spectrum region in which some interferences can be eliminated. The present work combines the advantages of chemiluminescence techniques such as sensitivity and simplicity, and the specificity of the fluorescein diacetate-lipase hydrolysis reaction to develop a chemiluminecent method for the assay of lipase. In this method the pro-enhancer fluorescein diacetate is converted into fluorescein by the enzyme lipase; the fluorescein released causes an enhancement of the luminol-H2O2-HRP system chemiluminescence emitting the energy transferred by the chemiluminescent system. The enhancement phenomenon allows us to obtain greater fluorescence intensities and so to get a
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chemiluminescent emission. This fact gives us the opportunity to use the emission at 525 nm as an analytical signal instead of the chemiluminescent emission at 425 nm, which is more exposed to spectral interferences. In addition to this, interferences from excitation energy are also avoided, because chemiluminescent energy transfer is the exciting source. Experimental parameters
Scheme 1 Chemical and photophysical pathways of the system
lower detection limit for the substrate analyses. Scheme 1 shows the chemical and photophysical pathways of the system. The utility of the system for the lipase determination was tested in serum rabbit. Other analytes, which participate in the chemical system, including hydrogen peroxide and HRP, are also determined.
Experimental Reagents. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), horseradish peroxidase (HRP) Type VI-A, RZ apr. 3.0, lipase (8 U/mg) and fluorescein diacetate were from Sigma (St. Louis, MO). Enzyme activities were taken from the commercial specifications. All other reagents used were analytical grades. Bidistilled and deionized water were used throughout. Fluorescein diacetate standard solution was prepared daily by weighing 8.250 mg of the pure compound and dissolving in 10 mL of 2-methoxyethanol. Luminol solution was prepared by dissolving 0.0913 g of luminol in a few drops of diluted NaOH solution; the volume was adjusted to 50 mL with 0.1 M tris-HCl buffer (pH 8.5). HRP was dissolved in 0.1 M tris-HCl, 8.5 pH buffer and lipase in bidistilled water. Apparatus. Luminescent measurements were performed in a Perkin-Elmer LS-50 spectrofluorometer (Beaconsfield, UK) with the light source switched off and running in the phosphorescence mode with 0.00 ms delay time. Emission slit was fitted at 20 nm and the photomultiplier voltage at 900 V. The reactants were introduced in a quartz cuvette and stirred with a magnetic intra-cuvette stirrer. The chemiluminescent reaction was triggered by injecting H2O2 with a syringe through a septum. The chemiluminescent or fluorescent emission was registered for 300 s. All the experiments were performed at 20 °C. Procedures. In a quartz cuvette 1 mL of 0.1 M tris-HCl buffer pH 8.5, lipase, fluorescein diacetate solution and bidistilled water up to 2 mL were introduced. After an incubation time of between 0 and 30 min, which depended on the analyte to be determined, luminol and HRP solutions were added and the reaction was triggered by the addition of H2O2. The final conditions for the indicated analytes are the following: FDA 1 × 10–4 M, lipase 3 U/mL, HRP 2.2 U/mL, luminol 7 × 10–5 M, H2O2 2 × 10–3 M.
Chemiluminescence intensity from the luminol-H2O2HRP system is affected by the pH because of the dependence of the fluorescence quantum yield of the emitter on pH [34]. To check the pH range, in order to obtain the maximum efficiency in the production of chemiluminescence, maximum intensity and area under peak from the time-emission profiles were measured. Figure 1 shows the results obtained from solutions with pHs between 6.5 and 9. From this figure it can be observed that maxima intensity and area were obtained working at pH around 8. The best selection to carry out the measurements is pH 8 because better precision can be obtained (the maximum chemiluminescence intensity is at pH 8.5 and at this point the measurements are more oscillating) and because such high pHs can affect the enzyme behavior (HRP and lipase). FDA acts as pre-intensifier of the luminol chemiluminescence. By the action of a hydrolase enzyme (lipase) the ester type links in the FDA molecule are broken down, releasing free fluorescein that acts as an enhancer. The extremely low solubility of FDA in water obliges us to solve it in an organic solvent. A high ratio of organic solvent in the luminol-H2O2-HRP-lipase system considerably diminishes enzyme activity, so the kind and properties of the organic solvent must be studied previously. Three solvents were assayed based on water compatibility and FDA solubility: tetrahydrofuran (THF), dioxane and 2-methoxyethanol (methyl cellosolve) [35]. Good solubility (5% of organic solvent) and maximum enzyme activity were obtained with methyl cellosolve. Increasing organic solvent percentage in the bulk solution diminishes lipase activity, depending on the solvent type. For 10–5 M DAF concentrations, the minimum organic phase percentage to avoid precipitation of the substract is
Results and discussion As depicted in Scheme 1, FDA is hydrolyzed by lipase releasing fluorescein that in turn acts as an enhancer of the chemiluminescent reaction luminol-H2O2-HRP and, at concentrations higher than 10–4 M, as an acceptor of the
Fig. 1 pH-effect on chemiluminescence maximum emission (A), and area under peak between 0 and 300 s (B). [luminol] = 6.67 × 10–5 M, [H2O2] = 2 × 10–3 M and [HRP] = 1.83 U/mL
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THF 95% (v/v) or dioxane 40% (v/v) or methyl cellosolve 2% (v/v). Thus, methyl cellosolve as solvent of the FDA substrate was selected because a lower ratio of the organic phase is established. Free fluorescein in the medium was provided by the lipase action on FDA. Since fluorescein acts as an acceptor in the energy transfer process, its concentration must be high enough to make the energy transfer process effective; thus the FDA concentration must be as high as possible. 10–3 M initial FDA concentration (5% organic phase) is not acceptable because some precipitation occurs and fluorescence quenching appears. 10–4 M and 10–5 M of FDA seem to be the most suitable initial concentrations to be assayed for the calibration graphs of lipase: we need a high concentration of FDA without precipitation to avoid fluorescence quenching. Another important experimental condition to be fixed is the incubation time needed to accomplish the hydrolytic break of FDA by lipase activity. Experiments were carried out with 10–5 M and 10–4 M FDA concentrations taking measurements in the time interval between 0–60 min. These measurements show that after 1 h the hydrolysis reaction of FDA is incomplete because the fluorescence intensity continues to increase weakly for times greater then 1 h. However, after 15 min of incubation, a high level of fluorescein is free in the solution. So, efficient energy transfer processes and stable measurements can be carried out from this time. Calibration curves Calibration curves of lipase obtained with 10–5 M and 10–4 M FDA concentrations show that although similar regres-
sion parameters were obtained, higher fluorescence signals (10%) can be obtained from 10–4 M FDA solutions. To obtain these calibration curves, maximum emission intensity was plotted versus lipase concentration. The maximum emission gives better analytical response than the area under peak or initial rate. Table 1 shows the analytical parameters of the calibration curve obtained under the conditions of luminol 7 × 10–5 M, H2O2 2 × 10–3 M, HRP 2.2 U/mL, FDA 10–4 M and incubation time of 15 min (it gives a high emission and better analytical parameters are not obtained with longer incubation times). Based on the system luminol-H2O2-HRP-lipase the determination of H2O2 has been developed. To avoid side effects of lipase concentration on the overall reaction, the influence of lipase concentration on the fluorescence signal was studied at two different incubation times. Figure 2 shows the plot of the data obtained. It can be observed that lipase concentrations between 3–5 U/mL do not significantly affect the emission (plot near zero order in respect to lipase). Thus, at these lipase concentrations, the signal does not depend on lipase and is proportional to the H2O2 concentration. An incubation time of 30 min and a lipase concentration of 3 U/mL were selected to set up the calibration curve for H2O2. The reaction was triggered by the addition of H2O2 20 s after the start of the recording. The curve slope is very sensitive to the H2O2 concentration changes. So, this parameter was selected for the calibration curve. The analytical figures of merit are shown in Table 1. For the HRP determination the same lipase concentration of 3 U/mL was used. An emission signal was observed without HRP (blank reaction). Table 1 gives analytical details about the calibration graph (maximum intensity versus HRP concentration) for the determination of HRP. Applications
Fig. 2 Plot of lipase concentration against emission intensity. Incubation time: A) 15 min. B) 30 min Table 1 Analytical parameters of lipase, H2O2 and HRP determinations
a significance
level 99.86%; limit of detection; c maximum intensity; d Vi = ∆I/∆t b
To assess the usefulness of the methodology for the determination of lipase activity, samples of rabbit serum were spiked with lipase and recovery experiments performed. The samples assayed do not require any previous treatment, and aliquots of the rabbit serum (previously spiked with standard lipase solution) were directly submitted to the general procedure for determination of lipase. The recovery experiments were made at two different concentrations, 0.4 and 0.8 U/mL of lipase. In the first case the lipase found was 0.39 ± 0.01 U/mL (n = 3) and in the second case it was 0.82 ± 0.04 U/mL (n = 3). The recoveries
Analytical parametera
Lipase determination
H2O2 determination
HRP determination
Linear range LDb RSD (n = 3), % Regression equation r (n = 5)
0.2–1.5 U/mL 0.1 U/mL 2.3 Ic = –8.0 + 184.6 [lipase] 0.99
0.5–2 mM 0.02 mM 6.9 Vid = –14.4 + 70049.8 [H2O2] 0.99
5.5–49.5 U/mL 2.8 U/mL 3.6 Ic = 20.7 + 2.3 [HRP] 0.99
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were 96.7 ± 3.3% and 102.9 ± 5.4%, respectively. Good recovery values and concordance between methods are obtained. A recovery assay for H2O2 determination was carried out in distilled water. A hydrogen peroxide concentration of 1 mM was selected and the obtained results were 0.91 ± 0.06 mM (n = 3). The recovery was 91.0 ± 5%. Determination of H2O2 was tested in disinfectant solution for contact lenses. In the case of lenses with endogenous H2O2 content, as indicated on the label, the general method was applied and the data obtained confirmed by comparing with the results from another alternative method based on a peroxidase biosensor [36]. The results were 1.48 M (RSD 4.7% for n = 3) and those obtained with the alternative method were 1.57 M (RSD 8.5% for n = 4). Thus, good recovery and concordance between the methods are obvious. The methodology gives promising perspectives to take advantage of the chemiluminescent system luminol-H2O2HRP for the determination of enhancers, enzymes and compound participants in the chemical system. Acknowledgement This research was supported by a grant from the DGICYT (Spain), project PB96–690.
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